Receptor-like proteins (RLPs) on plant cells have been implicated in immune responses and developmental processes. Although hundreds of RLP genes have been identified in plants, only a few RLPs have been functionally characterized in a limited number of plant species. Here, we identified RLPs in the pepper (Capsicum annuum) genome and performed comparative transcriptomics coupled with the analysis of conserved gene co-expression networks (GCNs) to reveal the role of core RLP regulators in pepper–pathogen interactions. A total of 102 RNA-seq datasets of pepper plants infected with four pathogens were used to construct CaRLP-targeted GCNs (CaRLP-GCNs). Resistance-responsive CaRLP-GCNs were merged to construct a universal GCN. Fourteen hub CaRLPs, tightly connected with defense-related gene clusters, were identified in eight modules. Based on the CaRLP-GCNs, we evaluated whether hub CaRLPs in the universal GCN are involved in the biotic stress response. Of the nine hub CaRLPs tested by virus-induced gene silencing, three genes (CaRLP264, CaRLP277, and CaRLP351) showed defense suppression with less hypersensitive response-like cell death in race-specific and non-host resistance response to viruses and bacteria, respectively, and consistently enhanced susceptibility to Ralstonia solanacearum and/or Phytophthora capsici. These data suggest that key CaRLPs are involved in the defense response to multiple biotic stresses and can be used to engineer a plant with broad-spectrum resistance. Together, our data show that generating a universal GCN using comprehensive transcriptome datasets can provide important clues to uncover genes involved in various biological processes.

Target activation-induced cytidine deaminase (Target-AID), a novel CRISPR/Cas9-based genome-editing tool, confers the base-editing capability on the Cas9 genome-editing system. It involves the fusion of cytidine deaminase (CDA), which catalyzes cytidine (C) to uridine (U) substitutions, to the mutated nickase-type nCas9 or deactivated-type dCas9. To confirm and extend the applicability of the Target-AID genome-editing system in tomatoes (Solanum lycopersicum L.), we transformed the model tomato cultivar “Micro-Tom” and commercial tomato cultivars using this system by targeting SlDELLA, which encodes a negative regulator of the plant phytohormone gibberellic acid (GA) signaling pathway. We confirmed that the nucleotide substitutions were induced by the Target-AID system, and we isolated mutants showing high GA sensitivity in both “Micro-Tom” and the commercial cultivars. Moreover, by successfully applying this system to ETHYLENE RECEPTOR 1 (SlETR1) with single sgRNA targeting, double sgRNA targeting, as well as dual-targeting of both SlETR1 and SlETR2 with a single sgRNA, we demonstrated that the Target-AID genome-editing system is a promising tool for molecular breeding in tomato crops. This study highlights an important aspect of the scientific and agricultural potential of the combinatorial use of the Target-AID and other base-editing systems.

The floral microbiome is of significant relevance to plant reproduction and crop productivity. While plant genotype is key to floral microbiome assembly, whether and how genotypic variation in floral traits and plant-level mutualistic and antagonistic interactions at the rhizosphere and phyllosphere influence the microbiome in the anthosphere remain little known. Using a factorial field experiment that manipulated biotic interactions belowground (mycorrhizae treatments) and aboveground (herbivory treatments) in three strawberry genotypes, we assessed how genotypic variation in flower abundance and size and plant-level biotic interactions influence the bidirectional relationships between floral volatile organic compounds (VOCs) and the floral microbiome using structural equation modeling. We found that plant genotype played a stronger role, overall, in shaping the floral microbiome than biotic interactions with mycorrhizae and herbivores. Genotypic variation in flower abundance and size influenced the emission of floral VOCs, especially terpenes (e.g. α- and β-pinene, ocimene isomers) and benzenoids (e.g. p-anisaldehyde, benzaldehyde), which in turn affected floral bacterial and fungal communities. While the effects of biotic interactions on floral traits including VOCs were weak, mycorrhizae treatments (mycorrhizae and herbivory + mycorrhizae) affected the fungal community composition in flowers. These findings improve our understanding of the mechanisms by which plant genotype influences floral microbiome assembly and provide the first evidence that biotic interactions in the rhizosphere and phyllosphere can influence the floral microbiome, and offer important insights into agricultural microbiomes.

MicroRNA172 (miR172) plays a role in regulating a diverse range of plant developmental processes, including flowering, fruit development and nodulation. However, its role in regulating flavonoid biosynthesis is unclear. In this study, we show that transgenic apple plants over-expressing miR172 show a reduction in red coloration and anthocyanin accumulation in various tissue types. This reduction was consistent with decreased expression of APETALA2 homolog MdAP2_1a (a miR172 target gene), MdMYB10, and targets of MdMYB10, as demonstrated by both RNA-seq and qRT-PCR analyses. The positive role of MdAP2_1a in regulating anthocyanin biosynthesis was supported by the enhanced petal anthocyanin accumulation in transgenic tobacco plants overexpressing MdAP2_1a, and by the reduction in anthocyanin accumulation in apple and cherry fruits transfected with an MdAP2_1a virus-induced-gene-silencing construct. We demonstrated that MdAP2_1a could bind directly to the promoter and protein sequences of MdMYB10 in yeast and tobacco, and enhance MdMYB10 promotor activity. In Arabidopsis, over-expression of miR172 reduced flavonoid (including anthocyanins and flavonols) concentration and RNA transcript abundance of flavonoid genes in plantlets cultured on medium containing 7% sucrose. The anthocyanin content and RNA abundance of anthocyanin genes could be partially restored by using a synonymous mutant of MdAP2_1a, which had lost the miR172 target sequences at mRNA level, but not restored by using a WT MdAP2_1a. These results indicate that miR172 inhibits flavonoid biosynthesis through suppressing the expression of an AP2 transcription factor that positively regulates MdMYB10.

Multipartite viral vectors provide a simple, inexpensive and effective biotechnological tool to transiently manipulate (i.e. reduce or increase) gene expression in planta and characterise the function of genetic traits. The development of virus-induced gene regulation (VIGR) systems usually involve the targeted silencing or overexpression of genes involved in pigment biosynthesis or degradation in plastids, thereby providing rapid visual assessment of success in establishing RNA- or DNA-based VIGR systems in planta. Carotenoids pigments provide plant tissues with an array of yellow, orange, and pinkish-red colours. VIGR-induced transient manipulation of carotenoid-related gene expression has advanced our understanding of carotenoid biosynthesis, regulation, accumulation and degradation, as well as plastid signalling processes. In this review, we describe mechanisms of VIGR, the importance of carotenoids as visual markers of technology development, and knowledge gained through manipulating carotenogenesis in model plants as well as horticultural crops not always amenable to transgenic approaches. We outline how VIGR can be utilised in plants to fast-track the characterisation of gene function(s), accelerate fruit tree breeding programs, edit genomes, and biofortify plant products enriched in carotenoid micronutrients for horticultural innovation.

Improving fruit quality traits such as metabolic composition remains a challenge for tomato breeders. To better understand the genetic architecture of these traits and decipher the demographic history of the loci controlling tomato quality traits, we applied an innovative approach using multiple haplotype-based analyses, aiming to test the potentials of haplotype based study in association and genomic prediction studies. We performed and compared haplotype vs SNP-based associations (hapQTL) with multi-locus mixed model (MLMM), focusing on tomato fruit weight and metabolite contents (i.e. sugars, organic acids and amino acids). Using a panel of 163 tomato accessions genotyped with 5995 SNPs, we detected a total of 784 haplotype blocks, with an average size of haplotype blocks ∼58 kb. A total of 108 significant associations for 26 traits were detected thanks to Haplotype/SNP-based Bayes models. Haplotype-based Bayes model (97 associations) outperformed SNP-based Bayes model (50 associations) and MLMM (53 associations) in identifying marker-trait associations as well as in genomic prediction (especially for those traits with moderate to low heritability). To decipher the demographic history, we identified 24 positive selective sweeps using the integrated haplotype score (iHS). Most of the significant associations for tomato quality traits were located within selective sweeps (54.63% and 71.7% in hapQTL and MLMM models, respectively). Promising candidate genes were identified controlling tomato fruit weight and metabolite contents. We thus demonstrated the benefits of using haplotypes for evolutionary and genetic studies, providing novel insights into tomato quality improvement and breeding history.

Over the past two centuries, introgression through repeated backcrossing has introduced disease resistance from wild grape species into the domesticated lineage Vitis vinifera subsp. sativa. Introgression lines are being cultivated over increasing vineyard surface areas, as their wines now rival in quality those obtained from preexisting varieties. There is, however, a lot of debate about whether and how wine laws defining commercial product categories, which are based on the classification of V. vinifera and interspecific hybrid grapes, should be revised to accommodate novel varieties that do not fit either category. Here, we developed a method of multilocus genotype analysis using short–read resequencing to identify haplotypic blocks of wild ancestry in introgression lines and quantify the physical length of chromosome segments free–of–introgression or with monoallelic and biallelic introgression. We used this genomic data to characterize species, hybrids and introgression lines and show that newly released resistant varieties contain 76.5–94.8% of V. vinifera DNA. We found that varietal wine ratings are not always commensurate with the percentage of V. vinifera ancestry and linkage drag of wild alleles around known resistance genes persists over at least 7.1–11.5 Mb, slowing down the recovery of the recurrent parental genome. This method also allowed us to identify the donor species of resistance haplotypes, define the ancestry of wild genetic background in introgression lines with complex pedigrees, validate the ancestry of the historic varieties Concord and Norton, and unravel sample curation errors in public databases.

Vitis has two subgenera: Euvitis, which includes commercially important Vitis vinifera and interspecific hybrid cultivars, and Muscadinia. Of note, the market for Muscadinia grapes remains small, and only Muscadinia rotundifolia is cultivated as a commercial crop. To establish a basis for the study of Muscadinia species, we generated chromosome-level whole-genome sequences of Muscadinia rotundifolia cv. Noble. A total of 393.8 Mb of sequences were assembled from 20 haploid chromosomes, and 26 394 coding genes were identified from the sequences. Comparative analysis with the genome sequence of V. vinifera revealed a smaller size of the M. rotundifolia genome but highly conserved gene synteny. A genome-wide association study of 12 Muscadinia berry-related traits was performed among 356 individuals from breeding populations of M. rotundifolia. For the transferability of markers between Euvitis and Muscadinia, we used 2000 core genome rhAmpSeq markers developed to allow marker transferability across Euvitis species. A total of 1599 (80%) rhAmpSeq markers returned data in Muscadinia. From the GWAS analyses, we identified a total of 52 quantitative trait nucleotides (QTNs) associated with the 12 berry-related traits. The transferable markers enabled the direct comparison of the QTNs with previously reported results. The whole-genome sequences along with the GWAS results provide a new basis for the extensive study of Muscadinia species.

Leptospermum scoparium J. R. Forst et G. Forst, known as mānuka by Māori, the indigenous people of Aotearoa (New Zealand), is a culturally and economically significant shrub species, native to New Zealand and Australia. Chemical, morphological and phylogenetic studies have indicated geographical variation of mānuka across its range in New Zealand, and genetic differentiation between New Zealand and Australia. We used pooled whole genome re-sequencing of 76 L. scoparium and outgroup populations from New Zealand and Australia to compile a dataset totalling ∼2.5 million SNPs. We explored the genetic structure and relatedness of L. scoparium across New Zealand, and between populations in New Zealand and Australia, as well as the complex demographic history of this species. Our population genomic investigation suggests there are five geographically distinct mānuka gene pools within New Zealand, with evidence of gene flow occurring between these pools. Demographic modelling suggests three of these gene pools have undergone expansion events, whilst the evolutionary histories of the remaining two have been subjected to contractions. Furthermore, mānuka populations in New Zealand are genetically distinct from populations in Australia, with coalescent modelling suggesting these two clades diverged ∼9–12 million years ago. We discuss the evolutionary history of this species and the benefits of using pool-seq for such studies. Our research will support the management and conservation of mānuka by landowners, particularly Māori, and the development of a provenance story for the branding of mānuka based products.

The endophytic microbiome of plants is believed to have a significant impact on its physiology and disease resistance, however, the role of host genotype in determining the composition of the endophytic microbiome of apple root systems remains an open question that has important implications for defining breeding objectives. In the current study, the bacterial and fungal microbiota associated with four different apple rootstocks planted in April, 2018 in the same soil environment and harvested in May, 2019 were evaluated to determine the role of genotype on the composition of both the bacterial and fungal communities. Results demonstrated a clear impact of genotype and root size on microbial composition and diversity. The fungal community was more affected by plant genotype whereas the bacterial community was shaped by root size. Fungal and bacterial abundance was equal between different-sized roots however, significantly higher microbial counts were detected in rhizosphere samples compared to root endosphere samples. This study provides information that can be used to develop a comprehensive and readily applicable understanding of the impact of genotype and environmental factors on the establishment of plant microbiome, as well as its potential function and impact on host physiology.

BABY BOOM (BBM) is a member of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family and its expression has been shown to improve herbaceous plant transformation and regeneration. However, this improvement has not been shown clearly for tree species. This study demonstrated that the efficiency of transgenic apple (Malus domestica Borkh.) plant production was dramatically increased by ectopic expression of the MdBBM1 gene. “Royal Gala” apple plants were first transformed with a CaMV 35S-MdBBM1 construct (MBM) under kanamycin selection. These MBM transgenic plants exhibited enhanced shoot regeneration from leaf explants on tissue culture media, with most plants displaying a close-to-normal phenotype compared with CaMV 35S-GUS transgenic plants when grown under greenhouse conditions, the exception being that some plants had slightly curly leaves. Thin leaf sections revealed the MBM plants produced more cells than the GUS plants, indicating that ectopic-expression of MdBBM1 enhanced cell division. Transcriptome analysis showed that mRNA levels for cell division activators and repressors linked to hormone (auxin, cytokinin and brassinosteroid) signalling pathways were enhanced and reduced, respectively, in the MBM plants compared with the GUS plants. Plants of eight independent MBM lines were compared with the GUS plants by re-transforming them with an herbicide-resistant gene construct. The number of transgenic plants produced per 100 leaf explants was 0–3% for the GUS plants, 3–8% for five MBM lines, and 20–30% for three MBM lines. Our results provided a solution for overcoming the barriers to transgenic plant production in apple, and possibly in other trees.

Trichomes are specialised epidermal cells developed in the aerial surface of almost every terrestrial plant. These structures form physical barriers, which combined with their capability of synthesis of complex molecules, prevent plagues from spreading and confer trichomes a key role in the defence against herbivores. In this work, the tomato gene HAIRPLUS (HAP) that controls glandular trichome density in tomato plants was characterised. HAP belongs to a group of proteins involved in histone tail modifications although some also bind methylated DNA. HAP loss of function promotes epigenomic modifications in the tomato genome reflected in numerous differentially methylated cytosines and causes transcriptomic changes in hap mutant plants. Taken together, these findings demonstrate that HAP links epigenome remodelling with multicellular glandular trichome development and reveal that HAP is a valuable genomic tool for pest resistance in tomato breeding.

In the current study, we used a grapevine cell line in which actin filaments are labeled by GFP to show that aluminum causes actin remodeling through activation of NADPH oxidase in the plasma membrane, followed by activation of phytoalexin synthesis genes. Elimination of actin filaments by latrunculin B disrupts gene activation and inhibition of MAPK signaling by the inhibitor PD98059. Interestingly, aluminum also induces the transcription of ISOCHORISMATE SYNTHASE, a key enzyme for the synthesis of salicylic acid, as well as PR1, a gene that is known to be responsive to salicylic acid. However, while salicylic acid responses are usually a hallmark of the hypersensitive response, aluminum-triggered defense is not accompanied by cell death. Both actin remodeling and gene activation in response to aluminum can be suppressed by the natural auxin indole acetic acid, suggesting that the actin response is not caused by nonspecific signaling. Further evidence for the specificity of the aluminum-triggered activation of phytoalexin synthesis genes comes from experiments in which plant peptide elicitors induce significant cellular mortality but do not evoke induction of these transcription. The response in grapevine cells can be recapitulated in grapevine leaf discs from two genotypes contrasting in stilbene inducibility. Here, aluminum can induce accumulation of the central grapevine phytoalexin, the stilbene aglycone trans-resveratrol; this is preceded by a rapid induction of transcription for RESVERATROL SYNTHASE and the regulating transcription factor MYB14. The amplitude of this induction reflects the general stilbene inducibility of these genotypes, indicating that the aluminum effect is not caused by nonspecific toxicity but by activation of specific signaling pathways. The findings are discussed in relation to a model in which actin filaments activate a specific branch of defense signaling, acting in concert with calcium-dependent PAMP-triggered immunity. This pathway links the apoplastic oxidative burst through MAPK signaling with the activation of defense-related transcription.

In citrus, 1,6-rhamnosytransferase (1,6RhaT) and 1,2-rhamnosytransferase (1,2RhaT) catalyze flavanone-7- O-glucosides to form nonbitter flavanone rutinosides (FRs) and bitter flavanone neohesperidosides (FNs), respectively. As revealed in this study of fruit peels from 36 citrus accessions, FRs varied from undetectable levels in pummelo and kumquat to being the dominant flavonoids in sweet orange and loose-skin mandarins. Furthermore, a previously annotated full-length 1,6RhaT-like gene was identified as another 1,6RhaT-encoding gene by in vitro experiments. In total, 28 alleles of full-length 1,6RhaTs were isolated and classified into A, B and C types with only type A alleles encoding a functional protein. Coincidently, only the accessions that contained FRs harbored type A alleles, as was further verified in two F1 hybrid populations. Moreover, the inferior substrate conversion efficiency of 1,6RhaTs in comparison with that of 1,2RhaT in vitro might partly explain the lower proportions of FRs to total flavanone disaccharides in citrus hybrids harboring both functional rhamnosyltransferases. Our findings provide a better understanding of FR content variations among citrus and are meaningful for a mechanistic illustration of citrus flavonoid metabolism and fruit quality improvement practices.

Rejuvenation refers to the transition from an adult state to a juvenile state. Trunk truncation at the base of the tree can result in tree rejuvenation. However, little is known about the association of rejuvenation with leaf biomass and flavonoid accumulation. The results of this study showed that, compared with control leaves, leaves of renewed Ginkgo biloba shoots were larger, thicker, and more lobed and had higher fresh/dry weights and chlorophyll contents. The leaf biomass per hectare of rejuvenated trees was twofold higher than that of the untruncated controls. Moreover, we observed a marked increase in the accumulation of flavonol glycosides via metabolomic analysis and detected upregulated expression of genes involved in flavonoid biosynthesis, including CHS, FLS, F3’H, DFR, and LAR. Overexpression of GbCHS in ginkgo calli confirmed that GbCHS plays an important role in flavonoid biosynthesis. Interestingly, the contents of gibberellins significantly increased in the rejuvenated leaves. Moreover, exogenous gibberellin treatment significantly increased GbCHS expression and flavonoid contents. Our findings show that truncation can stimulate tree rejuvenation by altering hormone levels, representing an effective and feasible approach for enhancing the biomass and flavonoid content of G. biloba leaves.

Gummosis, one of the most detrimental diseases to the peach industry worldwide, can be induced by Lasiodiplodia theobromae. Ethylene (ET) is known to trigger the production of gum exudates, but the mechanism underlying fungus-induced gummosis remains unclear. In this study, L. theobromae infection triggered the accumulation of ET and jasmonic acid (JA) but not salicylic acid (SA) in a susceptible peach variety. Gaseous ET and its biosynthetic precursor increased gum formation, whereas ET inhibitors repressed it. SA and methyl- jasmonate treatments did not influence gum formation. RNA-seq analysis indicated that L. theobromae infection and ET treatment induced a shared subset of 1808 differentially expressed genes, which were enriched in the category “starch and sucrose, UDP-sugars metabolism”. Metabolic and transcriptional profiling identified a pronounced role of ET in promoting the transformation of primary sugars (sucrose, fructose, and glucose) into UDP-sugars, which are substrates of gum polysaccharide biosynthesis. Furthermore, ethylene insensitive3-like1 (EIL1), a key transcription factor in the ET pathway, could directly target the promoters of the UDP-sugar biosynthetic genes UXS1a, UXE, RGP and MPI and activate their transcription, as revealed by firefly luciferase and yeast one-hybrid assays. On the other hand, the supply of SA and inhibitors of ET and JA decreased the lesion size. ET treatment reduced JA levels and the transcription of the JA biosynthetic gene OPR but increased the SA content and the expression of its biosynthetic gene PAL. Overall, we suggest that endogenous and exogenous ET aggravate gummosis disease by transactivating UDP-sugar metabolic genes through EIL1 and modulating JA and SA biosynthesis in L. theobromae-infected peach shoots. Our findings shed light on the molecular mechanism by which ET regulates plant defense responses in peach during L. theobromae infection.

There are several causes for the great diversity in floral terpenes. The terpene products are determined by the catalytic fidelity, efficiency and plasticity of the active sites of terpene synthases (TPSs). However, the molecular mechanism of TPS in catalyzing terpene biosynthesis and its evolutionary fate in wild plant species remain largely unknown. In this study, the functionality of terpene synthases and their natural variants were assessed in two Northeastern Asia endemic columbine species and their natural hybrid. Synoptically, TPS7, TPS8, and TPS9 were highly expressed in these Aquilegia species from the Zuojia population. The in vitro and in vivo enzymatic assays revealed that TPS7 and TPS8 mainly produced (+)-limonene and β-sesquiphellandrene, respectively, whereas TPS9 produced pinene, similar to the major components released from Aquilegia flowers. Multiple sequence alignment of Aquilegia TPS7 and TPS8 in the Zuojia population revealed amino acid polymorphisms. Domain swapping and amino acid substitution assays demonstrated that 413A, 503I and 529D had impacts on TPS7 catalytic activity, whereas 420G, 538F and 545 L affected the ratio of β-sesquiphellandrene to β-bisabolene in TPS8. Moreover, these key polymorphic amino acid residues were found in Aquilegia species from the Changbai Mountain population. Interestingly, amino acid polymorphisms in TPSs were present in individuals with low expression levels, and nonsynonymous mutations could impact the catalytic activity or product specificity of these genes. The results of this study will shed new light on the function and evolution of TPS genes in wild plant species and are beneficial to the modification of plant fragrances.

Cultivated chrysanthemum (Chrysanthemum × morifolium Ramat.) is a beloved ornamental crop due to the diverse capitula types among varieties, but the molecular mechanism of capitulum development remains unclear. Here, we report a 2.60 Gb chromosome-scale reference genome of C. lavandulifolium, a wild Chrysanthemum species found in China, Korea and Japan. The evolutionary analysis of the genome revealed that only recent tandem duplications occurred in the C. lavandulifolium genome after the shared whole genome triplication (WGT) in Asteraceae. Based on the transcriptomic profiling of six important developmental stages of the radiate capitulum in C. lavandulifolium, we found genes in the MADS-box, TCP, NAC and LOB gene families that were involved in disc and ray floret primordia differentiation. Notably, NAM and LOB30 homologs were specifically expressed in the radiate capitulum, suggesting their pivotal roles in the genetic network of disc and ray floret primordia differentiation in chrysanthemum. The present study not only provides a high-quality reference genome of chrysanthemum but also provides insight into the molecular mechanism underlying the diverse capitulum types in chrysanthemum.

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the causal agents of grapevine leafroll disease (GLD), which severely impacts grapevine production in most viticultural regions of the world. The development of virus-resistant plants is a desirable strategy for the efficient control of viral diseases. However, natural resistant resources have not been reported in the genus Vitis, and anti-GLRaV-3 research has been quite limited in grapevine. In this study, by expressing FnCas9 and LshCas13a, we established a highly effective transgenic construct screening system via an optimized Agrobacterium-mediated transient delivery system in grapevine plantlets. Our study indicated that CRISPR/FnCas9 and LshCas13a caused GLRaV-3 inhibition. Moreover, three vectors-pCR01-CP, pCR11-Hsp70h and pCR11-CP-exhibited the most robust inhibition efficiency compared to those targeting other sites and could be further engineered to generate GLRaV-3-resistant grapevine. In addition, the viral interference efficiency of FnCas9 was dependent on its RNA binding activity. The efficiency of virus inhibition was positively correlated with the level of Cas gene expression. Importantly, we demonstrated that LshCas13a had better interference efficiency against viruses than FnCas9. In summary, this study confirmed that these two RNA-targeting CRISPR mechanisms can confer immunity against viruses in grapevine, providing new avenues to control GLRaV-3 or other RNA viruses in fruit crops.

In some horticultural crops, such as Cucurbitaceae, Solanaceae, and Rosaceae species, fruit set and development can occur without the fertilization of ovules, a process known as parthenocarpy. Parthenocarpy is an important agricultural trait that can not only mitigate fruit yield losses caused by environmental stresses but can also induce the development of seedless fruit, which is a desirable trait for consumers. In the present review, the induction of parthenocarpic fruit by the application of hormones such as auxins (2,4 dichlorophenoxyacetic acid; naphthaleneacetic acid), cytokinins (forchlorfenuron; 6-benzylaminopurine), gibberellic acids, and brassinosteroids is first presented. Then, the molecular mechanisms of parthenocarpic fruit formation, mainly related to plant hormones, are presented. Auxins, gibberellic acids, and cytokinins are categorized as primary players in initiating fruit set. Other hormones, such as ethylene, brassinosteroids, and melatonin, also participate in parthenocarpic fruit formation. Additionally, synergistic and antagonistic crosstalk between these hormones is crucial for deciding the fate of fruit set. Finally, we highlight knowledge gaps and suggest future directions of research on parthenocarpic fruit formation in horticultural crops.

Cold acclimation (CA) and deacclimation (DA), which are often accompanied by changes in freezing tolerance (FT), carbohydrates and hormones, are crucial for winter survival, especially under global warming. Plants with weak CA and premature DA caused by warm winters and/or unseasonal warm spells can be easily injured by adverse reactions to cold. Thus, understanding the molecular mechanisms of FT is imperative. In this study, we used high-throughput RNA-seq to profile the CA and DA of leaves of overwintering Rhododendron “Miyo-no-Sakae” over time; these leaves do not undergo dormancy but do undergo photoprotection during CA, and they do not grow during DA. Using Mfuzz and weighted gene coexpression network analysis, we identified specific transcriptional characteristics in each phase of CA and DA and proposed networks involving coexpressed genes and physiological traits. In particular, we discovered that the circadian rhythm is critical for obtaining the strongest FT, and high expression of circadian rhythm-related genes might be linked to sugar accumulation during winter. Furthermore, evergreen leaves exhibited robust photoprotection during winter, as revealed by high values of nonphotochemical quenching, high expression of transcripts annotated as “early light-induced proteins”, loss of granum stacks and destacking of thylakoids, all of which were alleviated during DA. The strong requirement of photoprotection could be the reason for decreased abscisic acid (ABA) and jasmonic acid (JA) contents during CA, and decreases in ABA and JA contents may contribute to decreases in lignin content. Our data suggest that the molecular mechanisms of FT in overwintering leaves are unique, which may be due to the high requirements for photoprotection during winter.

Cuticular wax on plant aerial surfaces plays a vital role in the defense against various stresses, and the genes related to wax metabolism have been well documented in several model plants. However, there is very limited research on the key enzymes and transcription factors (TFs) associated with carbon chain distribution and wax biosynthesis in citrus fruit. In this study, an analysis of wax metabolites indicated that even carbon-chain (C₂₄-C₂₈) metabolites are the dominant wax components in citrus fruit, and a 3- ketoacyl-CoA synthase (KCS) family gene (CsKCS20) plays an important role in the carbon chain distribution during wax biosynthesis in a wax-deficient mutant (MT). Expression of CsKCS20 in yeast indicated that CsKCS20 can catalyze the biosynthesis of C₂₂ and C₂₄ very-long-chain fatty acids (VLCFAs). In addition, transcriptome and sequence analysis indicated that the differential expression of CsKCS20 between the wild-type (WT) and MT fruit can be partly attributed to the regulation of CsMYB96, which was further confirmed by yeast one-hybrid (Y1H) assays, electrophoretic mobility shift assays (EMSAs) and dual luciferase assays . The functions of CsMYB96 and CsKCS20 in wax biosynthesis were further validated by heterologous expression in Arabidopsis. In summary, this study elucidates the important roles of CsKCS20 and CsMYB96 in regulating VLCFA elongation and cuticular wax biosynthesis, which provides new directions for the improvement of citrus fruit wax quality in genetic breeding programs.

MYB transcription factors play important roles in plant responses to cold stress, but the associated underlying mechanisms remain unclear. In this study, a cold-induced MYB transcription factor, DgMYB2, was isolated from chrysanthemum (Chrysanthemum morifolium Ramat). DgMYB2 was localized to the nucleus and exhibited transactivational activity. Overexpression of DgMYB2 improved cold tolerance in chrysanthemum, while cold tolerance in the antisense suppression lines decreased compared to that of the wild type. Additionally, electrophoretic mobility shift assays, chromatin immunoprecipitation, luciferase complementary imaging analysis, and dual-luciferase reporter gene detection experiments confirmed that DgMYB2 directly targets DgGPX1 and increases the activity of glutathione peroxidase to reduce the accumulation of reactive oxygen species, thereby improving cold resistance in chrysanthemum.

Free amino acids are one of the main chemical components in tea, and they contribute to the pleasant flavor, function, and quality of tea, notably the level of theanine. Here, a high-density genetic map was constructed to characterize quantitative trait loci (QTLs) for free amino acid content. A total of 2688 polymorphic SNP markers were obtained using genotyping-by-sequencing (GBS) based on 198 individuals derived from a pseudotestcross population of “Longjing 43”× “Baijiguan”, which are elite and albino tea cultivars, respectively. The 1846.32 cM high-density map with an average interval of 0.69 cM was successfully divided into 15 linkage groups (LGs) ranging from 93.41 cM to 171.28 cM. Furthermore, a total of 4 QTLs related to free amino acid content (theanine, glutamate, glutamine, aspartic acid and arginine) identified over two years were mapped to LG03, LG06, LG11 and LG14. The phenotypic variation explained by these QTLs ranged from 11.8% to 23.7%, with an LOD score from 3.56 to 7.7. Furthermore, several important amino acid metabolic pathways were enriched based on the upregulated differentially expressed genes (DEGs) among the offspring. These results will be essential for fine mapping genes involved in amino acid pathways and diversity, thereby providing a promising avenue for the genetic improvement of tea plants.

Mulberry (Morus spp .) is an economically important plant as the main food plant used for rearing domesticated silkworm and it has multiple uses in traditional Chinese medicine. Two basic chromosome numbers (Morus notabilis, n = 7, and Morus alba, n = 14) have been reported in the genus Morus, but the evolutionary history and relationship between them remain unclear. In the present study, a 335-Mb high-quality chromosome-scale genome was assembled for the wild mulberry species M. notabilis. Comparative genomic analyses indicated high chromosomal synteny between the 14 chromosomes of cultivated M. alba and the six chromosomes of wild M. notabilis. These results were successfully verified by fluorescence in situ hybridization. Chromosomal fission/fusion events played crucial roles in the chromosome restructuring process between M. notabilis and M. alba. The activity of the centromere was another key factor that ensured the stable inheritance of chromosomes. Our results also revealed that long terminal repeat retrotransposons were a major driver of the genome divergence and evolution of the mulberry genomes after they diverged from each other. This study provides important insights and a solid foundation for studying the evolution of mulberry, allowing the accelerated genetic improvement of cultivated mulberry species.

Radish (Raphanus sativus L.) belongs to the family Brassicaceae. The Yunnan red radish variety contains relatively large amounts of anthocyanins, making them important raw materials for producing edible red pigment. However, the genetic mechanism underlying this pigmentation has not been fully characterized. Here, the radish inbred line YAAS-WR1 (white root skin and white root flesh) was crossed with the inbred line YAAS-RR1 (red root skin and red root flesh) to produce F₁ , F>₂ , BC>₁ P₁ , and BC₁ P₂ populations. Genetic analyses revealed that the pigmented/non-pigmented and purple/red traits were controlled by two genetic loci. The F₂ population and the specific-locus amplified fragment sequencing (SLAF-seq) technique were used to construct a high-density genetic map (1230.16 cM), which contained 4032 markers distributed in nine linkage groups, with a mean distance between markers of 0.31 cM. Additionally, two quantitative trait loci (QAC1 and QAC2) considerably affecting radish pigmentation were detected. A bioinformatics analysis of the QAC1 region identified 58 predicted protein-coding genes. Of these, RsF3 ^′ H , which is related to anthocyanin biosynthesis, was revealed as a likely candidate gene responsible for the purple/red trait. The results were further verified by analyzing gene structure and expression. Regarding QAC2, RsMYB1.3 was determined to be a likely candidate gene important for the pigmented/non-pigmented trait, with a 4-bp insertion in the first exon that introduced a premature termination codon in the YAAS-WR1 sequence. Assays demonstrated that RsMYB1.3 interacted with RsTT8 and activated RsTT8 and RsUFGT expression . These findings may help clarify the complex regulatory mechanism underlying radish anthocyanin synthesis. Furthermore, this study’s results may be relevant for the molecular breeding of radish to improve the anthocyanin content and appearance of the taproots.

The jujube witches’ broom (JWB) phytoplasma is associated with witches’ broom, dwarfism, and smaller leaves in jujube, resulting in yield losses. In this study, eight putative JWB effector proteins were identified from potential mobile units of the JWB genome. Among them, Zaofeng6 induced witches’ broom symptoms in Arabidopsis and jujube. Zaofeng6-overexpressing Arabidopsis and unrooted jujube transformants displayed witches’ broom-like shoot proliferation. Transient expression of Zaofeng6 induced hypersensitive response like cell death and expression of hypersensitive response marker genes, like harpin-induced gene 1 (H1N1), and the pathogenesis-related genes PR1, PR2, and PR3 in transformed Nicotiana benthamiana leaves, suggesting that Zaofeng6 could be a virulence effector. Yeast two-hybrid library screening and bimolecular fluorescence complementation confirmed that Zaofeng6 interacts with ZjTCP7 through its first two α-helix domains in the cell nuclei. ZjTCP7 mRNA and protein abundance decreased in Zaofeng6 transgenic jujube seedlings. The expression of some genes in the strigolactone signaling pathway (ZjCCD7, ZjCCD8, and CYP711A1) were down-regulated in jujube shoots overexpressing Zaofeng6 and in zjtcp7 CRISPR/Cas9 mutants. Zaofeng6 induces shoot proliferation through decreased expression of ZjTCP7 at the transcriptional and translational levels.

Grafting with pumpkin rootstock is commonly used not only to improve the quality of cucumber fruits but also to confer biotic or abiotic stress tolerance. However, the molecular mechanism of the response of grafted cucumbers to drought stress and the possible roles of mobile mRNAs in improving stress tolerance have remained obscure. Hence, we conducted transcriptome sequencing and combined it with morphophysiological experiments to compare the response of homografts (cucumber as scion and rootstock) and heterografts (cucumber as scion and pumpkin as rootstock) to drought stress. After applying drought stress, homografts and heterografts expressed 2960 and 3088 genes, respectively, in response to the stress. The differentially expressed genes identified in heterografts under drought stress were categorized into different stress-responsive groups, such as carbohydrate metabolism (involved in osmotic adjustment by sugar accumulation), lipid and cell wall metabolism (involved in cell membrane integrity by a reduction in lipid peroxidation), redox homeostasis (increased antioxidant enzyme activities), phytohormone (increased abscisic acid content), protein kinases, and transcription factors, using MapMan software. Earlier and greater H₂O₂ accumulation in xylem below the graft union was accompanied by leaf abscisic acid accumulation in heterografts in response to drought stress. Greater leaf abscisic acid helped heterografted cucumbers to sense and respond to drought stress earlier than homografts. The timely response of heterografts to drought stress led to the maintenance of higher water content in the leaves even in the late stage of drought stress. The mobile mRNAs identified in heterografts were mostly related to photosynthesis, which would be the possible reason for improved chlorophyll content and maximum photochemical efficiency of photosystem II (Fv/Fm). The existence of some stress-responsive pumpkin (rootstock) mRNAs in cucumber (scion), such as heat shock protein (HSP70, a well-known stress-responsive gene), led to higher proline accumulation than in homografts. Expression of mobile and immobile stress-responsive mRNAs and timely response of heterografts to drought stress could improve drought tolerance in pumpkin-rooted plants.

Pollen tube growth is critical for the sexual reproduction of flowering plants. Catharanthus roseus receptor-like kinases (CrRLK1L) play an important role in plant sexual reproduction, pollen tube growth, and male and female gametophyte recognition. Here, we identified a CrRLK1L protein in pear (Pyrus bretschneideri), PbrCrRLK1L13, which is necessary for normal tip growth of the pollen tube. When PbrCrRLK1L13 was knocked down, the pollen tube grew faster. Interaction analysis showed that the kinase domain of PbrCrRLK1L13 interacted with the C-terminal region of PbrGEF8, and PbrCrRLK1L13 activated the phosphorylation of PbrGEF8 in vitro. Furthermore, PbrROP1 and PbrROP2 were the downstream targets of PbrCrRLK1L13-PbrGEF8. When we knocked down the expression of PbrCrRLK1L13, PbrGEF8, or PbrROP1/2, the balance of cellulose deposition in the pollen tube wall was disrupted. Considering these factors, we proposed a model for a signaling event regulating pear pollen tube growth. During pear pollen tube elongation, PbrCrRLK1L13 acted as a surface regulator of the PbrROP1 and PbrROP2 signaling pathway via PbrGEF8 to affect the balance of cellulose deposition and regulate pear pollen tube growth.

Pueraria lobata var. thomsonii (hereinafter abbreviated as Podalirius thomsonii), a member of the legume family, is one of the important traditional Chinese herbal medicines, and its puerarin extract is widely used in the health and pharmaceutical industry. Here, we assembled a high-quality genome of P. thomsonii using long-read single-molecule sequencing and Hi-C technologies. The genome assembly is ∼1.37 Gb in size and consists of 5145 contigs with a contig N50 of 593.70 kb, further clustered into 11 pseudochromosomes. Genome structural annotation resulted in ∼869.33 Mb (∼62.70% of the genome) repeat regions and 45 270 protein-coding genes. Genome evolution analysis revealed that P. thomsonii is most closely related to soybean and underwent two ancient whole-genome duplication events; one was in the common ancestor shared by legume species and the other occurred independently at around 7.2 million years ago, after its speciation. A total of 2373 gene families were found to be unique in P. thomsonii compared with five other legume species. Genes and metabolites related to puerarin content in tuberous tissues were characterized. A total of 572 genes that were upregulated in the puerarin biosynthesis pathway were identified, and 235 candidate genes were further enriched by omics data. Furthermore, we identified six 8-C-glucosyltransferase (8-C-GT) candidate genes significantly involved in puerarin metabolism. Our study filled a key genomic gap in the legume family, and provided valuable multi-omic resources for the genetic improvement of P. thomsonii .

Somaclonal variation arising from tissue culture may provide a valuable resource for the selection of new germplasm, but may not preserve true-to-type characteristics, which is a major concern for germplasm conservation or genome editing. The genomic changes associated with dedifferentiation and somaclonal variation during long-term in vitro culture are largely unknown. Sweet orange was one of the earliest plant species to be cultured in vitro and induced via somatic embryogenesis. We compared four sweet orange callus lines after 30 years of constant tissue culture with newly induced calli by comprehensively determining the single-nucleotide polymorphisms, copy number variations, transposable element insertions, methylomic and transcriptomic changes. We identified a burst of variation during early dedifferentiation, including a retrotransposon outbreak, followed by a variation purge during long-term in vitro culture. Notably, CHH methylation showed a dynamic pattern, initially disappearing during dedifferentiation and then more than recovering after 30 years of in vitro culture. We also analyzed the effects of somaclonal variation on transcriptional reprogramming, and indicated subgenome dominance was evident in the tetraploid callus. We identified a retrotransposon insertion and DNA modification alternations in the potential regeneration-related gene CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 16. This study provides the foundation to harness in vitro variation and offers a deeper understanding of the variation introduced by tissue culture during germplasm conservation, somatic embryogenesis, gene editing, and breeding programs.

Overexpression and knockout (or knockdown) of gene of interest are two commonly used strategies for gene functional study. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system-mediated gene knockout has been applied in most plant species, including grapevine. However, CRISPR/dCas9 (deactivated Cas9)-based transcriptional activation is still unreported in fruit crops, although a few studies have been documented in Arabidopsis and rice. Here, we tested two transcriptional activators VP64 and TV for transcriptional activation of endogenous genes in grape. Both the dCas9-VP64 and dCas9-TV systems are efficient enough for transcriptional activation of the UDP-glucose flavonoid glycosyltransferases (UFGT) gene in grape cells. The effectiveness of the dCas9-VP64 system in UFGT activation was about 1.6- to 5.6-fold, while the efficiency of the dCas9-TV system was around 5.7- to 7.2- fold. Moreover, in grapevine plants, highly efficient activation of the cold-responsive transcription factor gene CBF4 was achieved by using the dCas9-TV system. The expression of CBF4 was increased 3.7- to 42.3-fold in transgenic plants. Compared with the wild-type plants, the CBF4-activated plants exhibited lower electrolyte leakage after cold treatment. Our results demonstrate the effectiveness of the dCas9-VP64 and dCas9-TV systems in gene activation in grape, which will facilitate application of transcriptional activation in this economically important species.

The jasmonic acid (JA) signaling pathway plays a vital role in mediating plant resistance to herbivores . The tea plant (Camellia sinensis) is one of the most important woody cash crops in the world. Due to the lack of genetic transformation systems for tea plants, how the JA signaling pathway works in tea plants has not yet been determined. Now, with the development of cross-disciplines, chemical biology provides new means for analyzing the JA signaling pathway. In the present study, the structure of the small-molecule isoquinoline compound ZINC71820901 (lyn3) was obtained from the ZINC molecular library through virtual screening based on the structure of the crystal COI1-JAZ1 co-receptor and was found to act as an inhibitor of the JA signaling pathway in both Arabidopsis and tea plants. Our results revealed that lyn3 repressed tea plant resistance to Ectropis grisescens mainly by decreasing the accumulation of (−)-epicatechin and (−)-epigallocatechin via repression of the JA signaling pathway, which functioned in a modulation manner different from that of the already known inhibitor salicylhydroxamic acid (SHAM). As a novel inhibitor of the JA signaling pathway, lyn3 provides a specific option for further research on the JA pathway.

Cytoplasmic male sterility (CMS) has long been used to economically produce hybrids that harness growth vigor through heterosis. Yet, how CMS systems operate within commercially viable seed production strategies in various economically important vegetable crops, and their underlying molecular mechanisms, are often overlooked details that could expand the utility of CMS as a cost-effective and stable system. We provide here an update on the nature of cytoplasmic–nuclear interplay for pollen sterility and fertility transitions in vegetable crops, based on the discovery of components of nuclear fertility restoration and reversion determinants. Within plant CMS systems, pollen fertility can be rescued by the introduction of nuclear fertility restorer genes (Rfs), which operate by varied mechanisms to countermand the sterility phenotype. By understanding these systems, it is now becoming feasible to achieve fertility restoration with Rfs designed for programmable CMS-associated open reading frames (ORFs). Likewise, new opportunities exist for targeted disruption of CMS-associated ORFs by mito-TALENs in crops where natural Rfs have not been readily identified, providing an alternative approach to recovering fertility of cytoplasmic male sterile lines in crops. Recent findings show that facultative gynodioecy, as a reproductive strategy, can coordinate the sterility and fertility transition in response to environmental cues and/or metabolic signals that reflect ecological conditions of reproductive isolation. This information is important to devising future systems that are more inherently stable.

Pear, belonging to the genus Pyrus, is one of the most economically important temperate fruit crops. Pyrus is an important genus of the Rosaceae family, subfamily Maloideae, and has at least 22 different species with over 5000 accessions maintained or identified worldwide. With the release of draft whole-genome sequences for Pyrus, opportunities for pursuing studies on the evolution, domestication, and molecular breeding of pear, as well as for conducting comparative genomics analyses within the Rosaceae family, have been greatly expanded. In this review, we highlight key advances in pear genetics, genomics, and breeding driven by the availability of whole-genome sequences, including whole-genome resequencing efforts, pear domestication, and evolution. We cover updates on new resources for undertaking gene identification and molecular breeding, as well as for pursuing functional validation of genes associated with desirable economic traits. We also explore future directions for “pear-omics”.

Although cytokinins (CKs) regulate fruit development, no direct genetic evidence supports the role of endogenous CKs in pericarp growth or development or fruit size. Here, we report that the reduction in levels of endogenous active CKs via overexpression of the CK-inactivating enzyme gene AtCKX2 specifically in fruit tissues resulted in reduced pericarp thickness and smaller fruit size compared with wild-type control fruits. Pericarp thickness and single fruit weight in transgenic plants were significantly reduced. Analysis of paraffin sections showed that the reduced pericarp thickness was due largely to a decreased number of cells, and thus decreased cell division. Transcriptome profiling showed that the expression of cell division- and expansion-related genes was reduced in AtCKX2-overexpressing fruits. In addition, the expression of auxin-signaling and gibberellin-biosynthetic genes was repressed, whereas that of gibberellin-inactivating genes was enhanced, in AtCKX2-overexpressing fruits. These results demonstrate that endogenous CKs regulate pericarp cell division and subsequently fruit size. They also suggest that CKs interact with auxin and gibberellins in regulating tomato pericarp thickness and fruit size.

In this study we investigated the transcriptome and epigenome dynamics of the tomato fruit during post-harvest in a landrace belonging to a group of tomatoes (Solanum lycopersicum L.) collectively known as “Piennolo del Vesuvio”, all characterized by a long shelf- life. Expression of protein-coding genes and microRNAs as well as DNA methylation patterns and histone modifications were analysed in distinct post-harvest phases. Multi-omics data integration contributed to the elucidation of the molecular mechanisms underlying processes leading to long shelf-life. We unveiled global changes in transcriptome and epigenome. DNA methylation increased and the repressive histone mark H3K27me3 was lost as the fruit progressed from red ripe to 150 days post-harvest. Thousands of genes were differentially expressed, about half of which were potentially epi-regulated as they were engaged in at least one epi-mark change in addition to being microRNA targets in ∼5% of cases. Down-regulation of the ripening regulator MADS-RIN and of genes involved in ethylene response and cell wall degradation was consistent with the delayed fruit softening. Large-scale epigenome reprogramming that occurred in the fruit during post-harvest likely contributed to delayed fruit senescence.

Most commercial peach [ Prunus persica (L.) Batsch] cultivars have leaves with extrafloral nectaries (EFNs). Breeders have selected this character over time, as they observed that the eglandular phenotype resulted in high susceptibility to peach powdery mildew, a major disease of peach trees. EFNs are controlled by a Mendelian locus (E), mapped on chromosome 7. However, the genetic factor underlying E was unknown. In order to address this point, we developed a mapping population of 833 individuals derived from the selfing of “Malo Konare”, a Bulgarian peach cultivar, heterozygous for the trait. This progeny was used to investigate the E-locus region, along with additional resources including peach genomic resequencing data, and 271 individuals from various origins used for validation. High-resolution mapping delimited a 40.6 kbp interval including the E-locus and four genes. Moreover, three double-recombinants allowed identifying Prupe.7G121100, a LMI1-like homeodomain leucine zipper (HD-Zip) transcription factor, as a likely candidate for the trait. By comparing peach genomic resequencing data from individuals with contrasted phenotypes, a MITE-like transposable element of the hAT superfamily (mMoshan) was identified in the third exon of Prupe.7G121100. It was associated with the absence or globose phenotype of EFNs. The insertion of the transposon was positively correlated with enhanced expression of Prupe.7G121100. Furthermore, a PCR marker designed from the sequence-variants, allowed to properly assign the phenotypes of all the individuals studied. These findings provide valuable information on the genetic control of a trait poorly known so far although selected for a long time in peach.

Carotenoid pigments confer photoprotection and visual attraction and serve as precursors for many important signaling molecules. Herein, the orange-fruited phenotype of a tomato elite inbred line resulting from sharply reduced carotenoid levels and an increased β-carotene-to-lycopene ratio in fruit was shown to be controlled by a single recessive gene, oft3. BSA-Seq combined with fine mapping delimited the oft3 gene to a 71.23 kb interval on chromosome 4, including eight genes. Finally, the oft3 candidate gene SlIDI1, harboring a 116 bp deletion mutation, was identified by genome sequence analysis. Further functional complementation and CRISPR–Cas9 knockout experiments confirmed that SlIDI1 was the gene underlying the oft3 locus. qRT–PCR analysis revealed that the expression of SlIDI1 was highest in flowers and fruit and increased with fruit ripening or flower maturation. SlIDI1 simultaneously produced long and short transcripts by alternative transcription initiation and alternative splicing. Green fluorescent protein fusion expression revealed that the long isoform was mainly localized in plastids and that an N-terminal 59-amino acid extension sequence was responsible for plastid targeting. Short transcripts were identified in leaves and fruit by 5’ RACE and in fruit by 3’ RACE, which produced corresponding proteins lacking transit peptides and/or putative peroxisome targeting sequences, respectively. In SlIDI1 mutant fruit, SlBCH1 transcription involved in β-carotenoid catabolism was obviously suppressed, which may be responsible for the higher β-carotene-to-lycopene ratio and suggested potential feedback regulatory mechanisms involved in carotenoid pathway flux.

Caffeine is an important functional substance and is abundant in tea plant, but little is known about how its biosynthesis is regulated by transcription factors . In this study, the NAC-like transcription factor-encoding gene CsNAC7, which is involved in caffeine synthesis, was isolated from a Yinghong 9 cDNA library using a yeast one-hybrid assay; this gene comprises 1371 bp nucleotides and is predicted to encode 456 amino acids. The expression of CsNAC7 at the transcriptional level in tea shoots shared a similar pattern with that of the caffeine synthase gene yhNMT1 in the spring and summer, and its expressed protein was localized in the nucleus. Assays of gene activity showed that CsNAC7 has self-activation activity in yeast, that the active region is at the N-terminus, and that the transient expression of CsNAC7 could significantly promote the expression of yhNMT1 in tobacco leaves. In addition, overexpression or silencing of CsNAC7 significantly increased or decreased the expression of yhNMT1 and the accumulation of caffeine in transgenic tea calli, respectively. Our data suggest that the isolated transcription factor CsNAC7 positively regulates the caffeine synthase gene yhNMT1 and promotes caffeine accumulation in tea plant .

Somatic embryogenesis is a preferred method for large-scale production of forest trees due to its high propagation efficiency. In this study, hybrid sweetgum leaves with phase changes from mature to embryogenic state were selected as experimental material to study somatic embryo initiation. Embryogenicity ranged from high to low, i.e. from 45%, 25%, and 12.5% to 0, with the samples of embryogenic callus (EC), whiten leaf edge (WLI), whiten leaf (WLII), and green leaf (GL) respectively. High correlations existed between embryogenicity and endogenous brassinosteroids (BRs) (r = 0.95, p < 0.05). Similarly, concentrations of endogenous BRs of the sample set correlated positively (r = 0.93, 0.99, 0.87, 0.99, 0.96 respectively, P < 0.05) to expression of somatic embryo (SE)-related genes, i.e. BBM, LEC2, ABI3, PLT2, and WOX2. Hierarchical cluster and weighted gene coexpression network analysis identified modules of coexpressed genes and network in 4820 differentially expressed genes (DEGs) from All-BR-Regulated Genes (ABRG). Moreover, exogenously-supplemented epiBR, together with 2,4-D and 6-BA, increased embryogenicity of GL-sourced callus, and expression of SE- and auxin-related genes, while brassinazole (BRZ), a BR biosynthesis inhibitor, reduced embryogenicity. Evidences obtained in this study revealed that BRs involved in phase change of leaf explants and may function in regulating gene expression and enhancing auxin effects. This study successfully established protocols for inducing somatic embryogenesis from leaf explants in hybrid sweetgum, which could facilitate the propagation process greatly, and provide theoretical basis for manipulating SE competence of explants in ornamental woody plants.

Wood formation involves sequential developmental events requiring the coordination of multiple hormones. Brassinosteroids (BRs) play a key role in wood development, but little is known about the cellular and molecular processes that underlie wood formation in tree species. Here, we generated transgenic poplar lines with edited PdBRI1 genes, which are orthologs of Arabidopsis vascular-enriched BR receptors, and showed how inhibition of BR signaling influences wood development at the mRNA and/or proteome level. Six Populus PdBRI1 genes formed three gene pairs, each of which was highly expressed in basal stems. Simultaneous mutation of PdBRI1–1, − 2, − 3 and − 6, which are orthologs of the Arabidopsis vascular-enriched BR receptors BRI1, BRL1 and BRL3, resulted in severe growth defects. In particular, the stems of these mutant lines displayed a discontinuous cambial ring and patterning defects in derived secondary vascular tissues. Abnormal cambial formation within the cortical parenchyma was also observed in the stems of pdbri1–1;2;3;6. Transgenic poplar plants expressing edited versions of PdBRI1–1 or PdBRI1–1;2;6 exhibited phenotypic alterations in stem development at 4.5 months of growth, indicating that there is functional redundancy among these PdBRI1 genes. Integrated analysis of the transcriptome and proteome of pdbri1–1;2;3;6 stems revealed differential expression of a number of genes/proteins associated with wood development and hormones. Concordant (16%) and discordant (84%) regulation of mRNA and protein expression, including wood-associated mRNA/protein expression, was found in pdbri1–1;2;3;6 stems. This study found a dual role of BRs in procambial cell division and xylem differentiation and provides insights into the multiple layers of gene regulation that contribute to wood formation in Populus.

Southwest China is a plant diversity hotspot. The near-cosmopolitan genus Ilex (c. 664 spp., Aquifoliaceae) reaches its maximum diversity in this region, with many narrow-range and a few widespread species. Divergent selection on widespread species leads to local adaptation, with consequences for both conservation and utilization, but is counteracted by geneflow. Many Ilex species are utilized as teas, medicines, ornamentals, honey plants, and timber, but variation below the species level is largely uninvestigated. We therefore studied the widespread Ilex polyneura, which occupies most of the elevational range available and is cultivated for its decorative leafless branches with persistent red fruits. We assembled a chromosome-scale genome using approximately 100x whole genome long-read and short-read sequencing combined with Hi-C sequencing. The genome is approximately 727.1 Mb, with a contig N50 size of 5 124 369 bp and a scaffold N50 size of 36 593 620 bp, for which the BUSCO score was 97.6%, and 98.9% of the assembly was anchored to 20 pseudochromosomes. Out of 32 838 genes predicted, 96.9% were assigned functions. Two whole genome duplication events were identified. Using this genome as a reference, we conducted a population genomics study of 112 individuals from 21 populations across the elevation range using restriction site-associated DNA sequencing (RADseq). Most populations clustered into four clades separated by distance and elevation. Selective sweep analyses identified 34 candidate genes potentially under selection at different elevations, with functions related to responses to abiotic and biotic stresses. This first high-quality genome in the Aquifoliales will facilitate the further domestication of the genus.

The genus Camelina (Brassicaceae) comprises 7–8 diploid, tetraploid, and hexaploid species. Of particular agricultural interest is the biofuel crop, C. sativa (gold-of-pleasure or false flax), an allohexaploid domesticated from the widespread weed, C. microcarpa. Recent cytogenetics and genomics work has uncovered the identity of the parental diploid species involved in ancient polyploidization events in Camelina. However, little is known about the maternal subgenome ancestry of contemporary polyploid species. To determine the diploid maternal contributors of polyploid Camelina lineages, we sequenced and assembled 84 Camelina chloroplast genomes for phylogenetic analysis. Divergence time estimation was used to infer the timing of polyploidization events. Chromosome counts were also determined for 82 individuals to assess ploidy and cytotypic variation. Chloroplast genomes showed minimal divergence across the genus, with no observed gene-loss or structural variation. Phylogenetic analyses revealed C. hispida as a maternal diploid parent to the allotetraploid Camelina rumelica, and C. neglecta as the closest extant diploid contributor to the allohexaploids C. microcarpa and C. sativa. The tetraploid C. rumelica appears to have evolved through multiple independent hybridization events. Divergence times for polyploid lineages closely related to C. sativa were all inferred to be very recent, at only ∼65 thousand years ago. Chromosome counts confirm that there are two distinct cytotypes within C. microcarpa (2n = 38 and 2n = 40). Based on these findings and other recent research, we propose a model of Camelina subgenome relationships representing our current understanding of the hybridization and polyploidization history of this recently-diverged genus.

While roses are today among the most popular ornamental plants, the petals and fruits of some cultivars have flavored foods for millennia. The genetic origins of these edible cultivars remain poorly investigated. We collected the major varieties of edible roses available in China, assembled their plastome sequences, and phased the haplotypes for internal transcribed spacers (ITS1/ ITS2) of the 18S-5.8S-26S nuclear ribosomal cistron. Our phylogenetic reconstruction using 88 plastid genomes, of primarily maternal origin, uncovered well-supported genetic relationships within Rosa, including all sections and all subgenera. We phased the ITS sequences to identify potential donor species ancestral to the development of known edible cultivars. The tri-parental Middle-Eastern origin of R. × damascena, the species most widely used in perfume products and food additives, was confirmed as a descendent of past hybridizations among R. moschata, R. gallica, and R. majalis/R. fedtschenkoana/R. davurica. In contrast, R. chinensis, R. rugosa, and R. gallica, in association with six other wild species, were the main donors for fifteen varieties of edible roses. The domesticated R. rugosa ‘Plena’ was shown to be a hybrid between R. rugosa and R. davurica, sharing a common origin with R. ‘Fenghua’. Only R. ‘Jinbian’ and R. ‘Crimson Glory’ featured continuous flowering. All remaining cultivars of edible roses bloomed only once a year. Our study provides important resources for clarifying the origin of edible roses and suggests a future for breeding new cultivars with unique traits, such as continuous flowering.

Vegetable soybean is one of the most important vegetables in China, and the demand for this vegetable has markedly increased worldwide over the past two decades. Here, we present a high-quality de novo genome assembly of the vegetable soybean cultivar Zhenong 6 (ZN6), which is one of the most popular cultivars in China. The 20 pseudochromosomes cover 94.57% of the total 1.01 Gb assembly size, with contig N50 of 3.84 Mb and scaffold N50 of 48.41 Mb. A total of 55 517 protein-coding genes were annotated. Approximately 54.85% of the assembled genome was annotated as repetitive sequences, with the most abundant long terminal repeat transposable elements. Comparative genomic and phylogenetic analyses with grain soybean Williams 82, six other Fabaceae species and Arabidopsis thaliana genomes highlight the difference of ZN6 with other species. Furthermore, we resequenced 60 vegetable soybean accessions. Alongside 103 previously resequenced wild soybean and 155 previously resequenced grain soybean accessions, we performed analyses of population structure and selective sweep of vegetable, grain, and wild soybean. They were clearly divided into three clades. We found 1112 and 1047 genes under selection in the vegetable soybean and grain soybean populations compared with the wild soybean population, respectively. Among them, we identified 134 selected genes shared between vegetable soybean and grain soybean populations. Additionally, we report four sucrose synthase genes, one sucrose-phosphate synthase gene, and four sugar transport genes as candidate genes related to important traits such as seed sweetness and seed size in vegetable soybean. This study provides essential genomic resources to promote evolutionary and functional genomics studies and genomically informed breeding for vegetable soybean.

Acidity is an important factor influencing the organoleptic quality of apple fruits. In this study, an apple pyrophosphate-energized proton pump (PEPP) gene was isolated and designated MdMa12. On the basis of a phylogenetic analysis in Rosaceae species, PEPP genes were divided into three groups, with apple PEPP genes most closely related to pear PEPP genes. Gene expression analysis revealed that high malic acid content was generally accompanied by high MdMa12 expression levels. Moreover, MdMa12 was mainly expressed in the fruit. A subcellular localization analysis suggested that MdMa12 is a mitochondrial protein. The ectopic expression and overexpression of MdMa12 in “Micro-Tom” tomato and apple calli, respectively, increased the malic acid content. One (MDH12) of four malate dehydrogenase genes highly expressed in transgenic apple calli was confirmed to encode a protein localized in mitochondria. The overexpression of MDH12 increased the malate content in apple calli. Furthermore, MdMa12 overexpression increased MdDTC1, MdMa1, and MdMa10 expression levels, which were identified to transport malate. These findings imply that MdMa12 has important functions related to apple fruit acidity. Our study explored the regulatory effects of mitochondria on the complex mechanism underlying apple fruit acidity.

Brassica juncea is an important vegetable and oil crop cultivated worldwide. To increase its genetic variation, we introgressed the A genome of Brassica rapa into B. juncea. We used three each of heading and semi-heading B. juncea accessions as recipient parents and a B. rapa line, B9008, as the donor parent. We obtained 101 BC₁S₁ lines in total with expanded phenotypic variations such as leafy head shapes. We developed 132 single-nucleotide polymorphism (SNP) markers that could distinguish the A genome of B. juncea from the B. rapa genome, and tracked the introgression of B. rapa segments in the new B. juncea germplasm. On average, 59.2% of the B. juncea A genome in the B. juncea introgression lines was covered by the donor segments. We also identified three markers whose donor genotype frequencies were significantly lower than the theoretical value, suggesting strong selection of the recipient genotype during the introgression process. We provide an effective strategy to evaluate the diversity of the new germplasm based on the combination of parental resequencing data and marker genotyping results. Further genetic analysis of 1642 SNPs showed that the genetic diversity of the new B. juncea germplasm with the introgressed B. rapa genome was significantly increased. This study illustrates the potential for expanding the genetic diversity of B. juncea through the introgression of the B. rapa genome.

Pathogen invasion leads to fast, local-to-systemic signal transduction that initiates plant defense responses. Despite tremendous progress in past decades, aspects of this process remain unknown, such as which cell types respond first and how signals are transferred among cell types. Here, we used single-cell RNA-seq of >50 000 single cells to document the gene expression landscape in leaves of woodland strawberry during infection by Botrytis cinerea and identify major cell types. We constructed a single-cell atlas and characterized the distinct gene expression patterns of hydathode, epidermal, and mesophyll cells during the incubation period of B. cinerea infection. Pseudotime trajectory analysis revealed signals of the transition from normal functioning to defense response in epidermal and mesophyll cells upon B. cinerea infection. Genes related to disease resistance showed different expression patterns among cell types: disease resistance-related genes and genes encoding transcription factors were highly expressed in individual cell types and interacted to trigger plant systemic immunity to B. cinerea. This is the first report to document the single-cell transcriptional landscape of the plant pathogenic invasion process; it provides new insights into the holistic dynamics of host–pathogen interactions and can guide the identification of genes and the formulation of strategies for resistant cultivar development.

In grafting, an agricultural technique for propagating flower species and fruit trees, two plants are combined to exploit their beneficial characteristics, such as rootstock disease tolerance and vigor. Grafting incompatibility has been observed, however, between distantly related plant combinations, which limits the availability of plant resources. A high grafting capacity has been found in Nicotiana, belonging to Solanaceae, but not in Ipomoea nil, a Convolvulaceae species. Here, we found that Petunia hybrida, another solanaceous species, has similar ability of interfamily grafting, which indicates that interfamily grafting capability in Solanaceae is not limited to the genus Nicotiana. RNA sequencing-based comparative time-series transcriptomic analyses of Nicotiana benthamiana, I. nil, and P. hybrida revealed that N. benthamiana and P. hybrida share a common gene expression pattern, with continued elevated expression of the β-1,4-glucanase subclade gene GH9B3 observed after interfamily grafting. During self-grafting, GH9B3 expression in each species was similarly elevated, thus suggesting that solanaceous plants have altered regulatory mechanisms for GH9B3 gene expression that allow tissue fusion even with other species. Finally, we tested the effect of the β-1,4-glucanase inhibitor D-glucono-1,5-lactone, using glucose as a control, on the interfamily grafting usability of P. hybrida with Arabidopsis rootstock. Strong inhibition of graft establishment was observed only with D-glucono-1,5-lactone, thus suggesting the important role of GH9B3 in P. hybrida grafting. The newly discovered grafting compatibility of Petunia with different families enhances the propagation techniques and the production of flower plants.

Cucurbitaceae is one of the most genetically diverse plant families in the world. Many of them are important vegetables or medicinal plants and are widely distributed worldwide. The rapid development of sequencing technologies and bioinformatic algorithms has enabled the generation of genome sequences of numerous important Cucurbitaceae species. This has greatly facilitated research on gene identification, genome evolution, genetic variation, and molecular breeding of cucurbit crops. So far, genome sequences of 18 different cucurbit species belonging to tribes Benincaseae, Cucurbiteae, Sicyoeae, Momordiceae, and Siraitieae have been deciphered. This review summarizes the genome sequence information, evolutionary relationships, and functional genes associated with important agronomic traits (e.g. fruit quality). The progress of molecular breeding in cucurbit crops and prospects for future applications of Cucurbitaceae genome information are also discussed.

Most potato cultivars are susceptible to late blight disease caused by the oomycete pathogen Phytophthora infestans. Here we report that the genetic loss of host susceptibility is a new source of resistance to prevent or diminish pathogen infection. Previously, we showed that RNAi-mediated silencing of the potato susceptibility (S) genes StDND1, StDMR1, and StDMR6 leads to increased late blight resistance. The mechanisms underlying this S-gene-mediated resistance have thus far not been identified. In this study, we examined the infection process of P. infestans in StDND1-, StDMR1-, and StDMR6-silenced potato lines. Microscopic analysis showed that penetration of P. infestans spores was hampered in StDND1-silenced plants. In StDMR1- and StDMR6-silenced plants, P. infestans infection was arrested at a primary infection stage by enhanced cell death responses . Histochemical staining revealed that StDMR1- and StDMR6-silenced plants display elevated ROS levels in cells at the infection sites. Resistance in StDND1-silenced plants, however, seems not to rely on a cell death response as ROS accumulation was found to be absent at most inoculated sites. Quantitative analysis of marker gene expression suggests that the increased resistance observed in StDND1- and StDMR6-silenced plants relies on an early onset of salicylic acid- and ethylene-mediated signaling pathways. Resistance mediated by silencing StDMR1 was found to be correlated with the early induction of salicylic acid-mediated signaling. These data provide evidence that different defense mechanisms are involved in late blight resistance mediated by functional impairment of different potato S-genes.

The crop wild relative Fragaria nilgerrensis is adapted to a variety of diverse habitats across its native range in China. Thus, discoveries made in this species could serve as a useful guide in the development of new superior strawberry cultivars that are resilient to new or variable environments. However, the genetic diversity and genetic architecture of traits in this species underlying important adaptive traits remain poorly understood. Here, we used whole-genome resequencing data from 193 F. nilgerrensis individuals spanning the distribution range in China to investigate the genetic diversity, population structure and genomic basis of local adaptation. We identified four genetic groups, with the western group located in Hengduan Mountains exhibiting the highest genetic diversity. Redundancy analysis suggested that both environment and geographic variables shaped a significant proportion of the genomic variation. Our analyses revealed that the environmental difference explains more of the observed genetic variation than geographic distance. This suggests that adaptation to distinct habitats, which present a unique combination of abiotic factors, likely drove genetic differentiation. Lastly, by implementing selective sweep scans and genome–environment association analysis throughout the genome, we identified the genetic variation associated with local adaptation and investigated the functions of putative candidate genes in F. nilgerrensis.

The potato tuber is the main nutrient supply and reproductive organ; however, tuber sprouting can reduce its commercial value. Snakin-2 (StSN2) was first reported as an antimicrobial peptide that positively regulates potato disease resistance. Our recent study suggested StSN2 overexpression inhibited sprout growth, while the sprouting process was accelerated in StSN2 RNAi lines. Cytoplasmic glyceraldehyde-3-phosphate dehydrogenase 1 (StGAPC1) was identified as a candidate protein that interacts with StSN2 in co-immunoprecipitation/mass spectrometry experiments. Here, we report that the expression levels of StSN2 and StGAPC1 decreased during sprouting compared with dormancy. Coexpression of StSN2 and StGAPC1 in bud eyes and apical buds was verified by immunofluorescence analysis of paraffin sections. In addition, interaction of StSN2 and StGAPC1 was confirmed by yeast two- hybrid, co-immunoprecipitation, and split luciferase complementation assays. Overexpression of StGAPC1 depressed sprout growth, which is similar to the function of StSN2 , and StSN2 - and StGAPC1 -overexpressing lines showed decreased glucose, fructose, and galactose contents. The interaction of StSN2 and StGAPC1 enhanced StGAPC1 activity and decreased its oxidative modification to inhibit sprout growth. Our results suggest that StSN2 plays a regulatory role in tuber sprout growth through interaction with StGAPC1.

Plants have a cuticular membrane (CM) and periderm membrane (PM), which act as barriers to terrestrial stresses. The CM covers primary organs with a continuous hydrophobic layer of waxes embedded in cutin, while the PM includes suberized cells stacked externally to the secondary tissues. The formation of native periderm is regulated by a postembryonic meristem phellogen that produces suberized phellem (cork) outwardly. However, the mechanism controlling phellogen differentiation to phellem remains to be clarified. Here, map-based cloning in a pear F₁ population with segregation for periderm development in fruit skin facilitated the identification of an aspartic acid repeat deletion in Pyrus Periderm Programmed Cell Death 1.1 (PyPPCD1.1) that triggers phellogen activity for cork formation in russet fruit skin of pear. PyPPCD1.1 showed preferential expression in pear fruit skin, and the encoded protein shares a structural similarity to that of the viral capsid proteins. Aspartic acid deletion in PyPPCD1.1 weakened its nuclear localization but increased its accumulation in the chloroplast. The products of both PyPPCD1.1 and its recessive allele directly interact with ADP-ribosylation factor 1 (ARF1). PyPPCD1.1 triggered programmed cell death in an ARF1-dependent manner. Thus, this study identified the switch gene for programmed cell death and periderm development and provided a new molecular regulatory mechanism underlying the development of this trait.

Pea (Pisum sativum L. subsp. sativum) is one of the oldest domesticated species and a widely cultivated legume. In this study, we combined next generation sequencing (NGS) data referring to two genotyping-by-sequencing (GBS) libraries, each one prepared from a different Pisum germplasm collection. The selection of single nucleotide polymorphism (SNP) loci called in both germplasm collections caused some loss of information; however, this did not prevent the obtainment of one of the largest datasets ever used to explore pea biodiversity, consisting of 652 accessions and 22 127 markers. The analysis of population structure reflected genetic variation based on geographic patterns and allowed the definition of a model for the expansion of pea cultivation from the domestication centre to other regions of the world. In genetically distinct populations, the average decay of linkage disequilibrium (LD) ranged from a few bases to hundreds of kilobases, thus indicating different evolutionary histories leading to their diversification. Genome-wide scans resulted in the identification of putative selective sweeps associated with domestication and breeding, including genes known to regulate shoot branching, cotyledon colour and resistance to lodging, and the correct mapping of two Mendelian genes. In addition to providing information of major interest for fundamental and applied research on pea, our work describes the first successful example of integration of different GBS datasets generated from ex situ collections – a process of potential interest for a variety of purposes, including conservation genetics, genome-wide association studies, and breeding.

Cucumber (Cucumis sativus L.) predominantly translocates raffinose family oligosaccharides (RFOs) in the phloem and accumulates RFOs in leaves. Galactinol synthase (GolS) catalyzes the critical step of RFO biosynthesis, and determining the functional diversity of multiple GolS isoforms in cucumber is of scientific significance. In this study, we found that all four isoforms of CsGolS in the cucumber genome were upregulated by different abiotic stresses. β -Glucuronidase staining and tissue separation experiments suggested that CsGolS1 is expressed in vascular tissues, whereas the other three CsGolS s are located in mesophyll cells. Further investigation indicates that CsGolS1 plays double roles in both assimilate loading and stress response in minor veins, which could increase the RFO concentration in the phloem sap and then improve assimilate transport under adverse conditions. Cold-induced minor vein-specific overexpression of CsGolS1 enhanced the assimilate translocation efficiency and accelerated the growth rates of sink leaves, fruits, and whole plants under cold stress. Finally, our results demonstrate a previously unknown response to adverse environments and provide a potential biotechnological strategy to improve the stress resistance of cucumber.

Powdery mildew (PM), caused by the fungal pathogen Erysiphe necator , is one of the most destructive diseases of grapevine (Vitis vinifera and other Vitis spp.). Resistance to PM is an important goal for cultivar improvement, and understanding the underlying molecular mechanisms conditioning resistance is critical. Here, we report that transgenic expression of the WRKY transcription factor gene VqWRKY31 from the PM-resistant species Vitis quinquangularis conferred resistance to PM in V. vinifera through promoting salicylic acid signaling and specific metabolite synthesis. VqWRKY31 belongs to the WRKY IIb subfamily, and expression of the VqWRKY31 gene was induced in response to E. necator inoculation. Transgenic V. vinifera plants expressing VqWRKY31 were substantially less susceptible to E. necator infection, and this was associated with increased levels of salicylic acid and reactive oxygen species. Correlation analysis of transcriptomic and metabolomic data revealed that VqWRKY31 promoted expression of genes in metabolic pathways and the accumulation of many disease resistance-related metabolites, including stilbenes, flavonoids, and proanthocyanidins. In addition, results indicated that VqWRKY31 can directly bind to the promoters of two structural genes in stilbene synthesis, STS9 and STS48, and activate their expression. Based on our results, we propose a model where VqWRKY31 enhances grapevine PM resistance through activation of salicylic acid defense signaling and promotion of specific disease resistance- related metabolite synthesis. These findings can be directly exploited for molecular breeding strategies to produce PM-resistant grapevine germplasm.

Morphological, genic and epigenetic differences often exist in separate sexes of dioecious and trioecious plants. However, the connections and relationships among them in different breeding systems are still unclear. Papaya has three sex types, which is genetically determined and epigenetically regulated, and was chosen as a model to study sex differentiation. Bisulfite sequencing of genomic DNA extracted from early-stage flowers revealed sex-specific genomic methylation landscapes and seasonally methylome reprogramming processes in dioecious and gynodioecious papaya grown in spring and summer. Extensive methylation of sex-determining region (SDR) was the distinguishing epigenetic characteristics of nascent XY sex chromosomes in papaya. Seasonal methylome reprogramming of early-stage flowers in both dioecy and gynodioecy systems were detected, resulting from transcriptional expression pattern alterations of methylation-modification-related and chromatin-remodeling-related genes, particularly from those genes involved in active demethylation. Genes involved in phytohormone signal transduction pathway in male flowers have played an important role in the formation of male-specific characteristics. These findings enhanced the understanding of the genetic and epigenetic contributions to sex differentiation and the complexity of sex chromosome evolution in trioecious plants.

Variation in solar ultraviolet radiation induces a wide-range of plant responses from the cellular to whole-plant scale. We demonstrate here for the first time that partial stomatal closure caused by ultraviolet radiation exposure results in significant increases in leaf temperature. Significant leaf warming in response to ultraviolet radiation was consistent in tomato (Solanum lycopersicum L.) across different experimental approaches. In field experiments where solar ultraviolet radiation was attenuated using filters, exposure to ultraviolet radiation significantly decreased stomatal conductance and increased leaf temperature by up to 1.5^◦C. Using fluorescent lamps to provide ultraviolet radiation treatments, smaller but significant increases in leaf temperature due to decreases in stomatal conductance occurred in both multi-day controlled environment growth room experiments and short-term (<2 hours) climate cabinet irradiance response experiments. We show that leaf warming due to partial stomatal closure is independent of any direct warming effects of ultraviolet radiation manipulations. We discuss the implications of ultraviolet radiation-induced warming both for horticultural crop production and understanding broader plant responses to ultraviolet radiation.

Grafting is an ancient method that has been intensively used for the clonal propagation of vegetables and woody trees. Despite its importance in agriculture the physiological and molecular mechanisms underlying phenotypic changes of plants following grafting are still poorly understood. In the present study, we analyse the populations of small RNAs in homo and heterografts and take advantage of the sequence differences in the genomes of heterograft partners to analyse the possible exchange of small RNAs. We demonstrate that the type of grafting per se dramatically influences the small RNA populations independently of genotypes but also show genotype specific effects. In addition, we demonstrate that bilateral exchanges of small RNAs, mainly short interfering RNAs, may occur in heterograft with the preferential transfer of small RNAs from the scion to the rootstock. Altogether, the results suggest that small RNAs may have an important role in the phenotype modifications observed in heterografts.

Flavonoids are the most widespread polyphenolic compounds and are important dietary constituents present in horticultural crops such as fruits, vegetables, and tea. Natural flavonoids are responsible for important quality traits, such as food colors and beneficial dietary antioxidants, and numerous investigations have shown that intake of flavonoids can reduce the incidence of various non-communicable diseases. Analysis of the thousands of flavonoids reported so far has shown that different hydroxylation modifications affect their chemical properties and nutritional values. These diverse flavonoids can be classified based on different hydroxylation patterns in the B, C, and A rings and multiple structure–activity analyses have shown that hydroxylation decoration at specific positions markedly enhances their bioactivities. This review focuses on current knowledge concerning hydroxylation of flavonoids catalyzed by several different types of hydroxylase enzymes. Flavonoid 3^′ -hydroxylase (F3^′ H) and flavonoid 3^′ 5^′ - hydroxylase (F3^′ 5^′ H) are important enzymes for the hydroxylation of the B ring of flavonoids. Flavanone 3-hydroxylase (F3H) is key for the hydroxylation of the C ring, while flavone 6-hydroxylase (F6H) and flavone 8-hydroxylase (F8H) are key enzymes for hydroxylation of the A ring. These key hydroxylases in the flavonoid biosynthesis pathway are promising targets for the future bioengineering of plants and mass production of flavonoids with designated hydroxylation patterns of high nutritional importance. In addition, hydroxylation in key places on the ring may help render flavonoids ready for degradation, and the catabolic turnover of flavonoids may open the door for new lines of inquiry.

Unordered parent-offspring (PO) relationships are an outstanding issue in pedigree reconstruction studies. Resolution of the order of these relationships would expand the results, conclusions, and usefulness of such studies; however, no such PO order resolution (POR) tests currently exist. This study describes two such tests, demonstrated using SNP array data in the outcrossing species apple (Malus × domestica) on a PO relationship of known order (‘Keepsake’ as a parent of ‘Honeycrisp’) and two PO relationships previously ordered only via provenance information. The first test, POR-1, tests whether some of the extended haplotypes deduced from homozygous SNP calls from one individual in an unordered PO duo are composed of recombinant haplotypes from accurately phased SNP genotypes from the second individual. If so, the first individual would be the offspring of the second individual, otherwise the opposite relationship would be present. The second test, POR-2, does not require phased SNP genotypes and uses similar logic as the POR-1 test, albeit in a different approach. The POR-1 and POR-2 tests determined the correct relationship between ‘Keepsake’ and ‘Honeycrisp’. The POR-2 test confirmed ‘Reinette Franche’ as a parent of ‘Nonpareil’ and ‘Brabant Bellefleur’ as a parent of ‘Court Pendu Plat’. The latter finding conflicted with the recorded provenance information, demonstrating the need for these tests. The successful demonstration of these tests suggests they can add insights to future pedigree reconstruction studies, though caveats, like extreme inbreeding or selfing, would need to be considered where relevant.

Grafting is an important horticultural technique used for many crop species. However, some scion/rootstock combinations are considered as incompatible due to poor graft union formation and subsequently high plant mortality. The early identification of graft incompatibility could allow the selection of non-viable plants before planting and would have a beneficial impact on research and development in the nursery sector. In general, visible phenotypes of grafted plants (size, root number, etc.) are poorly correlated with grafting success, but some studies have suggested that some polyphenols could be used as markers of graft incompatibility several months or years after grafting. However, much of the previous studies into metabolite markers of grafting success have not included all the controls necessary to unequivocally validate the markers proposed. In this study, we quantified 73 primary and secondary metabolites in nine hetero-grafts and six homo-grafted controls 33 days after grafting at the graft interface and in both the scion and rootstock woody tissues. Certain biomarker metabolites typical of a high stress status (such as proline, GABA and pallidol) were particularly accumulated at the graft interface of the incompatible scion/rootstock combination. We then used correlation analysis and generalized linear models to identify potential metabolite markers of grafting success measured one year after grafting. Here we present the first attempt to quantitatively predict graft compatibility and identify marker metabolites (especially asparagine, trans- resveratrol, trans-piceatannol and α-viniferin) 33 days after grafting, which was found to be particularly informative for homo-graft combinations.

Clubroot is one of the major diseases adversely affecting Chinese cabbage (Brassica rapa) yield and quality. To precisely characterize the Plasmodiophora brassicae infection of Chinese cabbage, we developed a dual fluorescent staining method for simultaneously examining the pathogen, cell structures, and starch grains. The number of starch (amylopectin) grains increased in B. rapa roots infected by P. brassicae, especially from 14 to 21 days after inoculation. Therefore, the expression levels of 38 core starch metabolism genes were investigated by quantitative real-time PCR. Most genes related to starch synthesis were up-regulated at 7 days after P. brassicae inoculation, whereas the expression levels of starch degradation-related genes were increased at 14 days after inoculation. Then, genes encoding the core enzymes involved in starch metabolism were investigated by assessing their chromosomal distributions, structures, duplication events, and synteny among Brassica species. Genome comparisons indicated that 38 non-redundant genes belonging to six core gene families related to starch metabolism are highly conserved among Arabidopsis thaliana, B. rapa, Brassica nigra, and Brassica oleracea. Previous genome sequencing projects have revealed that P. brassicae obtained host nutrients by manipulating plant metabolism. Starch may serve as a carbon source for P. brassicae colonization, as indicated by histological observations and transcriptomic analysis. Results of this study may elucidate the evolution and expression of core starch metabolism genes and provide researchers with novel insights into the pathogenesis of clubroot in B. rapa.

Grafting is a useful cultivation technology to resist abiotic and biotic stresses and is an integral part of citrus production. However, some widely utilized rootstocks may still exhibit graft incompatibility in the orchard. ‘Hongmian miyou’ (Citrus maxima (Burm.) Merrill) is mutated from ‘Guanxi miyou’, but these two scions showed different compatibility with available Poncirus trifoliata rootstock. Foliage etiolation is an observed symptom of graft incompatibility, but its mechanism remains poorly understood. This study is the first to investigate the morphological, physiological, and anatomical differences between compatible and incompatible grafts, and perform transcriptome profiling at crucial stages of the foliage etiolation process. Based on comprehensive analyses, hormonal balance was disordered, and two rate-limiting genes, NCED3 ( 9- cis -epoxycarotenoid dioxygenase 3) and NCED5 , being responsible for ABA (abscisic acid) accumulation, were highlighted. Further correlation analysis indicated that IAA (indole-3-acetic acid) and ABA were the most likely inducers of the expression of stress-related genes. In addition, excessive starch accumulation was observed in the lamina and midribs of leaves of incompatible grafts. These results provide a new insight into the role of hormonal balance and ABA biosynthesis genes in regulating and contributing to graft incompatibility, and will further define and deploy candidate genes to explore the mechanisms underlying citrus rootstock–scion interactions.

Shoot regeneration from leaf tissue requires the de-differentiation of cells from a highly differentiated state into an actively dividing state, but it remains unclear how this physiological transition occurs and is regulated, especially at the epigenetic level. Here, we characterized the DNA methylome represented by 5-methylcytosine (5mC) in leaf and callus tissue derived from leaf explants of woodland strawberry, Fragaria vesca. We detected an overall increase in DNA methylation and distinct 5mC enrichment patterns in the CG, CHG, and CHH sequence contexts in genes and transposable elements. Our analyses revealed an intricate relationship between DNA methylation and gene expression level in leaves or leaf-derived callus. However, when considering the genes involved in callus formation and shoot regeneration, e.g. FvePLT3/7, FveWIND3, FveWIND4, FveLOG4 and FveIAA14, their dynamic transcription levels were associated with differentially methylated regions located in the promoters or gene bodies, indicating a regulatory role of DNA methylation in the transcriptional regulation of pluripotency acquisition in strawberry. Furthermore, application of the DNA methyltransferase inhibitor 5^′-azacytidine (5^′-Aza) hampered both callus formation and shoot regeneration from the leaf explants. We further showed that 5^′-Aza downregulated the expression of genes involved in cell wall integrity, such as expansin, pectin lyase, and pectin methylesterase genes, suggesting an essential role of cell wall metabolism during callus formation. This study reveals the contribution of DNA methylation to callus formation capacity and will provide a basis for developing a strategy to improve shoot regeneration for basic and applied research applications.

Watermelons used for seed consumption tend to have larger seeds, whereas watermelons used for flesh consumption often require relatively small seeds. Therefore, watermelon seed size has received extensive attention from consumers and breeders. However, the natural variation and genetic mechanism of watermelon seed size remain unclear. In the present study, 100-seed weight, seed hilum length, seed length, seed width, and seed thickness were examined in 197 watermelon accessions. Furthermore, association analysis was performed between seed size traits and high-quality SNP data. The results revealed that there were strong correlations among the five seed traits, and seed enlargement was an important feature during watermelon seed size domestication. The seed-consumed biological species Citrullus mucosospermus and the edible seed watermelon Citrullus lanatus had significantly larger seeds than the other species. Eleven non-repeating significant SNPs above the threshold line were obtained from GWAS analysis. Four SNPs on chromosome 5 were considered to be closely associated with seed size traits (S5:32250307, S5:32250454, S5:32256177, and S5:32260870) and could be used as potential molecular markers for the breeding of watermelon cultivars with a target seed size. In addition, based on gene annotation information and previous reports, five genes near the four significant SNPs may regulate seed size. qRT-PCR analysis showed that two genes that may be involved in abscisic acid metabolism, Cla97C05G104360 and Cla97C05G104380, may play an important role in regulating watermelon seed size. Our findings provide molecular insights into natural variation in watermelon seed size and valuable information for molecular marker-assisted breeding.

Allopolyploids contain two or more sets of subgenomes. To establish a compatible relationship between subgenomes, a series of gene expression changes has occurred in allopolyploids. What evolutionary changes have taken place in transcripts of Brassica napus during its early establishment and subsequent evolution is a fascinating scientific question. Here, we study this issue using a set of materials (natural and resynthesized B. napus and their progenitors/parents) and long-read RNA sequencing technology. The results showed that more genes were upregulated in resynthesized B. napus compared with its two parents, and more upregulated expressed genes were observed in natural B. napus than in resynthesized B. napus. The presence of upregulated genes in an organism may help it to adapt to the influence of “genomic shock” and cope with the natural environment. Isoforms are produced from precursor mRNAs by alternative splicing (AS) events, and more than 60% of the isoforms identified in all materials were novel, potentially improving the reference genome information for B. napus. We found that the isoform numbers and the numbers of genes potentially involved in AS and alternative polyadenylation increased in B. napus after evolution, and they may have been involved in the adaptation of plants to the natural environment. In addition, all identified isoforms were functionally annotated by searching seven databases. In general, this study can improve our overall understanding of the full-length transcriptome of B. napus and help us to recognize the significant changes in gene expression and isoform abundance that have occurred in allopolyploid B. napus during evolution.

Abscisic acid (ABA) plays a major role in the regulation of strawberry fruit ripening; however, the origin of the ABA signal is largely unknown. Here, we report an autocatalytic mechanism for ABA biosynthesis and its synergistic interaction with auxin to regulate strawberry fruit ripening. We demonstrate that ABA biosynthesis is self-induced in the achenes but not in the receptacle, resulting in its substantial accumulation during ripening. ABA was found to regulate both IAA transport and biosynthesis, thereby modulating IAA content during both early fruit growth and later fruit ripening. Taken together, these results reveal the origins of the ABA signal and demonstrate the importance of its coordinated action with IAA in the regulation of strawberry fruit development and ripening.

Leaf senescence is a highly-programmed developmental process during the plant life cycle. ABA plays an important role in leaf senescence. However, the mechanism underlying ABA-mediated leaf senescence, particularly the upstream epigenetic regulatory network, remains largely unclear. Here, we demonstrated that SlJMJ4, a Jumonji C (jmjC) domain-containing protein in tomato (Solanum lycopersicum), specifically demethylates di- and trimethylations of lysine 27 of histone H3 (H3K27) in vitro and in vivo. Overexpression of SlJMJ4 results in a premature senescence phenotype and promotes dark- and ABA-induced leaf senescence in tomato. Under dark conditions, SlJMJ4-promoted leaf senescence is associated with upregulated expression of transcription factors (SlORE1 and SlNAP2) and senescence-associated genes (SlSAG113 and SlSAG12) via removal of H3K27me3. In response to ABA, overexpression of SlJMJ4 increases its binding at the loci of SlORE1, SlNAP2, SlSAG113, SlSAG12, SlABI5,and SlNCED3 and decreases their H3K27me3 levels,thereby activating their expression and mediating ABA-induced leaf senescence in tomato. Taken together, these results demonstrate that SlJMJ4 plays a positive role in leaf senescence in tomato and functions in ABA-induced leaf senescence by binding to many key genes related to ABA synthesis and signaling, transcription regulation, and senescence, thus promoting their H3K27me3 demethylation.

Phytohormones and their interactions are critical for fruit development and are key topics in horticulture research. Auxin, together with gibberellic acid (GA), promotes cell division and expansion, thereby regulating fruit development and enlargement after fertilization. Auxin- and GA-related mutants show parthenocarpy (fruit formation without fertilization of the ovule) in many plant species, indicating that these hormones and possibly their interactions play a key role in the regulation of fruit initiation and development. Recent studies have shown clear molecular and genetic evidence that ARF/IAA and DELLA proteins interact with one another and regulate both auxin and GA signaling pathways in response to auxin and GA during fruit growth in horticultural plants such as tomato (the most studied fleshy fruit) and strawberry (the model for Rosaceae). These recent findings provide new insights into the mechanisms by which the plant hormones auxin and GA regulate fruit development.

Acylsugars are a class of plant defense compounds produced across many distantly related families. Members of the horticulturally important morning glory (Convolvulaceae) family produce a diverse subclass of acylsugars called resin glycosides (RGs), which comprise oligosaccharide cores, hydroxyacyl chain(s), and decorating aliphatic and aromatic acyl chains. While many RG structures are characterized, the extent of structural diversity of this class in different genera and species is not known. In this study, we asked whether there has been lineage-specific diversification of RG structures in different Convolvulaceae species that may suggest diversification of the underlying biosynthetic pathways. Liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) was performed from root and leaf extracts of 26 species sampled in a phylogeny-guided manner. LC–MS/MS revealed thousands of peaks with signature RG fragmentation patterns with one species producing over 300 signals, mirroring the diversity in Solanaceae-type acylsugars. A novel RG from Dichondra argentea was characterized using nuclear magnetic resonance spectroscopy, supporting previous observations of RGs with open hydroxyacyl chains instead of closed macrolactone ring structures. Substantial lineage-specific differentiation in utilization of sugars, hydroxyacyl chains, and decorating acyl chains was discovered, especially among Ipomoea and Convolvulus-the two largest genera in Convolvulaceae. Adopting a computational, knowledge-based strategy, we further developed a high-recall workflow that successfully explained ∼72% of the MS/MS fragments, predicted the structural components of 11/13 previously characterized RGs, and partially annotated ∼45% of the RGs. Overall, this study improves our understanding of phytochemical diversity and lays a foundation for characterizing the evolutionary mechanisms underlying RG diversification.

The development of trichomes, which protect plants against herbivores, is affected by various stresses. In tomato, previous studies showed that stress-triggered jasmonic acid (JA) signaling influences trichome formation, but the underlying mechanism is not fully resolved. Here, we found that two C2H2 zinc finger proteins synergistically regulate JA-induced trichome formation in tomato. The naturally occurring mutations in the H gene and its close homolog H-like in a spontaneous mutant, LA3172, cause severely affected trichome development. Compared with the respective single mutant, the h/ hl double mutant displayed more severe trichome defects in all tissues. Despite their partially redundant function, the H and HL genes regulate trichome formation in a spatially distinct manner, with HL more involved in hypocotyls and leaves while H is more involved in stems and sepals. Furthermore, the activity of H/HL is essential for JA-triggered trichome formation. The JA signaling inhibitor SlJAZ2 represses the activity of H and HL via physical interaction, resulting in the activation of THM1, a negative regulator of trichome formation. Our results provide novel insight into the mechanism of trichome formation in response to stress-induced JA signaling in tomato.

Earliness and ripening behavior are important attributes of fruits on and off the vine, and affect quality and preference of both growers and consumers. Fruit ripening is a complex physiological process that involves metabolic shifts affecting fruit color, firmness, and aroma production. Melon is a promising model crop for the study of fruit ripening, as the full spectrum of climacteric behavior is represented across the natural variation. Using Recombinant Inbred Lines (RILs) population derived from the parental lines “Dulce” (reticulatus, climacteric) and “Tam Dew” (inodorus, non-climacteric) that vary in earliness and ripening traits, we mapped QTLs for ethylene emission, fruit firmness and days to flowering and maturity. To further annotate the main QTL intervals and identify candidate genes, we used Oxford Nanopore long-read sequencing in combination with Illumina short-read resequencing, to assemble the parental genomes de-novo. In addition to 2.5 million genome-wide SNPs and short InDels detected between the parents, we also highlight here the structural variation between these lines and the reference melon genome. Through systematic multi-layered prioritization process, we identified 18 potential polymorphisms in candidate genes within multi-trait QTLs. The associations of selected SNPs with earliness and ripening traits were further validated across a panel of 177 diverse melon accessions and across a diallel population of 190 F1 hybrids derived from a core subset of 20 diverse parents. The combination of advanced genomic tools with diverse germplasm and targeted mapping populations is demonstrated as a way to leverage forward genetics strategies to dissect complex horticulturally important traits.

Cucurbit crops are suitable models for studying long-distance signaling in horticultural plants. Although thousands of substances are graft transmissible in cucurbits, functional studies have been hampered by the lack of efficient genetic transformation systems. Here, we report a convenient and efficient root transformation method for several cucurbit crops that will facilitate studies of functional genes and shoot–root crosstalk. We obtained healthy plants with completely transformed roots and non-transgenic shoots within 6 weeks. Furthermore, we combined this root transformation method with grafting, which allowed for gene manipulation in the rootstock. We validated our system by exploring salt tolerance mechanisms using a cucumber (Cucumis sativus)/pumpkin (Cucurbita moschata Duch.) (scion/rootstock) graft in which the sodium transporter gene High-affinity K⁺ transporter1 (CmoHKT1;1) was edited in the pumpkin rootstock and by overexpressing the pumpkin tonoplast Na⁺/H⁺ antiporter gene Sodium hydrogen exchanger4 (CmoNHX4) in cucumber roots.

Camellia oil extracted from Camellia seeds is rich in unsaturated fatty acids and secondary metabolites beneficial to human health. However, no oil-tea tree genome has yet been published, which is a major obstacle to investigating the heredity improvement of oil-tea trees. Here, using both Illumina and PicBio sequencing technologies, we present the first chromosome-level genome sequence of the oil-tea tree species Camellia chekiangoleosa Hu. (CCH). The assembled genome consists of 15 pseudochromosomes with a genome size of 2.73 Gb and a scaffold N50 of 185.30 Mb. At least 2.16 Gb of the genome assembly consists of repetitive sequences, and the rest involves a high-confidence set of 64 608 protein-coding gene models. Comparative genomic analysis revealed that the CCH genome underwent a whole-genome duplication event shared across the Camellia genus at ~57.48 MYA and a γ-WGT event shared across all core eudicot plants at ~120 MYA. Gene family clustering revealed that the genes involved in terpenoid biosynthesis have undergone rapid expansion. Furthermore, we determined the expression patterns of oleic acid accumulation- and terpenoid biosynthesis-associated genes in six tissues. We found that these genes tend to be highly expressed in leaves, pericarp tissues, roots, and seeds. The first chromosome-level genome of oil-tea trees will provide valuable resources for determining Camellia evolution and utilizing the germplasm of this taxon.

Cucumber (Cucumis sativus L.) has been a model for the study of sex differentiation over the last two decades. Cucumber sex differentiation is mainly under genetic control, but plant growth regulators can also influence or even change it. However, the effect of exogenous auxin application on cucumber sex differentiation is not well understood at the physiological level. In this study, we explored the effects of different exogenous auxin concentrations on cucumber varieties with different mutant sex-controlling genotypes and found that there was a dosage effect of exogenous indole-3-acetic acid (IAA) on the enhancement of cucumber femaleness. Several ACC synthetase (ACS) family members responded directly to exogenous IAA, increasing endogenous ethylene synthesis, and this process appeared to be independent of the previously identified sex-related ACC oxidase CsACO2. We further demonstrated that ENHANCER OF SHOOT REGENERATION 2 (ESR2) responded to exogenous auxin induction by binding to ERE cis-acting element regions in the CsACS2 promoter, directly activating CsACS2 expression and thus increasing endogenous ethylene content, which may induce femaleness. These findings reveal that exogenous auxin increases cucumber femaleness by inducing a sex-controlling gene and promoting ethylene synthesis.

Fruits and vegetables in the Cucurbitaceae family, such as cucumber, melon, watermelon, and squash, contribute greatly to the human diet. The widespread use of genome editing technologies has greatly accelerated gene functional characterization and crop improvement. However, most economically important cucurbit plants, including melon and squash, remain recalcitrant to standard Agrobacterium tumefaciens-mediated transformation, limiting the effective use of genome editing technology. In this study, we used an “optimal infiltration intensity” strategy to establish an efficient genetic transformation system for melon and squash. We harnessed the power of this method to target homologs of the ERECTA family of receptor kinase genes and created alleles that resulted in a compact plant architecture with shorter internodes in melon, squash, and cucumber. The optimized transformation method presented here enables stable CRISPR/Cas9-mediated mutagenesis and provides a solid foundation for functional gene manipulation in cucurbit crops.

Plant specialized 1,4-naphthoquinones present a remarkable case of convergent evolution. Species across multiple discrete orders of vascular plants produce diverse 1,4-naphthoquinones via one of several pathways using different metabolic precursors. Evolution of these pathways was preceded by events of metabolic innovation and many appear to share connections with biosynthesis of photosynthetic or respiratory quinones. Here, we sought to shed light on the metabolic connections linking shikonin biosynthesis with its precursor pathways and on the origins of shikonin metabolic genes. Downregulation of Lithospermum erythrorhizon geranyl diphosphate synthase (LeGPPS), recently shown to have been recruited from a cytoplasmic farnesyl diphosphate synthase (FPPS), resulted in reduced shikonin production and a decrease in expression of mevalonic acid and phenylpropanoid pathway genes. Next, we used LeGPPS and other known shikonin pathway genes to build a coexpression network model for identifying new gene connections to shikonin metabolism. Integrative in silico analyses of network genes revealed candidates for biochemical steps in the shikonin pathway arising from Boraginales-specific gene family expansion. Multiple genes in the shikonin coexpression network were also discovered to have originated from duplication of ubiquinone pathway genes. Taken together, our study provides evidence for transcriptional crosstalk between shikonin biosynthesis and its precursor pathways, identifies several shikonin pathway gene candidates and their evolutionary histories, and establishes additional evolutionary links between shikonin and ubiquinone metabolism. Moreover, we demonstrate that global coexpression analysis using limited transcriptomic data obtained from targeted experiments is effective for identifying gene connections within a defined metabolic network.

Leucine-rich repeat receptor-like kinases (LRR-RLKs) are ubiquitous in higher plants and act as receptors of extracellular signals to trigger multiple physiological processes. However, the functions of the majority of LRR-RLKs remain largely unknown, especially in tomato (Solanum lycopersicum L.). Here, we found that MRK1 (Multiple resistance-associated kinase 1), encoding a novel tomato LRR-RLK, was significantly induced by temperature stresses and bacterial pathogen attacks. Knocking out MRK1 impaired tolerance to both cold and heat stress, accompanied by decreased transcript levels of the master regulators C-repeat binding factor 1 (CBF1) and Heat shock transcription factor a-1a (HsfA1a), respectively. In addition, mrk1 mutants were hypersensitive to Pseudomonas syringae pv. tomato DC3000 and Ralstonia solanacearum and showed compromised pattern-triggered immunity (PTI) responses, as evidenced by decreased production of reactive oxygen species and reduced upregulation of PTI marker genes. Moreover, bimolecular fluorescence complementation, split-luciferase assays, and co-immunoprecipitation supported the formation of a complex of MRK1, FLS2, and Somatic embryogenesis receptor kinase (SERK3A/SERK3B) in a ligand-independent manner. This work demonstrates that tomato MRK1 is a novel positive regulator of multiple stress responses and may be a potential breeding target for improving crop stress resistance.

Water submergence is an environmental stress with detrimental effects on plant growth and survival. As a wetland plant species, lotus (Nelumbo nucifera) is widely cultivated in flood-prone lowlands throughout Asian countries, but little is known about its endurance and acclimation mechanisms to complete submergence. Here, we performed a time-course submergence experiment and an RNA- sequencing transcriptome analysis of the two lotus varieties “Qiuxing” and “China Antique”. Both varieties showed low submergence tolerance, with a median lethal time of approximately 10 days. Differentially expressed gene (DEG) analysis and weighted gene co- expression network analysis (WGCNA) identified a number of key genes putatively involved in lotus submergence responses. Lotus plants under complete submergence developed thinned leaves and elongated petioles containing a high density of aerenchyma. All four lotus submergence-responsive ERF-VII genes and gene sets corresponding to the low oxygen “escape” strategy (LOES) were elevated. In addition, a number of lotus innate immunity genes were rapidly induced by submergence, probably to confer resistance to possible pathogen infections. Our data also reveal the probable involvement of jasmonic acid in the modulation of lotus submergence responses, although to a lesser extent than the gaseous hormone ethylene. These results suggest that lotus plants primarily use the LOES strategy to cope with complex submergence-induced stresses, and they will be valuable for understanding the molecular basis underlying plant submergence acclimation.

Low temperatures severely restrict melon seedling growth. However, the mechanisms by which melon adapts to cold stress are poorly understood. Arginine decarboxylase (ADC), a key synthetase, catalyzes putrescine biosynthesis in plants. In this study, we found that CmADC functions as a positive regulator of melon seedling cold tolerance. In addition, two transcription factors, abscisic acid-responsive element (ABRE)-binding factor 1 (CmABF1) and C-repeat binding factor 4 (CmCBF4), directly target CmADC to trigger its expression. Consistently, virus-induced gene silencing (VIGS) of CmABF1 or CmCBF4 downregulated CmADC abundance, decreased putrescine accumulation, and reduced cold tolerance. Furthermore, some other CBF and ABF members show at least partial functional redundancy and complementarity with CmABF1 and CmCBF4. Overall, our work reveals that the ABA, CBF, and polyamine pathways may form a cooperative regulatory network to participate in plant cold stress response.

Fruit yield estimation is crucial for establishing fruit harvest and marketing strategies. Recently, computer vision and deep learning techniques have been used to estimate citrus fruit yield and have exhibited notable fruit detection ability. However, computer-vision-based citrus fruit counting has two key limitations: inconsistent fruit detection accuracy and double-counting of the same fruit. Using oranges as the experimental material, this paper proposes a deep-learning-based orange counting algorithm using video sequences to help overcome these problems. The algorithm consists of two sub-algorithms, OrangeYolo for fruit detection and OrangeSort for fruit tracking. The OrangeYolo backbone network is partially based on the YOLOv3 algorithm, which has been improved upon to detect small objects (fruits) at multiple scales. The network structure was adjusted to detect small-scale targets while enabling multiscale target detection. A channel attention and spatial attention multiscale fusion module was introduced to fuse the semantic features of the deep network with the shallow textural detail features. OrangeYolo can achieve mean Average Precision (mAP) values of 0.957 in the citrus dataset, higher than the 0.905, 0.911, and 0.917 achieved with the YOLOv3, YOLOv4, and YOLOv5 algorithms. OrangeSort was designed to alleviate the double-counting problem associated with occluded fruits. A specific tracking region counting strategy and tracking algorithm based on motion displacement estimation were established. Six video sequences taken from two fields containing 22 trees were used as the validation dataset. The proposed method showed better performance (Mean Absolute Error (MAE) = 0.081, Standard Deviation (SD) = 0.08) than video-based manual counting and produced more accurate results than the existing standards Sort and DeepSort (MAE = 0.45 and 1.212; SD = 0.4741 and 1.3975).

Very-long-chain (VLC) alkanes are the main wax compounds of tomato fruits and leaves. ECERIFERUM1 (CER1) and ECERIFERUM3 (CER3) are the two key genes involved in VLC alkane biosynthesis in Arabidopsis thaliana. However, CER1 and CER3 homologs have not been investigated in tomato, and their exact biological functions remain unknown. We analyzed the wax profiles of tomato leaves and fruits at different growth stages and characterized the tomato CER1 and CER3 homologs. VLC alkanes were the predominant wax compounds in both leaves and fruits at all developmental stages. We identified five CER1 homologs and two CER3 homologs in tomato, which were designated SlCER1–1 to SlCER1–5 and SlCER3–1 and SlCER3–2, respectively. The genes exhibited tissue- and organ-specific expression patterns and were induced by abiotic stresses. SlCER1–1 was localized to the endoplasmic reticulum (ER), which is also the main site of wax biosynthesis. Silencing SlCER1–1 in tomato significantly reduced the amounts of n-alkanes and branched alkanes, whereas its overexpression in Arabidopsis had the opposite effect. Under drought stress, both n-alkanes and branched alkanes increased significantly in wild-type but not SlCER1–1 RNAi tomato plants. Furthermore, SlCER1–1 silencing also increased the cuticular permeability of leaves and fruits. In conclusion, SlCER1–1 is involved in wax alkane biosynthesis in tomato and plays an important role in drought tolerance and fruit storability.

Aristolochic acids (AAs) and their derivatives are present in multiple Aristolochiaceae species that have been or are being used as medicinal materials. During the past decades, AAs have received increasing attention because of their nephrotoxicity and carcinogenicity. Elimination of AAs from medicinal materials using biotechnological approaches is important for improving medication safety. However, it has not been achieved because of the limited information available on AA biosynthesis. Here, we report a high-quality, reference-grade genome assembly of the AA-containing vine Aristolochia contorta. The total size of the assembly is 209.27 Mb, and it is assembled into 7 pseudochromosomes. Synteny analysis, Ks distribution, and 4DTv suggest an absence of whole-genome duplication (WGD) events in Aristolochia contorta after the angiosperm-wide WGD. Based on genomic, transcriptomic, and metabolic data, pathways and candidate genes were proposed for benzylisoquinoline alkaloid (BIA) and AA biosynthesis in A. contorta. Five O-methyltransferase genes, including AcOMT1–3, AcOMT5, and AcOMT7, were cloned and functionally characterized. The results provide a high-quality reference genome for AA-containing species of Aristolochiaceae. They lay a solid foundation for further elucidation of AA biosynthesis and regulation and for the molecular breeding of Aristolochiaceae medicinal materials.

Tulip bulbs are modified underground stems that originate from axillary meristems of mother bulb scales. Hormones, including jasmonic acids (JAs), play key roles in the regulation of tulip bulb development. Here, we compared variations in daughter bulb development through transcriptomic profiling analysis and characterized the functions of JA biosynthesis-related genes during daughter bulb enlargement. The results showed that tulip cultivars exhibited contrasting bulb size variations. Transcriptomic analyses revealed that genes involved in plant hormones and development, including the two lipoxygenase genes TgLOX4 and TgLOX5, showed significant changes in expression following tulip bulb growth. Ectopic overexpression of TgLOX4 and TgLOX5 in Arabidopsis enhanced endogenous JA content, improved plant growth, and increased lateral root numbers. Silencing of these two genes in tulip repressed the growth of daughter bulbs. Furthermore, exogenous JA treatment promoted tulip bulb growth, whereas the JA biosynthesis inhibitor sodium diethyldithiocarbamate (DIECA) inhibited this process. This study offers supporting evidence for the involvement of tulip TgLOX4 and TgLOX5 in the regulation of daughter bulb growth and development.

Low temperature can affect the growth and development of plants through changes in DNA demethylation patterns. Another known effect of low temperature is the accumulation of anthocyanin pigments. However, it is not known whether the two phenomena are linked, specifically whether DNA demethylation participates in anthocyanin accumulation in response to low-temperature stress. The ROS1 gene is involved in plant DNA demethylation and influences methylation levels in response to low-temperature stress. In this study, using RNA sequencing, we detected the transcription levels of MdROS1, as well as those of anthocyanin biosynthesis-related genes, correlate with the anthocyanin content in apple (Malus domestica), at low temperature. Genomic bisulfite sequencing showed that the methylation levels of the promoters of the anthocyanin-related genes MdCHS, MdCHI, MdF3^′ H, MdANS, MdUFGT, and MdMYB10 decreased in apple leaves after low-temperature treatment. Similar expression and methylation results were found in apple fruit. Transiently silencing MdROS1 in the leaves and fruit of apple cultivars inhibited the accumulation of anthocyanins and led to decreased expression of anthocyanin biosynthetic genes, and the opposite results were detected in MdROS1-overexpressing leaves and fruit. A promoter binding assay showed that the conserved RRD-DME domains of MdROS1 bind directly to the promoters of MdF3^′ H and MdUFGT. Taken together, these results suggest that ROS1 affects the anthocyanin biosynthetic pathway by decreasing the methylation level of anthocyanin-related gene promoters, thereby increasing their expression and increasing anthocyanin accumulation.

Pomegranate fruit peels are highly abundant in metabolites derived from the shikimate pathway, such as hydrolyzable tannins (HTs) and flavonoids. These metabolites are beneficial to human health (commercial juice is enriched with peel metabolites), and also protect the fruit from environmental stresses. To understand the transcriptional control of shikimate pathway-related metabolites in pomegranate, we cloned and characterized a subgroup S4 R2R3 Myb transcription factor, PgMyb308-like. Overexpressing PgMyb308-like in pomegranate hairy roots increased the accumulation of shikimate, aromatic amino acids, isoferulic acid, and total lignins, but led to reduced gallic acid and its downstream products HTs, as well as multiple flavonoids. Changes in these metabolites are supported by the increased expression of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and shikimate dehydrogenase 1 (PgSDH1) (the SDH isoform associated with shikimate biosynthesis), and the reduced expression of PgSDH4 (the SDH isoform suggested to produce gallic acid). Transcriptome analysis of PgMyb308-like-overexpressing hairy roots further revealed reprogramming of cell wall-related genes, while overexpression of PgMyb308-like in Arabidopsis thaliana plants uncovered its distinct role in a different genetic and metabolic background. These results together suggest that PgMyb308-like activates genes in the shikimate pathway and lignin biosynthesis, but suppresses those involved in the production of HTs and flavonoids.

Malate is a major contributor to the sourness of grape berries (Vitis spp.) and their products, such as wine. Excessive malate at maturity, commonly observed in wild Vitis grapes, is detrimental to grape and wine quality and complicates the introgression of valuable disease resistance and cold hardy genes through breeding. This study investigated an interspecific Vitis family that exhibited strong and stable variation in malate at ripeness for five years and tested the separate contribution of accumulation, degradation, and dilution to malate concentration in ripe fruit in the last year of study. Genotyping was performed using transferable rhAmpSeq haplotype markers, based on the Vitis collinear core genome. Three significant QTL for ripe fruit malate on chromosomes 1, 7, and 17, accounted for over two-fold and 6.9 g/L differences, and explained 40.6% of the phenotypic variation. QTL on chromosomes 7 and 17 were stable in all and in three out of five years, respectively. Variation in pre-veraison malate was the major contributor to variation in ripe fruit malate (39%), and based on two and five years of data, respectively, their associated QTL overlapped on chromosome 7, indicating a common genetic basis. However, use of transferable markers on a closely related Vitis family did not yield a common QTL across families. This suggests that diverse physiological mechanisms regulate the levels of this key metabolite in the Vitis genus, a conclusion supported by a review of over a dozen publications from the past decade, showing malate-associated genetic loci on all 19 chromosomes.

Plant receptor-like kinases (RLKs) regulate many processes in plants. Many RLKs perform significant roles in plant immunity. Lectin receptor-like kinases (LecRLKs) are a large family of RLKs. However, the function of most LecRLKs is poorly understood. In this study, we show that a potato LecRLK, StLecRK-IV.1, is involved in plant immunity against Phytophthora infestans . As a negative regulator of immunity, StLecRK-IV.1 is downregulated by P. infestans and activated by abscisic acid. The transient expression of StLecRK-IV.1 in Nicotiana benthamiana enhanced P. infestans leaf colonization significantly. In contrast, the size of disease lesions caused by P. infestans was reduced by virus-induced gene silencing of the StLecRK-IV.1 ortholog in N. benthamiana , NbLecRK-IV.1 , as well as in potato plants with stable RNA interference of StLecRK-IV.1 . Tetraspanin-8 (StTET8) was identified to be interacting with StLecRK-IV.1 using a membrane yeast two-hybrid system, which was further verified by co-immunoprecipitation, a luciferase complementation assay, and a bimolecular fluorescence complementation test. StTET8 is a positive immune regulator that restrains P. infestans infection. The co-expression of StLecRK-IV.1 with StTET8 antagonized the positive roles of StTET8 against P. infestans. Moreover, the co-expression of StTET8 with StLecRK-IV.1 affected the stability of StTET8, which was confirmed by a western blot assay and confocal assay. Taken together, our work first reveal that a potato L-type lectin RLK, StLecRK-IV.1, negatively regulates plant immunity by targeting a positive regulator, StTET8, through affecting its stability.

Watercress is a nutrient-dense leafy green crop, traditionally grown in aquatic outdoor systems and increasingly seen as well-suited for indoor hydroponic systems. However, there is concern that this crop has a detrimental impact on the environment through direct phosphate additions causing environmental pollution. Phosphate-based fertilisers are supplied to enhance crop yield, but their use may contribute to eutrophication of waterways downstream of traditional watercress farms. One option is to develop a more phosphate use efficient (PUE) crop. This review identifies the key traits for this aquatic crop (the ideotype), for future selection, marker development and breeding. Traits identified as important for PUE are (i) increased root surface area through prolific root branching and adventitious root formation, (ii) aerenchyma formation and root hair growth. Functional genomic traits for improved PUE are (iii) efficacious phosphate remobilisation and scavenging strategies and (iv) the use of alternative metabolic pathways. Key genomic targets for this aquatic crop are identified as: PHT phosphate transporter genes, global transcriptional regulators such as those of the SPX family and genes involved in galactolipid and sulfolipid biosynthesis such as MGD2/3, PECP1, PSR2, PLD ζ 1/2 and SQD2. Breeding for enhanced PUE in watercress will be accelerated by improved molecular genetic resources such as a full reference genome sequence that is currently in development.

Sapindus mukorossi is an environmentally friendly plant and renewable energy source whose fruit has been widely used for biomedicine, biodiesel, and biological chemicals due to its richness in saponin and oil contents. Here, we report the first chromosome-scale genome assembly of S. mukorossi (covering ∼391 Mb with a scaffold N50 of 24.66 Mb) and characterize its genetic architecture and evolution by resequencing 104 S. mukorossi accessions. Population genetic analyses showed that genetic diversity in the southwestern distribution area was relatively higher than that in the northeastern distribution area. Gene flow events indicated that southwest species may be the donor population for the distribution areas in China. Genome-wide selective sweep analysis showed that a large number of genes are involved in defense responses, growth and development, including SmRPS2 , SmRPS4 , SmRPS7 , SmNAC2 , SmNAC23 , SmNAC102 , SmWRKY6 , SmWRKY26 , and SmWRKY33 . We also identified several candidate genes controlling six agronomic traits by genome-wide association studies, including SmPCBP2 , SmbHLH1 , SmCSLD1 , SmPP2C , SmLRR-RKs , and SmAHP . Our study not only provides a rich genomic resource for further basic research on Sapindaceae woody trees but also identifies several economically significant genes for genomics-enabled improvements in molecular breeding.

Fleshy fruits undergo a complex ripening process, developing organoleptic fruit traits that attract herbivores and maximize seed dispersal. Ripening is the terminal stage of fruit development and involves a series of physiological and biochemical changes. In fleshy fruits, ripening always involves a drastic color change triggered by the accumulation of pigments and degradation of chlorophyll, softening caused by cell wall remodeling, and flavor formation as acids and sugars accumulate alongside volatile compounds. The mechanisms underlying fruit ripening rely on the orchestration of ripening-related transcription factors, plant hormones, and epigenetic modifications. In this review, we discuss current knowledge of the transcription factors that regulate ripening in conjunction with ethylene and environmental signals (light and temperature) in the model plant tomato (Solanum lycopersicum) and other fleshy fruits. We emphasize the critical roles of epigenetic regulation, including DNA methylation and histone modification as well as RNA m⁶A modification, which has been studied intensively. This detailed review was compiled to provide a comprehensive description of the regulatory mechanisms of fruit ripening and guide new strategies for its effective manipulation.

Nitrate is the major nitrogen source for higher plants. In addition to serving as a nutrient, it is also a signaling molecule that regulates plant growth and development. Although membrane-bound nitrate transporter/peptide transporters (NRT/PTRs) have been extensively studied and shown to regulate nitrate uptake and movement, little is known about how these factors are regulated by the external nitrogen environment. Red-fleshed apple, whose coloration is determined by the transcription factor MdMYB10, had higher nitrate uptake efficiency than non-red-fleshed apple. Nitrate assimilation and utilization were higher in red-fleshed apple cultivars, and comparative transcriptome analysis showed that the expression of genes encoding the NRT2s was increased in red-fleshed apple. In vitro and in vivo experiments showed that MdMYB10 directly bound to the MdNRT2.4-1 promoter to transcriptionally activate its expression, resulting in enhanced nitrate uptake. MdMYB10 also controlled nitrate reallocation from old leaves to new leaves through MdNRT2.4-1. Overall, our findings provide novel insights into the mechanism by which MdMYB10 controls nitrate uptake and reallocation in apple, which facilitates adaptation to a low-nitrogen environment.

Soil microbiota has increasingly been shown to play an integral role in viticulture resilience. The emergence of new metagenomic and culturomic technologies has led to significant advances in the study of microbial biodiversity. In the agricultural sector, soil and plant microbiomes have been found to significantly improve resistance to environmental stressors and diseases, as well as influencing crop yields and fruit quality thus improving sustainability under shifting environments. Grapevines are usually cultivated as a scion grafted on rootstocks, which are selected according to pedoclimatic conditions and cultural practices, known as terroir. The rootstock connects the surrounding soil to the vine’s aerial part and impacts scion growth and berry quality. Understanding rootstock and soil microbiome dynamics is a relevant and important field of study, which may be critical to improve viticulture sustainability and resilience. This review aims to highlight the relationship between grapevine roots and telluric microbiota diversity and activity. In addition, this review explores the concept of core microbiome regarding potential applications of soil microbiome engineering with the goal of enhancing grapevine adaptation to biotic and abiotic stress.

Carotenoids are one of the most important pigments for the coloring of many plants, fruits, and flowers. Recently, significant progress has been made in carotenoid metabolism. However, our specific understanding of the transcriptional regulation that controls the expression of carotenoid metabolic genes remains extremely limited. Anemone-type chrysanthemums, a special group of chrysanthemum cultivars, contain elongated disc florets in the capitulum that usually differ in color from the ray florets because of their different carotenoid contents. In this study, the carotenoid composition and content of ray and disc florets from the anemone-type chrysanthemum cultivar “Dong Li Fen Gui” were analyzed by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS), and the key structural gene CmCCD4a-2, whose differential expression resulted in different carotenoid contents in these two types of florets, was identified. The promoter sequence of CmCCD4a-2 was then used as bait to screen a chrysanthemum flower cDNA library, and the transcription factors (TFs) CmAP3 and CmUIF1 were identified. Y2H, BiFC, and Y3H experiments demonstrated that these two TFs were connected by CmPI to form a CmAP3-CmPI-CmUIF1 TF complex. This TF complex regulated carotenoid metabolism by directly activating the expression of CmCCD4a-2. A large number of target genes regulated directly by the CmAP3-CmPI-CmUIF1 TF complex, including carotenoid biosynthetic genes, flavonoid biosynthetic genes, and flower development-related genes, were identified by DNA-affinity purification sequencing (DAP-seq). This result indicated that the CmAP3-CmPI-CmUIF1 TF complex may participate in multiple processes. These findings expand our knowledge of the transcriptional regulation of carotenoid metabolism in plants and will be helpful for manipulating carotenoid accumulation in chrysanthemum.

Longan (Dimocarpus longan) is a subtropical fruit tree best known for its nutritious fruit and regarded as a valuable tonic and traditional medicine since ancient times. A high-quality chromosome-scale genome assembly is valuable for functional genomic study and genetic improvement of longan. Here, we report a chromosome-level reference genome sequence for the longan cultivar JDB. The assembled genome is 455.5 Mb in size and anchored to fifteen chromosomes, representing a significant improvement in contiguity (contig N50 = 12.1 Mb, scaffold N50 = 29.5 Mb) over a previous draft assembly. A total of 40 420 protein-coding genes were predicted in the D. longan genome. Synteny analysis suggests that longan shares the widespread gamma event with core eudicots but has no other whole genome duplications. Comparative genomics showed that the D. longan genome experienced significant expansions of UDP-glucosyltransferase and phenylpropanoid biosynthesis-related gene families. Deep genome sequencing analysis of longan cultivars identified longan biogeography as a major contributing factor to its genetic diversity and revealed clear population admixture and introgression among cultivars of different geographic origins, suggesting a likely migration trajectory of longan that is confirmed by existing historical records. Finally, genome-wide association studies (GWAS) of longan cultivars identified quantitative trait loci (QTLs) for six different fruit quality traits and revealed a shared QTL that contained three genes for total soluble solids and seed weight. The chromosome-level reference genome assembly, annotation, and population genetic resources for D. longan will facilitate the molecular studies and breeding of desirable longan cultivars in the future.

Anthocyanins are plant secondary metabolites that have a variety of biological functions, including pigmentation. The accumulation of anthocyanins is regulated by both transcriptional activators and repressors. Studies have shown that bZIP family members act primarily as positive regulators of anthocyanin biosynthesis, but there are few reports of negative regulation. Here, we report that a grapevine (Vitis vinifera) bZIP gene from group K, VvbZIP36, acts as a negative regulator of anthocyanin biosynthesis. Knocking out one allele of VvbZIP36 in grapevine with CRISPR/Cas9 promoted anthocyanin accumulation. Correlation analysis of transcriptome and metabolome data showed that a range of anthocyanin biosynthesis genes were activated in VvbZIP36 mutant plants relative to the wild type, resulting in the accumulation of related metabolites, including naringenin chalcone, naringenin, dihydroflavonols, and cyanidin-3- O-glucoside. The synthesis of stilbenes (α-viniferin), lignans, and some flavonols (including quercetin-3- O-rhamnoside, kaempferol-3- O-rhamnoside, and kaempferol-7- O-rhamnoside) was significantly inhibited, and several genes linked to their metabolism were downregulated in the mutant plants. In summary, our results demonstrate that VvbZIP36 is a negative regulator of anthocyanin biosynthesis that plays a role in balancing the synthesis of stilbenes (α-viniferin), lignans, flavonols, and anthocyanins.

Non-conventional peptides (NCPs), which are peptides derived from previously unannotated coding sequences, play important biological roles in plants. In this study, we used peptidogenomic methods that integrated mass spectrometry (MS) peptidomics and a six-frame translation database to extensively identify NCPs in grape. In total, 188 and 2021 non-redundant peptides from the Arabidopsis thaliana and Vitis vinifera L. protein database at Ensembl/URGI and an individualized peptidogenomic database were identified. Unlike conventional peptides, these NCPs derived mainly from intergenic, intronic, upstream ORF, 5^′UTR, 3^′UTR, and downstream ORF regions. These results show that unannotated regions are translated more broadly than we thought. We also found that most NCPs were derived from regions related to phenotypic variations, LTR retrotransposons, and domestication selection, indicating that the NCPs have an important function in complex biological processes. We also found that the NCPs were developmentally specific and had transient and specific functions in grape berry development. In summary, our study is the first to extensively identify NCPs in grape. It demonstrated that there was a large amount of translation in the genome. These results lay a foundation for studying the functions of NCPs and also provide a reference for the discovery of new functional genes in grape.

Naturally occurring methylated catechins, especially methylated EGCG in tea leaves, are known to have many health benefits. Although the genes involved in methylated EGCG biosynthesis have been studied extensively, the transcription factors that control methylated EGCG biosynthesis are still poorly understood. In the present study, a WRKY domain-containing protein termed CsWRKY57like was identified, which belongs to group IIc of the WRKY family and contains one conserved WRKY motif. CsWRKY57like was found to localize in the nucleus and function as a transcriptional activator; its expression was positively correlated with methylated EGCG level. In addition, CsWRKY57like activated the transcription of three genes related to methylated EGCG biosynthesis (CCoAOMT, CsLAR, and CsDFR), specifically interacting with their promoters by binding to the cis-acting element (C/T)TGAC(T/C). Further assays revealed that CsWRKY57like physically interacts with CsVQ4 and participates in the metabolic regulation of O-methylated catechin biosynthesis. We conclude that CsWRKY57like may positively impact the biosynthesis of methylated EGCG in the tea plant. These results comprehensively enrich the regulatory network of WRKY TFs associated with methylated EGCG and provide a potential strategy for the breeding of specific tea plant cultivars with high methylated EGCG levels.

L-Theanine is a crucial secondary metabolite in tea and positively determines the potential quality and health benefits of tea products. Previous work found that the content of L-theanine decreased during the withering process, although the specific mechanism is still unknown. Here, weighted gene co-expression network analysis (WGCNA) was performed based on transcriptome data obtained previously. The key hydrolysis gene CsPDX2.1 in L-theanine metabolism and seven candidate transcription factors were screened out. Among these transcription factors, CsWRKY40 displayed the strongest activation on the CsPDX2.1 promoter (373.18-fold) by binding to the W box element, based on dual luciferase assay and EMSA results. The CsWRKY40 protein was located in the nucleoplasm, whereas CsPDX2.1 was found in both the nucleoplasm and cytoplasm. Analysis of withering, water-retention, and water-loss treatments confirmed that water loss from tea leaves was the critical factor that affected ABA and L-theanine contents by activating the expression of CsWRKY40 and CsPDX2.1. Our results provide a new insight into the regulatory mechanism of L-theanine hydrolysis metabolism.

Organic acid content in fruit is an important determinant of peach organoleptic quality, and undergoes considerable variations during development and maturation. However, its molecular mechanism remains largely unclear. In this study, an integrative approach of genome-wide association studies and comparative transcriptome analysis was applied to identify candidate genes involved in organic acid accumulation in peach. A key gene, PpTST1, encoding tonoplast sugar transporter, was identified and the genotype of PpTST1 with a single-base transversion (G1584T) in the third exon that leads to a single amino acid substitution (Q528H) was associated with a low level of organic acid content in peach. Overexpression of PpTST1^His resulted in reduced organic acid content along with increased sugar content both in peach and tomato fruits, suggesting its dual function in sugar accumulation and organic acid content reduction. Two V-type proton ATPases interacted with PpTST1 in a yeast two-hybrid assay. In addition, the G1584T transversion appeared and gradually accumulated during domestication and improvement, which indicated that PpTST1 was under selection. The identification and characterization of PpTST1 would facilitate the improvement of peach fruit quality.

The foliage of the native grape species Vitis riparia and certain cold-hardy hybrid grapes are particularly susceptible to the insect pest phylloxera, Daktulosphaira vitifoliae Fitch. A previous study using a cold-hardy hybrid grape biparental F₁ population (N ∼125) detected the first quantitative trait locus (QTL) for foliar resistance on chromosome 14, designated as resistance to Daktulosphaira vitifoliae 3 (Rdv3). This locus spans a ∼7-Mbp (10–20 cM) region and is too wide for effective marker-assisted selection or identification of candidate genes. Therefore, we fine mapped the QTL using a larger F₁ population, GE1783 (N ∼1023), and genome-wide rhAmpSeq haplotype markers. Through three selective phenotyping experiments replicated in the greenhouse, we screened 184 potential recombinants of GE1783 using a 0 to 7 severity rating scale among other phylloxera severity traits. A 500-kb fine mapped region at 4.8 Mbp on chromosome 14 was identified. The tightly linked rhAmpSeq marker 14_4 805 213 and flanking markers can be used for future marker-assisted breeding. This region contains 36 candidate genes with predicted functions in disease resistance (R genes and Bonzai genes) and gall formation (bifunctional 3-dehydroquinate dehydratase/shikimate dehydrogenase). Disease resistance genes suggest a traditional R-gene-mediated resistance mechanism often accompanied by a hypersensitive response, which has been widely studied in the plant pathology field. A novel resistance mechanism, non-responsiveness to phylloxera gall formation is proposed as a function of the bifunctional dehydratase gene, which plays a role in gallic acid biosynthesis and is important in gall formation. This study has implications for improvement of foliar phylloxera resistance in cold-hardy hybrid germplasm and is a starting place to understand the mechanism of resistance in crops to gall-forming insects.

Implementation of genomic tools is desirable to increase the efficiency of apple breeding. Recently, the multi-environment apple reference population (apple REFPOP) proved useful for rediscovering loci, estimating genomic predictive ability, and studying genotype by environment interactions (G × E). So far, only two phenological traits were investigated using the apple REFPOP, although the population may be valuable when dissecting genetic architecture and reporting predictive abilities for additional key traits in apple breeding. Here we show contrasting genetic architecture and genomic predictive abilities for 30 quantitative traits across up to six European locations using the apple REFPOP. A total of 59 stable and 277 location-specific associations were found using GWAS, 69.2% of which are novel when compared with 41 reviewed publications. Average genomic predictive abilities of 0.18–0.88 were estimated using main-effect univariate, main-effect multivariate, multi-environment univariate, and multi-environment multivariate models. The G × E accounted for up to 24% of the phenotypic variability. This most comprehensive genomic study in apple in terms of trait- environment combinations provided knowledge of trait biology and prediction models that can be readily applied for marker-assisted or genomic selection, thus facilitating increased breeding efficiency.

Lotus (Nelumbo Adans.), a relict plant, is testimony to long-term sustained ecological success, but the underlying genetic changes related to its survival strategy remain unclear. Here, we assembled the high-quality lotus genome, investigated genome variation of lotus mutation accumulation (MA) lines and reconstructed the demographic history of wild Asian lotus. We identified and validated 43 base substitutions fixed in MA lines, implying a spontaneous mutation rate of 1.4 × 10⁻⁹ bases/generation in lotus shoot stem cells. The past history of the lotus revealed that the ancestors of the lotus in eastern and southern Asia could be traced back to ∼20 million years ago and twice experienced significant bottlenecks and population splits. We further identified selected genes among three lotus groups in different habitats, suggesting that 453 differed genes between the tropical and temperate group and 410 differed genes between two subgroups from Northeastern China and the Yangtze River–Yellow River Basin might play important roles in natural selection in the lotus’s adaptation and resilience. Our findings not only improve understanding of the evolutionary history of the lotus and the genetic basis of its survival advantages, but also provide valuable data for addressing various questions in evolution and protection for relict plants.

A novel haplotype-based approach that uses Procrustes analysis and automatic classification was used to provide further insights into tomato history and domestication. Agrarian societies domesticated species of interest by introducing complex genetic modifications. For tomatoes, two species, one of which had two botanical varieties, are thought to be involved in its domestication: the fully wild Solanum pimpinellifolium (SP), the wild and semi-domesticated Solanum lycopersicum var. cerasiforme (SLC) and the cultivated S. l. var. lycopersicum (SLL). The Procrustes approach showed that SP evolved into SLC during a gradual migration from the Peruvian deserts to the Mexican rainforests and that Peruvian and Ecuadorian SLC populations were the result of more recent hybridizations. Our model was supported by independent evidence, including ecological data from the accession collection site and morphological data. Furthermore, we showed that photosynthesis-, and flowering time-related genes were selected during the latitudinal migrations.

Cucumber (Cucumis sativus L.) often experiences chilling stress that limits its growth and productivity. Grafting is widely used to improve abiotic stress resistance by exploiting a vigorous root system, suggesting there exists systemic signals communication between distant organs. mRNAs are reported to be evolving fortification strategies involving long-distance signaling when plants suffer from chilling stress. However, the potential function of mobile mRNAs in alleviating chilling stress in grafted cucumber is still unknown. Here, the physiological changes, mobile mRNA profiles, and transcriptomic and metabolomic changes in above- and underground tissues of all graft combinations of cucumber and pumpkin responding to chilling stress were established and analyzed comprehensively. The co-relationship between the cluster of chilling-induced pumpkin mobile mRNAs with differentially expressed genes and differentially intensive metabolites revealed that four key chilling-induced pumpkin mobile mRNAs were highly related to glycine, serine, and threonine synthesis and fatty acid β-oxidative degradation metabolism in cucumber tissues of heterografts. The verification of mobile mRNAs, potential transport of metabolites, and exogenous application of key metabolites of the glycerophospholipid metabolism pathway in cucumber seedlings confirmed the role of mobile mRNAs in regulating chilling responses in grafted cucumber. Our results build a link between the long-distance mRNAs of chilling-tolerant pumpkin and the fatty acid β-oxidative degradation metabolism of chilling-sensitive cucumber. They also help to uncover the mechanism of signaling interaction between scion and stock as a means of achieving chilling tolerance in grafted cucumber.

Grafting is a widely used practice for asexual propagation of fruit trees. Many physiological, biochemical, and molecular changes occur upon grafting that can influence important horticultural traits. This technology has many advantages, including avoidance of juvenility, modifying the scion architecture, improving productivity, adapting scion cultivars to unfavourable environmental conditions, and developing traits in resistance to insect pests, bacterial and fungal diseases. A limitation of grafting is scion-rootstock incompatibility. It may be caused by many factors, including insufficient genetic proximity, physiological or biochemical factors, lignification at the graft union, poor graft architecture, insufficient cell recognition between union tissues, and metabolic differences in the scion and the rootstock. Plant hormones, like auxin, ethylene (ET), cytokinin (CK), gibberellin (GA), abscisic acid (ABA), and jasmonic acid (JA) orchestrate several crucial physiological and biochemical processes happening at the site of the graft union. Additionally, epigenetic changes at the union affect chromatin architecture by DNA methylation, histone modification, and the action of small RNA molecules. The mechanism triggering these effects likely is affected by hormonal crosstalk, protein and small molecules movement, nutrients uptake, and transport in the grafted trees. This review provides an overview of the basis of physiological, biochemical, and molecular aspects of fruit tree grafting between scion and rootstock.

Brassica oleracea displays enormous phenotypic variation, including vegetables like cabbage, broccoli, cauliflower, kohlrabi, kales etc. Its domestication has not been clarified, despite several genetic studies and investigations of ancient literature. We used 14 152 high-quality SNP markers for population genetic studies and species-tree estimation (treating morphotypes as “species”) using SVD-quartets coalescent-modelling of a collection of 912 globally distributed accessions representing ten morphotypes of B. oleracea, wild B. oleracea accessions and nine related C9 Brassica species. Our genealogical tree provided evidence for two domestication lineages, the “leafy head” lineage (LHL) and the “arrested inflorescence” lineage (AIL). It also showed that kales are polyphyletic with regards to B. oleracea morphotypes, which fits ancient literature describing highly diverse kale types at around 400 BC. The SVD-quartets species tree topology showed that different kale clades are sister to either the LHL or the AIL. Cabbages from the middle-east formed the first-branching cabbage-clade, supporting the hypothesis that cabbage domestication started in the middle-east, which is confirmed by archeological evidence and historic writings. We hypothesize that cabbages and cauliflowers stem from kales introduced from Western Europe to the middle-east, possibly transported with the tin-trade routes in the Bronze age, to be re-introduced later into Europe. Cauliflower is the least diverse morphotype showing strong genetic differentiation with other morphotypes except broccoli, suggesting a strong genetic bottleneck. Genetic diversity reduced from landraces to modern hybrids for almost all morphotypes. This comprehensive Brassica C-group germplasm collection provides valuable genetic resources and a sound basis for B. oleracea breeding.

Ethylene regulates anthocyanin synthesis in ripening apple fruit via the antagonistic activities of the R2R3-MYB repressors and activators. However, the molecular mechanism underlying this process remains unknown. In this study, ethylene significantly induced the expression of the R2R3-MYB gene MdMYB17 in apple fruit. Moreover, MdMYB17 was revealed to be an important repressor of anthocyanin synthesis. Specifically, MdMYB17 binds directly to the promoters of the ethylene-induced genes MdMYB1 and MdEIL1, which encode positive regulators of anthocyanin synthesis, and represses their expression. Additionally, MdMYB1 and MdEIL1 bind to the MdMYB17 promoter to activate its expression. Thus, MdMYB17, MdMYB1, and MdEIL1 form a regulatory module that controls the expression of the corresponding genes. MdMYB17 interacts with MdEIL1. The interaction between MdMYB17 and MdEIL1 attenuates the regulatory effects of MdMYB17 on MdMYB1 and MdEIL1 as well as the regulatory effects of MdEIL1 on MdMYB17. Overall, our results reveal the molecular mechanisms by which MdMYB17, MdMYB1, and MdEIL1 finely mediate ethylene-regulated anthocyanin synthesis in apple fruit.

Heat shock transcription factor (Hsf) plays a critical role in regulating heat resistance. Here, 2950 Hsf family genes were identified from 111 horticultural and representative plants. More Hsf genes were detected in higher plants than in lower plants. Based on all Hsf genes, we constructed a phylogenetic tree, which indicated that Hsf genes of each branch evolved independently after species differentiation. Furthermore, we uncovered the evolutionary trajectories of Hsf genes by motif analysis. There were only six motifs (M1–M6) in lower plants, and then four novel motifs (M7–M10) appeared in higher plants. However, the motifs of some Hsf genes were lost in higher plants, indicating that Hsf genes have undergone sequence variation during their evolution. The number of Hsf genes lost was greater than the number of genes that were duplicated after whole-genome duplication in higher plants. The heat response network was constructed using 24 Hsf genes and 2421 downstream and 222 upstream genes of Arabidopsis. Further enrichment analysis revealed that Hsf genes and other transcription factors interacted with each other in the response to heat stress. Global expression maps were illustrated for Hsf genes under various abiotic and biotic stresses and several developmental stages in Arabidopsis. Syntenic and phylogenetic analyses were conducted using Hsf genes of Arabidopsis and the pan-genome of 18 Brassica rapa accessions. We also performed expression pattern analysis of Hsf and six Hsp family genes using expression values from different tissues and heat treatments in B. rapa. The interaction network between the Hsf and Hsp gene families was constructed in B. rapa, and several core genes were detected in the network. Finally, we constructed an Hsf database (http://hsfdb.bio2db.com) for researchers to retrieve Hsf gene family information. Therefore, our study will provide rich resources for the study of the evolution and function of Hsf genes.

Fruit growth is a complex mechanism resulting from biochemical and biophysical events leading water and dry matter to accumulate in the fruit tissues. Understanding how fruits choose their growth strategies can help growers optimizing their resource management for a more sustainable production and a higher fruit quality. This paper compares the growth strategies adopted by different fruit crops, at different times during the season and relates their fruit surface conductance to key physiological parameters for fruit growth such as phloem and xylem inflows as well transpiration losses. Our results show how fruits capacity to transpire (determined by their surface conductance) is a key driver in determining the growth strategy adopted by a species and explains the inter-species variability existing among different crops. Indeed, fruits change their surface conductance depending on the species and the phenological stage. This has an impact on the fruit’s ability to lose water due to transpiration, affecting fruit pressure potential and increasing the force with which the fruit is able to attract xylem and phloem flows, with a considerable impact on fruit growth rate.

Fruit weight is an integral part of fruit quality and directly influences the commodity value and economic return of fruit crops. Despite the importance of fruit weight, its underlying molecular mechanisms remain understudied, especially for perennial fruit tree crops such as cultivated loquat (Eriobotrya japonica Lindl.). Auxin is known to regulate fruit development, but its role and metabolism during the development of loquat fruit remain obscure. In this study, we used a multi-omics approach, integrating whole-genome resequencing-based quantitative trait locus (QTL) mapping with an F₁ population, population genomics analysis using germplasm accessions, transcriptome analysis, and metabolic profiling to identify genomic regions potentially associated with fruit weight in loquat. We identified three major loci associated with fruit weight, supported by both QTL mapping and comparative genomic analysis between small- and large-fruited loquat cultivars. Comparison between two genotypes with contrasting fruit weight performance by transcriptomic and metabolic profiling revealed an important role for auxin in the regulation of fruit development, especially at the fruit enlargement stage. The multi-omics approach identified homologs of ETHYLENE INSENSITIVE 4 (EjEIN4) and TORNADO 1 (EjTRN1) as promising candidates for the control of fruit weight. Three single nucleotide polymorphism (SNP) markers were also closely associated with fruit weight. Results from this study provide insights into the genetic and metabolic controls of fruit weight in loquat from multiple perspectives. The candidate genomic regions, genes, and sequence variants will facilitate our understanding of the molecular basis of fruit weight and lay a foundation for future breeding and manipulation of fruit weight in loquat.

The basic helix–loop–helix (bHLH) family of transcription factors (TFs) participate in a variety of biological regulatory processes in plants, and have undergone significant expansion during land plant evolution by gene duplications. In cucurbit crops, several bHLH genes have been found to be responsible for agronomic traits such as bitterness. However, the characterization of bHLH genes across the genomes of cucurbit species has not been reported, and how they have evolved and diverged remains largely unanswered. Here we identified 1,160 bHLH genes in seven cucurbit crops and performed a comprehensive comparative genomics analysis. We determined orthologous and paralogous bHLH genes across cucurbit crops by syntenic analysis between or within species. Orthology and phylogenetic analysis of the tandem-duplicated bHLH genes in the Bt cluster, which regulate the biosynthesis of cucurbitacins, suggest that this cluster is derived from three ancestral genes after the cucurbit-common tetraploidization event. Interestingly, we identified a new conserved cluster paralogous to the Bt cluster that includes two tandem bHLH genes, and the evolutionary history and expression profiles of these two genes in the new cluster suggest the involvement of one gene (Brp) in the regulation of cucurbitacin biosynthesis in roots. Further biochemical and transgenic assays in melon hairy roots supported the function of Brp. This study provides useful information for further investigating the functions of bHLH TFs and novel insights into the regulation of cucurbitacin biosynthesis in cucurbit crops and other plants.

Low temperatures are known to destroy cell membranes’ structural integrity by affecting the remodeling of their phospholipids. Fruits stored at low temperature are prone to chilling injury, characterized by discoloration, absence of ripening, surface pitting, growth inhibition, flavor loss, decay, and wilting. Phosphatidic acid, a vital second-messenger lipid in plants, is known to accumulate in response to different kinds of stress stimuli. However, the regulatory mechanism of its production from the degradation of phospholipids remains poorly understood. We identified two cold-responsive NAC (NAM/ATAF1/CUC2) transcription factors from bananas, namely, MaNAC25 and MaNAC28, which negatively regulated cold tolerance in banana fruits by upregulating the expression of phospholipid degradation genes in banana fruits. Furthermore, MaNAC25 and MaNAC28 formed a positive feedback loop to induce phospholipid degradation and produce phosphatidic acid. In contrast, ethylene directly inhibited the degradation of phospholipids in banana and transgenic tomato fruits. In addition, ethylene reduced the activity of MaNAC25 and MaNAC28, thereby inhibiting phospholipid degradation. To conclude, NAC-mediated membrane lipid remodeling negatively regulates the cold tolerance of banana and transgenic tomato fruits.

Although many genes and biological processes involved in abiotic stress responses have been identified, how they are regulated remains largely unclear. Here, to study the regulatory mechanism of birch (Betula platyphylla) responding to drought induced by polyethylene glycol 6000 (20%, w/v), a partial correlation coefficient-based algorithm for constructing a gene regulatory network (GRN) was proposed, and a three-layer hierarchical GRN was constructed, including 68 transcription factors and 252 structural genes. A total of 1448 predicted regulatory relationships are included, and most of them are novel. The reliability of the GRN was verified by chromatin immunoprecipitation (ChIP)–PCR and qRT–PCR based on transient transformation. About 55% of genes in the bottom layer of the GRN could confer drought tolerance. We selected two TFs, BpMADS11 and BpNAC090, from the top layer and characterized their function in drought tolerance. Overexpression of BpMADS11 and BpNAC090 reduces electrolyte leakage, reactive oxygen species (ROS) and malondialdehyde (MDA) contents, giving greater drought tolerance than wild-type birch. According to this GRN, the important biological processes involved in drought were identified, including ‘signaling hormone pathways’, ‘water transport’, ‘regulation of stomatal movement’, and ‘response to oxidative stress’. This work indicated that BpERF017, BpAGL61, and BpNAC090 are the key upstream regulators of birch drought tolerance. Our data clearly revealed that upstream regulators and transcription factor–DNA interaction regulate different biological processes to adapt to drought stress.

Crop breeding involves two selection steps: choosing progenitors and selecting individuals within progenies. Genomic prediction, based on genome-wide marker estimation of genetic values, could facilitate these steps. However, its potential usefulness in grapevine (Vitis vinifera L.) has only been evaluated in non-breeding contexts mainly through cross-validation within a single population. We tested across-population genomic prediction in a more realistic breeding configuration, from a diversity panel to ten bi-parental crosses connected within a half-diallel mating design. Prediction quality was evaluated over 15 traits of interest (related to yield, berry composition, phenology and vigour), for both the average genetic value of each cross (cross mean) and the genetic values of individuals within each cross (individual values). Genomic prediction in these conditions was found useful: for cross mean, average per-trait predictive ability was 0.6, while per-cross predictive ability was halved on average, but reached a maximum of 0.7. Mean predictive ability for individual values within crosses was 0.26, about half the within-half-diallel value taken as a reference. For some traits and/or crosses, these across-population predictive ability values are promising for implementing genomic selection in grapevine breeding. This study also provided key insights on variables affecting predictive ability. Per-cross predictive ability was well predicted by genetic distance between parents and when this predictive ability was below 0.6, it was improved by training set optimization. For individual values, predictive ability mostly depended on trait-related variables (magnitude of the cross effect and heritability). These results will greatly help designing grapevine breeding programs assisted by genomic prediction.

Flowering date is an important trait in Prunus fruit species, especially for their adaptation in a global warming context. Numerous quantitative trait loci (QTLs) have been identified and a major one was previously located on LG4. The objectives of this study were to fine-map this QTL in sweet cherry, to identify robust candidate genes by using the new sweet cherry genome sequence of the cultivar “Regina” and to define markers usable in marker-assisted selection (MAS). We performed QTL analyses on two populations derived from crosses using cultivars “Regina” and “Garnet” as parents. The first one (n = 117) was phenotyped over ten years, while the second one (n = 1386) was evaluated during three years. Kompetitive allele specific PCR (KASP) markers located within the QTL region on LG4 were developed and mapped within this region, consisting in the first fine mapping in sweet cherry. The QTL interval was narrowed from 380 kb to 68 kb and candidate genes were identified by using the genome sequence of “Regina”. Their expression was analyzed from bud dormancy period to flowering in cultivars “Regina” and “Garnet”. Several genes, such as PavBOI-E3, PavSR45a and PavSAUR71, were differentially expressed in these two cultivars and could be then considered as promising candidate genes. Two KASP markers were validated using a population derived from a cross between cultivars “Regina” and “Lapins” and two collections, including landraces and modern cultivars. Thanks to the high synteny within the Prunus genus, these results give new insights into the control of flowering date in Prunus species and pave the way for the development of molecular breeding strategies.

Arbuscular mycorrhizal symbiosis (AMS) is widespread mutualistic association between plants and fungi, which plays an essential role in nutrient exchange, enhancement in plant stress resistance, development of host, and ecosystem sustainability. Previous studies have shown that plant small secreted proteins (SSPs) are involved in beneficial symbiotic interactions. However, the role of SSPs in the evolution of AMS has not been well studied yet. In this study, we performed computational analysis of SSPs in 60 plant species and identified three AMS-specific ortholog groups containing SSPs only from at least 30% of the AMS species in this study and three AMS-preferential ortholog groups containing SSPs from both AMS and non-AMS species, with AMS species containing significantly more SSPs than non-AMS species. We found that independent lineages of monocot and eudicot plants contained genes in the AMS-specific ortholog groups and had significant expansion in the AMS-preferential ortholog groups. Also, two AMS-preferential ortholog groups showed convergent changes, between monocot and eudicot species, in gene expression in response to arbuscular mycorrhizal fungus Rhizophagus irregularis. Furthermore, conserved cis-elements were identified in the promoter regions of the genes showing convergent gene expression. We found that the SSPs, and their closely related homologs, in each of three AMS-preferential ortholog groups, had some local variations in the protein structural alignment. We also identified genes co-expressed with the Populus trichocarpa SSP genes in the AMS-preferential ortholog groups. This first plant kingdom-wide analysis on SSP provides insights on plant-AMS convergent evolution with specific SSP gene expression and local diversification of protein structures.

Brassica parachinensis is a popular leafy vegetable. It is able to accumulate high concentrations of cadmium (Cd), but the molecular mechanism of Cd accumulation is unknown. This study investigated the function and regulatory mechanism of the Cd-responsive metal ion transporter gene BrpHMA2 . BrpHMA2 was induced by Cd stress and specifically expressed in vascular tissues, and the protein was localized in the plasma membrane. Heterologous expression of BrpHMA2 enhanced Cd accumulation and Cd sensitivity in transgenic Arabidopsis and yeast. After Cd stress, the transcription factors BrpNAC895 and BrpABI449, which may recognize the abscisic acid-responsive elements in the BrpHMA2 promoter, were also differentially expressed. The transcriptional regulation of BrpHMA2 was further investigated using the chromatin immunoprecipitation–quantitative PCR (ChIP–qPCR) assay, the electrophoretic mobility shift assay (EMSA), and luciferase (LUC) reporter activity analysis employing the transient expression system of B. parachinensis protoplasts and tobacco leaves and the Escherichia coli expression system. By binding to the promoter, BrpNAC895 induced the transcription of BrpHMA2 . BrpABI449 might bind to the BrpHMA2 promoter or interact with BrpNAC895 to interfere with the action of BrpNAC895. The findings suggest that BrpHMA2 is a membrane-based afflux-type Cd transporter involved in Cd²⁺ uptake and long-distance transport in plants. BrpNAC895 and BrpABI449, which function as the transcription activator and repressor, respectively, coregulate BrpHMA2 expression.

Somatic embryogenesis (SE) is of great significance in Lilium bulb production, germplasm preservation, and genetic improvement. miRNAs are important regulators of plant growth and development at the transcriptional level. Previous research by our group has shown that lpu-miR171 and its target gene SCARECROW-LIKE 6 (SCL6) play an important regulatory role in lily SE, and we predicted and identified that endogenous target mimics (eTMs) can regulate lpu-miR171. However, the associated mechanism and internal regulatory network are not yet clear. In the present study, lpu-miR171 was used as an entry point to explore the regulatory network between its upstream eTMs and its downstream target gene LpSCL6, as well as to identify the mechanism of this regulatory network in Lilium SE. Tobacco transient transformation confirmed that miRNA171 significantly inhibited the expression of LpSCL6. On this basis, the Lilium stable genetic transformation system was used to demonstrate that silencing lpu-miR171a and lpu-miR171b and overexpressing LpSCL6-II and LpSCL6-I promoted starch accumulation in calli and the expression of key cell cycle genes, thus providing energy to meet preconditions for SE and accelerate the formation and development of Lilium somatic embryos. LpSCL6-II and LpSCL6-I are nuclear proteins with self-activation activity in yeast cells. In addition, we confirmed in Lilium that lpu-eTM171 is the eTM of lpu-miR171 that binds lpu-miR171 to prevent cleavage of the target gene LpSCL6, thereby promoting SE. Therefore, the present study established a new mechanism whereby the eTM-miR171- SCL6 module regulates SE in Lilium pumilum and provides new insights clarifying the mechanism of SE.

The tea plant is an economically important crop in China, but long-term monoculture and substantial chemical nitrogen fertilizer input cause soil acidification, which in turn affects the nutrient supply and tea quality. Intercropping has drawn more attention in tea gardens because this pattern is expected to improve soil fertility and tea quality and change the soil microbial community composition. However, the roles of some key microorganisms in rhizosphere soils have not been well characterized. Here, a “soybean in summer and smooth vetch in winter” strategy was used in a tea garden to investigate the effects of intercropped legumes on soil fertility, tea quality, and potential changes in beneficial bacteria such as Bacillus. Our data showed that when soybeans were turned into the soil, the intercropping system exhibited higher soil organic matter (SOM), total nitrogen (TN), tea quality indices, and expression of the Camellia sinensis glutamine synthetase gene (CsGS). Notably, intercropping significantly affected the bacterial communities, decreasing the relative abundance of Bacillus but increasing its absolute abundance. Bacillus amyloliquefaciens BM1 was isolated from intercropped soil and showed outstanding plant growth-promoting (PGP) properties when co-inoculated with rhizobia. In winter, intercropping with smooth vetch had a beneficial effect on soil properties and tea quality. Co-inoculation with strain BM1 and Rhizobium leguminosarum Vic5 on smooth vetch (Vicia villosa) produced huge improvements in SOM, TN, and tea leaf quality, which were accompanied by the highest level of amino acids and the lowest levels of polyphenols and caffeine (p < 0.05). Our findings demonstrate that intercropping with some legumes in the tea garden is a strategy that increases SOM, TN, and tea quality, and the optional use of some PGP Bacillus species produces an amplification effect.

Plant transformation and regeneration remain highly species- and genotype-dependent. Conventional hormone-based plant regeneration via somatic embryogenesis or organogenesis is tedious, time-consuming, and requires specialized skills and experience. Over the last 40 years, significant advances have been made to elucidate the molecular mechanisms underlying embryogenesis and organogenesis. These pioneering studies have led to a better understanding of the key steps and factors involved in plant regeneration, resulting in the identification of crucial growth and developmental regulatory genes that can dramatically improve regeneration efficiency, shorten transformation time, and make transformation of recalcitrant genotypes possible. Co-opting these regulatory genes offers great potential to develop innovative genotype-independent genetic transformation methods for various plant species, including specialty crops. Further developing these approaches has the potential to result in plant transformation without the use of hormones, antibiotics, selectable marker genes, or tissue culture. As an enabling technology, the use of these regulatory genes has great potential to enable the application of advanced breeding technologies such as genetic engineering and gene editing for crop improvement in transformation-recalcitrant crops and cultivars. This review will discuss the recent advances in the use of regulatory genes in plant transformation and regeneration, and their potential to facilitate genotype-independent plant transformation and regeneration.

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules regulating vegetative and reproductive development of plants. However, the molecular mechanisms of the SlMPK4 gene in tomato pollen and fruit development remain elusive. SlMPK4 is preferentially and highly expressed in tomato stamens and its mRNA levels increase during early flower development, peaking at the mature pollen stage. Either up- or downregulation of SlMPK4 expression had no significant effect on tomato vegetative growth. However, RNAi-mediated suppression of SlMPK4 caused defects in pollen development, resulting in pollen abortion. The aborted pollen grains were either malformed or collapsed and completely lacked viability, resulting in a predominantly reduced fruit set rate in RNAi lines compared with control and overexpressing transgenic plants. Interestingly, seed development was inhibited in RNAi lines. Moreover, >12% of emasculated RNAi flowers developed seedless fruits without pollination. Anthers can produce typical microspore mother cells as well as uninucleate microspores, according to cytological investigations, while binucleate pollen ceased to produce typical mature pollen. Pollen abortion was further confirmed by transmission electron microscopy analysis at the binucleate stage in RNAi plants. The exine layer in aberrant pollen had a normal structure, while the intine layer appeared thicker. Suppression of SlMPK4 affects the transcript level of genes related to cell wall formation and modification, cell signal transduction, and metabolic and biosynthetic processes. A subset of genes that may be putative substrates of plant MAPKs were also differentially changed in RNAi transgenic flowers. Taken together, these results suggest that SlMPK4 plays a critical role in regulating pollen development and fruit development in tomato plants.

Sugar metabolism and flavonoid biosynthesis vary with the development of tea leaves. In order to understand the regulatory mechanisms underlying the associations between them, a comprehensive transcriptomic analysis of naturally growing tea leaves at different stages of maturity was carried out. Based on weighted gene coexpression network analysis, the key gene modules (Modules 2 and 3) related to the varying relationship between sugar metabolism and flavonoid biosynthesis as well as the corresponding hub genes were obtained. KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis showed that the transcription factors (TFs) in Modules 2 and 3 were mainly enriched in the pathway of plant hormone signal transduction. An in vitro study showed that the transcriptional levels of ERF1B-like TF for hexokinase inhibitor and sucrose treatments were upregulated, being respectively 28.1- and 30.2-fold higher than in the control, suggesting that ERF1B-like TFs participate in the sugar-induced regulation of flavonoid biosynthesis. The results of yeast one-hybrid and dual-luciferase assays demonstrated that CsF3^′ H, encoding flavonoid 3^′-hydroxylase, was the target flavonoid biosynthetic gene for CsERF1B-like TF. Our study identified the potential key regulators participating in the metabolism of sugars and flavonoids, providing new insights into the crosstalk between sugar metabolism and flavonoid biosynthesis in tea plants.

Plant secondary metabolites make a great contribution to the agricultural and pharmaceutical industries. Their accumulation is determined by the integrated transport of target compounds and their biosynthesis-related RNA, protein, or DNA. However, it is hard to track the movement of these biomolecules in vivo. Grafting may be an ideal method to solve this problem. The differences in genetic and metabolic backgrounds between rootstock and scion, coupled with multiple omics approaches and other molecular tools, make it feasible to determine the movement of target compounds, RNAs, proteins, and DNAs. In this review, we will introduce methods of using the grafting technique, together with molecular biological tools, to reveal the differential accumulation mechanism of plant secondary metabolites at different levels. Details of the case of the transport of one diterpene alkaloid, fuziline, will be further illustrated to clarify how the specific accumulation model is shaped with the help of grafting and multiple molecular biological tools.

Root-knot nematodes (RKN; Meloidogyne spp.) cause a significant decrease in the yield of cucumber crops every year. Cucumis metuliferus is an important wild germplasm that has resistance to RKN in which plant root volatiles are thought to play a role. However, the underlying molecular mechanism is unclear. To investigate it, we used the resistant C. metuliferus line CM3 and the susceptible cucumber line Xintaimici (XTMC). CM3 roots repelled Meloidogyne incognita second-stage larvae (J2s), while the roots of XTMC plants attracted the larvae. CM3 and XTMC were found to contain similar amounts of root volatiles, but many volatiles, including nine hydrocarbons, three alcohols, two aldehydes, two ketones, one ester, and one phenol, were only detected in CM3 roots. It was found that one of these, (methoxymethyl)-benzene, could repel M. incognita , while creosol and (Z)-2-penten-1-ol could attract M. incognita . Interestingly, creosol and (Z)-2-penten-1-ol effectively killed M. incognita at high concentrations. Furthermore, we found that a mixture of CM3 root volatiles increased cucumber resistance to M. incognita . The results provide insights into the interaction between the host and plant-parasitic nematodes in the soil, with some compounds possibly acting as nematode biofumigation, which can be used to manage nematodes.

The shikimate pathway, the seven enzymatic steps that synthesize chorismate from phosphoenolpyruvate and erythrose 4-phosphate, produces the last common precursor of the three aromatic amino acids. It is firmly established that all seven enzymes are present in plastids, and it is generally accepted that this organelle is likely the sole location for production of chorismate in plants. However, recently a growing body of evidence has provided support for a previous proposal that at least portions of the pathway are duplicated in the cytosol, referred to as the Dual Pathway Hypothesis. Here I revisit this obscure hypothesis by reviewing the findings that provided the original basis for its formulation as well as more recent results that provide fresh support for a possible extra-plastidial shikimate pathway duplication. Similarities between this possible intercompartmental metabolic redundancy and that of terpenoid metabolism are used to discuss potential advantages of pathway duplication, and the translational implications of the Dual Pathway Hypothesis for metabolic engineering are noted.

Isolation by geographic distance is involved in the formation of potential genomic islands and the divergence of genomes, which often result in speciation. The mechanisms of sympatric and allopatric speciation associated with geographic distance remain a topic of interest to evolutionary biologists. Here, we examined genomic divergence in three Actinidia species from large-scale sympatric and allopatric regions. Genome sequence data revealed that hexaploid Actinidia deliciosa originated from Actinidia chinensis and supported the speciation-with-gene-flow model in sympatric regions. The common ancestor of Actinidia setosa and A. deliciosa migrated from the mainland to the Taiwan Island ∼2.91 Mya and formed A. setosa ∼0.92 Mya, and the speciation of A. setosa is consistent with the divergence-after-speciation model with selective sweeps. Geographic isolation resulted in population contraction and accelerated the process of lineage sorting and speciation due to natural selection. Genomic islands contained genes associated with organ development, local adaptation, and stress resistance, indicating selective sweeps on a specific set of traits. Our results highlight the patterns of genomic divergence in sympatric and allopatric speciation, with the mediation of geographic isolation in the formation of genomic islands during Actinidia speciation.

In viticulture, grafting has been practiced widely and influences grape development as well as berry and wine quality. However, there is limited understanding of the effects of rootstocks on grape phenolic compounds, which are located primarily in the berry skin and contribute to certain sensory attributes of wine. In this study, scion–rootstock interactions were investigated at the green-berry stage and the veraison stage when grapevines were hetero-grafted with three commonly used rootstock genotypes (5BB, 101-14MG, and SO4). Physiological investigations showed that hetero-grafts, especially CS/5BB, contained higher concentrations of total proanthocyanidins (PAs) and various PA components in berry skins compared with the auto-grafted grapevines. Further metabolomics analysis identified 105 differentially accumulated flavonoid compounds, the majority of which, including anthocyanins, PAs, and flavonols, were significantly increased in the berry skins of hetero-grafted grapevines compared with auto-grafted controls. In addition, transcriptomic analysis of the same samples identified several thousand differentially expressed genes between hetero-grafted and auto-grafted vines. The three rootstocks not only increased the transcript levels of stilbene, anthocyanin, PA, and flavonol synthesis genes but also affected the expression of numerous transcription factor genes. Taken together, our results suggest that hetero-grafting can promote phenolic compound accumulation in grape berry skin during development. These findings provide new insights for improving the application value of grafting by enhancing the accumulation of nutritious phenolic components in grape.

The long juvenile period of perennial woody plants is a major constraint in breeding programs. FLOWERING LOCUS T (FT) protein is an important mobile florigen signal that induces plant flowering. However, whether FT can be transported in woody plants to shorten the juvenile period is unknown, and its transport mechanism remains unclear. In this study, trifoliate orange FT (ToFT) and Arabidopsis FT (AtFT, which has been confirmed to be transportable in Arabidopsis) as a control were transformed into tomato and trifoliate orange, and early flowering was induced in the transgenic plants. Long-distance and two-way (upward and downward) transmission of ToFT and AtFT proteins was confirmed in both tomato and trifoliate orange using grafting and western blot analysis. However, rootstocks of transgenic trifoliate orange could not induce flowering in grafted wild-type juvenile scions because of the low accumulation of total FT protein in the grafted scions. It was further confirmed that endogenous ToFT protein was reduced in trifoliate orange, and the accumulation of the transported ToFT and AtFT proteins was lower than that in grafted juvenile tomato scions. Furthermore, the trifoliate orange FT-INTERACTING PROTEIN1 homolog (ToFTIP1) was isolated by yeast two-hybrid analysis. The FTIP1 homolog may regulate FT transport by interacting with FT in tomato and trifoliate orange. Our findings suggest that FT transport may be conserved between the tomato model and woody plants. However, in woody plants, the transported FT protein did not accumulate in significant amounts in the grafted wild-type juvenile scions and induce the scions to flower.

The ripening of fleshy fruits is highly dependent on the regulation of endogenous hormones, including ethylene, abscisic acid (ABA) and other phytohormones. However, the regulatory mechanism of ABA signaling and its interaction with ethylene signaling in fruit ripening are still unclear. In this study, multi-gene interference (RNAi) was applied to silence the ABA receptor genes in tomato for screening the specific receptors that mediate ABA signaling during fruit ripening. The results indicated that the ABA receptors, including SlRCAR9, SlRCAR12, SlRCAR11, and SlRCAR13, participate in the regulation of tomato fruit ripening. Comparative analysis showed that SlRCAR11 and SlRCAR13 play more important roles in mediating ABA signaling during tomato fruit ripening. Co-silencing of the four genes encoding these receptors could weaken the ethylene biosynthesis and signaling pathway at the early stage of tomato fruit ripening, leading to delayed fruit ripening. Meanwhile, co-silencing enhanced fruit firmness, and altered the shelf-life and susceptibility to Botrytis cinerea of the transgenic fruits. Furthermore, blocking ABA signaling did not affect the ability of ethylene to induce fruit ripening, whereas the block may inhibit the effectiveness of ABA in promoting fruit ripening. These results suggested that ABA signaling may be located upstream of ethylene signaling in regulating fruit ripening. Our findings provide a new insight into the complex regulatory network of phytohormones in regulating fruit ripening in tomato.

R2R3-MYB genes (R2R3-MYBs) form one of the largest transcription factor gene families in the plant kingdom, with substantial structural and functional diversity. However, the evolutionary processes leading to this amazing functional diversity have not yet been clearly established. Recently developed genomic and classical molecular technologies have provided detailed insights into the evolutionary relationships and functions of plant R2R3-MYBs. Here, we review recent genome-level and functional analyses of plant R2R3-MYBs, with an emphasis on their evolution and functional diversification. In land plants, this gene family underwent a large expansion by whole genome duplications and small-scale duplications. Along with this population explosion, a series of functionally conserved or lineage-specific subfamilies/groups arose with roles in three major plant-specific biological processes: development and cell differentiation, specialized metabolism, and biotic and abiotic stresses. The rapid expansion and functional diversification of plant R2R3-MYBs are highly consistent with the increasing complexity of angiosperms. In particular, recently derived R2R3-MYBs with three highly homologous intron patterns (a, b, and c) are disproportionately related to specialized metabolism and have become the predominant subfamilies in land plant genomes. The evolution of plant R2R3-MYBs is an active area of research, and further studies are expected to improve our understanding of the evolution and functional diversification of this gene family.

Because of their great therapeutic and economic value, medicinal plants have attracted increasing scientific attention. With the rapid development of high-throughput sequencing technology, the genomes of many medicinal plants have been sequenced. Storing and analyzing the increasing volume of genomic data has become an urgent task. To solve this challenge, we have proposed the T raditionalC hineseM edicineP lantG enome database (TCMPG,http://cbcb.cdutcm.edu.cn/TCMPG/),an integrative database for storing the scattered genomes of medicinal plants. TCMPG currently includes 160 medicinal plants, 195 corresponding genomes, and 255 herbal medicines. Detailed information on plant species, genomes, and herbal medicines is also integrated into TCMPG. Popular genomic analysis tools are embedded in TCMPG to facilitate the systematic analysis of medicinal plants. These include BLAST for identifying orthologs from different plants, SSR Finder for identifying simple sequence repeats, JBrowse for browsing genomes, Synteny Viewer for displaying syntenic blocks between two genomes, and HmmSearch for identifying protein domains. TCMPG will be continuously updated by integrating new data and tools for comparative and functional genomic analysis.

Grafting has been demonstrated to significantly enhance the salt tolerance of crops. However, breeding efforts to develop enhanced graft combinations are hindered by knowledge-gaps as to how rootstocks mediate scion-response to salt stress. We grafted the scion of cultivated M82 onto rootstocks of 254 tomato accessions and explored the morphological and metabolic responses of grafts under saline conditions (EC = 20 dS m⁻¹) as compared to self-grafted M82 (SG-M82). Correlation analysis and Least Absolute Shrinkage and Selection Operator were performed to address the association between morphological diversification and metabolic perturbation. We demonstrate that grafting the same variety onto different rootstocks resulted in scion phenotypic heterogeneity and emphasized the productivity efficiency of M82 irrespective of the rootstock. Spectrophotometric analysis to test lipid oxidation showed largest variability of malondialdehyde (MDA) equivalents across the population, while the least responsive trait was the ratio of fruit fresh weight to total fresh weight (FFW/TFW). Generally, grafts showed greater values for the traits measured than SG-M82, except for branch number and wild race-originated rootstocks; the latter were associated with smaller scion growth parameters. Highly responsive and correlated metabolites were identified across the graft collection including malate, citrate, and aspartate, and their variance was partly related to rootstock origin. A group of six metabolites that consistently characterized exceptional graft response was observed, consisting of sorbose, galactose, sucrose, fructose, myo-inositol, and proline. The correlation analysis and predictive modelling, integrating phenotype- and leaf metabolite data, suggest a potential predictive relation between a set of leaf metabolites and yield-related traits.

Taxus trees are major natural sources for the extraction of taxol, an anti-cancer agent used worldwide. Taxus media is a dioecious woody tree with high taxol yield. However, the sexually dimorphic accumulation of taxoids in T. media is largely unknown. Our study revealed high accumulation of taxoids in female T. media trees using a UPLC–MS/MS method. Thereafter, many differential metabolites and genes between female and male T. media trees were identified using metabolomic and transcriptomic analyses, respectively. Most of the taxol-related genes were predominantly expressed in female trees. A female-specific R2R3-MYB transcription factor gene, TmMYB39, was identified. Furthermore, bimolecular fluorescence complementation and yeast two-hybrid assays suggested the potential interaction between TmMYB39 and TmbHLH13. Several taxol biosynthesis-related promoter sequences were isolated and used for the screening of MYB recognition elements. The electrophoretic mobility shift assay indicated that TmMYB39 could bind to the promoters of the GGPPS, T10OH, T13OH, and TBT genes. Interaction between TmMYB39 and TmbHLH13 transactivated the expression of the GGPPS and T10OH genes. TmMYB39 might function in the transcriptional regulation of taxol biosynthesis through an MYB-bHLH module. Our results give a potential explanation for the sexually dimorphic biosynthesis of taxol in T. media.

Carotenoids are the pigment substances of yellow-fleshed kiwifruit, and among them β -cryptoxanthin has only been detected in the brighter yellow-fleshed variety ‘Jinshi 1’. β -Carotene hydroxylase (BCH) catalyzes the formation of β -cryptoxanthin and zeaxanthin, but its molecular characteristics and functions have not been fully explained. Here we isolated two β -carotene hydroxylase genes, AcBCH1 and AcBCH2 from kiwifruit (Actinidia chinensis), and their relative expression levels exhibited a close correlation with the content of β -cryptoxanthin. AcBCH1 catalyzed the formation of β -cryptoxanthin when transformed into β -carotene-accumulating yeast cells. Moreover, silenced expression of AcBCH1 in kiwifruit caused decreases in the contents of zeaxanthin, lutein, and β -cryptoxanthin, and an increase in β -carotene content. The content of β -carotene decreased significantly after the AcBCH1 / 2 genes were overexpressed in tomato. The content of zeaxanthin increased and β -carotene decreased in transgenic kiwifruit seedlings. The results will enrich our knowledge of the molecular mechanisms of carotenoid biosynthesis in kiwifruit.

The CRISPR/Cas9 system is a revolutionary genome editing technique and has been widely used in numerous plants. For plants (e.g. citrus) with very low transformation efficiency, how to optimize gene editing efficiency and induce large-fragment deletion has been the focus of research. Here, we report that CRISPR/Cas9 induces efficient deletion of 16–673 bp fragments in the genome of Fortunella hindsii. The ability of two binary vectors, pK7WG2D and pMDC32, to introduce specific mutations into the genome of F. hindsii was evaluated. Double single guide RNAs (sgRNAs) were designed to achieve precise editing of two sites of a gene and deletion of fragments between the two sites. The construction of vectors based on Golden Gate assembly and Gateway recombination cloning is simple and efficient. pK7WG2D is more suitable for F. hindsii genome editing than the pMDC32 vector. Editing efficiency using the pK7WG2D vector reached 66.7%. Allele mutation frequency was 7.14–100%. Plants with 100% allele mutations accounted for 39.4% (13 100% allele mutation plants/33 mutants). The proportion of mutant plants with fragment deletion induced by this editing system was as high as 52.6% (10 fragment-deletion mutants/19 FhNZZ mutants). Altogether, these data suggest that our CRISPR/Cas9 platform is capable of targeted genome editing in citrus and has broad application in research on the citrus functional genome and citrus molecular breeding.

The horticultural industry helps to enrich and improve the human diet while contributing to growth of the agricultural economy. However, fungal diseases of horticultural crops frequently occur during pre- and postharvest periods, reducing yields and crop quality and causing huge economic losses and wasted food. Outcomes of fungal diseases depend on both horticultural plant defense responses and fungal pathogenicity. Plant defense responses are highly sophisticated and are generally divided into preformed and induced defense responses. Preformed defense responses include both physical barriers and phytochemicals, which are the first line of protection. Induced defense responses, which include innate immunity (pattern-triggered immunity and effector-triggered immunity), local defense responses, and systemic defense signaling, are triggered to counterstrike fungal pathogens. Therefore, to develop regulatory strategies for horticultural plant resistance, a comprehensive understanding of defense responses and their underlying mechanisms is critical. Recently, integrated multi-omics analyses, CRISPR-Cas9-based gene editing, high-throughput sequencing, and data mining have greatly contributed to identification and functional determination of novel phytochemicals, regulatory factors, and signaling molecules and their signaling pathways in plant resistance. In this review, research progress on defense responses of horticultural crops to fungal pathogens and novel regulatory strategies to regulate induction of plant resistance are summarized, and then the problems, challenges, and future research directions are examined.

Hibiscus hamabo is a semi-mangrove species with strong tolerance to salt and waterlogging stress. However, the molecular basis and mechanisms that underlie this strong adaptability to harsh environments remain poorly understood. Here, we assembled a high-quality, chromosome-level genome of this semi-mangrove plant and analyzed its transcriptome under different stress treatments to reveal regulatory responses and mechanisms. Our analyses suggested that H. hamabo has undergone two recent successive polyploidy events, a whole-genome duplication followed by a whole-genome triplication, resulting in an unusually large gene number (107 309 genes). Comparison of the H. hamabo genome with that of its close relative Hibiscus cannabinus, which has not experienced a recent WGT, indicated that genes associated with high stress resistance have been preferentially preserved in the H. hamabo genome, suggesting an underlying association between polyploidy and stronger stress resistance. Transcriptomic data indicated that genes in the roots and leaves responded differently to stress. In roots, genes that regulate ion channels involved in biosynthetic and metabolic processes responded quickly to adjust the ion concentration and provide metabolic products to protect root cells, whereas no such rapid response was observed from genes in leaves. Using co-expression networks, potential stress resistance genes were identified for use in future functional investigations. The genome sequence, along with several transcriptome datasets, provide insights into genome evolution and the mechanism of salt and waterlogging tolerance in H. hamabo, suggesting the importance of polyploidization for environmental adaptation.

Autophagy is a primary process involved in the degradation and reuse of redundant or damaged cytoplasmic components in eukaryotes. Autophagy has been demonstrated to facilitate nutrient recycling and remobilization by delivering intracellular materials to the vacuole for degradation in plants under nutrient starvation. However, the role of autophagy in nitrogen (N) uptake and utilization remains unknown. Here, we report that the ATG6-dependent autophagic pathway regulates N utilization in tomato (Solanum lycopersicum) under low-nitrogen (LN) conditions. Autophagy-disrupted mutants exhibited weakened biomass production and N accumulation compared with wild-type (WT), while ATG6 overexpression promoted autophagy and biomass production under LN stress. The N content in atg6 mutants decreased while that in ATG6-overexpressing lines increased due to the control of N transporter gene expression in roots under LN conditions. Furthermore, ATG6 -dependent autophagy enhanced N assimilation efficiency and protein production in leaves. Nitrate reductase and nitrite reductase activities and expression were compromised in atg6 mutants but were enhanced in ATG6-overexpressing plants under LN stress. Moreover, ATG6-dependent autophagy increased plant carbon fixation and photosynthetic capacity. The quantum yield of photosystem II, photosynthetic N use efficiency and photosynthetic protein accumulation were compromised in atg6 mutants but were restored in ATG6-overexpressing plants. A WT scion grafted onto atg6 mutant rootstock and an atg6 scion grafted onto WT rootstock both exhibited inhibited LN-induced autophagy and N uptake and utilization. Thus, ATG6-dependent autophagy regulates not only N uptake and utilization as well as carbon assimilation but also nutrient recycling and remobilization in tomato plants experiencing LN stress.

White rust, caused by Albugo occidentalis, is one of the major yield-limiting diseases of spinach (Spinacia oleracea) in some major commercial production areas, particularly in southern Texas in the United States. The use of host resistance is the most economical and environment-friendly approach to managing white rust in spinach production. The objectives of this study were to conduct a genome-wide associating study (GWAS), to identify single nucleotide polymorphism (SNP) markers associated with white rust resistance in spinach, and to perform genomic prediction (GP) to estimate the prediction accuracy (PA). A GWAS panel of 346 USDA (US Dept. of Agriculture) germplasm accessions was phenotyped for white rust resistance under field conditions and GWAS was performed using 13 235 whole-genome resequencing (WGR) generated SNPs. Nine SNPs, chr2_53 049 132, chr3_58 479 501, chr3_95 114 909, chr4_9 176 069, chr4_17 807 168, chr4_83 938 338, chr4_87 601 768, chr6_1 877 096, and chr6_31 287 118, located on chromosomes 2, 3, 4, and 6 were associated with white rust resistance in this GWAS panel. Four scenarios were tested for PA using Pearson’s correlation coefficient (r) between the genomic estimation breeding value (GEBV) and the observed values: (1) different ratios between the training set and testing set (fold), (2) different GP models, (3) different SNP numbers in three different SNP sets, and (4) the use of GWAS-derived significant SNP markers. The results indicated that a 2- to 10-fold difference in the various GP models had similar, although not identical, averaged r values in each SNP set; using GWAS-derived significant SNP markers would increase PA with a high r-value up to 0.84. The SNP markers and the high PA can provide valuable information for breeders to improve spinach by marker-assisted selection (MAS) and genomic selection (GS).

Peach [ Prunus persica L. Batsch] is one of the major temperate fruit tree species, the commercial materials of which have a low level of genetic variability. Almond [ P. dulcis (Mill) DA Webb], a close relative of peach cultivated for its kernels, has a much higher level of diversity. The species are inter-compatible and often produce fertile hybrids, almond being a possible source of new genes for peach that could provide biotic and abiotic stress tolerance traits. In this paper we describe the development of a collection of peach-almond introgression lines (ILs) having a single fragment of almond (cv. Texas) in the peach background (cv. Earlygold). Lines with few introgressions were selected with markers from successive generations from a “Texas”× “Earlygold” F1 hybrid, initially using a set of SSRs and later with the 18 k peach SNP chip, allowing for the final extraction of 67 lines, 39 with almond heterozygous introgressions covering 99% of the genome, and 28 with homozygous introgressions covering 83% of the genome. As a proof of concept, four major genes and four quantitative characters were examined in the selected ILs giving results generally consistent with previous information on the genetics of these characters. This collection is the first of its kind produced in a woody perennial species and promises to be a valuable tool for genetic analyses, including dissection of quantitative traits, positional cloning, epistasis and as prebreeding material to introgress almond genes of interest into the peach commercial gene pool.

Cineraria (Senecio cruentus) is an ornamental plant with pure colour and bicolour cultivars, widely used for landscaping. Anthocyanin biosynthesis influences coloration patterns in cineraria. However, how anthocyanins accumulate and distribute in cineraria is poorly understood. This study investigated the molecular mechanisms underlying anthocyanin biosynthesis and bicolour formation in cineraria using pure colour and bicolour cultivars. Transcriptome and gene expression analysis showed that five genes, ScCHS2, ScF3H1, ScDFR3, ScANS, and ScbHLH17, were inhibited in the white cultivar and colourless regions of bicolour cultivars. In contrast, two MADS-box genes, ScAG and ScAGL11, showed significantly higher expression in the colourless regions of bicolour cultivars. ScAG and ScAGL11 were localized in the nucleus and co-expressed with the bicolour trait. Further functional analysis verified that ScAG inhibits anthocyanin accumulation in tobacco (Nicotiana tabacum). However, virus-induced gene silencing (VIGS) experiments showed that silencing of ScAG and ScAGL11 increases anthocyanin content in cineraria leaves. Similar results were observed when ScAG and ScAGL11 were silenced in the cineraria capitulum, accompanied by the smaller size of the colourless region, specifically in the ScAG/ ScAGL11-silenced plants. The expression of ScCHS2, ScDFR3, and ScF3H1 increased in silenced cineraria leaves and capitulum. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments demonstrated that ScAG interacts with ScAGL11. Moreover, ScAG directly inhibited the transcription of ScF3H1 while ScAGL11 inhibited ScDFR3 expression by binding to their promoters separately. The findings reported herein indicate that ScAG and ScAGL11 negatively regulate anthocyanin biosynthesis in cineraria ray florets, and their differential expression in ray florets influences the bicolour pattern appearance.

Selection of high-performance lines with respect to traits of interest is a key step in plant breeding. Genomic prediction allows to determine the genomic estimated breeding values of unseen lines for trait of interest using genetic markers, e.g. single-nucleotide polymorphisms (SNPs), and machine learning approaches, which can therefore shorten breeding cycles, referring to genomic selection (GS). Here, we applied GS approaches in two populations of Solanaceous crops, i.e. tomato and pepper, to predict morphometric and colorimetric traits. The traits were measured by using scoring-based conventional descriptors (CDs) as well as by Tomato Analyzer (TA) tool using the longitudinally and latitudinally cut fruit images. The GS performance was assessed in cross-validations of classification-based and regression-based machine learning models for CD and TA traits, respectively. The results showed the usage of TA traits and tag SNPs provide a powerful combination to predict morphology and color-related traits of Solanaceous fruits. The highest predictability of 0.89 was achieved for fruit width in pepper, with an average predictability of 0.69 over all traits. The multi-trait GS models are of slightly better predictability than single-trait models for some colorimetric traits in pepper. While model validation performs poorly on wild tomato accessions, the usage as many as one accession per wild species in the training set can increase the transferability of models to unseen populations for some traits (e.g. fruit shape for which predictability in unseen scenario increased from zero to 0.6). Overall, GS approaches can assist the selection of high-performance Solanaceous fruits in crop breeding.

Chrysanthemum, one of the most important commercial ornamental crops, is susceptible to salinity, which limits its cultivation and application in coastal and inland saline areas. Grafting is widely used to improve the salt tolerance of horticultural crops, but the mechanisms of grafted chrysanthemum responses to salt stress remain unclear. In this study, we showed that heterografted chrysanthemums with Artemisia annua as rootstock exhibited increased salt tolerance compared with self-grafted and self-rooted chrysanthemums. Under high salt stress, the roots of heterografted chrysanthemums enrich Na⁺, resulting in a reduction of Na⁺ toxicity in the scion, with only a small amount of Na⁺ being transported to the leaves. On the other hand, the roots of heterografted chrysanthemums alleviated high Na⁺ stress via enhanced catalase enzyme activity, downregulation of the expression of reactive oxygen species (ROS) accumulation-related genes, massive accumulation of soluble sugars and proline, and upregulation of the expression of heat shock protein-related genes to enhance salt tolerance. In addition, the leaves of heterografted chrysanthemums respond to low Na⁺ stress by increasing peroxidase enzyme activity and soluble sugar and proline contents, to maintain a healthy state. However, self-grafted and self-rooted plants could not integrate ROS, soluble sugars, and proline in response to salt stress, and thus exhibited a salt-sensitive phenotype. Our research reveals the mechanisms underlying the increased salt tolerance of heterografted chrysanthemums and makes it possible to have large-scale cultivation of chrysanthemums in saline areas.

Crocins and picrocrocin are high-value hydrophilic pigments produced in saffron and used commercially in the food and pharmaceutical industries. These apocarotenoids are derived from the oxidative cleavage of zeaxanthin by specific carotenoid cleavage dioxygenases. The pathway for crocins and picrocrocin biosynthesis was introduced into tomato using fruit specific and constitutive promoters and resulted in 14.48 mg/g of crocins and 2.92 mg/g of picrocrocin in the tomato DW, without compromising plant growth. The strategy involved expression of CsCCD2L to produce crocetin dialdehyde and 2,6,6-trimethyl-4-hydroxy-1-carboxaldehyde-1-cyclohexene, and of glycosyltransferases UGT709G1 and CsUGT2 for picrocrocin and crocins production, respectively. Metabolic analyses of the engineered fruits revealed picrocrocin and crocetin-( β-D-gentiobiosyl)-( β-D-glucosyl)-ester, as the predominant crocin molecule, as well as safranal, at the expense of the usual tomato carotenoids. The results showed the highest crocins content ever obtained by metabolic engineering in heterologous systems. In addition, the engineered tomatoes showed higher antioxidant capacity and were able to protect against neurological disorders in a Caenorhabditis elegans model of Alzheimer’s disease. Therefore, these new developed tomatoes could be exploited as a new platform to produce economically competitive saffron apocarotenoids with health-promoting properties.

Many of the world’s most important vegetables and medicinal crops, including carrot, celery, coriander, fennel, and cumin, belong to the Apiaceae family. In this review, we summarize the complex origins of Apiaceae and the current state of research on the family, including traditional and molecular breeding practices, bioactive compounds, medicinal applications, nanotechnology, and omics research. Numerous molecular markers, regulatory factors, and functional genes have been discovered, studied, and applied to improve vegetable and medicinal crops in Apiaceae. In addition, current trends in Apiaceae application and research are also briefly described, including mining new functional genes and metabolites using omics research, identifying new genetic variants associated with important agronomic traits by population genetics analysis and GWAS, applying genetic transformation, the CRISPR-Cas9 gene editing system, and nanotechnology. This review provides a reference for basic and applied research on Apiaceae vegetable and medicinal plants.

This review describes three decades of introduction, agro-technology development, breeding and selection of Hylocereus species, known as pitaya or dragon fruit, as an example of a holistic program aimed to develop the horticultural potential of a perennial underutilized fruit crop. Interspecific homoploid and interploid crosses and embryo rescue procedures produced improved hybrids, some of which have been released to farmers. Molecular tools and morphological and phenological comparisons between the parental species and the resulting hybrids provided valuable information on dominant/recessive traits and on genetic relationships that could be exploited for further hybridizations. In addition, Hylocereus were crossed with species of the closely related genus Selenicereus, producing valuable intergeneric hybrids. In situ chromosome doubling resulted in the production of autopolyploid lines, from which an understanding of the effect of increased ploidy on fruit traits and metabolomic profiles was obtained. Gamete-derived lines were produced, adding to our biobank homozygote lines that were subsequently used for further hybridization. Spontaneous chromosome doubling occurred in haploid gamete-derived Hylocereus monacanthus lines and in interspecific interploid Hylocereus megalanthus × H. undatus hybrids obtained from an embryo rescue procedure, resulting in plants with double the expected ploidy. Challenging technical problems were addressed by the development of protocols for DNA isolation, flow cytometry, in situ chromosome doubling, androgenesis, gynogenesis and embryo rescue following interspecific and interploidy crosses. Current research leading to the development of genomics and molecular tools, including a draft genome of H. undatus, is also presented. Perspectives for further development of Hylocereus species and hybrids are discussed.

Herbaceous peony is an ancient medicinal and ornamental crop, cultivated in China and Japan for thousands of years. Numerous varieties of different colors are popular garden plants in different continents and countries. In recent decades, peony has gained a new reputation as cut flowers. Only in Europe, in 30 years, trade in cut peony stems has increased 50 fold. Today, more than 25 countries produce cut peony flowers, with primary markets in Europe, Asia and the USA. This short review summarizes the contribution of research in plant physiology to the development of new technologies of peony production and flowering advancement. Despite the popularity of cut peonies, several factors still restrict their production: complicated flowering physiology, challenges in mass propagation, and postharvest handling. Further research of biochemical and molecular mechanisms, as well as breeding of new cultivars will promote the development of the peony industry and facilitate the creation of a Global Peony Chain for the successful marketing of this beautiful flower.

Adventitious shoot (AS) regeneration accelerates plant reproduction and genetic transformation. WOX11 is involved in many biological processes, but its regulation of AS regeneration has not been reported. Here, we showed that the genotype and CK/IAA ratio of apple leaves were the key factors that affected their capacity for AS formation. Moreover, the expression level of MdWOX11 was negatively correlated with the capacity for AS formation. Phenotypic analysis of MdWOX11 transgenic plants showed that overexpression of MdWOX11 inhibited AS formation. Endogenous hormone analysis demonstrated that the contents of auxin (IAA), cytokinin (CK), and abscisic acid (ABA) were higher in MdWOX11-RNAi plants than in MdWOX11-OE transgenic plants . We used RNA sequencing to examine the transcriptional responses of genes in MdWOX11-RNAi and MdWOX11-OE transgenic apple plants at different AS stages. We identified 8066 differentially expressed genes and focused our analysis on those involved in the IAA, CK, ABA, and gibberellin (GA) hormone signaling pathways. The expression of genes related to the CK signaling pathway and shoot development was higher in GL-3 than in MdWOX11-OE transgenic plants during the callus and AS emergence stages. However, the expression of MdCKX5 was higher in MdWOX11-OE transgenic plants than in GL3 and MdWOX11-RNAi transgenic plants. Yeast one-hybrid (Y1H) assays, dual-luciferase reporter assays, and ChIP-qPCR showed that MdWOX11 binds to the promoter of MdCKX5, and a dual-luciferase reporter assay showed that MdWOX11 enhanced the promoter activity of MdCKX5. We concluded that MdCKX5 acts downstream of MdWOX11 to control AS formation, and we built a regulatory model of the suppression of AS formation by MdWOX11 in apple.

DNA methylation participates widely in the regulation of gene expression in plants. To date, the regulation and function of DNA methylation is still unknown in tomato plants. Here, we generated SlCMT4 mutants using the CRISPR-Cas9 gene editing system. We observed severe developmental defects in CRISPR-Cas9-mediated SlCMT4 mutants, including small and thick leaves, increased lateral buds, defective stamens and pistils, small fruit size with reduced setting rate, and defective seed development. The alterations at hormonal levels (IAA, tZR, strigol) were consistent with the multibranching phenotype in SlCMT4 mutant plants. CRISPR-Cas9-mediated knockout of SlCMT4 induced the expression of two pollen-specific genes (PMEI and PRALF) that suppressed the development of pollen wall and pollen tube elongation, which is responsible for irregular and defective pollen. The small-sized fruit phenotype is probably associated with upregulated expression of the IMA gene and reduced seeds in the mutant lines. Furthermore, we performed whole-genome bisulfite sequencing (WGBS) of fruits and found that SlCMT4 knockout reduced genome-wide cytosine methylation. A reduction of methylation was also observed in a 2-kp region of the IMA and LOXB promoters in the SlCMT4-mutant fruits, indicating that the hypermethylation status of the CHH context is critical for the inhibition of IMA and LOXB promoter activity. Our results show that SlCMT4 is required for normal development of tomato vegetative and reproductive organs. This study illuminates the function of SlCMT4 and sheds light on the molecular regulatory mechanism of tomato plant architecture and fruit development and ripening.

The genus Vaccinium L. (Ericaceae) contains a wide diversity of culturally and economically important berry crop species. Consumer demand and scientific research in blueberry (Vaccinium spp.) and cranberry (Vaccinium macrocarpon) have increased worldwide over the crops’ relatively short domestication history (~100 years). Other species, including bilberry (Vaccinium myrtillus), lingonberry (Vaccinium vitis-idaea), and ohelo berry (Vaccinium reticulatum) are largely still harvested from the wild but with crop improvement efforts underway. Here, we present a review article on these Vaccinium berry crops on topics that span taxonomy to genetics and genomics to breeding. We highlight the accomplishments made thus far for each of these crops, along their journey from the wild, and propose research areas and questions that will require investments by the community over the coming decades to guide future crop improvement efforts. New tools and resources are needed to underpin the development of superior cultivars that are not only more resilient to various environmental stresses and higher yielding, but also produce fruit that continue to meet a variety of consumer preferences, including fruit quality and health related traits.

Lycopene content in tomato fruit is largely under genetic control and varies greatly among genotypes. Continued improvement of lycopene content in elite varieties with conventional breeding has become challenging, in part because little is known about the underlying molecular mechanisms in high-lycopene tomatoes (HLYs). We collected 42 HLYs with different genetic backgrounds worldwide. High-performance liquid chromatography (HPLC) analysis revealed lycopene contents differed among the positive control wild tomato Solanum pimpinellifolium, HLYs, the normal lycopene cultivar “Moneymaker”, and the non-lycopene cultivar NC 1Y at the pink and red ripe stages. Real-time RT-PCR analysis of expression of the 25 carotenoid biosynthesis pathway genes of each genotype showed a significantly higher expression in nine upstream genes (GGPPS1, GGPPS2, GGPPS3, TPT1, SSU II, PSY2, ZDS, CrtISO and CrtISO-L1 but not the well-studied PSY1, PDS and Z-ISO) at the breaker and/or red ripe stages in HLYs compared to Moneymaker, indicating a higher metabolic flux flow into carotenoid biosynthesis pathway in HLYs. Further conversion of lycopene to carotenes may be prevented via the two downstream genes ( β -LCY2 and ε -LCY), which had low-abundance transcripts at either or both stages. Additionally, the significantly higher expression of four downstream genes (BCH1, ZEP, VDE, and CYP97C11) at either or both ripeness stages leads to significantly lower fruit lycopene content in HLYs than in the wild tomato. This is the first systematic investigation of the role of the complete pathway genes in regulating fruit lycopene biosynthesis across many HLYs, and enables tomato breeding and gene editing for increased fruit lycopene content.

Volatile organic compounds (VOCs) derived from fatty acids are major contributors to fruit flavor and affect human preferences. The ω-3 fatty acid linolenic acid 3 (18:3) serves as an important precursor for synthesis of (E)-2-hexenal and (Z)-3-hexenol. These short-chain C6 VOCs provide unique fresh notes in multiple fruit species. Metabolic engineering to improve fruit aroma requires knowledge of the regulation of fatty acid-derived VOCs. Here, we determined that ripe fruit-specific expression of PpFAD3-1 contributes to 18:3 synthesis in peach fruit. However, no significant increases in (E)-2-hexenal and (Z)-3-hexenol were detected after overexpressing PpFAD3-1. Interestingly, overexpressing the PpNAC1 transcription factor increased the content of 18:3 and enhanced the production of its derived volatiles. Moreover, induced expression of genes responsible for downstream VOC synthesis was observed for transgenic tomato fruit overexpressing PpNAC1, but not for transgenic fruit overexpressing PpFAD3-1. Electrophoretic mobility shift and ChIP-Seq assays showed that PpNAC1 activated PpFAD3-1 expression via binding to its promoter. Therefore, PpNAC1 plays an important role in modulating fatty acid flux to produce fruit flavor-related VOCs. In addition to PpNAC1, PpFAD3-1 expression was also associated with epigenetic modifications during peach fruit ripening. Taken together, our results provide new insights into the molecular mechanisms regulating biosynthesis of fatty acid and short-chain VOCs in fruit.

The timing of bud flush (TBF) in the spring is one of the most important agronomic traits of tea plants (Camellia sinensis). In this study, we designed an open-pollination breeding program using ‘Emei Wenchun’ (EW, a clonal tea cultivar with extra-early TBF) as a female parent. A half-sib population (n = 388) was selected for genotyping using specific-locus amplified fragment sequencing. The results enabled the identification of paternity for 294 (75.8%) of the offspring, including 11 (2.8%) from EW selfing and 217 (55.9%) assigned to a common father, ‘Chuanmu 217’ (CM). The putative EW × CM full-sib population was used to construct a linkage map. The map has 4244 markers distributed in 15 linkage groups, with an average marker distance of 0.34 cM. A high degree of collinearity between the linkage map and physical map was observed. Sprouting index, a trait closely related to TBF, was recorded for the offspring population in 2020 and 2021. The trait had moderate variation, with coefficients of variation of 18.5 and 17.6% in 2020 and 2021, respectively. Quantitative trait locus (QTL) mapping that was performed using the linkage map identified two major QTLs and three minor QTLs related to the sprouting index. These QTLs are distributed on Chr3, Chr4, Chr5, Chr9, and Chr14 of the reference genome. A total of 1960 predicted genes were found within the confidence intervals of QTLs, and 22 key candidate genes that underlie these QTLs were preliminarily screened. These results are important for breeding and understanding the genetic base of the TBF trait of tea plants.

Oil tea trees produce high-quality edible oils with desirably high oleic acid (18:1) and low linoleic (18:2) and linolenic (18:3) fatty acid (FA) levels, but limited understanding of tea oil biosynthesis and regulation has become a significant obstacle for the breeding of high-yield and -quality oil tea varieties. By integrating metabolite and transcriptome analyses of developing oil tea seeds, we dissected the critical metabolic pathways, including glycolysis, fatty acid, and triacylglycerol (TAG) biosynthesis, as well as genes essential for tea seed oil production. Two plastidic stearoyl-acyl carrier protein desaturases (CoSAD1 and 2) and two endoplasmic reticulum-localized FA desaturases (CoFAD2 and 3) were functionally characterized as responsible for high 18:1 and low 18:2 and 18:3 proportions in tea oils. Two diacylglycerol O-acyltransferases (CoDGAT1 and 2) that may prefer to synthesize 18:1-TAG were functionally characterized and might be also important for high 18:1-TAG production. The highly expressed CoWRI1a and b were identified and characterized as activators of glycolysis and regulators of directing source carbon flux into FA biosynthesis in developing oil tea seeds. The upregulated CoSADs with downregulated CoFAD2 and CoFAD3 at the late seed developmental stages mainly accounted for high 18:1 levels. Two CoDGATs might be responsible for assembling TAGs with oleoyl acyl chains, whilst two CoWRI1s regulated carbons from parental sources, partitioning into oil production in oil tea embryo sinks. This study provides a deep understanding of the biosynthesis of tea seed oils and information on genes that may be used as molecular markers to breed oil tea varieties with higher oil yield and quality.

Iron-deficiency chlorosis is a common nutritional disorder in crops grown on alkaline or calcareous soils. Although the acclimation mechanism to iron deficiency has been investigated, the genetic regulation of iron acquisition is still unclear. Here, by comparing the iron uptake process between the iron-poor-soil-tolerant citrus species Zhique (ZQ) and the iron-poor-soil-sensitive citrus species trifoliate orange (TO), we discovered that enhanced root H⁺ efflux is crucial for the tolerance to iron deficiency in ZQ. The H⁺ efflux is mainly regulated by a plasma membrane-localized H⁺-ATPase, HA6, the expression of which is upregulated in plants grown in soil with low iron content, and significantly higher in the roots of ZQ than TO. Overexpression of the HA6 gene in the Arabidopsis thaliana aha2 mutant, defective in iron uptake, recovered the wild-type phenotype. In parallel, overexpression of the HA6 gene in TO significantly increased iron content of plants. Moreover, an iron deficiency-induced transcription factor, MYB308, was revealed to bind the promoter and activate the expression of HA6 in ZQ in yeast one-hybrid, electrophoretic mobility shift, and dual-luciferase assays. Overexpression of MYB308 in ZQ roots significantly increased the expression level of the HA6 gene. However, MYB308 cannot bind or activate the HA6 promoter in TO due to the sequence variation of the corresponding MYB308 binding motif. Taking these results together, we propose that the MYB308 could activate HA6 to promote root H⁺ efflux and iron uptake, and that the distinctive MYB308- HA6 transcriptional module may be, at least in part, responsible for the iron deficiency tolerance in citrus.

Abscisic acid (ABA) is a dominant regulator of ripening and quality in non-climacteric fruits. Strawberry is regarded as a model non-climacteric fruit due to its extensive genetic studies and proven suitability for transgenic approaches to understanding gene function. Strawberry research has contributed to studies on color, flavor development, and fruit softening, and in recent years ABA has been established as a core regulator of strawberry fruit ripening, whereas ethylene plays this role in climacteric fruits. Despite this major difference, several components of the interacting genetic regulatory network in strawberry, such as MADS-box and NAC transcription factors, are similar to those that operate in climacteric fruit. In this review, we summarize recent advances in understanding the role of ABA biosynthesis and signaling and the regulatory network of transcription factors and other phytohormones in strawberry fruit ripening. In addition to providing an update on its ripening, we discuss how strawberry research has helped generate a broader and more comprehensive understanding of the mechanism of non-climacteric fruit ripening and focus attention on the use of strawberry as a model platform for ripening studies.

Polyploidization or whole-genome duplication (WGD) is a well-known speciation and adaptation mechanism in angiosperms, while subgenome dominance is a crucial phenomenon in allopolyploids, established following polyploidization. The dominant subgenomes contribute more to genome evolution and homoeolog expression bias, both of which confer advantages for short-term phenotypic adaptation and long-term domestication. In this review, we firstly summarize the probable mechanistic basis for subgenome dominance, including the effects of genetic [transposon, genetic incompatibility, and homoeologous exchange (HE)], epigenetic (DNA methylation and histone modification), and developmental and environmental factors on this evolutionary process. We then move to Brassica rapa, a typical allopolyploid with subgenome dominance. Polyploidization provides the B. rapa genome not only with the genomic plasticity for adapting to changeable environments, but also an abundant genetic basis for morphological variation, making it a representative species for subgenome dominance studies. According to the ‘two-step theory’, B. rapa experienced genome fractionation twice during WGD, in which most of the genes responding to the environmental cues and phytohormones were over-retained, enhancing subgenome dominance and consequent adaption. More than this, the pangenome of 18 B. rapa accessions with different morphotypes recently constructed provides further evidence to reveal the impacts of polyploidization and subgenome dominance on intraspecific diversification in B. rapa. Above and beyond the fundamental understanding of WGD and subgenome dominance in B. rapa and other plants, however, it remains elusive why subgenome dominance has tissue- and spatiotemporal-specific features and could shuffle between homoeologous regions of different subgenomes by environments in allopolyploids. We lastly propose acceleration of the combined application of resynthesized allopolyploids, omics technology, and genome editing tools to deepen mechanistic investigations of subgenome dominance, both genetic and epigenetic, in a variety of species and environments. We believe that the implications of genomic and genetic basis of a variety of ecologically, evolutionarily, and agriculturally interesting traits coupled with subgenome dominance will be uncovered and aid in making new discoveries and crop breeding.

Paphiopedilum orchids have a high ornamental value, and their flower abundance and timing are both key horticultural traits regulated by phytohormones. All one-flowered Paphiopedilum have additional lateral buds in the apical bract that fail to develop. In this study, an exogenous gibberellin (GA₃) application promoted flowering of Pathiopedilum callosum by inducing its early bolting instead of the floral transition of dominant flowers. Applying GA₃ effectively promoted lateral flower differentiation, resulting in a two-flowered inflorescence. GA-promoted lateral flower formation involved GA interacting with indole-3-acetic acid (IAA) and cytokinins (CTKs), given the decreased CTK content and downregulated expression of CTK synthesis genes, the increased IAA content and downregulated expression of IAA degradation, and the upregulated expression of transport genes. Further, GA acted via PcDELLA, PcTCP15, and PcXTH9 expressed in stage 5 to promote bolting, and via expression of PcAP3, PcPI, and PcSEP to promote flowering. This study provides insight into mechanisms regulating flower development of P. callosum.

Suberized and/or lignified (i.e. lignosuberized) periderm tissue appears often on surface of fleshy fruit skin by mechanical damage caused following environmental cues or developmental programs. The mechanisms underlying lignosuberization remain largely unknown to date. Here, we combined an assortment of microscopical techniques with an integrative multi-omics approach comprising proteomics, metabolomics and lipidomics to identify novel molecular components involved in fruit skin lignosuberization. We chose to investigate the corky Sikkim cucumber (Cucumis sativus var. sikkimensis) fruit. During development, the skin of this unique species undergoes massive cracking and is coated with a thick corky layer, making it an excellent model system for revealing fundamental cellular machineries involved in fruit skin lignosuberization. The large-scale data generated provides a significant source for the field of skin periderm tissue formation in fleshy fruit and suberin metabolism.

The APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factors (TFs) are involved in the regulation of specialized terpenoid biosynthesis. However, the AP2/ERF TFs in Litsea cubeba have not been characterized and their role in the biosynthesis of terpenoids is unknown. Here, 174 LcAP2/ERF TFs were identified in L. cubeba and categorized into four subfamilies: 27 AP2, 7 RAV, 1 Soloist, and 139 ERF. Transcriptomic and qRT-PCR assays both showed that the expression levels of LcERF19 were similar to that of terpene synthase LcTPS42 in the pericarp, which is related to the synthesis of geranial and neral in L. cubeba. LcERF19 was further shown to encode a nuclear-localized protein and its expression was strongly induced by jasmonate. Yeast one-hybrid and dual-luciferase assays showed that LcERF19 associated with GCC box elements of the LcTPS42 promoter and promoted its activity. Transient overexpression of LcERF19 in L. cubeba and overexpression of LcERF19 in tomato resulted in a significant increase in geranial and neral. Our findings show that LcERF19 enhances geranial and neral biosynthesis through activation of LcTPS42 expression, which provides a strategy to improve the flavor of tomato and other fruits.

Melatonin (N-acetyl-5-methoxytryptamine) is an emerging biomolecule that influences horticultural crop growth, flowering, fruit ripening, postharvest preservation, and stress protection. It functions as a plant growth regulator, preservative and antimicrobial agent to promote seed germination, regulate root system architecture, influence flowering and pollen germination, promote fruit production, ensure postharvest preservation, and increase resistance to abiotic and biotic stresses. Here, we highlight the potential applications of melatonin in multiple aspects of horticulture, including molecular breeding, vegetative reproduction, production of virus-free plants, food safety, and horticultural crop processing. We also discuss its effects on parthenocarpy, autophagy, and arbuscular mycorrhizal symbiosis. Together, these many features contribute to the promise of melatonin for improving horticultural crop production and food safety. Effective translation of melatonin to the horticultural industry requires an understanding of the challenges associated with its uses, including the development of economically viable sources.

Volatile compounds produced during ripening of strawberry are key determinants of fruit quality and consumer preference. Strawberry volatiles are largely esters which are synthesized by alcohol acyltransferases (AATs) and degraded by carboxylesterases (CXEs). Although CXE activity can have a marked influence on volatile contents in ripe strawberry fruits, CXE function and regulation in them are poorly known. Here, we report the biochemical and functional characterization of the fruit receptacle-specific and ripening-related carboxylesterase FanCXE1. The expression of the corresponding gene was found to be antagonistically regulated by auxins and abscisic acid, key hormones that regulate fruit growth and ripening in strawberry. In vitro, FanCXE1 was able to hydrolyze artificial ester substrates similar to those produced by ripe strawberry fruits. Transient suppression of the FanCXE1 gene by RNAi resulted in an increase of important volatile esters such as methyl hexanoate, methyl butanoate and ethyl hexanoate as well as a decrease of the alcohols hexenol and linanool. The results of this work enhance our understanding of the molecular basis for volatile syntheses and facilitate production of better flavored strawberry fruits by introduction of the relevant alleles into common cultivars.

Linalool and ionone are two important aromatic components in sweet osmanthus petals, and the regulatory mechanisms that produce these two components remain unclear. In this study, we employed whole-genome methylation sequencing and ATAC-seq technology to analyze the genomic DNA methylation status and chromatin accessibility of the sweet osmanthus cultivars ‘Zaohuang’ and ‘Chenghong Dangui’. Results showed that the promoter region of TPS2, a key gene in the linalool synthesis pathway, was less methylated in ‘Chenghong Dangui’ than in ‘Zaohuang’. The chromatin was more accessible in ‘Chenghong Dangui’ than in ‘Zaohuang’, which resulted in a much stronger expression of this gene in ‘Chenghong Dangui’ than in ‘Zaohuang’. This eventually led to a high quantity of linalool and its oxides in the petals of ‘Chenghong Dangui’, but there were lower levels present in the petals of ‘Zaohuang’. These results suggest that DNA methylation and chromatin accessibility play major roles in linalool synthesis in sweet osmanthus. The methylation level of the promoter region of CCD4, a key gene for ionone synthesis, was higher in ‘Zaohuang’ than in ‘Chenghong Dangui’. The chromatin accessibility was lower in ‘Zaohuang’ than in ‘Chenghong Dangui’, although the expression of this gene was significantly higher in ‘Zaohuang’ than in ‘Chenghong Dangui’. ChIP-seq analysis and a series of experiments showed that the differential expression of CCD4 and CCD1 in the two cultivars may predominantly be the result of regulation by ERF2 and other transcription factors. However, a 183-bp deletion involving the CCD4 promoter region in ‘Chenghong Dangui’ may be the main reason for the low expression of this gene in its petals. This study provides an important theoretical basis for improving selective breeding of key floral fragrance components in sweet osmanthus.

The metabolic engineered Bronze tomato line is characterized by the constitutive over-expression of the VvStSy gene encoding a structural protein responsible for the stilbenoids biosynthesis and the fruit-specific over-expression of AmDel/ Rosea1 and AtMYB12 genes encoding transcription factors that activate the polyphenol biosynthetic pathway. This tomato line is known for the increased levels of polyphenols in ripe fruits and for beneficial health promoting antioxidant and anti-inflammatory effects. In this study we analyzed the transcriptional and metabolic profiling in mature green, breaker, orange and ripe fruits compared to the normal tomato counterparts during ripening, to unravel the effect of regulatory and structural transgenes on metabolic fluxes of primary and secondary metabolisms. Our results showed that the shikimate synthase (SK) gene was up-regulated in the Bronze fruit, and the transcriptional activation is consistent with the metabolic changes observed throughout fruit ripening. These results paralleled with a reduced level of simple sugars and malate, highlighting the consumption of primary metabolites to favor secondary metabolites production and accumulation. Finally, carotenoids quantification revealed a change in the lycopene/ β-carotene ratio in the Bronze fruit as a consequence of significant lower level of the first and higher levels of the latter. The high polyphenols and β-carotene content displayed by the Bronze fruit at the later stages of fruit ripening renders this line an interesting model to study the additive or synergic effects of these phyto-chemicals in the prevention of human pathologies.

Anthocyanins and proanthocyanidins (PAs) are important types of flavonoids, plant secondary metabolites with a wide range of industrial and pharmaceutical applications. DFR (dihydroflavonol 4-reductase) is a pivotal enzyme that plays an important role in the flavonoid pathway. Here, four CsDFR genes were isolated from Camellia sinensis, and their overexpression was analyzed in vitro and in vivo. Based on transcription and metabolic analyses, CsDFR expression was closely consistent with catechins and PAs accumulation. Moreover, enzyme activity analyses revealed that the two recombinant proteins CsDFRa and CsDFRc exhibited DFR activity, converting dihydroflavonols into leucoanthocyanins in vitro, but CsDFRb1 and CsDFRb3 did not. CsDFRa and CsDFRc overexpression in AtDFR mutants (tt3) revealed that CsDFRs are involved in the biosynthesis of anthocyanins and PAs, as CsDFRa and CsDFRc restored not only the purple petiole phenotype but also the seed coat color. Site-directed mutagenesis revealed that the two amino acid residues S117 and T123 of CsDFRa play a prominent role in controlling DFR reductase activity. Enzymatic assays indicated that CsDFRa and CsDFRc exhibited a higher affinity for DHQ and DHK, respectively, whereas CsDFRb1^N120S and CsDFRb1^C126T exhibited a higher affinity for DHM. Our findings comprehensively characterize the DFRs from C. sinensis and shed light on their critical role in metabolic engineering.

Camptothecin is a chemotherapeutic drug widely used to treat various cancers. Ophiorrhiza pumila is an ideal plant model for the study of camptothecin production, with various advantages for studying camptothecin biosynthesis and regulation. The DNA-binding WRKY transcription factors have a key regulatory role in secondary metabolite biosynthesis in plants. However, little is currently known about their involvement in camptothecin biosynthesis in O. pumila. We identified 46 OpWRKY genes unevenly distributed on the 11 chromosomes of O. pumila. Phylogenetic and multiple sequence alignment analyses divided the OpWRKY proteins into three subfamilies. Based on spatial expression and co-expression, we targeted the candidate gene OpWRKY6. Overexpression of OpWRKY6 significantly reduced the accumulation of camptothecin compared with the control. Conversely, camptothecin accumulation increased in OpWRKY6 knockout lines. Further biochemical assays showed that OpWRKY6 negatively regulates camptothecin biosynthesis from both the iridoid and shikimate pathways by directly downregulating the gene expression of OpGES, Op10HGO, Op7DLH, and OpTDC. Our data provide direct evidence for the involvement of WRKYs in the regulation of camptothecin biosynthesis and offer valuable information for enriching the production of camptothecin in plant systems.

Specialized metabolites not only play important roles in biotic and abiotic stress adaptation of tea plants (Camellia sinensis (L.) O. Kuntze) but also contribute to the unique flavor of tea, the most important nonalcoholic beverage. However, the molecular networks and major genes that regulate specialized metabolites in tea plants are not well understood. Here, we constructed a population-level pan-transcriptome of the tea plant leaf using second-leaf transcriptome data from 134 accessions to investigate global expression differences in the population, expression presence or absence variations (ePAVs), and differentially expressed genes (DEGs) between pure Camellia sinensis var. assamica (CSA) and pure Camellia sinensis var. sinensis (CSS) accessions. Next, we used a genome-wide association study, a quantitative trait transcript study, and a transcriptome-wide association study to integrate genotypes, accumulation levels of specialized metabolites, and expression levels of pan-transcriptome genes to identify candidate regulatory genes for flavor-related metabolites and to construct a regulatory network for specialized metabolites in tea plants. The pan-transcriptome contains 30 482 expressed genes, 4940 and 5506 of which were newly annotated from a de novo transcriptome assembly without a reference and a genome reference-based assembly, respectively. DEGs and ePAVs indicated that CSA and CSS were clearly differentiated at the population transcriptome level, and they were closely related to abiotic tolerance and secondary metabolite synthesis phenotypes of CSA and CSS based on gene annotations. The regulatory network contained 212 specialized metabolites, 3843 candidate genes, and 3407 eQTLs, highlighting many pleiotropic candidate genes, candidate gene-rich eQTLs, and potential regulators of specialized metabolites. These included important transcription factors in the AP2/ERF-ERF, MYB, WD40, and bHLH families. CsTGY14G0001296, an ortholog of AtANS, appeared to be directly related to variation in proanthocyanins in the tea plant population, and the CsTGY11G0002074 gene encoding F3^′5^′H was found to contribute to the biased distribution of catechins between pure CSAs and pure CSSs. Together, these results provide a new understanding of the metabolite diversity in tea plants and offer new insights for more effective breeding of better-flavored tea varieties.

Timely initiation of leaf senescence is an integral part of plant development and, importantly, an adaptive strategy by which plants cope with various stresses, e.g. to limit the spread of pathogens. Powdery mildew is a major cucumber disease that promotes the initiation/progression of leaf senescence and reduces leaf photosynthesis, resulting in severe losses of yield and quality. However, how powdery mildew induces leaf senescence and how cucumber plants respond to enhance their resistance remain unclear. Here, with established agrochemical induction and pathogen inoculation systems, we demonstrate that both probenazole (PBZ) and powdery mildew activate ethylene (ET) biosynthesis and signal transduction, consequently promoting leaf senescence and enhancing plant resistance to powdery mildew through CsEIN3 to directly upregulate the expression of CsCCGs and CsRBOHs. Our analysis convincingly suggests that the regulation of leaf senescence and powdery mildew resistance is interconnected and mediated mainly by ET in cucumber.

Mango (Mangifera indica L.) is a climacteric tropical fruit consumed around the world. Although ethylene and abscisic acid (ABA) have been considered to be stimulators that trigger mango fruit ripening, their regulation mechanisms in modulating mango fruit ripening remain uncertain. In this study, we performed integrative analyses of metabolome and transcriptome data combined with a series of physiological and experimental analyses in the ‘Keitt’ mango, and we characterized changes in accumulation of specific metabolites at different stages during fruit development and ripening, which were strongly correlated with transcriptional changes and embodied physiological changes as well as taste formation. Specifically, we found that ABA, rather than ethylene, was highly associated with mango ripening, and exogenous ABA application promoted mango fruit ripening. Transcriptomic analysis identified diverse ripening-related genes involved in sugar and carotenoid biosynthesis and softening-related metabolic processes. Furthermore, networks of ABA- and ripening-related genes (such as MiHY5, MiGBF4, MiABI5, and MibZIP9) were constructed, and the direct regulation by the key ABA-responsive transcription factor MiHY5 of ripening-related genes was experimentally confirmed by a range of evidence. Taken together, our results indicate that ABA plays a key role in directly modulating mango fruit ripening through MiHY5, suggesting the need to reconsider how we understand ABA function in modulating climacteric fruit ripening.

Grafting, which joins a scion from a cultivar with the stem of a rootstock from a grapevine wild relative, is commonly used in viticulture. Grafting has crucial effects on various phenotypes of the cultivar, including its phenology, biotic and abiotic resistance, berry metabolome, and coloration, but the underlying genetics and regulatory mechanisms are largely unexplored. In this study, we investigated the phenotypic, metabolomic, and transcriptomic profiles at three developmental stages (45, 75, and 105 days after flowering) of the Crimson Seedless cultivar (Vitis vinifera) grafted onto four rootstocks (three heterografts, CS/101-14, CS/SO4, and CS/110R and one self-graft, CS/CS) with own-rooted graft-free Crimson Seedless (CS) as the control. All the heterografts had a significant effect on berry reddening as early as ∼45 days after flowering. The grafting of rootstocks promoted anthocyanin biosynthesis and accumulation in grape berries. The metabolomic features showed that cyanidin 3-O-glucoside, delphinidin 3-O-glucoside, malvidin 3-O-glucoside, peonidin 3-O-glucoside, and petunidin 3-O-glucoside were the pigments responsible for the purplish-red peel color. Transcriptomic analyses revealed that anthocyanin biosynthesis-related genes, from upstream (phenylalanine ammonia-lyase) to downstream (anthocyanidin 3-O-glucosyltransferase and anthocyanidin synthase), were upregulated with the accumulation of anthocyanins in the heterografted plants. At the same time, all these genes were also highly expressed and more anthocyanin was accumulated in self-grafted CS/CS samples compared with own-rooted graft-free CS samples, suggesting that self-grafting may also have promoted berry reddening in grapevine. Our results reveal global transcriptomic and metabolomic features in berry color regulation under different grafting conditions that may be useful for improving berry quality in viticulture.

Hexaploids, a group of organisms containing three complete sets of chromosomes in a single nucleus, are of utmost importance to evolutionary studies and breeding programs. Many studies have focused on hexaploid linkage analysis and QTL mapping in controlled crosses, but little methodology has been developed to reveal how hexaploids diversify and evolve in natural populations. We formulate a general framework for studying the pattern of genetic variation in autohexaploid populations through testing deviation from Hardy– Weinberg equilibrium (HWE) at individual molecular markers. We confirm that hexaploids cannot reach exact HWE but can approach asymptotic HWE at 8–9 generations of random mating. We derive a statistical algorithm for testing HWE and the occurrence of double reduction for autopolyploids, a phenomenon that affects population variation during long evolutionary processes. We perform computer simulation to validate the statistical behavior of our test procedure and demonstrate its usefulness by analyzing a real data set for autohexaploid chrysanthemum. When extended to allohexaploids, our test procedure will provide a generic tool for illustrating the genome structure of hexaploids in the quest to infer their evolutionary status and design association studies of complex traits.

The bZIP transcription factor plays a very important role in abiotic stresses, e.g. drought, salt, and low-temperature stress, but the mechanism of action at low temperature is still unclear. In this study, overexpression of DgbZIP3 led to increased tolerance of chrysanthemum (Chrysanthemum morifolium Ramat.) to cold stress, whereas antisense suppression of DgbZIP3 resulted in decreased tolerance. Electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), luciferase complementary imaging analysis (LCI), and dual-luciferase reporter gene detection (DLA) experiments indicated that DgbZIP3 directly bound to the promoter of DgPOD and activated its expression. DgbZIP2 was identified as a DgbZIP3-interacting protein using yeast two-hybrid, co-immunoprecipitation, LCI, and bimolecular fluorescence complementation assays. Overexpression of DgbZIP2 led to increased tolerance of chrysanthemum to cold stress, whereas antisense suppression of DgbZIP2 resulted in decreased tolerance. A ChIP–qPCR experiment showed that DgbZIP2 was highly enriched in the promoter of DgPOD, while DLA, EMSA, and LCI experiments further showed that DgbZIP2 could not directly regulate the expression of DgPOD. The above results show that DgbZIP3 interacts with DgbZIP2 to regulate the expression of DgPOD to promote an increase in peroxidase activity, thereby regulating the balance of reactive oxygen species and improving the tolerance of chrysanthemum to low-temperature stress.

Pigeonpea (Cajanus cajan) is an important legume food crop and plays a crucial role in a secure food supply in many developing countries. Several previous studies have suggested that pigeonpea has great potential for phosphorus (P) deficiency tolerance, but little is known about the underlying mechanism. In this study, the physiological and molecular responses of pigeonpea roots to phosphate (Pi) starvation were investigated through integrating phenotypic, genomic, transcriptomic, metabolomic, and lipidomic analyses. The results showed that low-Pi treatment increased total root length, root surface area, and root acid phosphatase activity, and promoted the secretion of organic acids (e.g. citric acids, piscidic acids, and protocatechuic acids) and the degradation of phospholipids and other P-containing metabolites in the roots of pigeonpea. Consistent with the morphological, physiological, and biochemical changes, a large number of genes involved in these Pi-starvation responses were significantly upregulated in Pi-deficient pigeonpea roots. Among these Pi-starvation response genes upregulated by low-Pi treatment, four gene families were expanded through recent tandem duplication in the pigeonpea genome, namely phosphate transporter 1 (PHT1), phosphoethanolamine/ phosphocholine phosphatase (PECP), fasciclin-like arabinogalactan protein (FLA), and glutamate decarboxylase (GAD). These gene families may be associated with Pi uptake from the soil, phospholipid recycling, root morphological remodeling, and regulation of organic acid exudation. Taken together, our results suggest that pigeonpea employs complex Pi-starvation responses to strengthen P acquisition and utilization during low-Pi stress. This study provides new insights into the genome evolution and P deficiency adaptation mechanism of pigeonpea.

The production of ginseng, an important Chinese medicine crop, has been increasingly challenged by soil degradation and pathogenic disease under continuous cropping in Northeast China. In a field experiment, an Alfisol garden continuously cropped with Chinese ginseng (Panax ginseng C. A. Meyer) was treated with soil amendment at 20 t ha⁻¹ with maize (MB) and wood (WB) biochar, respectively, compared to conventional manure compost (MC). Two years after the amendment, the rooted topsoil and ginseng plants were sampled. The changes in soil fertility and health, particularly in the soil microbial community and root disease incidence, and in ginseng growth and quality were portrayed using soil physico-chemical assays, biochemical assays of extracellular enzyme activities and gene sequencing assays as well as ginsenoside assays. Topsoil fertility was improved by 23% and 39%, ginseng root biomass increased by 25% and 27%, and root quality improved by 6% and 18% with WB and MB, respectively, compared to MC. In the ginseng rhizosphere, fungal abundance increased by 96% and 384%, with a significant and insignificant increase in bacterial abundance, respectively, under WB and MB. Specifically, the abundance of Fusarium spp. was significantly reduced by 19–35%, while that of Burkholderia spp. increased by folds under biochar amendments over MC. Relevantly, there was a significant decrease in the abundance proportion of pathotrophic fungi but a great increase in that of arbuscular mycorrhizal fungi, along with an enhanced microbial community network complexity, especially fungal community complexity, under biochar amendments. Thus, biochar, particularly from maize residue, could promote ginseng quality production while enhancing soil health and ecological services, including carbon sequestration, in continuously cropped fields.

G proteins function directly in cold tolerance of plants. However, the framework of the G α subunit in regulating cold tolerance remains to be explored. Here, we used protein interaction techniques to elucidate cold-related pathways regulated by CsGPA1. Suppression of CsGPA1 decreased the cold tolerance of cucumber. Further protein interaction experiments showed that CsGPA1 interacted with Csa_4G663630.1 located in the cell membrane and nucleus and with CsCOR413PM2 located in the cell membrane. Csa_4G663630.1 was named CsCDL1 due to its 71% protein sequence similarity to AtCDL1, a positive brassinolide signal gene. Suppression of CsGPA1 decreased the expression of most of brassinolide-related genes (including CsCDL1) under cold stress. Principal component and linear regression analyses showed that expressions of CsGPA1 and brassinolide-related genes were positively correlated. Suppression of CsCOR413PM2 also decreased cold tolerance of cucumber. The expression and protein content of CsCOR413PM2 and CsGPA1 in CsGPA1-RNAi and CsCOR413PM2-RNAi lines were determined under cold tolerance. Only CsGPA1 silencing affected the expression and protein content of CsCOR413PM2 during cold stress. Moreover, suppression of CsGPA1 or CsCOR413PM2 decreased Ca²⁺ influx at low temperature and then decreased the expression of CsICE– CsCBF. These results indicated that the CsGPA1– CsCOR413PM2–Ca²⁺ axis regulated the expression of CsICE– CsCBF during cold stress. In conclusion, Our results provide the first framework of CsGPA1 in regulating cold tolerance of cucumber, laying the foundation for further mechanistic studies of cold tolerance for G α in cucumber.

The factors underlying the plant collapse of certain melon-pumpkin graft combinations are not fully understood. Our working hypothesis was that impairment of photoassimilates transport in incompatible combinations induces an imbalance in the homeostasis of root auxin (indole-3-acetic acid; IAA) and of cytokinins, probably triggering plant collapse. Root IAA and cytokinins levels in the presence and absence of fruit and changes in root and scion metabolites were investigated in compatible and incompatible combinations. We showed elevated levels of IAA, 2-oxoindole-3-acetic acid (IAA catabolite), indole-3-acetylaspartate (IAA conjugate), and cis-zeatin-type cytokinins, but low levels of trans-zeatin-type cytokinins in the roots of plants of the incompatible combination during fruit ripening. Similarly, during fruit ripening, the expression of the YUCCA genes, YUC2, YUC6, and YUC11 (required for auxin biosynthesis), the GRETCHEN-HAGEN3 gene (required for auxin conjugation), and the cytokinin oxidase/dehydrogenase 7 (CKX7) gene (regulates the irreversible degradation of cytokinin) was enhanced in the roots of plants of the incompatible combination. Moreover, in the incompatible combination the fruiting process restricted transport of photoassimilates to the rootstock and induces their accumulation in the scion. In addition, high levels of hydrogen peroxide and malondialdehyde and reduced activity of antioxidant enzymes were observed in the roots of the incompatible graft. Our results showed that the collapse of the incompatible graft combination during fruit ripening is closely associated with a dramatic accumulation of IAA in the roots, which probably elicits oxidative damage and disturbs the balance of IAA and cytokinins that is of critical importance in melon-pumpkin graft compatibility.

Pears (Pyrus) are one of the most economically important fruits worldwide. The Pyrus genus is characterized by a high degree of genetic variability between species and interspecific hybrids, and several studies have been performed to assess this variability for both cultivated and wild accessions. These studies have mostly been limited by the resolving power of traditional molecular markers, although in the recent past the availability of reference genome sequences or SNP arrays for pear have enhanced the capability of high-resolution genomics studies. These tools can also be applied to better understand the intra-varietal (or clonal) variability in pear. Here we report the first high resolution genomics analysis of a pear clonal population using whole genome sequencing (WGS). Results showed unique signatures for the accumulation of mutations and transposable element insertions in each clone, which are likely related to their history of propagation and cultivation. The nucleotide diversity remained low in the clonal collection with the exception of few genomic windows, suggesting that balancing selection may be occurring. These windows included mainly genes related to plant fertility. Regions with higher mutational load were partially associated with transcription factors, probably reflecting the distinctive phenotypes in the collection. The annotation of variants also revealed the theoretical disruption of relevant genes in pear. Taken together, the results from this study show that pear clones accumulate mutations differently, and that those mutations can play a role on pear phenotypes, meaning that the study of pear clonal populations can be relevant in genetic studies, mainly when comparing with traditional association studies.

The Mediterranean basin countries are considered secondary centres of tomato diversification. However, information on phenotypic and allelic variation of local tomato materials is still limited. Here we report on the evaluation of the largest traditional tomato collection, which includes 1499 accessions from Southern Europe. Analyses of 70 traits revealed a broad range of phenotypic variability with different distributions among countries, with the culinary end use within each country being the main driver of tomato diversification. Furthermore, eight main tomato types (phenoclusters) were defined by integrating phenotypic data, country of origin, and end use. Genome-wide association study (GWAS) meta-analyses identified associations in 211 loci, 159 of which were novel. The multidimensional integration of phenoclusters and the GWAS meta-analysis identified the molecular signatures for each traditional tomato type and indicated that signatures originated from differential combinations of loci, which in some cases converged in the same tomato phenotype. Our results provide a roadmap for studying and exploiting this untapped tomato diversity.

Salinity is a universal environmental stress that causes yield reduction in plants. WRKY33, which has been extensively studied in plant defense against necrotrophic pathogens, has recently been found to be important in salt-responsive pathways. However, the underlying molecular mechanisms controlling the involvement of WRKY33 in salt tolerance have not been fully characterized. Here, we explored the function of BcWRKY33A in non-heading Chinese cabbage (NHCC). Under salt stress, BcWRKY33A expression is significantly induced in roots. As a nuclear protein, BcWRKY33A has strong transcriptional activation activity. Overexpression of BcWRKY33A confers salt tolerance in Arabidopsis, whereas silencing of BcWRKY33A causes salt sensitivity in NHCC. Furthermore, BcHSFA4A, a protein that interacts with BcWRKY33A, could directly bind to the HSE motif within the promoters of BcZAT12 and BcHSP17.6A, which are involved in the plant response to salt stress. Finally, we found that BcWRKY33A could enhance the transcriptional activity of BcHSFA4A and affect its downstream genes (e.g. BcZAT12 and BcHSP17.6A), and co-overexpression of BcWRKY33A and BcHSFA4A could promote the expression of salt-related genes, suggesting that the regulatory interaction between BcWRKY33A and BcHSFA4A improves salt tolerance in plants. Overall, our results provide insight into the molecular framework of the BcWRKY33A-BcHSFA4A signaling pathway, which also aids in our understanding of the molecular mechanism of salt tolerance in plants.

Clivia miniata is renowned for its evergreen and strap-like leaves, whereas its floral color and scent are lacking diversity. Here, anthocyanin, volatile terpene, and carotenoid metabolisms were integrally investigated in C. miniata flowers. The results showed that pelargonidins and lutein might cooperate to confer orange or yellow color to C. miniata flowers, but only a trace amount of (+)-limonene was detected. The expression levels of CmF3^′ H and CmDFR appeared to be responsible for the ratio of cyanidin and pelargonidin derivatives in C. miniata, and the low expression of CmF3^′ H was responsible for the lack of cyanidins in flowers. Moreover, the CmF3^′ H promoter could not be activated by CmMYBAs, suggesting that it was controlled by novel regulators. Only two CmTPSs were functional, with CmTPS2 responsible for (+)-limonene synthesis, contributing to the monotonous flower volatile terpenes of C. miniata. CmCCD1a and CmCCD1b were able to cleave carotenoids at the 5,6 (5^′,6^′), and 9,10 (9^′,10^′) positions to generate volatile apocarotenoids, whereas the substrates found in low-quantities or specific subcellular localizations of CmCCD1s might constrain volatile apocarotenoid release. Consequently, activating F3^′ H and introducing novel F3^′ 5^′ H or versatile TPS may be effective ways to modify the floral color and scent, respectively. Alternatively, modifying the carotenoid flux or CCD1 localization might affect floral color and scent simultaneously. Taking these results together, the present study provides a preliminary deciphering of the genetic constraints underlying flower color and scent development, and proposes possible schemes for further genetic modification of ornamental traits in C. miniata and other plants.

Apple ring rot, which is caused by Botryosphaeria dothidea, is one of the most devastating diseases of apple. However, the lack of a known molecular resistance mechanism limits the development of resistance breeding. Here, the ‘Golden Delicious’ and ‘Fuji Nagafu No. 2’ apple cultivars were crossed, and a population of 194 F₁ individuals was generated. The hybrids were divided into five categories according to their differences in B. dothidea resistance during three consecutive years. Quantitative proteomic sequencing was performed to analyze the molecular mechanism of the apple response to B. dothidea infection. Hierarchical clustering and weighted gene coexpression network analysis revealed that photosynthesis was significantly correlated with the resistance of apple to B. dothidea. The level of chlorophyll fluorescence in apple functional leaves increased progressively as the level of disease resistance improved. However, the content of soluble sugar decreased with the improvement of disease resistance. Further research revealed that sorbitol, the primary photosynthetic product, played major roles in apple resistance to B. dothidea. Increasing the content of sorbitol by overexpressing MdS6PDH1 dramatically enhanced resistance of apple calli to B. dothidea by activating the expression of salicylic acid signaling pathway-related genes. However, decreasing the content of sorbitol by silencing MdS6PDH1 showed the opposite phenotype. Furthermore, exogenous sorbitol treatment partially restored the resistance of MdS6PDH1-RNAi lines to B. dothidea. Taken together, these findings reveal that sorbitol is an important metabolite that regulates the resistance of apple to B. dothidea and offer new insights into the mechanism of plant resistance to pathogens.

Levels of ethylene, implicated in the induction of fruit ripening in a diverse array of plants, are influenced by genetic and environmental factors, such as other plant hormones. Among these, salicylic acid (SA) and its derivative, acetylsalicylic acid (ASA), have been demonstrated to inhibit ethylene biosynthesis in fruit, yet the underlying regulatory mechanisms remain elusive. Here, we showed that treatment with exogenous ASA dramatically reduced ethylene production, as well as activities of ACC synthase (ACS) and ACC oxidase (ACO), in kiwifruit tissues. Comparative transcriptome analysis indicated the differential expression of ethylene biosynthetic genes (AdACS1/2 and AdACO5). A screen of transcription factors indicated that AdERF105L and AdWRKY29 were ASA-responsive regulators of AdACS1/2 and AdACO5, respectively. In addition to these genes, AdACS3 and AdACO3 were abundantly expressed in both ASA-treated and control tissues. AdACS3 protein was phosphorylated and stabilized by AdMPK16, a mitogen-activated protein kinase, while AdACO3 activity was enhanced by AdAP, an aspartic peptidase. Exogenous ASA downregulated AdMPK16 and AdAP, thereby influencing ethylene biosynthesis at a post-transcriptional level. These findings led us to propose a multidimensional system for inhibition of ethylene biosynthesis by ASA, inducing differential expression of some ethylene biosynthesis genes, as well as differential effects on protein activity on other targets.

The biosynthesis of catechins, a major type of flavonoids accumulated in tea, is mediated by developmental cues and environmental stimuli. Light enhances but shading treatment reduces catechin accumulation in tea leaves. However, the transcription factors involved in light-mediated catechin biosynthesis remain to be identified. Two GOLDEN2 LIKE genes from tea plant (CsGLK1 and CsGLK2) were isolated and characterized in both tomato and tea plants. Transcripts of both CsGLK1 and CsGLK2 were affected by light intensity in tea plants. Overexpression of CsGLK1 and CsGLK2 promoted chloroplast development and carotenoid accumulation in tomato fruits. An integrated metabolomic and transcriptomic approach revealed that both catechin content and related biosynthetic genes were upregulated in CsGLK-overexpressing tomato leaves. Our further studies in tea plants indicated that CsGLKs directly regulate the transcription of CsMYB5b, a transcription factor involved in catechin biosynthesis. Suppression of CsGLKs in tea leaves led to the reduction of both CsMYB5b expression and catechin accumulation. Taken together, the results show that CsGLKs are involved in light-regulated catechin accumulation in tea plants by regulating expression of CsMYB5b and have great potential for enhancing the accumulation of both carotenoids and flavonoids in fruits of horticultural crops.

Several vineyard techniques have been proposed to delay grape maturity in light of the advanced maturation driven by increasingly frequent water and heat stress events that are detrimental to grape quality. These studies differ in terms of their experimental conditions, and in the present work we have attempted to summarize previous observations in a quantitative, data-driven systematic review. A meta-analysis of quantitative data gathered across 43 relevant studies revealed the overall significance of the proposed treatments and evaluated the impact of different experimental conditions on the outcome of antitranspirants, delayed pruning and late source limitation. Antitranspirants were most effective when applied twice and closer to veraison, while di-1- p-menthene increased the ripening delay by about 1^◦Brix compared to kaolin. Larger ripening delays were achieved with delayed pruning of low-yielding vines or by pruning at later stages of apical bud development. Late defoliation or shoot trimming delayed ripening in high-yielding vines and represent suitable solutions for late-harvested varieties, but became ineffective where the treatment decreased yield. This quantitative meta-analysis of 242 primary observations uncovers factors affecting the efficacy of vineyard practices to delay ripening, which should be carefully considered by grape growers attempting to achieve this outcome.

Lettuce is one of the economically important leaf vegetables and is cultivated mainly in temperate climate areas. Cultivar identification based on the distinctness, uniformity, and stability (DUS) test is a prerequisite for new cultivar registration. However, DUS testing based on morphological features is time-consuming, labor-intensive, and costly, and can also be influenced by environmental factors. Thus, molecular markers have also been used for the identification of genetic diversity as an effective, accurate, and stable method. Currently, genome-wide single nucleotide polymorphisms (SNPs) using next-generation sequencing technology are commonly applied in genetic research on diverse plant species. This study aimed to establish an effective and high-throughput cultivar identification system for lettuce using core sets of SNP markers developed by genotyping by sequencing (GBS). GBS identified 17 877 high-quality SNPs for 90 commercial lettuce cultivars. Genetic differentiation analyses based on the selected SNPs classified the lettuce cultivars into three main groups. Core sets of 192, 96, 48, and 24 markers were further selected and validated using the Fluidigm platform. Phylogenetic analyses based on all core sets of SNPs successfully discriminated individual cultivars that have been currently recognized. These core sets of SNP markers will support the construction of a DNA database of lettuce that can be useful for cultivar identification and purity testing, as well as DUS testing in the plant variety protection system. Additionally, this work will facilitate genetic research to improve breeding in lettuce.

Grapevine trunk diseases (GTDs) affect grape production and reduce vineyard longevity worldwide. Since the causative fungi also occur in asymptomatic trunks, we address disease outbreak in terms of altered chemical communication between host and endophyte. Here, we identified four chemically similar secondary metabolites secreted by the GTD-associated fungus Eutypa lata to analyse their modes of action in a grapevine cell culture of Vitis rupestris, where microtubules were tagged by GFP. Treatment with the metabolite eutypine activated defence responses, evident from extracellular alkalinisation and induction of defence genes. Eutypinol, instead, eliminated microtubules, in contrast to the other three compounds. Furthermore, we evaluated the effect of four corresponding chemical analogues of these compounds, sharing the phenolic but lacking the alkyne moiety. These analogues were able to induce similar defence responses in V. rupestris cells, albeit at reduced amplitude. Since closely related moieties differing only in details of the side groups at the phenolic ring differ significantly with respect to the response of the host cell, we propose that these fungal compounds act through a specific binding site at the membrane of grapevine cells. We corroborate this specificity by combination experiments, where the eutypine and the eutypinol analogues behave competitively with respect to the elicited responses. In summary, Eutypa lata secretes compounds that elicit host defence in a specific manner by interfering with early events of immunity signalling. This supports the notion that a real understanding of GTDs has to address inter-organismic chemical communication.

Fruit shape is an important biological trait that is also of special commercial value in tomato. The SUN gene has been known as a key regulator of tomato fruit elongation for years, but the molecular mechanisms underlying its transcriptional regulation remain little understood. Here, a unique BZR1-like transcription factor, BZR1.7, was identified as a trans-acting factor of the SUN gene promoter that bound to the conserved E-box of the promoter to promote SUN gene expression. Overexpression of BZR1.7 in tomato led to elevated SUN gene expression and formation of elongated fruits. Plants of the BZR1.7 knockout mutant created by gene editing did not exhibit an observable fruit shape phenotype, suggesting possible functional redundancy of BZR1-like genes in tomato. There were seven BZR1-like genes in the tomato genome and overexpression of BZR1.5 and BZR1.6 led to elongated fruit phenotypes similar to those observed in the BZR1.7 overexpression lines, further supporting the notion of functional redundancy of BZR1-like genes in tomato fruit shape specification. Microscopic analysis revealed that there was a decreased number of cell layers in the fruit pericarp in the BZR1.7 overexpression lines. These findings offer new insights into the regulatory mechanism by which BZR1.7 promotes SUN gene expression and regulates fruit elongation in tomato.

Drought poses a major threat to apple fruit production and quality. Because of the apple’s long juvenile phase, developing varieties with improved drought tolerance using biotechnology approaches is needed. Here, we used the RNAi approach to knock down six GH3 genes in the apple. Under prolonged drought stress, the MdGH3 RNAi plants performed better than wild-type plants and had stronger root systems, higher root-to-shoot ratio, greater hydraulic conductivity, increased photosynthetic capacity, and increased water use efficiency. Moreover, MdGH3 RNAi plants promoted the drought tolerance of the scion when they were used as rootstock, compared with wild-type and M9-T337 rootstocks. Scions grafted onto MdGH3 RNAi plants showed increased plant height, stem diameter, photosynthetic capacity, specific leaf weight, and water use efficiency. The use of MdGH3 RNAi plants as rootstocks can also increase the C/N ratio of the scion and achieve the same effect as the M9-T337 rootstock in promoting the flowering and fruiting of the scion. Notably, using MdGH3 RNAi plants as rootstocks did not reduce fruit weight and scion quality compared with using M9-T337 rootstock. Our research provides candidate genes and demonstrates a general approach that could be used to improve the drought tolerance of fruit trees without sacrificing the yield and quality of scion fruits.

Brassica rapa includes various vegetables with high economic value. Among them, green petiole type pakchoi (B. rapa ssp. chinensis) is one of the major vegetables grown in southern China. Compared with other B. rapa varieties, green petiole type pakchoi shows a higher level of heat resistance, which is partially derived from the rich epicuticular wax. Here we sequence a high-quality genome of green petiole type pakchoi, which has been widely used as the parent in breeding. Our results reveal that long terminal repeat retrotransposon insertion plays critical roles in promoting the genome expansion and transcriptional diversity of pakchoi genes through preferential insertions, particularly in cuticle biosynthetic genes. After whole-genome triplication, over-retained pakchoi genes escape stringent selection pressure, and among them a set of cuticle-related genes are retained. Using bulked-segregant analysis of a heat-resistant pakchoi cultivar, we identify a frame-shift deletion across the third exon and the subsequent intron of BrcCER1 in candidate regions . Using Nanopore long-read sequencing, we analyze the full-length transcriptome of two pakchoi cultivars with opposite sensitivity to high temperature. We find that the heat-resistant pakchoi cultivar can mitigate heat-caused leaf damage by activating an unfolded protein response, as well as by inhibiting chloroplast development and energy metabolism, which are presumably mediated by both transcriptional regulation and splicing factors. Our study provides valuable resources for Brassica functional genomics and breeding research, and deepens our understanding of plant stress resistance.

Uncovering the genetic basis of photosynthetic trait variation under drought stress is essential for breeding climate-resilient walnut cultivars. To this end, we examined photosynthetic capacity in a diverse panel of 150 walnut families (1500 seedlings) from various agro-climatic zones in their habitats and grown in a common garden experiment. Photosynthetic traits were measured under well-watered (WW), water-stressed (WS) and recovery (WR) conditions. We performed genome-wide association studies (GWAS) using three genomic datasets: genotyping by sequencing data (~43 K SNPs) on both mother trees (MGBS) and progeny (PGBS) and the Axiom™ Juglans regia 700 K SNP array data (~295 K SNPs) on mother trees (MArray). We identified 578 unique genomic regions linked with at least one trait in a specific treatment, 874 predicted genes that fell within 20 kb of a significant or suggestive SNP in at least two of the three GWAS datasets (MArray, MGBS, and PGBS), and 67 genes that fell within 20 kb of a significant SNP in all three GWAS datasets. Functional annotation identified several candidate pathways and genes that play crucial roles in photosynthesis, amino acid and carbohydrate metabolism, and signal transduction. Further network analysis identified 15 hub genes under WW, WS and WR conditions including GAPB, PSAN, CRR1, NTRC, DGD1, CYP38, and PETC which are involved in the photosynthetic responses. These findings shed light on possible strategies for improving walnut productivity under drought stress.

The cultivated strawberry (Fragaria × ananassa) arose through a hybridization of two wild American octoploid strawberry species in a French garden in the 1750s. Since then, breeders have developed improved cultivars adapted to different growing regions. Diverse germplasm is crucial to meet the challenges strawberry breeders will continue to address. The USDA-ARS National Clonal Germplasm Repository (NCGR) in Corvallis, Oregon maintains the U.S. strawberry collection. Recent developments in high-throughput genotyping for strawberry can provide new insights about the diversity and structure of the collection, germplasm management, and future breeding strategies. Genotyping was conducted on 539 F. × ananassa accessions using either the iStraw35 or FanaSNP 50 K Axiom array. Data for markers shared by the two arrays were curated for call quality, missing data, and minor allele frequency resulting in 4033 markers for structure assessment, diversity analysis, pedigree confirmation, core collection development, and the identification of haplotypes associated with desirable traits. The F. × ananassa collection was equally diverse across the different geographic regions represented. K-means clustering, sNMF, and UPGMA hierarchal clustering revealed seven to nine sub-populations associated with different geographic breeding centers. Two 100 accession core collections were created. Pedigree linkages within the collection were confirmed. Finally, accessions containing disease resistance-associated haplotypes for FaRCa1, FaRCg1, FaRMp1, and FaRPc2 were identified. These new core collections will allow breeders and researchers to more efficiently utilize the F. × ananassa collection. The core collections and other accessions of interest can be requested for research from the USDA-ARS NCGR via the Germplasm Resources Information Network (https://www.ars-grin.gov/).

Hybridization has been widely used in breeding of cultivated species showing low genetic variability, such as peach (Prunus persica). The merging of two different genomes in a hybrid often triggers a so-called “genomic shock” with changes in DNA methylation and in the induction of transposable element expression and mobilization. Here, we analysed the DNA methylation and transcription levels of transposable elements and genes in leaves of Prunus persica and Prunus dulcis and in an F1 hybrid using high-throughput sequencing technologies. Contrary to the “genomic shock” expectations, we found that the overall levels of DNA methylation in the transposable elements in the hybrid are not significantly altered compared with those of the parental genomes. We also observed that the levels of transcription of the transposable elements in the hybrid are in most cases intermediate as compared with that of the parental species and we have not detected cases of higher transcription in the hybrid. We also found that the proportion of genes whose expression is altered in the hybrid compared with the parental species is low. The expression of genes potentially involved in the regulation of the activity of the transposable elements is not altered. We can conclude that the merging of the two parental genomes in this Prunus persica x Prunus dulcis hybrid does not result in a “genomic shock” with significant changes in the DNA methylation or in the transcription. The absence of major changes may facilitate using interspecific peach x almond crosses for peach improvement.

Onion bulb color is a key breeding trait. The red bulb color is caused by the presence of anthocyanins, which are products of the flavonoid synthesis pathway. Research on flavonoid regulation in onion is lagging compared with that in other crops. AcB2 encodes a basic helix–loop–helix (bHLH) transcription factor, and its transcription is positively associated with anthocyanin accumulation and correlated with the expression of AcMYB1, which is an activator in the flavonoid biosynthetic pathway in onion. Phylogenetic analysis showed that AcB2 was grouped into the TRANSPARENT TESTA 8 (TT8) clade of the bHLH IIIf subgroup. The AcB2 protein contained an MYB-interacting region and physically interacted with AcMYB1 in yeast and tobacco leaves. AcMYB1 directly bound to the promoters of anthocyanidin synthase (AcANS) and flavonoid 3-hydroxylase 1 (AcF3H1) and activated their expression. The coexpression of AcB2 with AcMYB1 in Arabidopsis thaliana protoplasts dramatically increased the expression of AcANS and AcF3H1 compared with that under the expression of AcMYB1 alone. Transient co-overexpression of AcB2 with AcMYB1 induced anthocyanin accumulation in the epithelial cells of onion bulbs. Complementation of the Arabidopsis tt8-1 mutant with AcB2 restored pigmentation defects in tt8-1. In addition, AcB2 physically interacted with AtTT2 in yeast cells and tobacco leaves, indicating that the functions of AcB2 were similar to those of AtTT8. Together, these results demonstrated that AcB2 enhanced the function of AcMYB1 in upregulating anthocyanin biosynthesis in onion, which provides a theoretical basis for breeding onions with higher anthocyanin contents.

Although autophagy is a conserved mechanism operating across eukaryotes, its effects on crops and especially their metabolism has received relatively little attention. Indeed, whilst a few recent studies have used systems biology tools to look at the consequences of lack of autophagy in maize these focused on leaf tissues rather than the kernels. Here we utilized RNA interference (RNAi) to generate tomato plants that were deficient in the autophagy-regulating protease ATG4. Plants displayed an early senescence phenotype yet relatively mild changes in the foliar metabolome and were characterized by a reduced fruit yield phenotype. Metabolite profiling indicated that metabolites of ATG4-RNAi tomato leaves just exhibited minor alterations while that of fruit displayed bigger difference compared to the WT. In detail, many primary metabolites exhibited decreases in the ATG4-RNAi lines, such as proline, tryptophan and phenylalanine, while the representative secondary metabolites (quinic acid and 3 -trans-caffeoylquinic acid) were present at substantially higher levels in ATG4-RNAi green fruits than in WT. Moreover, transcriptome analysis indicated that the most prominent differences were in the significant upregulation of organelle degradation genes involved in the proteasome or chloroplast vesiculation pathways, which was further confirmed by the reduced levels of chloroplastic proteins in the proteomics data. Furthermore, integration analysis of the metabolome, transcriptome and proteome data indicated that ATG4 significantly affected the lipid metabolism, chlorophyll binding proteins and chloroplast biosynthesis. These data collectively lead us to propose a more sophisticated model to explain the cellular co-ordination of the process of autophagy.

Abscisic acid (ABA) is a phytohormone essential for plants to respond to various environmental stresses, and abscisic acid-insensitive 5 (ABI5) is a basic leucine zipper transcription factor of the ABA signaling pathway. Exogenous ABA induces cold tolerance in bananas; however, the role of MaABI5-like in ABA-induced cold tolerance remains unexplored. The present study found that exogenous ABA alleviated chilling injury of ‘Fenjiao’ banana, induced the accumulation of endogenous ABA, unsaturated fatty acids, and flavonoid content, and reduced the saturated fatty acid content. Moreover, ABA treatment upregulated the transcription levels of MaABI5-like, fatty acid desaturation genes, and flavonoid synthesis-related genes during cold storage. More interestingly, MaABI5-like directly interacted with the promoter of genes related to fatty acid desaturation (MaFAD3-1, MaFAD3-4, MaFAD3-5, MaFAD6-2, MaFAD6-3) and flavonoid synthesis (MaPAL-like, MaPAL-like1, MaC4H-like3, Ma4CL-like1, Ma4CL-like10, MaCHS6-4-like, and MaFLS) and activated their expressions. Furthermore, the transient overexpression of MaABI5-like in ‘Fenjiao’ banana fruit and ectopic expression in tomato plants enhanced cold tolerance and upregulated fatty acid desaturation and flavonoid synthesis-related gene transcript levels. The reduced expression of MaABI5-like by virus-induced gene silencing in ‘Fenjiao’ banana increased chilling injury and downregulated the expression of fatty acid desaturation and flavonoid synthesis-related genes. Thus, the study indicates that MaABI5-like regulates ABA-induced cold tolerance by increasing unsaturated fatty acid and flavonoid content.

Fruit ripening is one of the main processes affecting fruit quality and shelf life. In melon there are both climacteric and non-climacteric genotypes, making it a suitable species to study fruit ripening. In the current study, in order to fine tune ripening, we have pyramided three climacteric QTLs in the non-climacteric genotype “Piel de Sapo”: ETHQB3.5, ETHQV6.3 and ETHQV8.1. The results showed that the three QTLs interact epistatically, affecting ethylene production and ripening-related traits such as aroma profile. Each individual QTL has a specific role in the ethylene production profile. ETHQB3.5 accelerates the ethylene peak, ETHQV6.3 advances the ethylene production and ETHQV8.1 enhances the effect of the other two QTLs. Regarding aroma, the three QTLs independently activated the production of esters changing the aroma profile of the fruits, with no significant effects in fruit firmness, soluble solid content and fruit size. Understanding the interaction and the effect of different ripening QTLs offers a powerful knowledge for candidate gene identification as well as for melon breeding programs, where fruit ripening is one of the main objectives.

Plant tissues are capable of developing unorganized cell masses termed calluses in response to the appropriate combination of auxin and cytokinin. Revealing the potential epigenetic mechanisms involved in callus development can improve our understanding of the regeneration process of plant cells, which will be beneficial for overcoming regeneration recalcitrance in peach. In this study, we report on single-base resolution mapping of DNA methylation and reprogramming of the pattern of trimethylation of histone H3 at lysine 27 (H3K27me3) at the genome-wide level during the leaf-to-callus transition in peach. Overall, mCG and mCHH were predominant at the genome-wide level and mCG was predominant in genic regions. H3K27me3 deposition was mainly detected in the gene body and at the TSS site, and GAGA repetitive sequences were prone to recruit H3K27me3 modification. H3K27me3 methylation was negatively correlated with gene expression. In vitro culture of leaf explants was accompanied by DNA hypomethylation and H3K27me3 demethylation, which could activate auxin- and cytokinin-related regulators to induce callus development. The DNA methylation inhibitor 5-azacytidine could significantly increase callus development, while the H3K27me3 demethylase inhibitor GSK-J4 dramatically reduced callus development. These results demonstrate the roles of DNA methylation and H3K27me3 modification in mediating chromatin status during callus development. Our study provides new insights into the epigenetic mechanisms through which differentiated cells acquire proliferative competence to induce callus development in plants.

VviAGL11, the Arabidopsis SEEDSTICK homolog, has been proposed to have a causative role in grapevine stenospermocarpy. An association between a mutation in the coding sequence (CDS) and the seedless phenotype was reported, however, no working mechanisms have been demonstrated yet. We performed a deep investigation of the full VviAGL11 gene sequence in a collection of grapevine varieties belonging to several seedlessness classes that revealed three different promoter-CDS combinations. By investigating the expression of the three VviAGL11 alleles, and by evaluating their ability to activate the promoter region, we observed that VviAGL11 self-activates in a specific promoter-CDS combination manner. Furthermore, by transcriptomic analyses on ovule and developing seeds in seeded and seedless varieties and co-expression approaches, candidate VviAGL11 targets were identified and further validated through luciferase assay and in situ hybridization. We demonstrated that VviAGL11 Wild Type CDS activates Methyl jasmonate esterase and Indole-3-acetate beta-glucosyltransferase, both involved in hormone signaling and Isoflavone reductase, involved in secondary metabolism. The dominant-negative effect of the mutated CDS was also functionally ectopically validated in target induction. VviAGL11 was shown to co-localize with its targets in the outer seed coat integument, supporting its direct involvement in seed development, possibly by orchestrating the crosstalk among MeJA, auxin, and isoflavonoids synthesis. In conclusion, the VviAGL11 expression level depends on the promoter-CDS allelic combination, and this will likely affect its ability to activate important triggers of the seed coat development. The dominant-negative effect of the mutated VviAGL11 CDS on the target genes activation was molecularly validated. A new regulatory mechanism correlating VviAGL11 haplotype assortment and seedlessness class in grapevine is proposed.

Tomato ripening is a complex and dynamic process coordinated by many regulatory elements, including plant hormones, transcription factors, and numerous ripening-related RNAs and proteins. Although recent studies have shown that some RNA-binding proteins are involved in the regulation of the ripening process, understanding of how RNA-binding proteins affect fruit ripening is still limited. Here, we report the analysis of a glycine-rich RNA-binding protein, RZ1A-Like (RZ1AL), which plays an important role in tomato ripening, especially fruit coloring. To analyze the functions of RZ1AL in fruit development and ripening, we generated knockout cr-rz1al mutant lines via the CRISPR/Cas9 gene-editing system. Knockout of RZ1AL reduced fruit lycopene content and weight in the cr-rz1al mutant plants. RZ1AL encodes a nucleus-localized protein that is associated with Cajal-related bodies. RNA-seq data demonstrated that the expression levels of genes that encode several key enzymes associated with carotenoid biosynthesis and metabolism were notably downregulated in cr-rz1al fruits. Proteomic analysis revealed that the levels of various ribosomal subunit proteins were reduced. This could affect the translation of ripening-related proteins such as ZDS. Collectively, our findings demonstrate that RZ1AL may participate in the regulation of carotenoid biosynthesis and metabolism and affect tomato development and fruit ripening.

The capacity of plants to resist abiotic stresses is of great importance to agricultural, ecological and environmental sustainability, but little is known about its genetic underpinnings. Existing genetic tools can identify individual genetic variants mediating biochemical, physiological, and cellular defenses, but fail to chart an overall genetic atlas behind stress resistance. We view stress response as an eco-evo-devo process by which plants adaptively respond to stress through complex interactions of developmental canalization, phenotypic plasticity, and phenotypic integration. As such, we define and quantify stress response as the developmental change of adaptive traits from stress-free to stress-exposed environments. We integrate composite functional mapping and evolutionary game theory to reconstruct omnigenic, information-flow interaction networks for stress response. Using desert-adapted Euphrates poplar as an example, we infer salt resistance-related genome-wide interactome networks and trace the roadmap of how each SNP acts and interacts with any other possible SNPs to mediate salt resistance. We characterize the previously unknown regulatory mechanisms driving trait variation; i.e. the significance of a SNP may be due to the promotion of positive regulators, whereas the insignificance of a SNP may result from the inhibition of negative regulators. The regulator-regulatee interactions detected are not only experimentally validated by two complementary experiments, but also biologically interpreted by their encoded protein–protein interactions. Our eco-evo-devo model of genetic interactome networks provides an approach to interrogate the genetic architecture of stress response and informs precise gene editing for improving plants’ capacity to live in stress environments.

Hull-less pumpkins (Cucurbita pepo L.) are naturally occurring novel variants known as oilseed or naked-seeded pumpkins, and are characterized by the absence of a normal lignified seed coat. Due to a specialized seed coat structure, these variants serve as a good model for studying seed coat formation and simplify the processing of pumpkin seeds. However, causal genes for this hull-less trait still remain unknown. Here, by bulked segregant analysis and fine mapping, we found that mutation of a single gene, NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1 (NST1), accounts for the hull-less trait. A 14-bp sequence insertion in the CpNST1 gene causes premature termination of CpNST1 translation, leading to lack of secondary cell wall (SCW) biosynthesis in hull-less seed coats. In situ hybridization analysis provided further evidence for the role of CpNST1 in pumpkin seed coat SCW biosynthesis. Interestingly, through secondary cell wall compositional analysis, we found that the main SCW components differed among cell layers in the seed coat. RNA-seq analysis indicated an upstream role of CpNST1 in the SCW biosynthesis network. Collectively, our findings provide mechanistic insight into seed coat SCW biosynthesis, and a target gene for breeders to introduce this hull-less trait for commercial exploitation.

The morphological diversity of leaf margin shapes is an identifying characteristic of many plant species. In our previous work, BoALG10 (α-1,2 glycosyltransferase) was predicted to be a key regulator of leaf margin shape in ornamental kale (Brassica oleracea var. acephala). An alanine and a leucine residue in the conserved domain of the smooth-margined S0835 were replaced by an aspartate and a phenylalanine, respectively, in the corresponding positions of the feathered-margined F0819. However, the expression pattern and function of this gene remain unclear. Here, we examined the expression patterns of BoALG10 using quantitative real-time PCR, and found that statistically significant differences in expression existed between F0819 and S0835 in nine developmental stages. The BoALG10 protein localized to the endoplasmic reticulum. The function of BoALG10 was then examined using complementary mutant assays. The overexpression strains phenocopied the smooth leaf margin after introduction of BoALG10^S0835 into the feathered-margined inbred line F0819. Simultaneously, irregular dissections appeared in the leaf margins of knockout mutants KO-1 and KO-2, which were generated by CRISPR/Cas9 technology from the smooth-margined inbred line S0835. Microscopic observation showed that the leaf margin cells of the smooth-margined plants S0835 and OE-3 were arranged regularly, while the cells of the feathered-margined plants F0819 and KO-1 were of inconsistent size and distributed in an irregular manner, particularly around the indentations of the leaf. This elucidation of BoALG10 function provides a novel insight into the morphological regulation of leaf margin shape.

Flavonol glycosides are health-promoting phytochemicals important for human nutrition and plant defense against environmental stresses. Glycosylation modification greatly enriches the diversity of flavonols. Morella rubra, a member of the Myricaceae, contains high amounts of myricetin 3- O-rhamnoside (M3Rha), quercetin 3- O-rhamnoside (Q3Rha), and quercetin 3- O-galactoside (Q3Gal). In the present study, MrUGT78R1 and MrUGT78R2 were identified as two functional UDP-rhamnosyltransferases, while MrUGT78W1 was identified as a UDP-galactosyltransferase. Site-directed mutagenesis identified Pro143 and Asn386 as important residues for rhamnosyl transfer activity of MrUGT78R1, while the two corresponding positions in MrUGT78W1 (i.e. Ser147 and Asn370) also play important roles in galactosyl transfer activity. Transient expression data for these three MrUGTs in Nicotiana benthamiana tested the function of MrUGT78R1 and MrUGT78R2 as rhamnosyltransferases and MrUGT78W1 as a galactosyltransferase in glycosylation of flavonols. This work enriches knowledge of the diversity of UDP-rhamnosyltransferase in planta and identifies two amino acid positions important for both rhamnosyltransferase and galactosyltransferase.

Isatis indigotica accumulates several active substances, including C-glycosylflavonoids, which have important pharmacological activities and health benefits. However, enzymes catalyzing the methylation step of C-glycosylflavonoids in I. indigotica remain unknown. In this study, three O-methyltransferases (OMTs) were identified from I. indigotica that have the capacity for O-methylation of the C-glycosylflavonoid isoorientin. The Type II OMTs IiOMT1 and IiOMT2 efficiently catalyze isoorientin to form isoscoparin, and decorate one of the aromatic vicinal hydroxyl groups on flavones and methylate the C6, C8, and 3^′-hydroxyl positions to form oroxylin A, wogonin, and chrysoeriol, respectively. However, the Type I OMT IiOMT3 exhibited broader substrate promiscuity and methylated the C7 and 3^′-hydroxyl positions of flavonoids. Further site-directed mutagenesis studies demonstrated that five amino acids of IiOMT1/IiOMT2 (D121/D100, D173/D149, A174/A150R, N200/N176, and D248/D233) were critical residues for their catalytic activity. Additionally, only transient overexpression of Type II OMTs IiOMT1 and IiOMT2 in Nicotiana benthamiana significantly increased isoscoparin accumulation, indicating that the Type II OMTs IiOMT1 and IiOMT2 could catalyze the methylation step of C-glycosylflavonoid, isoorientin at the 3^′-hydroxyl position. This study provides insights into the biosynthesis of methylated C-glycosylflavonoids, and IiOMTs could be promising catalysts in the synthesis of bioactive compounds.

Pear (Pyrus spp.) is one of the most common fruit crops grown in temperate regions worldwide. Genetic enhancement of fruit quality is a fundamental goal of pear breeding programs. The genetic control of pear fruit quality traits is highly quantitative, and development of high-density genetic maps can facilitate fine-mapping of quantitative trait loci (QTLs) and gene identification. Bin-mapping is a powerful method of constructing high-resolution genetic maps from large-scale genotyping datasets. We performed whole-genome sequencing of pear cultivars ‘Niitaka’ and ‘Hongxiangsu’ and their 176 F₁ progeny to identify genome-wide single-nucleotide polymorphism (SNP) markers for constructing a high-density bin-map of pear. This analysis yielded a total of 1.93 million SNPs and a genetic bin-map of 3190 markers spanning 1358.5 cM, with an average adjacent interval of 0.43 cM. This bin-map, along with other high-density genetic maps in pear, improved the reference genome assembly from 75.5 to 83.7% by re-anchoring the scaffolds. A quantitative genetic analysis identified 148 QTLs for 18 fruit-related traits; among them, QTLs for stone cell content, several key monosaccharides, and fruit pulp acids were identified for the first time in pear. A gene expression analysis of six pear cultivars identified 399 candidates in the identified QTL regions, which showed expression specific to fruit developmental stages in pear. Finally, we confirmed the function of PbrtMT1, a tonoplast monosaccharide transporter-related gene responsible for the enhancement of fructose accumulation in pear fruit on linkage group 16, in a transient transformation experiment. This study provides genomic and genetic resources as well as potential candidate genes for fruit quality improvement in pear.

Ethylene and jasmonic acid (JA) are crucial hormones that promote anthocyanin synthesis in apple (Malus × domestica) . However, the mechanism by which these hormones cooperate to modulate anthocyanin production in apple is unclear. According to our results, MdERF1B expression was strongly induced by ethylene and JA. Physiological phenotypes and the results of molecular biological analyses indicated that MdERF1B encodes a positive regulator of anthocyanin synthesis. Specifically, MdERF1B was capable of combining directly with the MdMYC2 promoter to promote gene expression. Additionally, MdERF1B interacted with two JA signaling pathway inhibitors, namely MdJAZ5 and MdJAZ10. The MdERF1B–MdJAZ5/10 protein complex decreased the ability of MdERF1B to activate the MdMYC2 promoter. Furthermore, MdEIL1, which is a crucial protein for ethylene signal transduction, was observed to bind directly to the MdERF1B promoter, thereby upregulating gene expression. These results suggest that MdERF1B is a core gene responsive to JA and ethylene signals. The encoded protein, together with MdMYC2, MdJAZ5/10, and MdEIL1, modulates anthocyanin synthesis in apple. This study clarifies the synergistic mechanism by which JA and ethylene regulate anthocyanin production in apple.

Target of rapamycin (TOR) is a highly conserved master regulator in eukaryotes; it regulates cell proliferation and growth by integrating different signals. However, little is known about the function of TOR in perennial woody plants. Different concentrations of AZD8055 (an inhibitor of TOR) were used in this study to investigate the role of TOR in the response to low nitrogen (N) stress in the wild apple species Malus hupehensis. Low N stress inhibited the growth of M. hupehensis plants, and 1 μM AZD alleviated this effect. Plants supplied with 1 μM AZD had higher photosynthetic capacity, which promoted the accumulation of biomass, as well as higher contents of N and anthocyanins and lower content of starch. Exogenous application of 1 μM AZD also promoted the development of the root system. Plants supplied with at least 5 μM AZD displayed early leaf senescence. RNA-seq analysis indicated that TOR altered the expression of genes related to the low N stress response, such as genes involved in photosystem, starch metabolism, autophagy, and hormone metabolism. Further analysis revealed altered autophagy in plants supplied with AZD under low N stress; the metabolism of plant hormones also changed following AZD supplementation. In sum, our findings revealed that appropriate inhibition of TOR activated autophagy and jasmonic acid signaling in M. hupehensis, which allowed plants to cope with low N stress. Severe TOR inhibition resulted in the excessive accumulation of salicylic acid, which probably led to programmed cell death in M. hupehensis.

The interaction between jasmonic acid (JA) and salicylic acid (SA) pathways, which affects plant stress resistance, is mainly considered to be antagonistic. Using an established theoretical model, we investigated how tea plant (Camellia sinensis) volatiles induced by exogenous elicitors of the JA and SA pathways are affected by the sequence of elicitor application, elicitor identity, and the applied concentrations. We also examined the effects of the volatiles mediated by the JA–SA synergistic interaction on the behaviors of a tea leaf-chewing herbivore (Ectropis grisescens) and its parasitic wasp (Apanteles sp.). The JA and SA pathway interactions were almost always reciprocally synergistic when the two pathways were elicited at different times, except at high JA elicitor concentrations. However, the JA pathway antagonized the SA pathway when they were elicited simultaneously. The elicitor identity affected the degree of JA–SA interaction. The volatiles induced by the JA pathway in the JA–SA reciprocal synergism treatments included up to 11 additional compounds and the total amount of volatiles was up to 7.9-fold higher. Similarly, the amount of emitted volatiles induced by the SA pathway in the reciprocal synergism treatments increased by up to 4.2-fold. Compared with the volatiles induced by either pathway, the enriched volatiles induced by the JA–SA reciprocal synergism similarly repelled E. grisescens, but attracted Apanteles sp. more strongly. Thus, non-simultaneous activation is important for optimizing the JA–SA reciprocal synergism. This reciprocal synergism enables plants to induce multifarious responses, leading to increased biotic stress resistance.

Early accurate detection of crop disease is extremely important for timely disease management. Huanglongbing (HLB), one of the most destructive citrus diseases, has brought about severe economic losses for the global citrus industry. The direct strategies for HLB identification, such as quantitative real-time polymerase chain reaction (qPCR) and chemical staining, are robust for the symptomatic plants but powerless for the asymptomatic ones at the early stage of affection. Thus, it is very necessary to develop a practical method used for the early detection of HLB. In this study, a novel method combining ultra-high performance liquid chromatography/mass spectrometry (UHPLC/MS)-based nontargeted metabolomics and machine learning (ML) was developed for conducting the early detection of HLB for the first time. Six ML algorithms were selected to build the classifiers. Regularized logistic regression (LR-L2) and gradient-boosted decision tree (GBDT) outperformed with the highest average accuracy of 95.83% to not only classify healthy and infected plants but identify significant features. The proposed method proved to be practical for early detection of HLB, which tackled the shortcomings of low sensitivity in the conventional methods and avoid the problems such as lighting condition interference in spectrum/image recognition-based ML methods. Additionally, the discovered biomarkers were verified by the metabolic pathway analysis and content change analysis, which was remarkably consistent with the previous reports.

Fruit glossiness is an important external fruit quality trait for fresh-consumed cucumber fruit, affecting its marketability. Dull fruit appearance is mainly controlled by a single gene, D (for dull fruit) that is dominant to glossy fruit (dd), but the molecular mechanism controlling fruit glossiness is unknown. In the present study, we conducted map-based cloning of the D locus in cucumber and identified a candidate gene (Csa5G577350) that encodes a C₂H₂-type zinc finger transcription factor, CsDULL. A 4895-bp deletion including the complete loss of CsDULL resulted in glossy fruit. CsDULL is highly expressed in the peel of cucumber fruit, and its expression level is positively correlated with the accumulation of cutin and wax in the peel. Through transcriptome analysis, yeast one-hybrid and dual-luciferase assays, we identified two genes potentially targeted by CsDULL for regulation of cutin and wax biosynthesis/transportation that included CsGPAT4 and CsLTPG1. The possibility that CsDULL controls both fruit glossiness and wart development in cucumber is discussed. The present work advances our understanding of regulatory mechanisms of fruit epidermal traits, and provides a useful tool for molecular breeding to improve external fruit quality in cucumber.

MicroRNAs (miRNAs) are small noncoding RNAs, which modulate the abundance and spatiotemporal accumulation of target mRNAs at transcriptional and post-transcriptional levels and through that play important roles in several biological processes in plants. Here we show that in polyploid species, CRISPR/Cas9 system can be used for fine-tuning of miRNA expression, which can have broader range of applications compared to knock-out mutants. We established the complete pipeline for CRISPR-Cas9-mediated modulation of miRNA expression in potato. It consists of (1) design and assembly of dual sgRNA CRISPR/Cas9 constructs, (2) transient transfection of protoplasts following fast and efficient screening by high resolution melting analysis to select functional sgRNAs, and (3) stable transformation of potato explants with functional sgRNAs and selection of regenerated transgenic lines with desired mutations and desired miRNA abundance based on sequencing and RT-qPCR. We show that miRNA-editing using dual sgRNA approach results in different types of mutations among transgenic lines but also in different alleles of the same plant, which are target site-dependent. The most frequent were short deletions, but we also detected 1-nt insertions (T or G), deletions between two sgRNAs and larger deletions. miRNA abundance correlates with the frequency and type of introduced mutations, as more extensive mutations in more alleles result in lower miRNA abundance. Interestingly, some mutated loci can generate alternative miRNAs, now novel targets were however predicted for those. In all transgenic lines with Cas9 expression, we detected mutations, suggesting high efficiency of Cas9-editing. We confirmed the miRNA-editing efficiency of our optimised approach in two different potato genotypes and three different loci.

CRISPR/Cas systems have been widely used for genome engineering in many plant species. However, their potentials have remained largely untapped in fruit crops, particularly in pear, due to the high levels of genomic heterozygosity and difficulties in tissue culture and stable transformation. To date, only a few reports on the application of the CRISPR/Cas9 system in pear have been documented, and have shown very low editing efficiency. Here we report a highly efficient CRISPR toolbox for loss-of-function and gain-of-function research in pear. We compared four different CRISPR/Cas9 expression systems for loss-of-function analysis and identified a potent system that showed nearly 100% editing efficiency for multi-site mutagenesis. To expand the targeting scope, we further tested different CRISPR/Cas12a and Cas12b systems in pear for the first time, albeit with low editing efficiency. In addition, we established a CRISPR activation (CRISPRa) system for multiplexed gene activation in pear calli for gain-of-function analysis. Furthermore, we successfully engineered the anthocyanin and lignin biosynthesis pathways using both CRISPR/Cas9 and CRISPRa systems in pear calli. Taking these results together, we have built a highly efficient CRISPR toolbox for genome editing and gene regulation, paving the way for functional genomics studies as well as molecular breeding in pear.

Apomixis is the phenomenon of clonal reproduction by seed. As apomixis can produce clonal progeny with exactly the same genotype as the maternal plant, it has an important application in genotype fixation and accelerating agricultural breeding strategies. The introduction of apomixis to major crops would bring many benefits to agriculture, including permanent fixation of superior genotypes and simplifying the procedures of hybrid seed production, as well as purification and rejuvenation of crops propagated vegetatively. Although apomixis naturally occurs in more than 400 plant species, it is rare among the major crops. Currently, with better understanding of apomixis, some achievements have been made in synthetic apomixis. However, due to prevailing limitations, there is still a long way to go to achieve large-scale application of apomixis to crop breeding. Here, we compare the developmental features of apomixis and sexual plant reproduction and review the recent identification of apomixis genes, transposons, epigenetic regulation, and genetic events leading to apomixis. We also summarize the possible strategies and potential genes for engineering apomixis into crop plants.

The fresh and unique flavor of cucumber fruits, mainly composed of aldehydes and alcohols, is one of its most important fruit qualities. However, little is known about the genetic basis of aroma compounds in cucumber fruit and the related quantitative trait loci (QTLs). In this study, genomic screening of QTLs underlying aroma compounds was performed based on the genetic linkage map constructed using 1301 single-nucleotide polymorphism (SNP) markers from genotyping-by-sequencing of a recombinant inbred line (RIL) population developed from Q16 × Q24. Significant genetic variations of aroma compounds in the RIL population were observed, and a total of 28 QTLs were screened. A major QTL (qol8-2.1) related to (E, Z)-2,6-nonadien-1-ol was detected with a markedly high LOD score (10.97 in 2020 and 3.56 in 2019) between mk190 and mk204 on chromosome 2. Genome scans identified a cluster of nine lipoxygenase genes in this region. A significant positive correlation was detected between CsaV3_2G005360 (CsLOX08) and (E, Z)-2,6-nonadien-1-ol, and five amino acid variations were detected between the CsLOX08 protein sequences of the two parental lines. Based on the genome variation of CsLOX08, we developed an InDel marker. Genotyping of InDel markers was consistent with the content of (E, Z)-2,6-nonadien-1-ol in RILs, which were also verified in nine cucumber inbred lines. The results will give breeders guidance for obtaining better flavor in cucumber.

O-methyltransferases play essential roles in producing structural diversity and improving the biological properties of benzylisoquinoline alkaloids (BIAs) in plants. In this study, Corydalis yanhusuo, a plant used in traditional Chinese medicine due to the analgesic effects of its BIA-active compounds, was employed to analyze the catalytic characteristics of O-methyltransferases in the formation of BIA diversity. Seven genes encoding O-methyltransferases were cloned, and functionally characterized using seven potential BIA substrates. Specifically, an O-methyltransferase (CyOMT2) with highly efficient catalytic activity of both 4′- and 6- O-methylations of 1-BIAs was found. CyOMT6 was found to perform two sequential methylations at both 9- and 2-positions of the essential intermediate of tetrahydroprotoberberines, (S)-scoulerine. Two O-methyltransferases (CyOMT5 and CyOMT7) with wide substrate promiscuity were found, with the 2-position of tetrahydroprotoberberines as the preferential catalytic site for CyOMT5 (named scoulerine 2- O-methyltransferase) and the 6-position of 1-BIAs as the preferential site for CyOMT7. In addition, results of integrated phylogenetic molecular docking analysis and site-directed mutation suggested that residues at sites 172, 306, 313, and 314 in CyOMT5 are important for enzyme promiscuity related to O-methylations at the 6- and 7-positions of isoquinoline. Cys at site 253 in CyOMT2 was proved to promote the methylation activity of the 6-position and to expand substrate scopes. This work provides insight into O-methyltransferases in producing BIA diversity in C. yanhusuo and genetic elements for producing BIAs by metabolic engineering and synthetic biology.

Chrysanthemum (Chrysanthemum morifolium Ramat.) is an economically important plant species growing worldwide. However, its origin, especially as revealed by biogeographic and metabolomics research, remains unclear. To understand the geographic distribution of species diversity and metabolomics in three genera (Chrysanthemum, Ajania, and Phaeostigma), geographic information systems and gas chromatography–mass spectrometry were used in 19, 15, and 4 species respectively. China and Japan were two potential panbiogeographic nodes and diverse hotspots of Chrysanthemum, with species richness ratios of 58.97 and 33.33%. We studied different species from two hotspots which in similar geographical environments had closer chemotaxonomic relationships under the same cultivation conditions based on a cluster of 30 secondary metabolites. The average distribution altitude (ADA) differed significantly among Chrysanthemum, Ajania, and Phaeostigma in which it was 1227.49, 2400.12, and 3760.53 m.a.s.l. respectively, and the presence/absence of ray florets (RF) was significantly correlated with ADA (−0.62). Mountain landform was an important contributor to global Chrysanthemum diversity, playing a key role in the divergence and distribution pattern of Chrysanthemum and its allies. The Hengduan Mountains–Qinling Mountains (HDQ) in China was a potential secondary radiation and evolution center of Chrysanthemum and its related genera in the world. During the Quaternary glacial–interglacial cycles, this region became their refuge, and they radiated and evolved from this center.

CRISPR-mediated genome editing has become a powerful tool for the genetic modification of biological traits. However, developing an efficient, site-specific, gene knock-in system based on homology-directed DNA repair (HDR) remains a significant challenge in plants, especially in woody species like poplar. Here, we show that simultaneous inhibition of non-homologous end joining (NHEJ) recombination cofactor XRCC4 and overexpression of HDR enhancer factors CtIP and MRE11 can improve HDR efficiency for gene knock-in. Using this approach, the BleoR gene was integrated onto the 3^′ end of the MKK2 MAP kinase gene to generate a BleoR-MKK2 fusion protein. Based on fully edited nucleotides evaluated by TaqMan real-time PCR, the HDR-mediated knock-in efficiency was up to 48% when using XRCC4 silencing incorporated with a combination of CtIP and MRE11 overexpression compared with no HDR enhancement or NHEJ silencing. Furthermore, this combination of HDR enhancer overexpression and NHEJ repression also increased genome targeting efficiency and gave 7-fold fewer CRISPR-induced insertions and deletions (InDels), resulting in no functional effects on MKK2-based salt stress responses in poplar. Therefore, this approach may be useful not only in poplar and plants or crops but also in mammals for improving CRISPR-mediated gene knock-in efficiency.

This study aims to: (i) identify the Rosa S-locus controlling self-incompatibility (SI); (ii) test the genetic linkage of the S-locus with other loci controlling important ornamental traits, such as the continuous-flowering (CF) characteristic; (iii) identify the S-alleles (S_C) of old Chinese CF cultivars (e.g, Old Blush, Slater’s Crimson China) and examine the changes in the frequency of cultivars with Sc through the history of breeding; (iv) identify wild species carrying the Sc-alleles to infer wild origins of CF cultivars. We identified a new S- RNase (S_C2) of Rosa chinensis in a contig from a genome database that has not been integrated into one of the seven chromosomes yet. Genetic mapping indicated that S_C2 is allelic to the previously-identified S-RNase (S_C1) in chromosome 3. Pollination experiments with half-compatible pairs of roses confirmed that they are the pistil-determinant of SI. The segregation analysis of an F₁-population indicated genetic linkage between the S-locus and the floral repressor gene KSN. The non-functional allele ksn is responsible for the CF characteristic. A total of five S-alleles (S_C1–5) were identified from old CF cultivars. The frequency of cultivars with S_C dramatically increased after the introgression of ksn from Chinese to European cultivars and remains high (80%) in modern cultivars, suggesting that S-genotyping is helpful for effective breeding. Wild individuals carrying S_C were found in Rosa multiflora (S_C1), Rosa chinensis var . spontanea (S_C3), and Rosa gigantea (S_C2, S_C4), supporting the hypothesis of hybrid origins of CF cultivars and providing a new evidence for the involvement of Rosa multiflora.

The ripening of climacteric fruits is mainly controlled by the plant hormone ethylene. The regulatory effect of sucrose on ethylene biosynthesis in fruits remains unclear. Here we examined ethylene production in two Ussurian pear (Pyrus ussuriensis) varieties, ‘Nanguo’ (NG) pear and its bud sport variety (BNG), which has a higher sucrose content. We found that the peak of ethylene release occurred earlier in BNG fruit than in NG fruit during ripening. The expression of the transcription factor PuWRKY31 was higher in BNG fruit than in NG fruit, and was induced by sucrose treatment. Furthermore, PuWRKY31 bound to the promoters of ethylene biosynthetic genes and upregulated their transcription. Our findings suggest a mechanism by which sucrose regulates ethylene biosynthesis in pears.

Avocado (Persea americana) is a member of the magnoliids, an early branching lineage of angiosperms that has high value globally with the fruit being highly nutritious. Here, we report a chromosome-level genome assembly for the commercial avocado cultivar Hass, which represents 80% of the world’s avocado consumption. The DNA contigs produced from Pacific Biosciences HiFi reads were further assembled using a previously published version of the genome supported by a genetic map. The total assembly was 913 Mb with a contig N50 of 84 Mb. Contigs assigned to the 12 chromosomes represented 874 Mb and covered 98.8% of benchmarked single-copy genes from embryophytes. Annotation of protein coding sequences identified 48 915 avocado genes of which 39 207 could be ascribed functions. The genome contained 62.6% repeat elements. Specific biosynthetic pathways of interest in the genome were investigated. The analysis suggested that the predominant pathway of heptose biosynthesis in avocado may be through sedoheptulose 1,7 bisphosphate rather than via alternative routes. Endoglucanase genes were high in number, consistent with avocado using cellulase for fruit ripening. The avocado genome appeared to have a limited number of translocations between homeologous chromosomes, despite having undergone multiple genome duplication events. Proteome clustering with related species permitted identification of genes unique to avocado and other members of the Lauraceae family, as well as genes unique to species diverged near or prior to the divergence of monocots and eudicots. This genome provides a tool to support future advances in the development of elite avocado varieties with higher yields and fruit quality.

Herbivore-induced plant volatiles (HIPVs) help the tea plant (Camellia sinensis) adapt to environmental stress, and they are also quality-related components of tea. However, the upstream mechanism regulating the herbivore-induced expression of volatile biosynthesis genes is unclear, especially at the level of epigenetic regulation. In this study, similar to the effects of a tea green leafhopper infestation, treatments with exogenous jasmonic acid (JA) and histone deacetylase inhibitors significantly increased the (E)-nerolidol content in tea and induced the expression of the associated biosynthesis gene CsNES. Furthermore, a key transcription factor related to JA signaling, myelocytomatosis 2 (CsMYC2), interacted with histone deacetylase 2 (CsHDA2) in vitro and in vivo. A tea green leafhopper infestation inhibited CsHDA2 expression and decreased CsHDA2 abundance. Moreover, the tea green leafhopper infestation increased H3 and H4 acetylation levels in the promoter region of CsNES, which in turn upregulated the expression of CsNES and increased the (E)-nerolidol content. In this study, we revealed the effects of histone acetylations on the accumulation of HIPVs, while also confirming that CsHDA2–CsMYC2 is an important transcriptional regulatory module for the accumulation of (E)-nerolidol induced by tea green leafhoppers. The results of this study may be useful for characterizing plant aromatic compounds and the main upstream stress-responsive signaling molecules. Furthermore, the study findings will assist researchers clarify the epigenetic regulation influencing plant secondary metabolism in response to external stress.

Plant viruses recruit multiple host factors for translation, replication, and movement in the infection process. The loss-of-function mutation of the susceptibility genes will lead to the loss of susceptibility to viruses, which is referred to as ‘recessive resistance’. Essential for potexvirus Accumulation 1 (EXA1) has been identified as a susceptibility gene required for potexvirus, lolavirus, and bacterial and oomycete pathogens. In this study, EXA1 knockdown in potato (StEXA1) was found to confer novel resistance to potato virus Y (PVY, potyvirus) in a strain-specific manner. It significantly compromised PVY^O accumulation but not PVY^N:O and PVY^NTN. Further analysis revealed that StEXA1 is associated with the HC-Pro of PVY through a member of eIF4Es (StnCBP). HC-Pro^O and HC-Pro^N, two HC-Pro proteins from PVY^O and PVY^N, exhibited strong and weak interactions with StnCBP, respectively, due to their different spatial conformation. Moreover, the accumulation of PVY^O was mainly dependent on the stress granules (SGs) induced by StEXA1 and StnCBP, whereas PVY^N:O and PVY^NTN could induce SGs by HC-Pro^N independently through an unknown mechanism. These results could explain why StEXA1 or StnCBP knockdown conferred resistance to PVY^O but not to PVY^N:O and PVY^NTN. In summary, our results for the first time demonstrate that EXA1 can act as a susceptibility gene for PVY infection. Finally, a hypothetical model was proposed for understanding the mechanism by which StEXA1 interacts with StnCBP to facilitate PVY accumulation in potato through the SG-dependent RNA regulatory pathway.

In the past years, breeding programs have been mainly addressed on pushing the commercial features, forgetting important traits, such as those related to environmental stress resilience, that are instead present in wild relatives. Among the traits neglected by breeding processes, the ability to recruit beneficial microorganisms that recently is receiving a growing attention due to its potentiality. In this context, this review will provide a spotlight on critical issues of the anthropocentric point of view that, until now, has characterized the selection of elite plant genotypes. Its effects on the plant-microbiome interactions, and the possibility to develop novel strategies mediated by the exploitation of beneficial root-microbe interactions, will be discussed. More sustainable microbial-assisted strategies might in fact foster the green revolution and the achievement of a more sustainable agriculture in a climatic change scenario.

The heat shock transcription factors (Hsfs) play critical roles in plant responses to abiotic stresses. However, the mechanism of Hsfs in the regulation of pollen thermotolerance and their specific biological functions and signaling remain unclear. Herein, we demonstrate that HsfA1a played a key role in tomato pollen thermotolerance. Pollen thermotolerance was reduced in hsfA1a mutants but was increased by hsfA1a overexpression, based on pollen viability and germination. Analyzing the whole transcriptome by RNA-seq data, we found that HsfA1a mainly regulated the genes involved in oxidative stress protection, protein homeostasis regulation and protein modification, as well as the response to biological stress in anthers under heat stress. The accumulation of reactive oxygen species in anthers was enhanced in hsfA1a mutants but decreased in HsfA1a-overexpressing lines. Furthermore, HsfA1a bound to the promoter region of genes involved in redox regulation (Cu/Zn-SOD, GST8, and MDAR1), protein repair (HSP17.6A, HSP70-2, HSP90-2, and HSP101) and degradation (UBP5, UBP18, RPN10a, and ATG10) and regulated the expression of these genes in tomato anthers under heat stress. Our findings suggest that HsfA1a maintains pollen thermotolerance and cellular homeostasis by enhancing antioxidant capacity and protein repair and degradation, ultimately improving pollen viability and fertility.

Viruses can interfere with the ability of plants to overcome abiotic stresses, indicating the existence of common molecular networks that regulate stress responses. A begomovirus causing the tomato yellow leaf curl disease was recently shown to enhance heat tolerance in tomato and drought tolerance in tomato and Nicotiana benthamiana and experimental evidence suggested that the virus-encoded protein C4 is the main trigger of drought responses. However, the physiological and molecular events underlying C4-induced drought tolerance need further elucidation. In this study, transgenic tomato plants expressing the tomato yellow leaf curl Sardinia virus (TYLCSV) C4 protein were subjected to severe drought stress, followed by recovery. Morphometric parameters, water potential, gas exchanges, and hormone contents in leaves were measured, in combination with molecular analysis of candidate genes involved in stress response and hormone metabolism. Collected data proved that the expression of TYLCSV C4 positively affected the ability of transgenic plants to tolerate water stress, by delaying the onset of stress-related features, improving the plant water use efficiency and facilitating a rapid post-rehydration recovery. In addition, we demonstrated that specific anatomical and hydraulic traits, rather than biochemical signals, are the keynote of the C4-associated stress resilience. Our results provide novel insights into the biology underpinning drought tolerance in TYLCSV C4-expressing tomato plants, paving the way for further deepening the mechanism through which such proteins tune the plant-virus interaction.

Diosgenin saponins isolated from Dioscorea species such as D. zingiberensis exhibit a broad spectrum of pharmacological activities. Diosgenin, the aglycone of diosgenin saponins, is an important starting material for the production of steroidal drugs. However, how plants produce diosgenin saponins and the origin and evolution of the diosgenin saponin biosynthetic pathway remain a mystery. Here we report a high-quality, 629-Mb genome of D. zingiberensis anchored on 10 chromosomes with 30 322 protein-coding genes. We reveal that diosgenin is synthesized in leaves (‘source’), then converted into diosgenin saponins, and finally transported to rhizomes (‘sink’) for storage in plants. By evaluating the distribution and evolutionary patterns of diosgenin saponins in Dioscorea species, we find that diosgenin saponin-containing may be an ancestral trait in Dioscorea and is selectively retained. The results of comparative genomic analysis indicate that tandem duplication coupled with a whole-genome duplication event provided key evolutionary resources for the diosgenin saponin biosynthetic pathway in the D. zingiberensis genome. Furthermore, comparative transcriptome and metabolite analysis among 13 Dioscorea species suggests that specific gene expression patterns of pathway genes promote the differential evolution of the diosgenin saponin biosynthetic pathway in Dioscorea species. Our study provides important insights and valuable resources for further understanding the biosynthesis, evolution, and utilization of plant specialized metabolites such as diosgenin saponins.

Jasmonate (JA) signaling regulates plant growth and development, biotic and abiotic stress tolerance, and primary and secondary metabolism biosynthesis. It is extensively modulated by JA-ZIM-domain (JAZ) family genes. In previous work, we obtained nine SmJAZ genes of Salvia miltiorrhiza and proved that SmJAZ8 was the core repressor of JA-induced tanshinone and phenolic acid biosynthesis. Here, we demonstrate that SmJAZ3 and SmJAZ4 act as repressors of JA-induced biosynthesis of tanshinones and salvianolic acid B (Sal B). This suggests that SmJAZ3/4 are functionally redundant in tanshinone and Sal B biosynthesis. SmJAZ1/2/5/6/9 are activators of JA-induced tanshinone biosynthesis and repressors of JA-induced Sal B biosynthesis. This demonstrates the redundancy and diversity of SmJAZ1/2/5/6/9 functions. Besides, SmJAZ10 inhibited JA-induced Sal B synthesis, but had no effect on the synthesis of tanshinone. Two-hybrid screening (Y2H) showed that SmJAZs formed homologous or heterogeneous dimers. Y2H and firefly luciferase complementation imaging (LCI) assays revealed that SmJAZs also formed a complex regulatory network with SmMYC2a, SmMYC2b, SmMYB39, and SmPAP1. Quantitative reverse transcription-PCR (qRT-PCR) indicated that SmJAZs regulated each other at the transcriptional level. Herein, we prove that SmJAZs have functional pleiotropism, diversity, and redundancy in JA-induced tanshinone and phenolic acid biosynthesis. This study provides an important clue for further understanding the inherent biological significance and molecular mechanisms of the JAZ family as the gene number increases during plant evolution.

Leafy head is an important agronomic trait that determines the yield and quality of Chinese cabbage. The molecular mechanism underlying heading in Chinese cabbage has been the focus of research, and wide leaves are a prerequisite for leafy head formation. In our study, two allelic leafy heading-deficient mutants (lhd1 and lhd2) with narrow leaf phenotypes were screened in an ethyl methanesulfonate mutagenized population from a heading Chinese cabbage double haploid line ‘FT’. Genetic analysis revealed that the mutant trait was controlled by a recessive nuclear gene, which was found to be BraA10g000480.3C by MutMap and Kompetitive allele-specific PCR analyses. As BraA10g000480.3C was the ortholog of ANGUSTIFOLIA in Arabidopsis, which has been found to regulate leaf width by controlling cortical microtubule arrangement and pavement cell shape, we named it BrAN. BrAN in mutant lhd1 carried an SNP (G to A) on intron 2 that co-segregated with the mutant phenotype, and disrupted the exon-intron splice junction generating intron retention and a putative truncated protein. BrAN in mutant lhd2 carried an SNP (G to A) on exon 4 leading to a premature stop codon. The ectopic overexpression of BrAN restored normal leaf phenotype due to abnormal cortical microtubule arrangement and pavement cell shape in the Arabidopsis an-t1 mutant. However, transformation of Bran did not rescue the an-t1 phenotype. These results indicate that BrAN contributes to leafy head formation of Chinese cabbage.

Floral traits often show correlated variation within and among species. For species with fused petals, strong correlations among corolla tube, stamen, and pistil length are particularly prevalent, and these three traits are considered an intra-floral functional module. Pleiotropy has long been implicated in such modular integration of floral traits, but empirical evidence based on actual gene function is scarce. We tested the role of pleiotropy in the expression of intra-floral modularity in the monkeyflower species Mimulus verbenaceus by transgenic manipulation of a homolog of Arabidopsis PRE1. Downregulation of MvPRE1 by RNA interference resulted in simultaneous decreases in the lengths of corolla tube, petal lobe, stamen, and pistil, but little change in calyx and leaf lengths or organ width. Overexpression of MvPRE1 caused increased corolla tube and stamen lengths, with little effect on other floral traits. Our results suggest that genes like MvPRE1 can indeed regulate multiple floral traits in a functional module but meanwhile have little effect on other modules, and that pleiotropic effects of these genes may have played an important role in the evolution of floral integration and intra-floral modularity.

White rust caused by Puccinia horiana is one of the most serious diseases of chrysanthemum (Chrysanthemum × morifolium). In this study, we report the DNA markers associated with resistance against P. horiana via a simple approach using the genome of a wild diploid relative, Chrysanthemum seticuspe. First, we identified the important region of the genome in the resistant cultivar “Ariesu” via a genome-wide association study. Simplex single nucleotide polymorphism (SNP) markers mined from ddRAD-Seq were used in a biparental population originating from crosses between resistant “Ariesu” and susceptible “Yellow Queen”. The C. seticuspe genome was used as a reference. For the fine mapping of P. horiana resistance locus 2 (Phr2), a comparative whole genome sequencing study was conducted. Although the genome sequences of chrysanthemum cultivars assembled via the short-read approach were fragmented, reliable genome alignments were reconstructed by mapping onto the chromosome level of the C. seticuspe pseudomolecule. Base variants were then identified by comparing the assembled genome sequences of resistant “Ariesu” and susceptible “Yellow Queen”. Consequently, SNP markers that were closer to Phr2 compared with ddRAD-Seq markers were obtained. These SNP markers co-segregated with resistance in F₁ progenies originating from resistant “Ariesu” and showed robust transferability for detecting Phr2-conferring resistance among chrysanthemum genetic resources. The wild C. seticuspe pseudomolecule, a de facto monoploid genome used for ddRAD-Seq analysis and assembled genome sequence comparison, demonstrated this method’s utility as a model for developing DNA markers in hexaploid chrysanthemum cultivars.

Cold stress limits plant growth, geographical distribution, and crop yield. The MYC-type bHLH transcription factor ICE1 is recognized as the core positive regulator of the cold-stress response. However, how ICE1 protein levels are regulated remains to be further studied. In this study, we observed that a U-box-type E3 ubiquitin ligase, MdPUB23, positively regulated the cold-stress response in apple. The expression of MdPUB23 increased at both the transcriptional and post-translational levels in response to cold stress. Overexpression of MdPUB23 in transgenic apple enhanced sensitivity to cold stress. Further study showed that MdPUB23 directly interacted with MdICE1, promoting the ubiquitination-mediated degradation of the MdICE1 protein through the 26S-proteasome pathway and reducing the MdICE1-improved cold-stress tolerance in apple. Our results reveal that MdPUB23 regulates the cold-stress response by directly mediating the stability of the positive regulator MdICE1. The PUB23–ICE1 ubiquitination module may play a role in maintaining ICE1 protein homeostasis and preventing overreactions from causing damage to plants. The discovery of the ubiquitination regulatory pathway of ICE1 provides insights for the further exploration of plant cold-stress-response mechanisms.

High pollen fertility can ensure the yield and efficiency of breeding work, but factors that affect the fertility of pepper pollen have not been studied extensively. In this work, we screened the reduced pollen fertility 1 (rpf1) mutant of Capsicum annuum with reduced pollen fertility and yellow anthers from an EMS (ethyl methanesulfonate)-mutagenized pepper population. Through construction of an F2 population followed by BSA (bulked segregant analysis) mapping and KASP genotyping, we identified CabHLH1 as a candidate gene for control of this trait. A G → A mutation at a splice acceptor site in CabHLH1 causes a frameshift mutation in the mutant, and the translated protein is terminated prematurely. Previous studies on CabHLH1 have focused on the regulation of flavonoid synthesis. Here, we found that CabHLH1 also has an important effect on pollen fertility. Pollen vigor, anther flavonoid content, and seed number were lower in CabHLH1-silenced pepper plants, whereas anther H₂O₂ and MDA (malondialdehyde) contents were higher. RNA-seq analyses showed that expression of the flavonoid synthesis genes DFR, ANS, and RT was significantly reduced in anthers of CabHLH1-silenced plants and rpf1 plants, as was the expression of DTX35, a gene related to pollen fertility and flavonoid transport. Yeast one-hybrid and dual-luciferase reporter assays showed that CabHLH1 can directly bind to the promoters of DTX35 and DFR and activate their expression. These results indicate that CabHLH1 regulates reactive oxygen species homeostasis by promoting the synthesis of anther flavonoids and acts as a positive regulator of pepper pollen fertility.

As the largest known tree-borne fruit in the world, jackfruit (Artocarpus heterophyllus) is an important cultivated crop in tropical regions of South and Southeast Asia. The species has been cultivated in China for more than 1000 years, but the history of its introduction to the country remains unclear. We assembled a high-quality chromosome-level genome of jackfruit into 985.63 Mb with scaffold N50 of 32.81 Mb. We analyzed whole-genome resequencing data of 295 landraces to investigate the domestication history in China and agronomic trait evolution of jackfruit. Population structure analysis revealed that jackfruits of China could be traced back to originate from Southeast Asia and South Asia independently. Selection signals between jackfruit and its edible congener, cempedak (Artocarpus integer), revealed several important candidate genes associated with fruit development and ripening. Moreover, analyses of selective sweeps and gene expression revealed that the AhePG1 gene may be the major factor in determining fruit texture. This study not only resolves the origins of jackfruit of China, but also provides valuable genomic resources for jackfruit breeding improvement and offers insights into fruit size evolution and fruit texture changes.

Environmental stresses can result in a wide range of physiological and molecular responses in plants. These responses can also impact epigenetic information in genomes, especially at the level of DNA methylation (5-methylcytosine). DNA methylation is the hallmark heritable epigenetic modification and plays a key role in silencing transposable elements (TEs). Although DNA methylation is an essential epigenetic mechanism, fundamental aspects of its contribution to stress responses and adaptation remain obscure. We investigated epigenome dynamics of wild strawberry (Fragaria vesca) in response to variable ecologically relevant environmental conditions at the DNA methylation level. F. vesca methylome responded with great plasticity to ecologically relevant abiotic and hormonal stresses. Thermal stress resulted in substantial genome-wide loss of DNA methylation. Notably, all tested stress conditions resulted in marked hot spots of differential DNA methylation near centromeric or pericentromeric regions, particularly in the non-symmetrical DNA methylation context. Additionally, we identified differentially methylated regions (DMRs) within promoter regions of transcription factor (TF) superfamilies involved in plant stress-response and assessed the effects of these changes on gene expression. These findings improve our understanding on stress-response at the epigenome level by highlighting the correlation between DNA methylation, TEs and gene expression regulation in plants subjected to a broad range of environmental stresses.

Citric acid and anthocyanin contents were co-selected during Citrus domestication. Pummelo is a founding species in the Citrus genus, but the domestication of pummelo has not been well studied. Here, we compared the citric acid and anthocyanin contents of a low citric acid pummelo (Citrus maxima LCA) and its high citric acid variety (HCA) from the same cultivation area in China. Our study revealed that, unlike the LCA type, the HCA variety accumulated anthocyanin in the pericarp early in fruit development. To investigate the genetic basis of acid and anthocyanin enrichment in HCA pulp and pericarp, respectively, we generated a chromosome-scale HCA genome using long-read sequence reads and Hi-C sequencing data. Transcriptome analysis and transient overexpression assays showed that the accumulation of citric acid and anthocyanin was associated with high expression of CgANTHOCYANIN1 (CgAN1), and two different MYBs transcription factors (CgPH4 and CgRuby1), respectively. Moreover, the CgAN1 promoter was more methylated in the LCA pulp than in the HCA pulp. Treatment with a DNA methylation inhibitor, 5-azacytidine, alleviated the CgAN1 promoter hypermethylation in the LCA pulp, leading to increased CgAN1 expression and citric acid content. This study provides a new high-quality pummelo genome and insight into the molecular mechanism behind the change in tissue-specific citric acid and anthocyanin accumulation during pummelo domestication and provides a conceptual basis for precise genetic manipulation in fruit flavor breeding.

Gypsophila paniculata, belonging to the Caryophyllaceae of the Caryophyllales, is one of the most famous worldwide cut flowers. It is commonly used as dried flowers, whereas the underlying mechanism of flower senescence has not yet been addressed. Here, we present a chromosome-scale genome assembly for G. paniculata with a total size of 749.58 Mb. Whole-genome duplication signatures unveil two major duplication events in its evolutionary history: an ancient one occurring before the divergence of Caryophyllaceae and a more recent one shared with Dianthus caryophyllus. The integrative analyses combining genomic and transcriptomic data reveal the mechanisms regulating floral development and ethylene response of G. paniculata. The reduction of AGAMOUS expression probably caused by sequence polymorphism and the mutation in miR172 binding site of PETALOSA are associated with the double flower formation in G. paniculata. The low expression of ETHYLENE RESPONSE SENSOR (ERS) and the reduction of downstream ETHYLENE RESPONSE FACTOR (ERF) gene copy number collectively lead to the ethylene insensitivity of G. paniculata, affecting flower senescence and making it capable of making dried flowers. This study provides a cornerstone for understanding the underlying principles governing floral development and flower senescence, which could accelerate the molecular breeding of the Caryophyllaceae species.

Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in regulating plant immunity. MAPKs usually transduce signals and regulate plant immunity by phosphorylating the downstream defence-related components. Our previous study indicates that StMPK7 positively regulates plant defence to Phytophthora pathogens via SA signalling pathway. However, the downstream component of StMPK7 remains unknown. In this study, we employed GFP-StMPK7 transgenic potato and performed immunoprecipitation-mass spectrometry (IP-MS) to identify the downstream component of StMPK7. We found that an RNA binding protein StUBA2a/b interacted with StMPK7, as revealed by luciferase complementation imaging (LCI) and coimmunoprecipitation (co-IP) assays. Transient expression of StUBA2a/b in Nicociana benthamiana enhanced plant resistance to Phytophthora pathogens, while silencing of UBA2a/b decreased the resistance, suggesting a positive regulator role of UBA2a/b in plant immunity. Similar to StMPK7, StUBA2a/b was also involved in SA signalling pathway and induced SGT1-dependent cell death as constitutively activated (CA)-StMPK7 did. Immune blotting indicated that StMPK7 phosphorylates StUBA2a/b at thr248 and thr408 (T248/408) sites and stabilizes StUBA2a/b. Silencing of MPK7 in N. benthamiana suppressed StUBA2a/b-induced cell death, while co-expression with StMPK7 enhanced the cell death. Besides, StUBA2a/bT248/408A mutant showed decreased ability to trigger cell death and elevate the expression of PR genes, indicating the phosphorylation by StMPK7 enhances the functions of StUBA2a/b. Moreover, CA-StMPK7-induced cell death was largely suppressed by silencing of NbUBA2a/b, genetically implying UBA2a/b acts as the downstream component of StMPK7. Collectively, our results reveal that StMPK7 phosphorylates and stabilizes its downstream substrate StUBA2a/b to enhance plant immunity via the SA signalling pathway.

Pyrethrins constitute a class of terpene derivatives with high insecticidal activity and are mainly synthesized in the capitula of the horticulturally important plant, Tanacetum cinerariifolium. Treatment of T. cinerariifolium with methyl jasmonate (MeJA) in the field induces pyrethrin biosynthesis, but the mechanism linking MeJA with pyrethrin biosynthesis remains unclear. In this study, we explored the transcription factors involved in regulating MeJA-induced pyrethrin biosynthesis. A single spray application of MeJA to T. cinerariifolium leaves rapidly upregulated the expression of most known pyrethrin biosynthesis genes and subsequently increased the total pyrethrin content in the leaf. A continuous 2-week MeJA treatment resulted in enhanced pyrethrin content and increased trichome density. TcMYC2, a key gene in jasmonate signaling, was screened at the transcriptome after MeJA treatment. TcMYC2 positively regulated expression of the pyrethrin biosynthesis genes TcCHS, TcAOC, and TcGLIP by directly binding to E-box/G-box motifs in the promoters. The stable overexpression of TcMYC2 in T. cinerariifolium hairy roots significantly increased the expression of TcAOC and TcGLIP. Further transient overexpression and viral-induced gene-silencing experiments demonstrated that TcMYC2 positively promoted pyrethrin biosynthesis. Collectively, the results reveal a novel molecular mechanism for MeJA-induced pyrethrin biosynthesis in T. cinerariifolium involving TcMYC2.

Apple bud sports offer a rich resource for clonal selection of numerous elite cultivars. The accumulation of somatic mutations as plants develop may potentially impact the emergence of bud sports. Previous studies focused on somatic mutation in the essential genes associated with bud sports. However, the rate and function of genome-wide somatic mutations that accumulate when a bud sport arises remain unclear. In this study, we identified a branch from a 10-year-old tree of the apple cultivar ‘Oregon Spur II’ as a bud sport. The mutant branch showed reduced red coloration on fruit skin. Using this plant material, we assembled a high-quality haplotype reference genome consisting of 649.61 Mb sequences with a contig N50 value of 2.04 Mb. We then estimated the somatic mutation rate of the apple tree to be 4.56 × 10⁻⁸ per base per year, and further identified 253 somatic single-nucleotide polymorphisms (SNPs), including five non-synonymous SNPs, between the original type and mutant samples. Transcriptome analyses showed that 69 differentially expressed genes between the original type and mutant fruit skin were highly correlated with anthocyanin content. DNA methylation in the promoter of five anthocyanin-associated genes was increased in the mutant compared with the original type as determined using DNA methylation profiling. Among the genetic and epigenetic factors that directly and indirectly influence anthocyanin content in the mutant apple fruit skin, the hypermethylated promoter of MdMYB10 is important. This study indicated that numerous somatic mutations accumulated at the emergence of a bud sport from a genome-wide perspective, some of which contribute to the low coloration of the bud sport.

Ongoing climate change has considerably reduced the seasonal window for crop vernalization, concurrently expanding cultivation area into northern latitudes with long-day photoperiod. To address these changes, cool season legume breeders need to understand molecular control of vernalization and photoperiod. A key floral transition gene integrating signals from these pathways is the Flowering locus T (FT). Here, a recently domesticated grain legume, yellow lupin (Lupinus luteus L.), was explored for potential involvement of FT homologues in abolition of vernalization and photoperiod requirements. Two FTa (LlutFTa1a and LlutFTa1b) and FTc (LlutFTc1 and LlutFTc2) homologues were identified and sequenced for two contrasting parents of a reference recombinant inbred line (RIL) population, an early-flowering cultivar Wodjil and a late-flowering wild-type P28213. Large deletions were detected in the 5′ promoter regions of three FT homologues. Quantitative trait loci were identified for flowering time and vernalization response in the RIL population and in a diverse panel of wild and domesticated accessions. A 2227 bp deletion found in the LlutFTc1 promoter was linked with early phenology and vernalization independence, whereas LlutFTa1a and LlutFTc2 indels with photoperiod responsiveness. Comparative mapping highlighted convergence of FTc1 indel evolution in two Old World lupin species, addressing both artificial selection during domestication and natural adaptation to short season environmental conditions. We concluded that rapid flowering in yellow lupin is associated with the de-repression of the LlutFTc1 homologue from the juvenile phase, putatively due to the elimination of all binding sites in the promoter region for the AGAMOUS-like 15 transcription factor.

BBX transcription factors play vital roles in plant growth, development, and stress responses. Although BBX proteins have been studied in great detail in the model plant Arabidopsis, their roles in crop plants such as chrysanthemum are still largely uninvestigated. Here, we cloned CmBBX22 and further determined the function of CmBBX22 in response to drought treatment. Subcellular localization and transactivation assay analyses revealed that CmBBX22 was localized in the nucleus and possessed transactivation activity. Overexpression of CmBBX22 in chrysanthemum was found to reduce plant drought tolerance, whereas expression of the chimeric repressor CmBBX22-SRDX was found to promote a higher drought tolerance than that shown by wild-type plants, indicating that CmBBX22 negatively regulates drought tolerance in chrysanthemum. Transcriptome analysis and physiological measurements indicated the potential involvement of the CmBBX22-mediated ABA response, stomatal conductance, and antioxidant responses in the negative regulation of drought tolerance in chrysanthemum. Based on the findings of this study, we were thus able to establish the mechanisms whereby the transcriptional activator CmBBX22 negatively regulates drought tolerance in chrysanthemum via the regulation of the abscisic acid response, stomatal conductance, and antioxidant responses.

The Brassicaceae family includes many economically important crop species, as well as cosmopolitan agricultural weed species. In addition, Arabidopsis thaliana, a member of this family, is used as a molecular model plant species. The genus Brassica is mesopolyploid, and the genus comprises comparatively recently originated tetrapolyploid species. With these characteristics, Brassicas have achieved the commonly accepted status of model organisms for genomic studies. This paper reviews the rapid research progress in the Brassicaceae family from diverse omics studies, including genomics, transcriptomics, epigenomics, and three-dimensional (3D) genomics, with a focus on cultivated crops. The morphological plasticity of Brassicaceae crops is largely due to their highly variable genomes. The origin of several important Brassicaceae crops has been established. Genes or loci domesticated or contributing to important traits are summarized. Epigenetic alterations and 3D structures have been found to play roles in subgenome dominance, either in tetraploid Brassica species or their diploid ancestors. Based on this progress, we propose future directions and prospects for the genomic investigation of Brassicaceae crops.

Black spot disease (BSD) (Diplocarpon rosae) is the most common and damaging fungal disease in garden roses (Rosa sp.). Although qualitative resistance to BSD has been extensively investigated, the research on quantitative resistance lags behind. The goal of this research was to study the genetic basis of BSD resistance in two multi-parental populations (TX2WOB and TX2WSE) through a pedigree-based analysis approach (PBA). Both populations were genotyped and evaluated for BSD incidence over five years in three locations in Texas. A total of 28 QTLs, distributed over all linkage groups (LGs), were detected across both populations. Consistent minor effect QTLs included two on LG1 and LG3 (TX2WOB and TX2WSE), two on LG4 and LG5 (TX2WSE), and one QTL on LG7 (TX2WOB). In addition, one major QTL detected in both populations was consistently mapped on LG3. This QTL was localized to an interval ranging from 18.9 to 27.8 Mbp on the Rosa chinensis genome and explained 20 and 33% of the phenotypic variation. Furthermore, haplotype analysis showed that this QTL had three distinct functional alleles. The parent PP-J14–3 was the common source of the LG3 BSD resistance in both populations. Taken together, this research presents the characterization of new SNP-tagged genetic determinants of BSD resistance, the discovery of marker-trait associations to enable parental choice based on their BSD resistance QTL haplotypes, and substrates for the development of trait-predictive DNA tests for routine use in marker-assisted breeding for BSD resistance.

The mapping and introduction of sustainable resistance to viruses in crops is a major challenge in modern breeding, especially regarding vegetables. We hence assembled a panel of cucumber elite lines and landraces from different horticultural groups for testing with six virus species. We mapped 18 quantitative trait loci (QTL) with a multiloci genome wide association studies (GWAS), some of which have already been described in the literature. We detected two resistance hotspots, one on chromosome 5 for resistance to the cucumber mosaic virus (CMV), cucumber vein yellowing virus (CVYV), cucumber green mottle mosaic virus (CGMMV) and watermelon mosaic virus (WMV), colocalizing with the RDR1 gene, and another on chromosome 6 for resistance to the zucchini yellowing mosaic virus (ZYMV) and papaya ringspot virus (PRSV) close to the putative VPS4 gene location. We observed clear structuring of resistance among horticultural groups due to plant virus coevolution and modern breeding which have impacted linkage disequilibrium (LD) in resistance QTLs. The inclusion of genetic structure in GWAS models enhanced the GWAS accuracy in this study. The dissection of resistance hotspots by local LD and haplotype construction helped gain insight into the panel’s resistance introduction history. ZYMV and CMV resistance were both introduced from different donors in the panel, resulting in multiple resistant haplotypes at same locus for ZYMV, and in multiple resistant QTLs for CMV.

Pollen development is crucial for the fruit setting process of tomatoes, but the underlying regulatory mechanism remains to be elucidated. Here, we report the isolation of one HD-Zip III family transcription factor, SlHB8, whose expression levels decreased as pollen development progressed. SlHB8 knockout using CRISPR/Cas9 increased pollen activity, subsequently inducing fruit setting, whereas overexpression displayed opposite phenotypes. Overexpression lines under control of the 35 s and p2A11 promoters revealed that SlHB8 reduced pollen activity by affecting early pollen development. Transmission electron microscopy and TUNEL analyses showed that SlHB8 accelerated tapetum degradation, leading to collapsed and infertile pollen without an intine and an abnormal exine. RNA-seq analysis of tomato anthers at the tetrad stage showed that SlHB8 positively regulates SPL/NZZ expression and the tapetum programmed cell death conserved genetic pathway DYT1–TDF1–AMS–MYB80 as well as other genes related to tapetum and pollen wall development. In addition, DNA affinity purification sequencing, electrophoretic mobility shift assay, yeast one-hybrid assay and dual-luciferase assay revealed SlHB8 directly activated the expression of genes related to pollen wall development. The study findings demonstrate that SlHB8 is involved in tapetum development and degradation and plays an important role in anther development.

Most of WRKY transcription factors play important roles in plant development, protection against disease, and response to abiotic stress; however, their roles in lily are largely unknown. Transcriptome analysis in lily (Lilium longiflorum) led to the identification and isolation of a WRKY-IIe gene, LlWRKY22, which was found to be activated at high temperature and play a positive role in thermotolerance regulation. LlWRKY22 expression was continuously activated by heat stress. We further found that LlWRKY22 protein localized to the nucleus and exhibited transactivation activity in both yeast and plant cells, and that its C terminus contributed to its transactivation activity. Meanwhile, overexpression of LlWRKY22 in lily improved thermotolerance and activated the expression of heat-related LlDREB2B gene; however, silencing of LlWRKY22 exerted the opposite effects. Further analysis revealed that LlWRKY22 directly activated the expression of LlDREB2B by binding to two tandem W-box elements on its promoter. Simultaneously, we also found that LlWRKY22 can directly bind its own promoter, thereby activating its own expression and forming a positive regulatory loop. Combined, our findings demonstrated that LlWRKY22 may be a new regulator of heat stress response and positively participates in the establishment of thermotolerance by activating itself and LlDREB2B.

Imaging-based high throughput phenotyping (HTP) systems have demonstrated promising solutions to enhance genetic understanding of grapevine powdery mildew (PM) resistance and have accelerated PM-resistant cultivar breeding. The accuracy and throughput of extracting phenotypic traits from images are still the bottleneck of modern HTP systems, especially at the microscopic level. The goal of this study was to develop a saliency-based processing pipeline for the quantification of PM infection in microscopic images and comprehensively evaluate its performance for genetic analyses. An input image was segregated into subimages that were classified as infected or healthy by a pretrained CNN classifier. Saliency maps from the classification were generated post-hoc and used for the quantification of PM infection in the input image at the pixel level without the use of mask annotations. A total of seven phenotypic traits were extracted from images collected for a biparental population. Experimental results showed that optimal combinations of convolutional neural network and saliency methods achieved strong measurement correlations (r = 0.74 to 0.75) with human assessments at the image patch level, and the traits calculated by the saliency-based processing pipeline were highly correlated (r = 0.87 to 0.88) with reference PM infection ratings at the leaf image level. The high quantification accuracy of the saliency-based pipeline led to the increased explanation of phenotypic variance and reliable identification of quantitative trait loci. Therefore, the saliency-based processing pipeline can be used as an effective and efficient analysis tool for PM disease research and breeding programs in the future, especially agricultural and life science studies requiring microscopic image analysis.

The Actinidia (kiwifruit) is an emerging fruit plant that is severely affected by salt stress in northern China. Plants have evolved several signaling network mechanisms to cope with the detrimental effects of salt stress. To date, no reported work is available on metabolic and molecular mechanisms involved in kiwifruit salt tolerance. Therefore, the present study aims to decipher intricate adaptive responses of two contrasting salt tolerance kiwifruit species Actinidia valvata [ZMH (an important genotype), hereafter referred to as R] and Actinidia deliciosa [‘Hayward’ (an important green-fleshed cultivar), hereafter referred to as H] under 0.4% (w/w) salt stress for time courses of 0, 12, 24, and 72 hours (hereafter refered to as h) by combined transcriptome and metabolome analysis. Data revealed that kiwifruit displayed specific enrichment of differentially expressed genes (DEGs) under salt stress. Interestingly, roots of R plants showed a differential expression pattern for up-regulated genes. The KEGG pathway analysis revealed the enrichment of DEGs related to plant hormone signal transduction, glycine metabolism, serine and threonine metabolism, glutathione metabolism, and pyruvate metabolism in the roots of R under salt stress. The WGCNA resulted in the identification of five candidate genes related to glycine betaine (GB), pyruvate, total soluble sugars (TSS), and glutathione biosynthesis in kiwifruit. An integrated study of transcriptome and metabolome identified several genes encoding metabolites involved in pyruvate metabolism. Furthermore, several genes encoding transcription factors were mainly induced in R under salt stress. Functional validation results for overexpression of a candidate gene betaine aldehyde dehydrogenase (AvBADH, R_transcript_80484 ) from R showed significantly improved salt tolerance in Arabidopsis thaliana (hereafter referred to as At) and Actinidia chinensis [‘Hongyang’ (an important red-fleshed cultivar), hereafter referred to as Ac] transgenic plants than in WT plants. All in all, salt stress tolerance in kiwifruit roots is an intricate regulatory mechanism that consists of several genes encoding specific metabolites.

Fleshy fruit ripening involves changes in numerous cellular processes and metabolic pathways, resulting from the coordinated actions of diverse classes of structural and regulatory proteins. These include enzymes, transporters and complex signal transduction systems. Many aspects of the signaling machinery that orchestrates the ripening of climacteric fruits, such as tomato (Solanum lycopersicum), have been elucidated, but less is known about analogous processes in non-climacteric fruits. The latter include strawberry (Fragaria x ananassa) and grape (Vitis vinifera), both of which are used as non-climacteric fruit experimental model systems, although they originate from different organs: the grape berry is a true fruit derived from the ovary, while strawberry is an accessory fruit that is derived from the floral receptacle. In this article, we summarize insights into the signal transduction events involved in strawberry and grape berry ripening. We highlight the mechanisms underlying non-climacteric fruit ripening, the multiple primary signals and their integrated action, individual signaling components, pathways and their crosstalk, as well as the associated transcription factors and their signaling output.

Theacrine (1,3,7,9-tetramethyluric acid) is a natural product with remarkable pharmacological activities such as antidepressant, sedative and hypnotic activities, while caffeine (1,3,7-trimethylxanthine) has certain side effects to special populations. Hence, breeding tea plants with high theacrine and low caffeine will increase tea health benefits and promote consumption. In this study, we construct an F1 population by crossing ‘Zhongcha 302’ (theacrine-free) and a tea germplasm ‘Ruyuan Kucha’ (RY, theacrine-rich) to identify the causal gene for accumulating theacrine. The results showed that the content of theacrine was highly negatively correlated with caffeine (R² > 0.9). Bulked segregant RNA sequencing analysis, molecular markers and gene expression analysis indicated that the theacrine synthase ( TcS) gene was the candidate gene. The TcS was located in the nucleus and cytoplasm, and the theacrine can be detected in stably genetic transformed tobacco by feeding the substrate 1,3,7-trimethyluric acid. Moreover, an in vitro enzyme activity experiment revealed that the 241st amino acid residue was the key residue. Besides, we amplified the promoter region in several tea accessions with varied theacrine levels, and found a 234-bp deletion and a 271-bp insertion in RY. Both GUS histochemical analysis and dual-luciferase assay showed that TcS promoter activity in RY was relatively high. Lastly, we developed a molecular marker that is co-segregate with high-theacrine individuals in RY’s offspring. These results demonstrate that the novel TcS allele in RY results in the high-theacrine and low-caffeine traits and the developed functional marker will facilitate the breeding of characteristic tea plants.

Under drought stress, reactive oxygen species (ROS) overaccumulate as a secondary stress that impairs plant performance and thus severely reduces crop yields. The mitigation of ROS levels under drought stress is therefore crucial for drought tolerance. MicroRNAs (miRNAs) are critical regulators of plant development and stress responses. However, the complex molecular regulatory mechanism by which they function during drought stress, especially in drought-triggered ROS scavenging, is not fully understood. Here, we report a newly identified drought-responsive miRNA, miR164g, in the wild apple species Malus sieversii and elucidate its role in apple drought tolerance. Our results showed that expression of miR164g is significantly inhibited under drought stress and it can specifically cleave transcripts of the transcription factor MsNAC022 in M. sieversii. The heterologous accumulation of miR164g in Arabidopsis thaliana results in enhanced sensitivity to drought stress, while overexpression of MsNAC022 in Arabidopsis and the cultivated apple line ‘GL-3’ ( Malus domestica Borkh.) lead to enhanced tolerance to drought stress by raising the ROS scavenging enzymes activity and related genes expression levels, particularly PEROXIDASE (MsPOD). Furthermore, we showed that expression of MsPOD is activated by MsNAC022 in transient assays. Interestingly, Part1 (P1) region is the key region for the positive regulation of MsPOD promoter by MsNAC022, and the different POD expression patterns in M. sieversii and M. domestica is attributed to the specific fragments inserted in P1 region of M. sieversii. Our findings reveal the function of the miR164g- MsNAC022 module in mediating the drought response of M. sieversii and lay a foundation for breeding drought-tolerant apple cultivars.

Carotene hydroxylase plays an important role in catalyzing the hydroxylation of carotene to xanthopylls, including two types: non-heme carotene hydroxylase (BCH type) and heme-containing cytochrome P450 hydroxylase (P450 type). Two BCH-encoding genes were annotated in the carrot genome. However, the role of BCHs and whether there are functional interactions between the duplicated BCHs in carrot remains unclear. In this study, two BCH encoding genes, DcBCH1 and DcBCH2, were cloned from carrot. The relative expression level of DcBCH1 was much higher than that of DcBCH2 in carrot taproots with different carotene accumulation levels. Overexpression of DcBCH1 in ‘KRD’ (high carotene accumulated) carrot changed the taproot color from orange to yellow, accompanied by substantial reductions in α-carotene and β-carotene. There was no obvious change in taproot color between transgenic ‘KRD’ carrot overexpressing DcBCH2 and control carrot. Simultaneously, the content of α-carotene in the taproot of DcBCH2-overexpressing carrot decreased, but the content of β-carotene did not change significantly in comparison with control carrot. Using the CRISPR/Cas9 system to knock out DcBCH1 in ‘KRD’ carrot lightened the taproot color from orange to pink-orange; the content of α-carotene in the taproot increased slightly, while the β-carotene content was still significantly decreased, compared with control carrot. In DcBCH1-knockout carrot, the transcript level of DcBCH2 was significantly increased. These results indicated that in carrot taproot, DcBCH1 played the main function of BCH enzyme, which could hydroxylate α-carotene and β-carotene; DcBCH1 and DcBCH2 had functional redundancy, and these two DcBCHs could partially compensate for each other.

The types and proportions of soluble sugar and organic acid in fruit significantly affect flavor quality. However, there are few reports on the crosstalk regulation between metabolism of organic acid and sugar in fruit. Here, we found that the overexpression of cytoplasmic malate dehydrogenase genes (MdcyMDHs) not only increased the malate content but also increased the sucrose concentration in transgenic apple calli and mature fruit. Enzyme activity assays indicated that the overexpression of MdcyMDH1 and MdcyMDH5 enhanced sucrose phosphate synthase (SPS) activity in transgenic materials. RNA-seq and expression analysis showed that the expression levels of SPS genes were up-regulated in MdcyMDH1-overexpressed apple fruit and MdcyMDH5-overexpressed apple calli. Further study showed that the inhibition of MdSPSB2 or MdSPSC2 expression in MdcyMDH1 transgenic fruit could reduce or eliminate, respectively, the positive effect of MdcyMDH1 on sucrose accumulation. Moreover, some starch cleavage-related genes (MdBAM6.1/6.2, MdBMY8.1/8.2, MdISA1) and the key gluconeogenesis-related phosphoenolpyruvate carboxykinase MdPEPCK1 gene were significantly up-regulated in the transcriptome differentially expressed genes of mature fruit overexpressing MdcyMDH1. These results indicate that alteration of malate metabolism mediated by MdcyMDH might regulate the expression of MdSPSs and SPS activity via affecting starch metabolism or gluconeogenesis, and thus accelerate sucrose synthesis and accumulation in fruit.

Clubroot is a soil-borne disease in cabbage (Brassica oleracea L. var. capitata L.) caused by Plasmodiophora brassicae, which poses a great threat to cabbage production. However, clubroot resistance (CR) genes in Brassica rapa could be introduced into the cabbage via breeding to make it clubroot resistant. In this study, CR genes from B. rapa were introduced into the cabbage genome and the mechanism of gene introgression was explored. Two methods were used to create CR materials: (i) The fertility of CR Ogura CMS cabbage germplasms containing CRa was restored by using an Ogura CMS restorer. After cytoplasmic replacement and microspore culture, CRa-positive microspore individuals were obtained. (ii) Distant hybridization was performed between cabbage and B. rapa, which contained three CR genes (CRa, CRb, and Pb8.1). Finally, BC2 individuals containing all three CR genes were obtained. Inoculation results showed that both CRa-positive microspore individuals and BC2 individuals containing three CR genes were resistant to race 4 of P. brassicae. Sequencing results from CRa-positive microspore individuals with specific molecular markers and genome-wide association study (GWAS) showed penetration at the homologous position of the cabbage genome by a 3.42 Mb CRa containing a fragment from B. rapa; indicating homoeologous exchange (HE) as the theoretical basis for the introgression of CR resistance. The successful introduction of CR into the cabbage genome in the present study can provide useful clues for creating introgression lines within other species of interest.

Excessive nitrogen (N) application may induce powdery mildew (PM) in perennial grasses, but the resistance mechanisms to PM remain unclear. This study evaluated the physiological and molecular mechanisms of PM resistance affected by N supplies in Kentucky bluegrass (Poa pratensis L.). Cultivar ‘Bluemoon’ (N tolerant) and ‘Balin’ (N sensitive) were treated with low N (0.5 mM), normal N (15 mM), and high N (30 mM) for 21 d in a greenhouse. With increasing N levels, the disease growth was more severe in ‘Balin’ than in ‘Bluemoon’. RNA-seq and weighted gene coexpression network analysis revealed that the PpGS1.1 gene encoding glutamine synthetase was a potential hub gene for PM resistance after comparisons across cultivars and N treatments. The N metabolism pathway was connected with the plant–pathogen interaction pathway via PpGS1.1. The expression of PpGS1.1 in rice protoplasts indicated that the protein was located in the nucleus and cytoplasm. Overexpression of PpGS1.1 in wild-type Kentucky bluegrass increased carbon and N contents, and the transgenic plants became more susceptible to PM with a lower wax density. The most differentially expressed genes (DEGs) for N metabolism were upregulated and DEGs for fatty acid metabolism pathway were downregulated in the overexpression lines. The results elucidated mechanisms of PM resistance in relation to N metabolism in Kentucky bluegrass.

Parasitic root-knot nematodes (RKNs) cause a severe reduction in crop yield and seriously threaten agricultural production. The phytohormones jasmonates (JAs) are important signals regulating resistance to multiple biotic and abiotic stresses. However, the molecular mechanism for JAs-regulated defense against RKNs in tomato remains largely unclear. In this study, we found that the transcription factor SlWRKY45 interacted with most JA-ZIM domain family proteins (JAZs), key repressors of the JA signaling. After infection by the RKN Meloidogyne incognita, the slwrky45 mutants exhibited lower gall numbers and egg numbers per gram of roots than wild type, whereas overexpression of SlWRKY45 attenuated resistance to Meloidogyne incognita. Under M. incognita infection, the contents of jasmonic acid (JA) and JA-isoleucine (JA-Ile) in roots were repressed by SlWRKY45-overexpression. Furthermore, SlWRKY45 bound to and inhibited the promoter of the JA biosynthesis gene ALLENE OXIDE CYCLASE (AOC), and repressed its expression. Overall, our findings revealed that the SlJAZ-interaction protein SlWRKY45 attenuated RKN-regulated JA biosynthesis and repressed defense against the RKN M. incognita in tomato.

Drought stress caused by water deficit reduces plant productivity in many regions of the world. In plants, basic helix–loop–helix (bHLH) transcription factors regulate a wide range of cellular activities related to growth, development and stress response; however, the role of tomato SlbHLHs in drought stress responses remains elusive. Here, we used reverse genetics approaches to reveal the function of SlbHLH96, which is induced by drought and abscisic acid (ABA) treatment. We found that SlbHLH96 functions as a positive regulator of drought tolerance in tomato. Overexpression of SlbHLH96 in tomato improves drought tolerance by stimulating the expression of genes encoding antioxidants, ABA signaling molecules and stress-related proteins. In contrast, silencing of SlbHLH96 in tomato reduces drought tolerance. SlbHLH96 physically interacts with an ethylene-responsive factor, SlERF4, and silencing of SlERF4 in tomato also decreases drought tolerance. Furthermore, SlbHLH96 can repress the expression of the ABA catabolic gene, SlCYP707A2, through direct binding to its promoter. Our results uncover a novel mechanism of SlbHLH96-mediated drought tolerance in tomato plants, which can be exploited for breeding drought-resilient crops.