Postharvest waste and loss of horticultural crops exacerbates the agricultural problems facing humankind and will continue to do so in the next decade. Fruits and vegetables provide us with a vast spectrum of healthful nutrients, and along with ornamentals, enrich our lives with a wide array of pleasant sensory experiences. These commodities are, however, highly perishable. Approximately 33% of the produce that is harvested is never consumed since these products naturally have a short shelf-life, which leads to postharvest loss and waste. This loss, however, could be reduced by breeding new crops that retain desirable traits and accrue less damage over the course of long supply chains. New gene-editing tools promise the rapid and inexpensive production of new varieties of crops with enhanced traits more easily than was previously possible. Our aim in this review is to critically evaluate gene editing as a tool to modify the biological pathways that determine fruit, vegetable, and ornamental quality, especially after storage. We provide brief and accessible overviews of both the CRISPR–Cas9 method and the produce supply chain. Next, we survey the literature of the last 30 years, to catalog genes that control or regulate quality or senescence traits that are “ripe” for gene editing. Finally, we discuss barriers to implementing gene editing for postharvest, from the limitations of experimental methods to international policy. We conclude that in spite of the hurdles that remain, gene editing of produce and ornamentals will likely have a measurable impact on reducing postharvest loss and waste in the next 5–10 years.

In plants, alternative splicing (AS) is markedly induced in response to environmental stresses, but it is unclear why plants generate multiple transcripts under stress conditions. In this study, RNA-seq was performed to identify AS events in cucumber seedlings grown under different light intensities. We identified a novel transcript of the gibberellin (GA)-deactivating enzyme Gibberellin 2-beta-dioxygenase 8 (CsGA2ox8). Compared with canonical CsGA2ox8.1, the CsGA2ox8.2 isoform presented intron retention between the second and third exons. Functional analysis proved that the transcript of CsGA2ox8.1 but not CsGA2ox8.2 played a role in the deactivation of bioactive GAs. Moreover, expression analysis demonstrated that both transcripts were upregulated by increased light intensity, but the expression level of CsGA2ox8.1 increased slowly when the light intensity was >400 µmol·m⁻²·s⁻¹ PPFD (photosynthetic photon flux density), while the CsGA2ox8.2 transcript levels increased rapidly when the light intensity was >200 µmol·m⁻²·s⁻¹ PPFD. Our findings provide evidence that plants might finely tune their GA levels by buffering against the normal transcripts of CsGA2ox8 through AS.

GRAS proteins are plant-specific transcription factors that play crucial roles in plant development and stress responses. However, their involvement in the ripening of economically important fruits and their transcriptional regulatory mechanisms remain largely unclear. Here, we demonstrated that SlGRAS4, encoding a transcription factor of the GRAS family, was induced by the tomato ripening process and regulated by ethylene. Overexpression of SlGRAS4 accelerated fruit ripening, increased the total carotenoid content and increased PSY1 expression in SlGRAS4-OE fruit compared to wild-type fruit. The expression levels of key ethylene biosynthesis genes (SlACS2, SlACS4, SlACO1, and SlACO3) and crucial ripening regulators (RIN and NOR) were increased in SlGRAS4-OE fruit. The negative regulator of tomato fruit ripening, SlMADS1, was repressed in OE fruit. Exogenous ethylene and 1-MCP treatment revealed that more endogenous ethylene was derived in SlGRAS4-OE fruit. More obvious phenotypes were observed in OE seedlings after ACC treatment. Yeast one-hybrid and dual-luciferase assays confirmed that SlGRAS4 can directly bind SlACO1 and SlACO3 promoters to activate their transcription, and SlGRAS4 can also directly repress SlMADS1 expression. Our study identified that SlGRAS4 acts as a new regulator of fruit ripening by regulating ethylene biosynthesis genes in a direct manner. This provides new knowledge of GRAS transcription factors involved in regulating fruit ripening.

Water deficit is a major worldwide constraint to common bean (Phaseolus vulgaris L.) production, being photosynthesis one of the most affected physiological processes. To gain insights into the genetic basis of the photosynthetic response of common bean under water-limited conditions, a collection of 158 Portuguese accessions was grown under both well-watered and water-deficit regimes. Leaf gas-exchange parameters were measured and photosynthetic pigments quantified. The same collection was genotyped using SNP arrays, and SNP-trait associations tested considering a linear mixed model accounting for the genetic relatedness among accessions. A total of 133 SNP-trait associations were identified for net CO₂ assimilation rate, transpiration rate, stomatal conductance, and chlorophylls a and b, carotenes, and xanthophyll contents. Ninety of these associations were detected under water-deficit and 43 under well-watered conditions, with only two associations common to both treatments. Identified candidate genes revealed that stomatal regulation, protein translocation across membranes, redox mechanisms, hormone, and osmotic stress signaling were the most relevant processes involved in common bean response to water-limited conditions. These candidates are now preferential targets for common bean water-deficit-tolerance breeding. Additionally, new sources of water-deficit tolerance of Andean, Mesoamerican, and admixed origin were detected as accessions valuable for breeding, and not yet explored.

Opium poppy (Papaver somniferum) is a source of morphine, codeine, and semisynthetic derivatives, including oxycodone and naltrexone. Here, we report the de novo assembly and genomic analysis of P. somniferum traditional landrace ‘Chinese Herbal Medicine’. Variations between the 2.62 Gb CHM genome and that of the previously sequenced high noscapine 1 (HN1) variety were also explored. Among 79,668 protein-coding genes, we functionally annotated 88.9%, compared to 68.8% reported in the HN1 genome. Gene family and 4DTv comparative analyses with three other Papaveraceae species revealed that opium poppy underwent two whole-genome duplication (WGD) events. The first of these, in ancestral Ranunculales, expanded gene families related to characteristic secondary metabolite production and disease resistance. The more recent species-specific WGD mediated by transposable elements resulted in massive genome expansion. Genes carrying structural variations and large-effect variants associated with agronomically different phenotypes between CHM and HN1 that were identified through our transcriptomic comparison of multiple organs and developmental stages can enable the development of new varieties. These genomic and transcriptomic analyses will provide a valuable resource that informs future basic and agricultural studies of the opium poppy.

Chrysanthemum white rust disease, which is caused by the fungus Puccinia horiana Henn., severely reduces the ornamental quality and yield chrysanthemum. WRKY transcription factors function in the disease-resistance response in a variety of plants; however, it is unclear whether members of this family improve resistance to white rust disease in chrysanthemum. In this study, using PCR, we isolated a WRKY15 homologous gene, CmWRKY15-1, from the resistant chrysanthemum cultivar C029. Real-time quantitative PCR (RT-qPCR) revealed that CmWRKY15-1 exhibited differential expression patterns between the immune cultivar C029 and the susceptible cultivar Jinba upon P. horiana infection. In addition, salicylic acid (SA) treatment strongly induced CmWRKY15-1 expression. Overexpression of CmWRKY15-1 in the chrysanthemum-susceptible cultivar Jinba increased tolerance to P. horiana infection. Conversely, silencing CmWRKY15-1 via RNA interference (RNAi) in C029 increased sensitivity to P. horiana infection. We also determined that P. horiana infection increased both the endogenous SA content and the expression of salicylic acid biosynthesis genes in CmWRKY15-1-overexpressing plants, whereas CmWRKY15-1 RNAi plants exhibited the opposite effects under the same conditions. Finally, the transcript levels of pathogenesis-related (PR) genes involved in the SA pathway were positively associated with CmWRKY15-1 expression levels. Our results demonstrated that CmWRKY15-1 plays an important role in the resistance of chrysanthemum to P. horiana by influencing SA signaling.

The limited bioavailability of plant-derived natural products with anticancer activity poses major challenges to the pharmaceutical industry. An example of this is camptothecin, a monoterpene indole alkaloid with potent anticancer activity that is extracted at very low concentrations from woody plants. Recently, camptothecin biosynthesis has been shown to become biotechnologically amenable in hairy-root systems of the natural producer Ophiorrhiza pumila. Here, time-course expression and metabolite analyses were performed to identify novel transcriptional regulators of camptothecin biosynthesis in O. pumila. It is shown here that camptothecin production increased over cultivation time and that the expression pattern of the WRKY transcription factor encoding gene OpWRKY2 is closely correlated with camptothecin accumulation. Overexpression of OpWRKY2 led to a more than three-fold increase in camptothecin levels. Accordingly, silencing of OpWRKY2 correlated with decreased camptothecin levels in the plant. Further detailed molecular characterization by electrophoretic mobility shift, yeast one-hybrid and dual-luciferase assays showed that OpWRKY2 directly binds and activates the central camptothecin pathway gene OpTDC. Taken together, the results of this study demonstrate that OpWRKY2 acts as a direct positive regulator of camptothecin biosynthesis. As such, a feasible strategy for the over-accumulation of camptothecin in a biotechnologically amenable system is presented.

‘HoneySweet’ plum (Prunus domestica) is resistant to Plum pox potyvirus, through an RNAi-triggered mechanism. Determining the precise nature of the transgene insertion event has been complicated due to the hexaploid genome of plum. DNA blots previously indicated an unintended hairpin arrangement of the Plum pox potyvirus coat protein gene as well as a multicopy insertion event. To confirm the transgene arrangement of the insertion event, ‘HoneySweet’ DNA was subjected to whole genome sequencing using Illumina short-read technology. Results indicated two different insertion events, one containing seven partial copies flanked by putative plum DNA sequence and a second with the predicted inverted repeat of the coat protein gene driven by a double 35S promoter on each side, flanked by plum DNA. To determine the locations of the two transgene insertions, a phased plum genome assembly was developed from the commercial plum ‘Improved French’. A subset of the scaffolds (2447) that were >10 kb in length and representing, >95% of the genome were annotated and used for alignment against the ‘HoneySweet’ transgene reads. Four of eight matching scaffolds spanned both insertion sites ranging from 157,704 to 654,883 bp apart, however we were unable to identify which scaffold(s) represented the actual location of the insertion sites due to potential sequence differences between the two plum cultivars. Regardless, there was no evidence of any gene(s) being interrupted as a result of the insertions. Furthermore, RNA-seq data verified that the insertions created no new transcriptional units and no dramatic expression changes of neighboring genes.

The apple (Malus domestica) is one of the world’s most commercially important perennial crops and its improvement has been the focus of human effort for thousands of years. Here, we genetically characterise over 1000 apple accessions from the United States Department of Agriculture (USDA) germplasm collection using over 30,000 single-nucleotide polymorphisms (SNPs). We confirm the close genetic relationship between modern apple cultivars and their primary progenitor species, Malus sieversii from Central Asia, and find that cider apples derive more of their ancestry from the European crabapple, Malus sylvestris, than do dessert apples. We determine that most of the USDA collection is a large complex pedigree: over half of the collection is interconnected by a series of first-degree relationships. In addition, 15% of the accessions have a first-degree relationship with one of the top 8 cultivars produced in the USA. With the exception of ‘Honeycrisp’, the top 8 cultivars are interconnected to each other via pedigree relationships. The cultivars ‘Golden Delicious’ and ‘Red Delicious’ were found to have over 60 first-degree relatives, consistent with their repeated use by apple breeders. We detected a signature of intense selection for red skin and provide evidence that breeders also selected for increased firmness. Our results suggest that Americans are eating apples largely from a single family tree and that the apple’s future improvement will benefit from increased exploitation of its tremendous natural genetic diversity.

Water-soluble phenolic acids are major bioactive compounds in the medicinal plant species Salvia miltiorrhiza. Phenolic acid biosynthesis is induced by methyl jasmonate (MeJA) in this important Chinese herb. Here, we investigated the mechanism underlying this induction by analyzing a transcriptome library of S. miltiorrhiza in response to MeJA. Global transcriptome analysis identified the MeJA-responsive R2R3-MYB transcription factor-encoding gene SmMYB1. Overexpressing SmMYB1 significantly promoted phenolic acid accumulation and upregulated the expression of genes encoding key enzymes in the phenolic acid biosynthesis pathway, including cytochrome P450-dependent monooxygenase (CYP98A14). Dual-luciferase (dual-LUC) assays and/or an electrophoretic mobility shift assays (EMSAs) indicated that SmMYB1 activated the expression of CYP98A14, as well as the expression of genes encoding anthocyanin biosynthesis pathway enzymes, including chalcone isomerase (CHI) and anthocyanidin synthase (ANS). In addition, SmMYB1 was shown to interact with SmMYC2 to additively promote CYP98A14 expression compared to the action of SmMYB1 alone. Taken together, these results demonstrate that SmMYB1 is an activator that improves the accumulation of phenolic acids and anthocyanins in S. miltiorrhiza. These findings lay the foundation for in-depth studies of the molecular mechanism underlying MeJA-mediated phenolic acid biosynthesis and for the metabolic engineering of bioactive ingredients in S. miltiorrhiza.

Loss of genetic variability is an increasing challenge in tree breeding programs due to the repeated use of a reduced number of founder genotypes. However, in almond, little is known about the genetic variability in current breeding stocks, although several cases of inbreeding depression have been reported. To gain insights into the genetic structure in modern breeding programs worldwide, marker-verified pedigree data of 220 almond cultivars and breeding selections were analyzed. Inbreeding coefficients, pairwise relatedness, and genetic contribution were calculated for these genotypes. The results reveal two mainstream breeding lines based on three cultivars: “Tuono”, “Cristomorto”, and “Nonpareil”. Descendants from “Tuono” or “Cristomorto” number 76 (sharing 34 descendants), while “Nonpareil” has 71 descendants. The mean inbreeding coefficient of the analyzed genotypes was 0.041, with 14 genotypes presenting a high inbreeding coefficient, over 0.250. Breeding programs from France, the USA, and Spain showed inbreeding coefficients of 0.075, 0.070, and 0.037, respectively. According to their genetic contribution, modern cultivars from Israel, France, the USA, Spain, and Australia trace back to a maximum of six main founding genotypes. Among the group of 65 genotypes carrying the S_f allele for self-compatibility, the mean relatedness coefficient was 0.125, with “Tuono” as the main founding genotype (24.7% of total genetic contribution). The results broaden our understanding about the tendencies followed in almond breeding over the last 50 years and will have a large impact into breeding decision-making process worldwide. Increasing current genetic variability is required in almond breeding programs to assure genetic gain and continuing breeding progress.

Sweet cherry (Prunus avium L.) is a stone fruit widely consumed and appreciated for its organoleptic properties, as well as its nutraceutical potential. We here investigated the characteristics of six non-commercial Tuscan varieties of sweet cherry maintained at the Regional Germplasm Bank of the CNR-IBE in Follonica (Italy) and sampled ca. 60 days post-anthesis over three consecutive years (2016-2017-2018). We adopted an approach merging genotyping and targeted gene expression profiling with metabolomics. To complement the data, a study of the soluble proteomes was also performed on two varieties showing the highest content of flavonoids. Metabolomics identified the presence of flavanols and proanthocyanidins in highest abundance in the varieties Morellona and Crognola, while gene expression revealed that some differences were present in genes involved in the phenylpropanoid pathway during the 3 years and among the varieties. Finally, proteomics on Morellona and Crognola showed variations in proteins involved in stress response, primary metabolism and cell wall expansion. To the best of our knowledge, this is the first multi-pronged study focused on Tuscan sweet cherry varieties providing insights into the differential abundance of genes, proteins and metabolites.

Soft rot disease caused by Pectobacterium spp. is responsible for severe agricultural losses in potato, vegetables, and ornamentals. The genus Zantedeschia includes two botanical groups of tuberous ornamental flowers that are highly susceptible to the disease. Previous studies revealed that Z. aethiopica, a member of the section Zantedeschia, is significantly more resistant to Pectobacterium spp. than members of the same genus that belong to the section Aestivae. During early infection, we found different patterns of bacterial colonization on leaves of hosts belonging to the different sections. Similar patterns of bacterial colonization were observed on polydimethylsiloxane (PDMS) artificial inert replicas of leaf surfaces. The replicas confirmed the physical effect of leaf texture, in addition to a biochemical plant–bacterium interaction. The differential patterns may be associated with the greater roughness of the abaxial leaf surfaces of Aestivae group that have evolutionarily adapted to mountainous environments, as compared to Zantedeschia group species that have adapted to warm, marshy environments. Transverse leaf sections also revealed compact aerenchyma and reduced the total volume of leaf tissue air spaces in Aestivae members. Finally, an analysis of defense marker genes revealed differential expression patterns in response to infection, with significantly higher levels of lipoxygenase 2 (lox2) and phenylalanine ammonia lyase (pal) observed in the more resistant Z. aethiopica, suggesting greater activation of induced systemic resistance (ISR) mechanisms in this group. The use of Zantedeschia as a model plant sheds light on how natural ecological adaptations may underlay resistance to bacterial soft rot in cultivated agricultural environments.

Passion fruit (Passiflora edulis Sims) is an economically valuable fruit that is cultivated in tropical and subtropical regions of the world. Here, we report an ~1341.7 Mb chromosome-scale genome assembly of passion fruit, with 98.91% (~1327.18 Mb) of the assembly assigned to nine pseudochromosomes. The genome includes 23,171 protein-coding genes, and most of the assembled sequences are repetitive sequences, with long-terminal repeats (LTRs) being the most abundant. Phylogenetic analysis revealed that passion fruit diverged after Brassicaceae and before Euphorbiaceae. Ks analysis showed that two whole-genome duplication events occurred in passion fruit at 65 MYA and 12 MYA, which may have contributed to its large genome size. An integrated analysis of genomic, transcriptomic, and metabolomic data showed that ‘alpha-linolenic acid metabolism’, ‘metabolic pathways’, and ‘secondary metabolic pathways’ were the main pathways involved in the synthesis of important volatile organic compounds (VOCs) in passion fruit, and this analysis identified some candidate genes, including GDP-fucose Transporter 1-like, Tetratricopeptide repeat protein 33, protein NETWORKED 4B isoform X1, and Golgin Subfamily A member 6-like protein 22. In addition, we identified 13 important gene families in fatty acid pathways and eight important gene families in terpene pathways. Gene family analysis showed that the ACX, ADH, ALDH, and HPL gene families, especially ACX13/14/15/20, ADH13/26/33, ALDH1/4/21, and HPL4/6, were the key genes for ester synthesis, while the TPS gene family, especially PeTPS2/3/4/24, was the key gene family for terpene synthesis. This work provides insights into genome evolution and flavor trait biology and offers valuable resources for the improved cultivation of passion fruit.

Almond [ Prunus dulcis Miller (D.A. Webb)] is the main tree nut species worldwide. Here, genotyping-by-sequencing (GBS) was applied to 149 almond cultivars from the ex situ collections of the Italian Council for Agricultural Research (CREA) and the Spanish National Research Council (CSIC), leading to the detection of 93,119 single-nucleotide polymorphisms (SNPs). The study of population structure outlined four distinct genetic groups and highlighted diversification between the Mediterranean and Californian gene pools. Data on SNP diversity and runs of homozygosity (ROHs) allowed the definition of kinship, inbreeding, and linkage disequilibrium (LD) decay in almond cultivated germplasm. Four-year phenotypic observations, gathered on 98 cultivars of the CREA collection, were used to perform a genome-wide association study (GWAS) and, for the first time in a crop species, homozygosity mapping (HM), resulting in the identification of genomic associations with nut, shell, and seed weight. Both GWAS and HM suggested that loci controlling nut and seed weight are mostly independent. Overall, this study provides insights on the almond cultivation history and delivers information of major interest for almond genetics and breeding. In a broader perspective, our results encourage the use of ROHs in crop science to estimate inbreeding, choose parental combinations minimizing the risk of inbreeding depression, and identify genomic footprints of selection for specific traits.

Corydalis yanhusuo W.T. Wang is a classic herb that is frequently used in traditional Chinese medicine and is efficacious in promoting blood circulation, enhancing energy, and relieving pain. Benzylisoquinoline alkaloids (BIAs) are the main bioactive ingredients in Corydalis yanhusuo. However, few studies have investigated the BIA biosynthetic pathway in C. yanhusuo, and the biosynthetic pathway of species-specific chemicals such as tetrahydropalmatine remains unclear. We performed full-length transcriptomic and metabolomic analyses to identify candidate genes that might be involved in BIA biosynthesis and identified a total of 101 full-length transcripts and 19 metabolites involved in the BIA biosynthetic pathway. Moreover, the contents of 19 representative BIAs in C. yanhusuo were quantified by classical targeted metabolomic approaches. Their accumulation in the tuber was consistent with the expression patterns of identified BIA biosynthetic genes in tubers and leaves, which reinforces the validity and reliability of the analyses. Full-length genes with similar expression or enrichment patterns were identified, and a complete BIA biosynthesis pathway in C. yanhusuo was constructed according to these findings. Phylogenetic analysis revealed a total of ten enzymes that may possess columbamine-O-methyltransferase activity, which is the final step for tetrahydropalmatine synthesis. Our results span the whole BIA biosynthetic pathway in C. yanhusuo. Our full-length transcriptomic data will enable further molecular cloning of enzymes and activity validation studies.

Mango fruit exposed to sunlight develops red skin and are more resistant to biotic and abiotic stresses. Here we show that harvested red mango fruit that was exposed to sunlight at the orchard is more resistant than green fruit to Colletotrichum gloeosporioides. LCMS analysis showed high amounts of antifungal compounds, as glycosylated flavonols, glycosylated anthocyanins, and mangiferin in red vs. green mango skin, correlated with higher antioxidant and lower ROS. However, also the green side of red mango fruit that has low levels of flavonoids was resistant, indicated induced resistance. Transcriptomes of red and green fruit inoculated on their red and green sides with C. gloeosporioides were analyzed. Overall, in red fruit skin, 2,187 genes were upregulated in response to C. gloeosporioides. On the green side of red mango, upregulation of 22 transcription factors and 33 signaling-related transcripts indicated induced resistance. The RNA-Seq analysis suggests that resistance of the whole red fruit involved upregulation of ethylene, brassinosteroid, and phenylpropanoid pathways. To conclude, red fruit resistance to fungal pathogen was related to both flavonoid toxicity and primed resistance of fruit that was exposed to light at the orchard.

The knowledge on post-transcriptional regulation mechanisms implicated in seed development (SD) is still limited, particularly in one of the most consumed grain legumes, Phaseolus vulgaris L. We explore for the first time the miRNA expression dynamics in P. vulgaris developing seeds. Seventy-two known and 39 new miRNAs were found expressed in P. vulgaris developing seeds. Most of the miRNAs identified were more abundant at 10 and 40 days after anthesis, suggesting that late embryogenesis/early filling and desiccation were SD stages in which miRNA action is more pronounced. Degradome analysis and target prediction identified targets for 77 expressed miRNAs. While several known miRNAs were predicted to target HD-ZIP, ARF, SPL, and NF-Y transcription factors families, most of the predicted targets for new miRNAs encode for functional proteins. MiRNAs-targets expression profiles evidenced that these miRNAs could tune distinct seed developmental stages. MiRNAs more accumulated at early SD stages were implicated in regulating the end of embryogenesis, postponing the seed maturation program, storage compound synthesis and allocation. MiRNAs more accumulated at late SD stages could be implicated in seed quiescence, desiccation tolerance, and longevity with still uncovered roles in germination. The miRNAs herein described represent novel P. vulgaris resources with potential application in future biotechnological approaches to modulate the expression of genes implicated in legume seed traits with impact in horticultural production systems.

Heading Chinese cabbage (Brassica rapa L. syn. B. campestris L. ssp. chinensis Makino var. pekinensis (Rupr.) J. Cao et Sh. Cao) is a cruciferous Brassica vegetable that has a triplicate genome, owing to an ancient genome duplication event. It is unclear whether the duplicated homologs have conserved or diversified functions. Hydrogen sulfide (H₂S) is a plant gasotransmitter that plays important physiological roles in growth, development, and responses to environmental stresses. The modification of cysteines through S-sulfhydration is an important mechanism of H₂S, which regulates protein functions. H₂S promotes flowering in Arabidopsis and heading Chinese cabbage. Here we investigated the molecular mechanisms of H₂S used to promote flowering in the latter. Four, five, and four BraFLC, BraSOC1, and BraFT homologs were identified in heading Chinese cabbage. Different BraFLC proteins were bound to different CArG boxes in the promoter regions of the BraSOC1 and BraFT homologs, producing different binding patterns. Thus, there may be functionally diverse BraFLC homologs in heading Chinese cabbage. Exogenous H₂S at 100 μmol L⁻¹ significantly promoted flowering by compensating for insufficient vernalization. BraFLC1 and BraFLC3 underwent S-sulfhydration by H₂S, after which their abilities to bind most BraSOC1 or BraFT promoter probes weakened or even disappeared. These changes in binding ability were consistent with the expression pattern of the BraFT and BraSOC1 homologs in seedlings treated with H₂S. These results indicated that H₂S signaling regulates flowering time. In summary, H₂S signaling promoted plant flowering by weakening or eliminating the binding abilities of BraFLCs to downstream promoters through S-sulfhydration.

Storage roots of sweet potato are important sink organs for photoassimilates and energy, and carbohydrate metabolism in storage roots affects yield and starch production. Our previous study showed that sweet potato H⁺-pyrophosphatase (IbVP1) plays a vital role in mitigating iron deficiency and positively controls fibrous root growth. However, its roles in regulating starch production in storage roots have not been investigated. In this study, we found that IbVP1 overexpression in sweet potato improved the photosynthesis ability of and sucrose content in source leaves and increased both the starch content in and total yield of sink tissues. Using¹³C-labeled sucrose feeding, we determined that IbVP1 overexpression promotes phloem loading and sucrose long-distance transport and enhances Pi-use efficiency. In sweet potato plants overexpressing IbVP1, the expression levels of starch biosynthesis pathway genes, especially AGPase and GBSSI, were upregulated, leading to changes in the structure, composition, and physicochemical properties of stored starch. Our study shows that the IbVP1 gene plays an important role in regulating starch metabolism in sweet potato. Application of the VP1 gene in genetic engineering of sweet potato cultivars may allow the improvement of starch production and yield under stress or nutrient-limited conditions.

Trichomes that cover the epidermis of aerial plant organs play multiple roles in plant protection. Compared with a unicellular trichome in model plants, the development mechanism of the multicellular trichome is largely unclear. Notably, variations in trichome development are often accompanied by defects in the biosynthesis of cuticle and secondary metabolites; however, major questions about the interactions between developmental differences in trichomes and defects in metabolic pathways remain unanswered. Here, we characterized the glabrous mutant mict/csgl1/cstbh via combined metabolomic and transcriptomic analyses to extend our limited knowledge regarding multicellular trichome development and metabolism in cucumber. Mict was found to be explicitly expressed within trichome cells. Transcriptomic analysis indicated that genes involved in flavonoid and cuticle metabolism are significantly downregulated in mict mutants. Further metabolomic analysis confirmed that flavonoids, lipids, and cuticle compositions are dramatically altered in mict mutants. Additional studies revealed that Mict regulates flavonoid, lipid, and cuticle biosynthesis by likely directly binding to downstream functional genes, such as CsTT4, CsFLS1, CsCER26, and CsMYB36. These findings suggest that specific metabolic pathways (e.g., flavonoids and cuticle components) are co-regulated by Mict and provide insights into transcriptional regulation mechanisms of multicellular trichome development and its specific metabolism in cucumber.

Nitrogen is an important factor that affects plant anthocyanin accumulation. In apple, the nitrate-responsive BTB/TAZ protein MdBT2 negatively regulates anthocyanin biosynthesis. In this study, we found that MdBT2 undergoes posttranslational modifications in response to nitrate deficiency. Yeast two-hybrid, protein pull-down, and bimolecular fluorescence complementation (BiFC) assays showed that MdBT2 interacts with MdGRF11, a 14-3-3 protein; 14-3-3 proteins compose a family of highly conserved phosphopeptide-binding proteins involved in multiple physiological and biological processes. The interaction of MdGRF11 negatively regulated the stability of the MdBT2 protein via a 26S proteasome-dependent pathway, which increased the abundance of MdMYB1 proteins to activate the expression of anthocyanin biosynthesis-related genes. Taken together, the results demonstrate the critical role of 14-3-3 proteins in the regulation of nitrate deficiency-induced anthocyanin accumulation. Our results provide a novel avenue to elucidate the mechanism underlying the induction of anthocyanin biosynthesis in response to nitrate deficiency.

Apomixis is a reproductive model that bypasses sexual reproduction, so it does not require the combination of paternal and maternal gametes but instead results in the production of offspring directly from maternal tissues. This reproductive mode results in the same genetic material in the mother and the offspring and has significant applications in agricultural breeding. Molecular and cytological methods were used to identify the reproductive type of Zanthoxylum bungeanum (ZB). Fluorescence detection of the amplified products of 12 pairs of polymorphic SSR primers showed consistent fluorescence signals for mother and offspring, indicating that no trait separation occurred during reproduction. In addition, the cytological observation results showed differentiation of ZB embryos (2n) from nucellar cells (2n) to form indefinite embryonic primordia and then form adventitious embryos (2n), indicating that the apomictic type of ZB is sporophytic apomixis. The MADS-box transcription factor ZbAGL11 was highly expressed during the critical period of nucellar embryo development in ZB. Unpollinated ZbAGL11-OE Arabidopsis produced fertile offspring and exhibited an apomictic phenotype. The overexpression of ZbAGL11 increased the callus induction rate of ZB tissue. In addition, the results of the yeast two-hybrid experiment showed that ZbAGL11 could interact with the ZbCYP450 and ZbCAD11 proteins. Our results demonstrate that ZbAGL11 can cause developmental disorders of Arabidopsis flower organs and result in apomixis-like phenotypes.

Cowpea is a nutrient-dense legume that significantly contributes to the population’s diet in sub-Saharan Africa and other regions of the world. Improving cowpea cultivars to be more resilient to abiotic stress such as drought would be of great importance. The use of a multi-parent advanced generation intercross (MAGIC) population has been shown to be efficient in increasing the frequency of rare alleles that could be associated with important agricultural traits. In addition, drought tolerance index has been reported to be a reliable parameter for assessing crop tolerance to water-deficit conditions. Therefore, the objectives of this study were to evaluate the drought tolerance index for plant growth habit, plant maturity, flowering time, 100-seed weight, and grain yield in a MAGIC cowpea population, to conduct genome-wide association study (GWAS) and identify single nucleotide polymorphism (SNP) markers associated with the drought tolerance indices, to investigate the potential relationship existing between the significant loci associated with the drought tolerance indices, and to conduct genomic selection (GS). These analyses were performed using the existing phenotypic and genotypic data published for the MAGIC population which consisted of 305 F₈ recombinant inbred lines (RILs) developed at University of California, Riverside. The results indicated that: (1) large variation in drought tolerance indices existed among the cowpea genotypes, (2) a total of 14, 18, 5, 5, and 35 SNPs were associated with plant growth habit change due to drought stress, and drought tolerance indices for maturity, flowering time, 100-seed weight, and grain yield, respectively, (3) the network-guided approach revealed clear interactions between the loci associated with the drought tolerance traits, and (4) the GS accuracy varied from low to moderate. These results could be applied to improve drought tolerance in cowpea through marker-assisted selection (MAS) and genomic selection (GS). To the best of our knowledge, this is the first report on marker loci associated with drought tolerance indices in cowpea.

Flavonols and other phenylpropanoids protect plants from biotic and abiotic stress and are dietarily desirable because of their health-promoting properties. The ability to develop new potatoes (Solanum tuberosum) with optimal types and amounts of phenylpropanoids is limited by lack of knowledge about the regulatory mechanisms. Exogenous sucrose increased flavonols, whereas overexpression of the MYB StAN1 induced sucrolytic gene expression. Heterologous StAN1 protein bound promoter fragments from sucrolytic genes (SUSY1 and INV1). Two additional MYBs and one microRNA were identified that regulated potato flavonols. Overexpression analysis showed MYB12A and C increased amounts of flavonols and other phenylpropanoids. Endogenous flavonol amounts in light-exposed organs were much higher those in the dark. Expression levels of StMYB12A and C were high in flowers but low in tubers. Transient overexpression of miR858 altered potato flavonol metabolism. Endogenous StmiR858 expression was much lower in flowers than leaves and correlated with flavonol amounts in these organs. Collectively, these findings support the hypothesis that sucrose, MYBs, and miRNA control potato phenylpropanoid metabolism in a finely tuned manner that includes a feedback loop between sucrose and StAN1. These findings will aid in the development of potatoes with phenylpropanoid profiles optimized for crop performance and human health.

Rose (Rosa spp.) plants flower via two contrasting methods: once flowering (OF) and continuous flowering (CF). Purple branch is a rare continuously flowering variety of Rosa rugosa that is extensively cultivated in China. However, the genetic basis of its CF behavior is unknown. We demonstrated that Purple branch is heterozygous for the TFL1 homolog KSN. One KSN allele with a 9 kb Copia insertion was found to be identical to that from continuously flowering Rosa chinensis Old blush. The other allele was found to be a functional wild-type allele. The overall expression of KSN was closely linked to the floral transition, and it was significantly repressed in continuously flowering Purple branch compared with OF Plena. The promoter region of the normal KSN allele was hypermethylated, and histone methylation at H3H4, H3K9, and H3K27 of the KSN gene locus was modified in continuously flowering Purple branch. Silencing of the DNA methyltransferase genes MET1 and CMT3 and the histone methyltransferase gene SUVR5 in Purple branch led to enhanced KSN expression, but silencing of the histone demethylase gene JMJ12 suppressed KSN expression. Therefore, the CF habit of Purple branch may be due to reduced expression of KSN caused by the halved dose and may be associated with epigenetic modifications together with retrotransposon insertions along the chromosome. Our study revealed a novel mechanism underlying the CF behavior of rose plants.

Almond is appreciated for its nutraceutical value and for the aromatic profile of the kernels. In this work, an almond collection composed of 96 Sicilian accessions complemented with 10 widely cultivated cultivars was phenotyped for the production of volatile organic compounds using a proton-transfer time-of-flight mass spectrometer and genotyped using the Illumina Infinium^®18 K Peach SNP array. The profiling of the aroma was carried out on fresh and roasted kernels enabling the detection of 150 mass peaks. Sixty eight, for the most related with sulfur compounds, furan containing compounds, and aldehydes formed by Strecker degradation, significantly increased during roasting, while the concentration of fifty-four mass peaks, for the most belonging to alcohols and terpenes, significantly decreased. Four hundred and seventy-one robust SNPs were selected and employed for population genetic studies. Structure analysis detected three subpopulations with the Sicilian accessions characterized by a different genetic stratification compared to those collected in Apulia (South Italy) and the International cultivars. The linkage-disequilibrium (LD) decay across the genome was equal to r² = 0.083. Furthermore, a high level of collinearity (r² = 0.96) between almond and peach was registered confirming the high synteny between the two genomes. A preliminary application of a genome-wide association analysis allowed the detection of significant marker-trait associations for 31 fresh and 33 roasted almond mass peaks respectively. An accurate genetic and phenotypic characterization of novel germplasm can represent a valuable tool for the set-up of marker-assisted selection of novel cultivars with an enhanced aromatic profile.

Breeding apple cultivars with resistance offers a potential solution to fire blight, a damaging bacterial disease caused by Erwinia amylovora. Most resistance alleles at quantitative trait loci (QTLs) were previously characterized in diverse Malus germplasm with poor fruit quality, which reduces breeding utility. This study utilized a pedigree-based QTL analysis approach to elucidate the genetic basis of resistance/susceptibility to fire blight from multiple genetic sources in germplasm relevant to U.S. apple breeding programs. Twenty-seven important breeding parents (IBPs) were represented by 314 offspring from 32 full-sib families, with ‘Honeycrisp’ being the most highly represented IBP. Analyzing resistance/susceptibility data from a two-year replicated field inoculation study and previously curated genome-wide single nucleotide polymorphism data, QTLs were consistently mapped on chromosomes (Chrs.) 6, 7, and 15. These QTLs together explained ~28% of phenotypic variation. The Chr. 6 and Chr. 15 QTLs colocalized with previously reported QTLs, while the Chr. 7 QTL is possibly novel. ‘Honeycrisp’ inherited a rare reduced-susceptibility allele at the Chr. 6 QTL from its grandparent ‘Frostbite’. The highly resistant IBP ‘Enterprise’ had at least one putative reduced-susceptibility allele at all three QTLs. In general, lower susceptibility was observed for individuals with higher numbers of reduced-susceptibility alleles across QTLs. This study highlighted QTL mapping and allele characterization of resistance/susceptibility to fire blight in complex pedigree-connected apple breeding germplasm. Knowledge gained will enable more informed parental selection and development of trait-predictive DNA tests for pyramiding favorable alleles and selection of superior apple cultivars with resistance to fire blight.

Seedlessness represents a highly appreciated trait in table grapes. Based on an interesting case of seedless fruit production described in the crop species Annona squamosa, we focused on the Vitis vinifera INNER NO OUTER (INO) gene as a candidate. This gene encodes a transcription factor belonging to the YABBY family involved in the determination of abaxial identity in several organs. In Arabidopsis thaliana, this gene was shown to be essential for the formation and asymmetric growth of the ovule outer integument and its mutation leads to a phenotypic defect of ovules and failure in seed formation. In this study, we identified in silico the V. vinifera orthologue and investigated its phylogenetic relationship to INO genes from other species and its expression in different organs in seeded and seedless varieties. Applying cross-species complementation, we have tested its functionality in the Arabidopsis ino-1 mutant. We show that the V. vinifera INO successfully rescues the ovule outer integument growth and seeds set and also partially complements the outer integument asymmetric growth in the Arabidopsis mutant, differently from orthologues from other species. These data demonstrate that VviINO retains similar activity and protein targets in grapevine as in Arabidopsis. Potential implications for grapevine breeding are discussed.

Solanum lycopersicum var. cerasiforme accession PI 114490 has broad-spectrum resistance to bacterial spot caused by several species of Xanthomonas. Resistance is quantitatively inherited, and a common quantitative trait locus QTL-11B on chromosome 11 has been identified previously. In this study, the SlPub24 gene was characterized in QTL-11B. SlPub24 in PI 114490 was upregulated by infection with X. euvesicatoria pv. perforans race T3, but its transcription was low in the susceptible line OH 88119 whether or not it was infected by the pathogen. The differential expression of SlPub24 between PI 114490 and OH 88119 was due to great sequence variation in the promoter region. The promoter of SlPub24 in OH 88119 had very low activity and did not respond to pathogen infection. Transgenic lines of OH 88119 overexpressing SlPub24 isolated from PI 114490 showed significantly enhanced resistance, while mutants of Slpub24 generated by CRISPR/Cas9 editing showed more susceptibility to race T3 and to other races. The mutants also showed spontaneous cell death in leaves. The expression of the salicylic acid (SA) pathway gene phenylalanine ammonia-lyase (PAL) and signaling-related genes pathogenesis-related (PR1) and nonexpresser of PR1 (NPR1) were influenced by SlPub24. The content of SA in tomato plants was consistent with the level of SlPub24 expression. Furthermore, SlPUB24 interacted with the cell wall protein SlCWP and could regulate the degradation of SlCWP. The expression levels of SlCWP and SlCWINV1, a cell wall invertase gene, showed opposite patterns during pathogen infection. The activity of SlCWINV1 was lower in mutants than in PI 114490. The results are discussed in terms of the roles of the abovementioned genes, and a potential model for SlPUB24-mediated resistance to bacterial spot is proposed.

Mitochondria are crucial for the production of primary and secondary metabolites, which largely determine the quality of fruit. However, a method for isolating high-quality mitochondria is currently not available in citrus fruit, preventing high-throughput characterization of mitochondrial functions. Here, based on differential and discontinuous Percoll density gradient centrifugation, we devised a universal protocol for isolating mitochondria from the pulp of four major citrus species, including satsuma mandarin, ponkan mandarin, sweet orange, and pummelo. Western blot analysis and microscopy confirmed the high purity and intactness of the isolated mitochondria. By using this protocol coupled with a label-free proteomic approach, a total of 3353 nonredundant proteins were identified. Comparison of the four mitochondrial proteomes revealed that the proteins commonly detected in all proteomes participate in several typical metabolic pathways (such as tricarboxylic acid cycle, pyruvate metabolism, and oxidative phosphorylation) and pathways closely related to fruit quality (such as γ-aminobutyric acid (GABA) shunt, ascorbate metabolism, and biosynthesis of secondary metabolites). In addition, differentially abundant proteins (DAPs) between different types of species were also identified; these were found to be mainly involved in fatty acid and amino acid metabolism and were further confirmed to be localized to the mitochondria by subcellular localization analysis. In summary, the proposed protocol for the isolation of highly pure mitochondria from different citrus fruits may be used to obtain high-coverage mitochondrial proteomes, which can help to establish the association between mitochondrial metabolism and fruit storability or quality characteristics of different species and lay the foundation for discovering novel functions of mitochondria in plants.

The aroma of peach fruit is predominantly determined by the accumulation of γ-decalactone and ester compounds. A previous study showed that the biosynthesis of these aroma compounds in peach fruit is catalyzed by PpAAT1, an alcohol acyltransferase. In this work, we investigated the key active site residues responsible for γ-decalactone and ester biosynthesis. A total of 14 candidate amino acid residues possibly involved in internal esterification and 9 candidate amino acid residues possibly involved in esterification of PpAAT1 were assessed via site-directed mutagenesis. Analyses of the in vitro enzyme activities of PpAAT1 and its site-directed mutant proteins (PpAAT1-SMs) with different amino acid residue mutations as well as the contents of γ-decalactone in transgenic tobacco leaves and peach fruits transiently expressing PpAAT1 and PpAAT1-SMs revealed that site-directed mutation of H165 in the conserved HxxxD motif led to lost enzymatic activity of PpAAT1 in both internal esterification and its reactions, whereas mutation of the key amino acid residue D376 led to the total loss of γ-decalactone biosynthesis activity of PpAAT1. Mutations of 9 and 7 other amino acid residues also dramatically affected the enzymatic activity of PpAAT1 in the internal esterification and esterification reactions, respectively. Our findings provide a biochemical foundation for the mechanical biosynthesis of γ-decalactone and ester compounds catalyzed by PpAAT1 in peach fruits, which could be used to guide the molecular breeding of new peach species with more favorable aromas for consumers.

Akebia trifoliata subsp. australis is a well-known medicinal and potential woody oil plant in China. The limited genetic information available for A. trifoliata subsp. australis has hindered its exploitation. Here, a high-quality chromosome-level genome sequence of A. trifoliata subsp. australis is reported. The de novo genome assembly of 682.14 Mb was generated with a scaffold N50 of 43.11 Mb. The genome includes 25,598 protein-coding genes, and 71.18% (485.55 Mb) of the assembled sequences were identified as repetitive sequences. An ongoing massive burst of long terminal repeat (LTR) insertions, which occurred ~1.0 million years ago, has contributed a large proportion of LTRs in the genome of A. trifoliata subsp. australis. Phylogenetic analysis shows that A. trifoliata subsp. australis is closely related to Aquilegia coerulea and forms a clade with Papaver somniferum and Nelumbo nucifera, which supports the well-established hypothesis of a close relationship between basal eudicot species. The expansion of UDP-glucoronosyl and UDP-glucosyl transferase gene families and β-amyrin synthase-like genes and the exclusive contraction of terpene synthase gene families may be responsible for the abundant oleanane-type triterpenoids in A. trifoliata subsp. australis. Furthermore, the acyl-ACP desaturase gene family, including 12 stearoyl-acyl-carrier protein desaturase (SAD) genes, has expanded exclusively. A combined transcriptome and fatty-acid analysis of seeds at five developmental stages revealed that homologs of SADs, acyl-lipid desaturase omega fatty acid desaturases (FADs), and oleosins were highly expressed, consistent with the rapid increase in the content of fatty acids, especially unsaturated fatty acids. The genomic sequences of A. trifoliata subsp. australis will be a valuable resource for comparative genomic analyses and molecular breeding.

Regulation of protein function by reversible S-nitrosation, a post-translational modification based on the attachment of nitroso group to cysteine thiols, has emerged among key mechanisms of NO signalling in plant development and stress responses. S-nitrosoglutathione is regarded as the most abundant low-molecular-weight S-nitrosothiol in plants, where its intracellular concentrations are modulated by S-nitrosoglutathione reductase. We analysed modulations of S-nitrosothiols and protein S-nitrosation mediated by S-nitrosoglutathione reductase in cultivated Solanum lycopersicum (susceptible) and wild Solanum habrochaites (resistant genotype) up to 96 h post inoculation (hpi) by two hemibiotrophic oomycetes, Phytophthora infestans and Phytophthora parasitica. S-nitrosoglutathione reductase activity and protein level were decreased by P. infestans and P. parasitica infection in both genotypes, whereas protein S-nitrosothiols were increased by P. infestans infection, particularly at 72 hpi related to pathogen biotrophy–necrotrophy transition. Increased levels of S-nitrosothiols localised in both proximal and distal parts to the infection site, which suggests together with their localisation to vascular bundles a signalling role in systemic responses. S-nitrosation targets in plants infected with P. infestans identified by a proteomic analysis include namely antioxidant and defence proteins, together with important proteins of metabolic, regulatory and structural functions. Ascorbate peroxidase S-nitrosation was observed in both genotypes in parallel to increased enzyme activity and protein level during P. infestans pathogenesis, namely in the susceptible genotype. These results show important regulatory functions of protein S-nitrosation in concerting molecular mechanisms of plant resistance to hemibiotrophic pathogens.

Chayote (Sechium edule) is an agricultural crop in the Cucurbitaceae family that is rich in bioactive components. To enhance genetic research on chayote, we used Nanopore third-generation sequencing combined with Hi–C data to assemble a draft chayote genome. A chromosome-level assembly anchored on 14 chromosomes (N50 contig and scaffold sizes of 8.40 and 46.56 Mb, respectively) estimated the genome size as 606.42 Mb, which is large for the Cucurbitaceae, with 65.94% (401.08 Mb) of the genome comprising repetitive sequences; 28,237 protein-coding genes were predicted. Comparative genome analysis indicated that chayote and snake gourd diverged from sponge gourd and that a whole-genome duplication (WGD) event occurred in chayote at 25 ± 4 Mya. Transcriptional and metabolic analysis revealed genes involved in fruit texture, pigment, flavor, flavonoids, antioxidants, and plant hormones during chayote fruit development. The analysis of the genome, transcriptome, and metabolome provides insights into chayote evolution and lays the groundwork for future research on fruit and tuber development and genetic improvements in chayote.

WRKY transcription factors (TFs) are of great importance in plant responses to different abiotic stresses. However, research on their roles in the regulation of thermotolerance remains limited. Here, we investigated the function of LlWRKY39 in the thermotolerance of lily (Lilium longiflorum ‘white heaven’). According to multiple alignment analyses, LlWRKY39 is in the WRKY IId subclass and contains a potential calmodulin (CaM)-binding domain. Further analysis has shown that LlCaM3 interacts with LlWRKY39 by binding to its CaM-binding domain, and this interaction depends on Ca²⁺. LlWRKY39 was induced by heat stress (HS), and the LlWRKY39-GFP fusion protein was detected in the nucleus. The thermotolerance of lily and Arabidopsis was increased with the ectopic overexpression of LlWRKY39. The expression of heat-related genes AtHSFA1, AtHSFA2, AtMBF1c, AtGolS1, AtDREB2A, AtWRKY39, and AtHSP101 was significantly elevated in transgenic Arabidopsis lines, which might have promoted an increase in thermotolerance. Then, the promoter of LlMBF1c was isolated from lily, and LlWRKY39 was found to bind to the conserved W-box element in its promoter to activate its activity, suggesting that LlWRKY39 is an upstream regulator of LlMBF1c. In addition, a dual-luciferase reporter assay showed that via protein interaction, LlCaM3 negatively affected LlWRKY39 in the transcriptional activation of LlMBF1c, which might be an important feedback regulation pathway to balance the LlWRKY39-mediated heat stress response (HSR). Collectively, these results imply that LlWRKY39 might participate in the HSR as an important regulator through Ca²⁺-CaM and multiprotein bridging factor pathways.

Betula L. (birch) is a pioneer hardwood tree species with ecological, economic, and evolutionary importance in the Northern Hemisphere. We sequenced the Betula platyphylla genome and assembled the sequences into 14 chromosomes. The Betula genome lacks evidence of recent whole-genome duplication and has the same paleoploidy level as Vitis vinifera and Prunus mume. Phylogenetic analysis of lignin pathway genes coupled with tissue-specific expression patterns provided clues for understanding the formation of higher ratios of syringyl to guaiacyl lignin observed in Betula species. Our transcriptome analysis of leaf tissues under a time-series cold stress experiment revealed the presence of the MEKK1–MKK2–MPK4 cascade and six additional mitogen-activated protein kinases that can be linked to a gene regulatory network involving many transcription factors and cold tolerance genes. Our genomic and transcriptome analyses provide insight into the structures, features, and evolution of the B. platyphylla genome. The chromosome-level genome and gene resources of B. platyphylla obtained in this study will facilitate the identification of important and essential genes governing important traits of trees and genetic improvement of B. platyphylla.

Magnolia biondii Pamp. (Magnoliaceae, magnoliids) is a phylogenetically, economically, and medicinally important ornamental tree species widely grown and cultivated in the north-temperate regions of China. Determining the genome sequence of M. biondii would help resolve the phylogenetic uncertainty of magnoliids and improve the understanding of individual trait evolution within the Magnolia genus. We assembled a chromosome-level reference genome of M. biondii using ~67, ~175, and ~154 Gb of raw DNA sequences generated via Pacific Biosciences single-molecule real-time sequencing, 10X Genomics Chromium, and Hi-C scaffolding strategies, respectively. The final genome assembly was ~2.22 Gb, with a contig N50 value of 269.11 kb and a BUSCO complete gene percentage of 91.90%. Approximately 89.17% of the genome was organized into 19 chromosomes, resulting in a scaffold N50 of 92.86 Mb. The genome contained 47,547 protein-coding genes, accounting for 23.47% of the genome length, whereas 66.48% of the genome length consisted of repetitive elements. We confirmed a WGD event that occurred very close to the time of the split between the Magnoliales and Laurales. Functional enrichment of the Magnolia-specific and expanded gene families highlighted genes involved in the biosynthesis of secondary metabolites, plant–pathogen interactions, and responses to stimuli, which may improve the ecological fitness and biological adaptability of the lineage. Phylogenomic analyses revealed a sister relationship of magnoliids and Chloranthaceae, which are sister to a clade comprising monocots and eudicots. The genome sequence of M. biondii could lead to trait improvement, germplasm conservation, and evolutionary studies on the rapid radiation of early angiosperms.

Heterosis is a complex phenomenon in which hybrids show better phenotypic characteristics than their parents do. Chinese cabbage (Brassica rapa L. spp. pekinensis) is a popular leafy crop species, hybrids of which are widely used in commercial production; however, the molecular basis of heterosis for biomass of Chinese cabbage is poorly understood. We characterized heterosis in a Chinese cabbage F₁ hybrid cultivar and its parental lines from the seedling stage to the heading stage; marked heterosis of leaf weight and biomass yield were observed. Small RNA sequencing revealed 63 and 50 differentially expressed microRNAs (DEMs) at the seedling and early-heading stages, respectively. The expression levels of the majority of miRNA clusters in the F₁ hybrid were lower than the mid-parent values (MPVs). Using degradome sequencing, we identified 1,819 miRNA target genes. Gene ontology (GO) analyses demonstrated that the target genes of the MPV-DEMs and low parental expression level dominance (ELD) miRNAs were significantly enriched in leaf morphogenesis, leaf development, and leaf shaping. Transcriptome analysis revealed that the expression levels of photosynthesis and chlorophyll synthesis-related MPV-DEGs (differentially expressed genes) were significantly different in the F₁ hybrid compared to the parental lines, resulting in increased photosynthesis capacity and chlorophyll content in the former. Furthermore, expression of genes known to regulate leaf development was also observed at the seedling stage. Arabidopsis plants overexpressing BrGRF4.2 and bra-miR396 presented increased and decreased leaf sizes, respectively. These results provide new insight into the regulation of target genes and miRNA expression patterns in leaf size and heterosis for biomass of B. rapa.

Cucumis hystrix Chakr. (2n = 2x = 24) is a wild species that can hybridize with cultivated cucumber (C. sativus L., 2n = 2x = 14), a globally important vegetable crop. However, cucumber breeding is hindered by its narrow genetic base. Therefore, introgression from C. hystrix has been anticipated to bring a breakthrough in cucumber improvement. Here, we report the chromosome-scale assembly of C. hystrix genome (289 Mb). Scaffold N50 reached 14.1 Mb. Over 90% of the sequences were anchored onto 12 chromosomes. A total of 23,864 genes were annotated using a hybrid method. Further, we conducted a comprehensive comparative genomic analysis of cucumber, C. hystrix, and melon (C. melo L., 2n = 2x = 24). Whole-genome comparisons revealed that C. hystrix is phylogenetically closer to cucumber than to melon, providing a molecular basis for the success of its hybridization with cucumber. Moreover, expanded gene families of C. hystrix were significantly enriched in “defense response,” and C. hystrix harbored 104 nucleotide-binding site–encoding disease resistance gene analogs. Furthermore, 121 genes were positively selected, and 12 (9.9%) of these were involved in responses to biotic stimuli, which might explain the high disease resistance of C. hystrix. The alignment of whole C. hystrix genome with cucumber genome and self-alignment revealed 45,417 chromosome-specific sequences evenly distributed on C. hystrix chromosomes. Finally, we developed four cucumber– C. hystrix alien addition lines and identified the exact introgressed chromosome using molecular and cytological methods. The assembled C. hystrix genome can serve as a valuable resource for studies on Cucumis evolution and interspecific introgression breeding of cucumber.

Cultivated strawberry (Fragaria × ananassa) is an important fruit crop species whose fruits are enjoyed by many worldwide. An octoploid of hybrid origin, the complex genome of this species was recently sequenced, serving as a key reference genome for cultivated strawberry and related species of the Rosaceae family. The current annotation of the F. ananassa genome mainly relies on ab initio predictions and, to a lesser extent, transcriptome data. Here, we present the structure and functional reannotation of the F. ananassa genome based on one PacBio full-length RNA library and ninety-two Illumina RNA-Seq libraries. This improved annotation of the F. ananassa genome, v1.0.a2, comprises a total of 108,447 gene models, with 97.85% complete BUSCOs. The models of 19,174 genes were modified, 360 new genes were identified, and 11,044 genes were found to have alternatively spliced isoforms. Additionally, we constructed a strawberry genome database (SGD) for strawberry gene homolog searching and annotation downloading. Finally, the transcriptome of the receptacles and achenes of F. ananassa at four developmental stages were reanalyzed and qualified, and the expression profiles of all the genes in this annotation are also provided. Together, this study provides an updated annotation of the F. ananassa genome, which will facilitate genomic analyses across the Rosaceae family and gene functional studies in cultivated strawberry.

The characteristic secondary metabolites in tea (theanine, caffeine, and catechins) are important factors contributing to unique tea flavors. However, there has been relatively little research on molecular markers related to these metabolites. Thus, we conducted a genome-wide association analysis of the levels of these tea flavor-related metabolites in three seasons. The theanine, caffeine, and catechin levels in Population 1 comprising 191 tea plant germplasms were examined, which revealed that their heritability exceeded 0.5 in the analyzed seasons, with the following rank order (highest to lowest heritabilities): (+)-catechin > (−)-gallocatechin gallate > caffeine = (−)-epicatechin > (−)-epigallocatechin-3-gallate > theanine > (−)-epigallocatechin > (−)-epicatechin-3-gallate > catechin gallate > (+)-gallocatechin. The SNPs detected by amplified-fragment SNP and methylation sequencing divided Population 1 into three groups and seven subgroups. An association analysis yielded 307 SNP markers related to theanine, caffeine, and catechins that were common to all three seasons. Some of the markers were pleiotropic. The functional annotation of 180 key genes at the SNP loci revealed that FLS, UGT, MYB, and WD40 domain-containing proteins, as well as ATP-binding cassette transporters, may be important for catechin synthesis. KEGG and GO analyses indicated that these genes are associated with metabolic pathways and secondary metabolite biosynthesis. Moreover, in Population 2 (98 tea plant germplasm resources), 30 candidate SNPs were verified, including 17 SNPs that were significantly or extremely significantly associated with specific metabolite levels. These results will provide a foundation for future research on important flavor-related metabolites and may help accelerate the breeding of new tea varieties.

The interplay between melatonin and ethylene in the regulation of fruit metabolism and the underlying molecular mechanism of this interplay remain largely unclear. Here, widely targeted metabolomics analysis revealed a total of 464 metabolites present in berry skin. Among them, 27 significantly differentially accumulated metabolites (DAMs) were produced in response to melatonin treatment in the presence or absence of 1-MCP. Most of the DAMs were secondary metabolites, including flavonoids, phenolic acids, stilbenes, and flavonols. Additionally, the accumulation of 25 DAMs was regulated by melatonin via ethylene. RNA-seq analysis indicated that melatonin primarily regulated the pathways of plant hormone signal transduction and secondary metabolite biosynthesis via ethylene. Gene-metabolite association analysis showed that melatonin regulated the expression of the VvSTS1, VvF3H, VvLAR2, and VvDFR genes, suggesting that these genes may play key roles in regulating secondary metabolites in the skin; additionally, VvMYB14 and VvACS1 were suggested to be involved in the regulation of secondary metabolites. Further experiments revealed that melatonin induced the expression of VvMYB14 and that VvMYB14 increased ethylene production by transcriptionally activating VvACS1, thereby affecting the accumulation of secondary metabolites. Collectively, melatonin promotes ethylene biosynthesis and alters secondary metabolite accumulation through the regulation of VvACS1 by VvMYB14.

Brassica downy mildew, a severe disease caused by Hyaloperonospora brassicae, can cause enormous economic losses in Chinese cabbage (Brassica rapa L. ssp. pekinensis) production. Although some research has been reported recently concerning the underlying resistance to this disease, no studies have identified or characterized long noncoding RNAs involved in this defense response. In this study, using high-throughput RNA sequencing, we analyzed the disease-responding mRNAs and long noncoding RNAs in two resistant lines (T12–19 and 12–85) and one susceptible line (91–112). Clustering and Gene Ontology analysis of differentially expressed genes (DEGs) showed that more DEGs were involved in the defense response in the two resistant lines than in the susceptible line. Different expression patterns and proposed functions of differentially expressed long noncoding RNAs among T12–19, 12–85, and 91–112 indicated that each has a distinct disease response mechanism. There were significantly more cis- and trans-functional long noncoding RNAs in the resistant lines than in the susceptible line, and the genes regulated by these RNAs mostly participated in the disease defense response. Furthermore, we identified a candidate resistance-related long noncoding RNA, MSTRG.19915, which is a long noncoding natural antisense transcript of a MAPK gene, BrMAPK15. Via an agroinfiltration-mediated transient overexpression system and virus-induced gene silencing technology, BrMAPK15 was indicated to have a greater ability to defend against pathogens. MSTRG.19915-silenced seedlings showed enhanced resistance to downy mildew, probably because of the upregulated expression of BrMAPK15. This research identified and characterized long noncoding RNAs involved in resistance to downy mildew, laying a foundation for future in-depth studies of disease resistance mechanisms in Chinese cabbage.

Small RNAs (sRNAs) are essential regulatory molecules, and there are three major sRNA classes in plants: microRNAs (miRNAs), phased small interfering RNAs (phased siRNAs or phasiRNAs), and heterochromatic siRNAs (hc-siRNAs). Excluding miRNAs, the other two classes are not well annotated or available in public databases for most sequenced plant genomes. We performed a comprehensive sRNA annotation of 143 plant species that have fully sequenced genomes and next-generation sequencing sRNA data publicly available. The results are available via an online repository called sRNAanno (www.plantsRNAs.org). Compared with other public plant sRNA databases, we obtained was much more miRNA annotations, which are more complete and reliable because of the consistent and highly stringent criteria used in our miRNA annotations. sRNAanno also provides free access to genomic information for >22,721 PHAS loci and >22 million hc-siRNA loci annotated from these 143 plant species. Both miRNA and PHAS loci can be easily browsed to view their main features, and a collection of archetypal trans-acting siRNA 3 (TAS3) genes were annotated separately for quick access. To facilitate the ease of sRNA annotation, sRNAanno provides free service for sRNA annotations to the community. In summary, the sRNAanno database is a great resource to facilitate genomic and genetic research on plant small RNAs.

Plant pathogenic oomycetes deliver a troop of effector proteins into the nucleus of host cells to manipulate plant cellular immunity and promote colonization. Recently, researchers have focused on identifying how effectors are transferred into the host cell nucleus, as well as the identity of the nuclear targets. In this study, we found that the RxLR effector PvAVH53 from the grapevine (Vitis vinifera) oomycete pathogen Plasmopara viticola physically interacts with grapevine nuclear import factor importin alphas (VvImpα and VvImpα4), localizes to the nucleus and triggers cell death when transiently expressed in tobacco (Nicotiana benthamiana) cells. Deletion of a nuclear localization signal (NLS) sequence from PvAVH53 or addition of a nuclear export signal (NES) sequence disrupted the nuclear localization of PvAVH53 and attenuated its ability to trigger cell death. Suppression of two tobacco importin-α genes, namely, NbImp-α1 and NbImp-α2, by virus-induced gene silencing (VIGS) also disrupted the nuclear localization and ability of PvAVH53 to induce cell death. Likewise, we transiently silenced the expression of VvImpα/α4 in grape through CRISPR/Cas13a, which has been reported to target RNA in vivo. Finally, we found that attenuating the expression of the Importin-αs genes resulted in increased susceptibility to the oomycete pathogen Phytophthora capsici in N. benthamiana and P. viticola in V. vinifera. Our results demonstrate that importin-αs are required for the nuclear localization and function of PvAVH53 and are essential for host innate immunity. The findings provide insight into the functions of importin-αs in grapevine against downy mildew.

Medicago polymorpha is a nutritious and palatable forage and vegetable plant that also fixes nitrogen. Here, we reveal the chromosome-scale genome sequence of M. polymorpha using an integrated approach including Illumina, PacBio and Hi-C technologies. We combined PacBio full-length RNA-seq, metabolomic analysis, structural anatomy analysis and related physiological indexes to elucidate the important agronomic traits of M. polymorpha for forage and vegetable usage. The assembled M. polymorpha genome consisted of 457.53 Mb with a long scaffold N50 of 57.72 Mb, and 92.92% (441.83 Mb) of the assembly was assigned to seven pseudochromosomes. Comparative genomic analysis revealed that expansion and contraction of the photosynthesis and lignin biosynthetic gene families, respectively, led to enhancement of nutritious compounds and reduced lignin biosynthesis in M. polymorpha. In addition, we found that several positively selected nitrogen metabolism-related genes were responsible for crude protein biosynthesis. Notably, the metabolomic results revealed that a large number of flavonoids, vitamins, alkaloids, and terpenoids were enriched in M. polymorpha. These results imply that the decreased lignin content but relatively high nutrient content of M. polymorpha enhance its edibility and nutritional value as a forage and vegetable. Our genomic data provide a genetic basis that will accelerate functional genomic and breeding research on M. polymorpha as well as other Medicago and legume plants.

Long noncoding RNAs (lncRNAs) are widely present in different species and play critical roles in response to abiotic stresses. However, the functions of lncRNAs in Chinese cabbage under heat stress remain unknown. Here, we first conducted a global comparative analysis of 247,242 lncRNAs among 37 species. The results indicated that lncRNAs were poorly conserved among different species, and only 960 lncRNAs were homologous to 524 miRNA precursors. We then carried out lncRNA sequencing for a genome-wide analysis of lncRNAs and their target genes in Chinese cabbage at different stages of heat treatment. In total, 18,253 lncRNAs were identified, of which 1229 differentially expressed (DE) lncRNAs were characterized as being heat-responsive. The ceRNA network revealed that 38 lncRNAs, 16 miRNAs, and 167 mRNAs were involved in the heat response in Chinese cabbage. Combined analysis of the cis- and trans-regulated genes indicated that the targets of DE lncRNAs were significantly enriched in the “protein processing in endoplasmic reticulum” and “plant hormone signal transduction” pathways. Furthermore, the majority of HSP and PYL genes involved in these two pathways exhibited similar expression patterns and responded to heat stress rapidly. Based on the networks of DE lncRNA-mRNAs, 29 and 22 lncRNAs were found to interact with HSP and PYL genes, respectively. Finally, the expression of several critical lncRNAs and their targets was verified by qRT-PCR. Overall, we conducted a comparative analysis of lncRNAs among 37 species and performed a comprehensive analysis of lncRNAs in Chinese cabbage. Our findings expand the knowledge of lncRNAs involved in the heat stress response in Chinese cabbage, and the identified lncRNAs provide an abundance of resources for future comparative and functional studies.

Haplotypes provide useful information for genomics-based approaches, genomic prediction, and genome-wide association study. As a small number of superior founders have contributed largely to the breeding history of fruit trees, the information of founder haplotypes may be relevant for performing the genomics-based approaches in these plants. In this study, we proposed a method to estimate 14 haplotypes from 7 founders and automatically trace the haplotypes forward to apple parental (185 varieties) and breeding (659 F₁ individuals from 16 full-sib families) populations based on 11,786 single-nucleotide polymorphisms, by combining multiple algorithms. Overall, 92% of the single-nucleotide polymorphisms information in the parental and breeding populations was characterized by the 14 founder haplotypes. The use of founder haplotype information improved the accuracy of genomic prediction in 7 traits and the resolution of genome-wide association study in 13 out of 27 fruit quality traits analyzed in this study. We also visualized the significant propagation of the founder haplotype with the largest genetic effect in genome-wide association study over the pedigree tree of the parental population. These results suggest that the information of founder haplotypes can be useful for not only genetic improvement of fruit quality traits in apples but also for understanding the selection history of founder haplotypes in the breeding program of Japanese apple varieties.

Pathological hypertrophy (cell enlargement) plays an important role in the development of citrus canker, but its regulators are largely unknown. Although WRKY22 is known to be involved in pathogen-triggered immunity and positively regulates resistance to bacterial pathogens in Arabidopsis, rice and pepper, the CRISPR/Cas9-mediated partial knockout of CsWRKY22 improves resistance to Xanthomonas citri subsp. citri (Xcc) in Wanjincheng orange (Citrus sinensis Osbeck). Here, we demonstrate that CsWRKY22 is a nucleus-localized transcriptional activator. CsWRKY22-overexpressing plants exhibited dwarf phenotypes that had wrinkled and thickened leaves and were more sensitive to Xcc, whereas CsWRKY22-silenced plants showed no visible phenotype changes and were more resistant to Xcc. Microscopic observations revealed that the overexpression of CsWRKY22 increased cell size in the spongy mesophyll. Transcriptome analysis showed that cell growth-related pathways, such as the auxin and brassinosteroid hormonal signaling and cell wall organization and biogenesis pathways, were significantly upregulated upon CsWRKY22 overexpression. Interestingly, CsWRKY22 activated the expression of CsLOB1, which is a key gene regulating susceptibility to citrus canker. We further confirmed that CsWRKY22 bound directly to the W-boxes just upstream of the transcription start site of CsLOB1 in vivo and in vitro. We conclude that CsWRKY22 enhances susceptibility to citrus canker by promoting host hypertrophy and CsLOB1 expression. Thus, our study provides new insights into the mechanism regulating pathological hypertrophy and the function of WRKY22 in citrus.

Cytokinin and gibberellic acid (GA) are growth regulators used to increase berry size in seedless grapes and it is of interest to understand their effects on the phenylpropanoid pathway and on ripening processes. GA₃ and synthetic cytokinin forchlorfenuron (N-(2-chloro-4-pyridyl)-N′-phenylurea, CPPU) and their combination were applied to 6 mm diameter fruitlets of ‘Sable Seedless’, and berries were sampled 51 and 70 days (d) following application. All treatments increased berry size and delayed sugar accumulation and acid degradation with a stronger effect of CPPU. CPPU, but not GA, reduced berry color and the levels of anthocyanins. While CPPU reduced the levels of anthocyanins by more than 50%, the combined treatment of GA+CPPU reduced the levels by about 25% at 51 d. CPPU treatment had minor effects on flavonols content but increased the levels of monomeric flavan-3-ols by more than two-fold. Phloroglucinol analysis using HPLC showed that proanthocyanidin content was significantly increased by CPPU, whereas mean degree of polymerization was reduced from 26 to 19. Volatile analysis by GC-MS showed changes in composition with CPPU or GA treatment with potential impact on flavor. RNA-seq analysis showed that GA had a minor overall effect on the transcriptome whereas CPPU had pronounced effects on gene expression at both 51 and 70 d. Comparing the control and CPPU at similar Brix of ca. 19.7°, a reduced expression of stilbene synthases (STSs) including their regulators MYB14 and MYB15, and other phenylpropanoid-related genes was observed in CPPU-treated grapes. Overall, our study shows that CPPU had a major influence on the phenylpropanoid pathway and affected multiple ripening-related processes.

The efficacy of the CRISPR/Cas9 system in grapevine (Vitis vinifera L.) has been documented, but the optimization of this system, as well as CRISPR/Cas9-mediated multiplex genome editing, has not been explored in this species. Herein, we identified four VvU3 and VvU6 promoters and two ubiquitin (UBQ) promoters in grapevine and demonstrated that the use of the identified VvU3/U6 and UBQ2 promoters could significantly increase the editing efficiency in grape by improving the expression of sgRNA and Cas9, respectively. Furthermore, we conducted multiplex genome editing using the optimized CRISPR/Cas9 vector that contained the conventional multiple sgRNA expression cassettes or the polycistronic tRNA-sgRNA cassette (PTG) by targeting the sugar-related tonoplastic monosaccharide transporter (TMT) family members TMT1 and TMT2, and the overall editing efficiencies were higher than 10%. The simultaneous editing of TMT1 and TMT2 resulted in reduced sugar levels, which indicated the role of these two genes in sugar accumulation in grapes. Moreover, the activities of the VvU3, VvU6, and UBQ2 promoters in tobacco genome editing were demonstrated by editing the phytoene desaturase (PDS) gene in Nicotiana benthamiana leaves. Our study provides materials for the optimization of the CRISPR/Cas9 system. To our knowledge, our simultaneous editing of the grape TMT family genes TMT1 and TMT2 constitutes the first example of multiplex genome editing in grape. The multiplex editing systems described in this manuscript expand the toolbox of grape genome editing, which would facilitate basic research and molecular breeding in grapevine.

The aromatic shrub Lavandula angustifolia produces various volatile terpenoids that serve as resources for essential oils and function in plant-insect communication. To better understand the genetic basis of the terpenoid diversity in lavender, we present a high-quality reference genome for the Chinese lavender cultivar ‘Jingxun 2’ using PacBio and Hi-C technologies to anchor the 894.50 Mb genome assembly into 27 pseudochromosomes. In addition to the γ triplication event, lavender underwent two rounds of whole-genome duplication (WGD) during the Eocene–Oligocene (29.6 MYA) and Miocene–Pliocene (6.9 MYA) transitions. As a result of tandem duplications and lineage-specific WGDs, gene families related to terpenoid biosynthesis in lavender are substantially expanded compared to those of five other species in Lamiaceae. Many terpenoid biosynthesis transcripts are abundant in glandular trichomes. We further integrated the contents of ecologically functional terpenoids and coexpressed terpenoid biosynthetic genes to construct terpenoid-gene networks. Typical gene clusters, including TPS-TPS, TPS-CYP450, and TPS-BAHD, linked with compounds that primarily function as attractants or repellents, were identified by their similar patterns of change during flower development or in response to methyl jasmonate. Comprehensive analysis of the genetic basis of the production of volatiles in lavender could serve as a foundation for future research into lavender evolution, phytochemistry, and ecology.

Hazelnut is popular for its flavor, and it has also been suggested that hazelnut is beneficial to cardiovascular health because it is rich in oleic acid. Here, we report the first high-quality chromosome-scale genome for the hazelnut species Corylus mandshurica (2n = 22), which has a high concentration of oleic acid in its nuts. The assembled genome is 367.67 Mb in length, and the contig N50 is 14.85 Mb. All contigs were assembled into 11 chromosomes, and 28,409 protein-coding genes were annotated. We reconstructed the evolutionary trajectories of the genomes of Betulaceae species and revealed that the 11 chromosomes of the hazelnut genus were derived from the most ancestral karyotype in Betula pendula, which has 14 protochromosomes, by inferring homology among five Betulaceae genomes. We identified 96 candidate genes involved in oleic acid biosynthesis, and 10 showed rapid evolution or positive selection. These findings will help us to understand the mechanisms of lipid synthesis and storage in hazelnuts. Several gene families related to salicylic acid metabolism and stress responses experienced rapid expansion in this hazelnut species, which may have increased its stress tolerance. The reference genome presented here constitutes a valuable resource for molecular breeding and genetic improvement of the important agronomic properties of hazelnut.

In plants and algae, PGR5-dependent cyclic electron flow (CEF) is an important regulator of acclimation to fluctuating environments, but how PGR5 participates in CEF is unclear. In this work, we analyzed two PGR5s in cucumber (Cucumis sativus L.) under different conditions and found that CsPGR5a played the dominant role in PGR5-dependent CEF. The results of yeast two-hybrid, biomolecular fluorescence complementation (BiFC), blue native PAGE, and coimmunoprecipitation (CoIP) assays showed that PGR5a interacted with PetC, Lhcb3, and PsaH. Furthermore, the intensity of the interactions was dynamic during state transitions, and the abundance of PGR5 attached to cyt b₆f decreased during the transition from state 1 to state 2, which revealed that the function of PGR5a is related to the state transition. We proposed that PGR5 is a small mobile protein that functions when attached to protein complexes.

Deciphering the genetic basis of plant secondary metabolism will provide useful insights for genetic improvement and enhance our fundamental understanding of plant biological processes. Although citrus plants are among the most important fruit crops worldwide, the genetic basis of secondary metabolism in these plants is largely unknown. Here, we use a high-density linkage map to dissect large-scale flavonoid metabolic traits measured in different tissues (young leaf, old leaf, mature pericarp, and mature pulp) of an F₁ pseudo-testcross citrus population. We detected 80 flavonoids in this population and identified 138 quantitative trait loci (QTLs) for 57 flavonoids in these four tissues. Based on transcriptional profiling and functional annotation, twenty-one candidate genes were identified, and one gene encoding flavanone 3-hydroxylase (F3H) was functionally verified to result in naturally occurring variation in dihydrokaempferol content through genetic variations in its promoter and coding regions. The abundant data resources collected for diverse citrus germplasms here lay the foundation for complete characterization of the citrus flavonoid biosynthetic pathway and will thereby promote efficient utilization of metabolites in citrus quality improvement.

Root–shoot communication has a critical role in plant adaptation to environmental stress. Grafting is widely applied to enhance the abiotic stress tolerance of many horticultural crop species; however, the signal transduction mechanism involved in this tolerance remains unknown. Here, we show that pumpkin- or figleaf gourd rootstock-enhanced cold tolerance of watermelon shoots is accompanied by increases in the accumulation of melatonin, methyl jasmonate (MeJA), and hydrogen peroxide (H₂O₂). Increased melatonin levels in leaves were associated with both increased melatonin in rootstocks and MeJA-induced melatonin biosynthesis in leaves of plants under cold stress. Exogenous melatonin increased the accumulation of MeJA and H₂O₂ and enhanced cold tolerance, while inhibition of melatonin accumulation attenuated rootstock-induced MeJA and H₂O₂ accumulation and cold tolerance. MeJA application induced H₂O₂ accumulation and cold tolerance, but inhibition of JA biosynthesis abolished rootstock- or melatonin- induced H₂O₂ accumulation and cold tolerance. Additionally, inhibition of H₂O₂ production attenuated MeJA-induced tolerance to cold stress. Taken together, our results suggest that melatonin is involved in grafting-induced cold tolerance by inducing the accumulation of MeJA and H₂O₂. MeJA subsequently increases melatonin accumulation, forming a self-amplifying feedback loop that leads to increased H₂O₂ accumulation and cold tolerance. This study reveals a novel regulatory mechanism of rootstock-induced cold tolerance.

The strawberry Fra a 1 proteins belong to the class 10 Pathogenesis-Related (PR-10) superfamily. In strawberry, a large number of members have been identified, but only a limited number is expressed in the fruits. In this organ, Fra a 1.01 and Fra a 1.02 are the most abundant Fra proteins in the green and red fruits, respectively, however, their function remains unknown. To know the function of Fra a 1.02 we have generated transgenic lines that silence this gene, and performed metabolomics, RNA-Seq, and hormonal assays. Previous studies associated Fra a 1.02 to strawberry fruit color, but the analysis of anthocyanins in the ripe fruits showed no diminution in their content in the silenced lines. Gene ontology (GO) analysis of the genes differentially expressed indicated that oxidation/reduction was the most represented biological process. Redox state was not apparently altered since no changes were found in ascorbic acid and glutathione (GSH) reduced/oxidized ratio, but GSH content was reduced in the silenced fruits. In addition, a number of glutathione-S-transferases (GST) were down-regulated as result of Fra a 1.02-silencing. Another highly represented GO category was transport which included a number of ABC and MATE transporters. Among the regulatory genes differentially expressed WRKY33.1 and WRKY33.2 were down-regulated, which had previously been assigned a role in strawberry plant defense. A reduced expression of the VQ23 gene and a diminished content of the hormones JA, SA, and IAA were also found. These data might indicate that Fra a 1.02 participates in the defense against pathogens in the ripe strawberry fruits.

Numerous environmental and endogenous signals control the highly orchestrated and intricate process of plant senescence. Ethylene, a well-known inducer of senescence, has long been considered a key endogenous regulator of leaf and flower senescence, but the molecular mechanism of ethylene-induced ovule senescence has not yet been elucidated. In this study, we found that blockage of fertilization caused ovule abortion in the pear cultivar ‘1913’. According to transcriptome and phytohormone content data, ethylene biosynthesis was activated by pollination. At the same time, ethylene overaccumulated in ovules, where cells were sensitive to ethylene signals in the absence of fertilization. We identified a transcription factor in the ethylene signal response, ethylene-insensitive 3-like (EIL1), as a likely participant in ovule senescence. Overexpression of PbEIL1 in tomato caused precocious onset of ovule senescence. We further found that EIL1 could directly bind to the promoter of the SENESCENCE-ASSOCIATED CYSTEINE PROTEINASE 1 (PbCysp1) gene and act upstream of senescence. Yeast one-hybrid and dual-luciferase assays revealed the interaction of the transcription factor and the promoter DNA sequence and demonstrated that PbEIL1 enhanced the action of PbCysp1. Collectively, our results provide new insights into how ethylene promotes the progression of unfertilized ovule senescence.

An effector–reporter system is a powerful tool used to study cellular signal transduction, but this technique has been traditionally used in protoplasts. A similar system to study cellular signal transduction in fruits has not yet been established. In this study, we aimed to establish an effector–reporter system for strawberry fruit, a model nonclimacteric fruit. We first investigated the characteristics of transient gene expression in strawberry fruits and found marked variation in gene expression levels among individual fruits, and this variation has complicated the establishment of a technical system. To overcome this difficulty, we investigated a sampling strategy based on a statistical analysis of the activity pattern of four different reporters (GUS, GFP, FLuc, and RLuc) among individual fruits and combinations of pairs of reporters (GUS/GFP and RLuc/FLuc). Based on an optimized sampling strategy, we finally established a step-by step protocol for the effector/reporter assay. Using FaMYB10 and FaWRKY71 as the effectors and GUS driven by the FaCHS promoter as the reporter, we demonstrated that this effector/reporter system was practical and reliable. This effector/reporter technique will contribute to an in-depth exploration of the signaling mechanism for the regulation of strawberry fruit ripening.

Lignification is a major cell wall modification that often results in the formation of sophisticated subcellular patterns during plant development or in response to environmental stresses. Precise localization of the spatiotemporal deposition of lignin is of great importance for revealing the lignification regulatory mechanism of individual cells. In loquat fruits, lignification typically increases the flesh lignin content and firmness, reducing their edibility and processing quality. However, the precise localization of the spatiotemporal active zones of lignification inside loquat fruit flesh remains poorly understood, and little is known about the contribution of patterned lignification to cell wall structure dynamics and the subsequent fruit-quality deterioration. Here, we performed an emerging bioorthogonal chemistry imaging technique to trace the in vivo patterned lignification dynamics in cells of loquat fruit flesh during development and storage. In developing fruits, lignified cells (LCs) and vascular bundles (VBs) were the zones of active lignification, and ring-like LCs deposited lignin at both the inner wall layer and the outer periphery sides. The domino effect of the generation of LCs was preliminarily visualized. In mature fruits, the newly formed lignin in the flesh of fruits during storage was specifically deposited in the corners and middle lamellae of parenchyma cells surrounding the VBs, resulting in the development of a reticular structure. Based on the findings, distinct spatiotemporal patterned lignification modes for different flesh cells in loquat fruits were proposed. These findings provide loquat lignification dynamics together with spatiotemporal data that can improve our understanding of the lignification process in planta.

The nonrandom three-dimensional organization of chromatin plays an important role in the regulation of gene expression. However, it remains unclear whether this organization is conserved and whether it is involved in regulating gene expression during speciation after whole-genome duplication (WGD) in plants. In this study, high-resolution interaction maps were generated using high-throughput chromatin conformation capture (Hi-C) techniques for two poplar species, Populus euphratica and Populus alba var. pyramidalis, which diverged ~14 Mya after a common WGD. We examined the similarities and differences in the hierarchical chromatin organization between the two species, including A/B compartment regions and topologically associating domains (TADs), as well as in their DNA methylation and gene expression patterns. We found that chromatin status was strongly associated with epigenetic modifications and gene transcriptional activity, yet the conservation of hierarchical chromatin organization across the two species was low. The divergence of gene expression between WGD-derived paralogs was associated with the strength of chromatin interactions, and colocalized paralogs exhibited strong similarities in epigenetic modifications and expression levels. Thus, the spatial localization of duplicated genes is highly correlated with biased expression during the diploidization process. This study provides new insights into the evolution of chromatin organization and transcriptional regulation during the speciation process of poplars after WGD.

Dragon fruits are tropical fruits economically important for agricultural industries. As members of the family of Cactaceae, they have evolved to adapt to the arid environment. Here we report the draft genome of Hylocereus undatus, commercially known as the white-fleshed dragon fruit. The chromosomal level genome assembly contains 11 longest scaffolds corresponding to the 11 chromosomes of H. undatus. Genome annotation of H. undatus found ~29,000 protein-coding genes, similar to Carnegiea gigantea (saguaro). Whole-genome duplication (WGD) analysis revealed a WGD event in the last common ancestor of Cactaceae followed by extensive genome rearrangements. The divergence time between H. undatus and C. gigantea was estimated to be 9.18 MYA. Functional enrichment analysis of orthologous gene clusters (OGCs) in six Cactaceae plants found significantly enriched OGCs in drought resistance. Fruit flavor-related functions were overrepresented in OGCs that are significantly expanded in H. undatus. The H. undatus draft genome also enabled the discovery of carbohydrate and plant cell wall-related functional enrichment in dragon fruits treated with trypsin for a longer storage time. Lastly, genes of the betacyanin (a red-violet pigment and antioxidant with a very high concentration in dragon fruits) biosynthetic pathway were found to be co-localized on a 12 Mb region of one chromosome. The consequence may be a higher efficiency of betacyanin biosynthesis, which will need experimental validation in the future. The H. undatus draft genome will be a great resource to study various cactus plants.

Olive (Olea europaea L.) is internationally renowned for its high-end product, extra virgin olive oil. An incomplete genome of O. europaea was previously obtained using shotgun sequencing in 2016. To further explore the genetic and breeding utilization of olive, an updated draft genome of olive was obtained using Oxford Nanopore third-generation sequencing and Hi-C technology. Seven different assembly strategies were used to assemble the final genome of 1.30 Gb, with contig and scaffold N50 sizes of 4.67 Mb and 42.60 Mb, respectively. This greatly increased the quality of the olive genome. We assembled 1.1 Gb of sequences of the total olive genome to 23 pseudochromosomes by Hi-C, and 53,518 protein-coding genes were predicted in the current assembly. Comparative genomics analyses, including gene family expansion and contraction, whole-genome replication, phylogenetic analysis, and positive selection, were performed. Based on the obtained high-quality olive genome, a total of nine gene families with 202 genes were identified in the oleuropein biosynthesis pathway, which is twice the number of genes identified from the previous data. This new accession of the olive genome is of sufficient quality for genome-wide studies on gene function in olive and has provided a foundation for the molecular breeding of olive species.

The term ‘ornamental plant’ refers to all plants with ornamental value, which generally have beautiful flowers or special plant architectures. China is rich in ornamental plant resources and known as the “mother of gardens”. Genomics is the science of studying genomes and is useful for carrying out research on genome evolution, genomic variations, gene regulation, and important biological mechanisms based on detailed genome sequence information. Due to the diversity of ornamental plants and high sequencing costs, the progress of genome research on ornamental plants has been slow for a long time. With the emergence of new sequencing technologies and a reduction in costs since the whole-genome sequencing of the first ornamental plant (Prunus mume) was completed in 2012, whole-genome sequencing of more than 69 ornamental plants has been completed in <10 years. In this review, whole-genome sequencing and resequencing of ornamental plants will be discussed. We provide analysis with regard to basic data from whole-genome studies of important ornamental plants, the regulation of important ornamental traits, and application prospects.

Breeding crops for improved flavor is challenging due to the high cost of sensory evaluation and the difficulty of connecting sensory experience to chemical composition. The main goal of this study was to identify the chemical drivers of sweetness and consumer liking for fresh strawberries (Fragaria × ananassa). Fruit of 148 strawberry samples from cultivars and breeding selections were grown and harvested over seven years and were subjected to both sensory and chemical analyses. Each panel consisted of at least 100 consumers, resulting in more than 15,000 sensory data points per descriptor. Three sugars, two acids and 113 volatile compounds were quantified. Consumer liking was highly associated with sweetness intensity, texture liking, and flavor intensity, but not sourness intensity. Partial least square analyses revealed 20 volatile compounds that increased sweetness perception independently of sugars; 18 volatiles that increased liking independently of sugars; and 15 volatile compounds that had positive effects on both. Machine learning-based predictive models including sugars, acids, and volatiles explained at least 25% more variation in sweetness and liking than models accounting for sugars and acids only. Volatile compounds such as γ-dodecalactone; 5-hepten-2-one, 6-methyl; and multiple medium-chain fatty acid esters may serve as targets for breeding or quality control attributes for strawberry products. A genetic association study identified two loci controlling ester production, both on linkage group 6 A. Co-segregating makers in these regions can be used for increasing multiple esters simultaneously. This study demonstrates a paradigm for improvement of fruit sweetness and flavor in which consumers drive the identification of the most important chemical targets, which in turn drives the discovery of genetic targets for marker-assisted breeding.

Ultraviolet-B (UV-B) light (280–315 nm) is an important environmental signal that regulates plant development and photomorphogenesis, while also affecting the flavonoid pathway, including anthocyanin biosynthesis. Regarding the effects of UV-B radiation on fruits, the effects of a short-term or postharvest irradiation on fruit quality have been well-documented, but the effects of a long-term preharvest UV-B irradiation on fruit growth and coloration remain unclear. Thus, in this study, we investigated the effects of a long-term treatment involving an environmentally relevant UV-B dose on highbush blueberry ( Vaccinium corymbosum) fruit. The preharvest UV-B treatment quickly promoted fruit growth and sugar accumulation, which is not commonly observed in other fruit tree species. The UV-B exposure also accelerated fruit ripening and coloration. The dual-luciferase assay proved that in blueberries, expression of VcUFGT encoding anthocyanin biosynthesis key enzyme, is positively and negatively regulated by VcMYBA1 and VcMYBC2, respectively. Throughout the fruit development stage, the UV-B treatment up-regulated VcMYBPA1 expression, which increased VcUFGT expression via VcMYBA1. In the green fruit stage, the UV-B treatment increased HY5 encoding UV receptor, which up-regulates VcMYBPA1 and down-regulates VcMYBC2, thereby promotes the accumulation of anthocyanins. On the other hand, excessive anthocyanin synthesis was inhibited by increased VcMYBC2 levels in mature fruits when exposed to UV-B light through HY5-independent pathway. In conclusion, anthocyanin-related MYB activators and repressor may coordinately balance the accumulation of anthocyanins in blueberry fruits, with UV-B treatments possibly influencing their effects in a stage-specific manner. The potential utility of preharvest UV-B treatments for improving blueberry fruit quality is discussed herein.

The leafy head characteristic is a special phenotype of Chinese cabbage resulting from artificial selection during domestication and breeding. BREVIS RADIX (BRX) has been suggested to control root elongation, shoot growth, and tiller angle in Arabidopsis and rice. In Brassica rapa, three BrBRX homoeologs have been identified, but only BrBRX.1 and BrBRX.2 were found to be under selection in leaf-heading accessions, indicating their functional diversification in leafy head formation. Here, we show that these three BrBRX genes belong to a plant-specific BRX gene family but that they have significantly diverged from other BRX-like members on the basis of different phylogenetic classifications, motif compositions and expression patterns. Moreover, although the expression of these three BrBRX genes differed, compared with BrBRX.3, BrBRX.1, and BrBRX.2 displayed similar expression patterns. Arabidopsis mutant complementation studies showed that only BrBRX.1 could rescue the brx root phenotype, whereas BrBRX.2 and BrBRX.3 could not. However, overexpression of each of the three BrBRX genes in Arabidopsis resulted in similar pleiotropic leaf phenotypes, including epinastic leaf morphology, with an increase in leaf number and leaf petiole length and a reduction in leaf angle. These leaf traits are associated with leafy head formation. Further testing of a SNP (T/C) in BrBRX.2 confirmed that this allele in the heading accessions was strongly associated with the leaf-heading trait of B. rapa. Our results revealed that all three BrBRX genes may be involved in the leaf-heading trait, but they may have functionally diverged on the basis of their differential expression.

Grafting is an ancient technique used for plant propagation and improvement in horticultural crops for at least 1,500 years. Citrus plants, with a seed-to-seed cycle of 5–15 years, are among the fruit crops that were probably domesticated by grafting. Poncirus trifoliata, a widely used citrus rootstock, can promote early flowering, strengthen stress tolerance, and improve fruit quality via scion–rootstock interactions. Here, we report its genome assembly using PacBio sequencing. We obtained a final genome of 303 Mb with a contig N50 size of 1.17 Mb and annotated 25,680 protein-coding genes. DNA methylome and transcriptome analyses indicated that the strong adaptability of P. trifoliata is likely attributable to its special epigenetic modification and expression pattern of resistance-related genes. Heterografting by using sweet orange as scion and P. trifoliata as rootstock and autografting using sweet orange as both scion and rootstock were performed to investigate the genetic effects of the rootstock. Single-base methylome analysis indicated that P. trifoliata as a rootstock caused DNA demethylation and a reduction in 24-nt small RNAs (sRNAs) in scions compared to the level observed with autografting, implying the involvement of sRNA-mediated graft-transmissible epigenetic modifications in citrus grafting. Taken together, the assembled genome for the citrus rootstock and the analysis of graft-induced epigenetic modifications provide global insights into the genetic effects of rootstock–scion interactions and grafting biology.

Abscisic acid (ABA) is a critical regulator of seed development and germination. β-glucosidases (BGs) have been suggested to be contributors to increased ABA content because they catalyze the hydrolysis of ABA-glucose ester to release free ABA. However, whether BGs are involved in seed development is unclear. In this study, a candidate gene, ClBG1, in watermelon was selected for targeted mutagenesis via the CRISPR/Cas9 system. Seed size and weight were significantly reduced in the Clbg1-mutant watermelon lines, which was mainly attributed to decreased cell number resulting from decreased ABA levels. A transcriptome analysis showed that the expression of 1015 and 1429 unique genes was changed 10 and 18 days after pollination (DAP), respectively. Cytoskeleton- and cell cycle-related genes were enriched in the differentially expressed genes of wild type and Clbg1-mutant lines during seed development. Moreover, the expression of genes in the major signaling pathways of seed size control was also changed. In addition, seed germination was promoted in the Clbg1-mutant lines due to decreased ABA content. These results indicate that ClBG1 may be critical for watermelon seed size regulation and germination mainly through the modulation of ABA content and thereby the transcriptional regulation of cytoskeleton-, cell cycle- and signaling-related genes. Our results lay a foundation for dissecting the molecular mechanisms of controlling watermelon seed size, a key agricultural trait of significant economic importance.

Nitric oxide (NO) regulates plant growth, enhances nutrient uptake, and activates disease and stress tolerance mechanisms in most plants, making NO a potential tool for use in improving the yield and quality of horticultural crop species. Although the use of NO in horticulture is still in its infancy, research on NO in model plant species has provided an abundance of valuable information on horticultural crop species. Emerging evidence implies that the bioactivity of NO can occur through many potential mechanisms but occurs mainly through S-nitrosation, the covalent and reversible attachment of NO to cysteine thiol. In this context, NO signaling specifically affects crop development, immunity, and environmental interactions. Moreover, NO can act as a fumigant against a wide range of postharvest diseases and pests. However, for effective use of NO in horticulture, both understanding and exploring the biological significance and potential mechanisms of NO in horticultural crop species are critical. This review provides a picture of our current understanding of how NO is synthesized and transduced in plants, and particular attention is given to the significance of NO in breaking seed dormancy, balancing root growth and development, enhancing nutrient acquisition, mediating stress responses, and guaranteeing food safety for horticultural production.

Most molecularly characterized plant resistance genes (R genes) belong to the nucleotide-binding-site-leucine-rich-repeat (NLR) receptor family and are prone to duplication and transposition with high sequence diversity. In this family, the Vat gene in melon is one of the few R genes known for conferring resistance to insect, i.e., Aphis gossypii, but it has been misassembled and/or mispredicted in the whole genomes of Cucurbits. We examined 14 genomic regions (about 400 kb) derived from long-read assemblies spanning Vat-related genes in Cucumis melo, Cucumis sativus, Citrullus lanatus, Benincasa hispida, Cucurbita argyrosperma, and Momordica charantia. We built the phylogeny of those genes. Investigating the paleohistory of the Vat gene cluster, we revealed a step by step process beginning from a common ancestry in cucurbits older than 50 my. We highlighted Vat exclusively in the Cucumis genera, which diverged about 20 my ago. We then focused on melon, evaluating a minimum duplication rate of Vat in 80 wild and cultivated melon lines using generalist primers; our results suggested that duplication started before melon domestication. The phylogeny of 44 Vat-CDS obtained from 21 melon lines revealed gain and loss of leucine-rich-repeat domains along diversification. Altogether, we revealed the high putative recognition scale offered in melon based on a combination of SNPs, number of leucine-rich-repeat domains within each homolog and number of homologs within each cluster that might jointly confer resistance to a large pest and pathogen spectrum. Based on our findings, we propose possible avenues for breeding programs.

Abiotic stresses have a negative effect on crop production, affecting both vegetative and reproductive development. Ethylene plays a relevant role in plant response to environmental stresses, but the specific contribution of ethylene biosynthesis and signalling components in the salt stress response differs between Arabidopsis and rice, the two most studied model plants. In this paper, we study the effect of three gain-of-function mutations affecting the ethylene receptors CpETR1B, CpETR1A, and CpETR2B of Cucurbita pepo on salt stress response during germination, seedling establishment, and subsequent vegetative growth of plants. The mutations all reduced ethylene sensitivity, but enhanced salt tolerance, during both germination and vegetative growth, demonstrating that the three ethylene receptors play a positive role in salt tolerance. Under salt stress, etr1b, etr1a, and etr2b germinate earlier than WT, and the root and shoot growth rates of both seedlings and plants were less affected in mutant than in WT. The enhanced salt tolerance response of the etr2b plants was associated with a reduced accumulation of Na⁺ in shoots and leaves, as well as with a higher accumulation of compatible solutes, including proline and total carbohydrates, and antioxidant compounds, such as anthocyanin. Many membrane monovalent cation transporters, including Na⁺/H⁺ exchangers (NHXs), K⁺ efflux antiporters (KEAs), high-affinity K⁺ transporters (HKTs), and K⁺ uptake transporters (KUPs) were also highly upregulated by salt in etr2b in comparison with WT. In aggregate, these data indicate that the enhanced salt tolerance of the mutant is led by the induction of genes that exclude Na⁺ in photosynthetic organs, while maintaining K⁺/Na⁺ homoeostasis and osmotic adjustment. If the salt response of etr mutants occurs via the ethylene signalling pathway, our data show that ethylene is a negative regulator of salt tolerance during germination and vegetative growth. Nevertheless, the higher upregulation of genes involved in Ca²⁺ signalling (CpCRCK2A and CpCRCK2B) and ABA biosynthesis (CpNCED3A and CpNCED3B) in etr2b leaves under salt stress likely indicates that the function of ethylene receptors in salt stress response in C. pepo can be mediated by Ca²⁺ and ABA signalling pathways.

Grafting is a highly useful technique, and its success largely depends on graft union formation. In this study, we found that root-specific expression of the auxin biosynthetic gene iaaM in tobacco, when used as rootstock, resulted in more rapid callus formation and faster graft healing. However, overexpression of the auxin-inactivating iaaL gene in rootstocks delayed graft healing. We observed increased endogenous auxin levels and auxin-responsive DR5::GUS expression in scions of WT/ iaaM grafts compared with those found in WT/WT grafts, which suggested that auxin is transported upward from rootstock to scion tissues. A transcriptome analysis showed that auxin enhanced graft union formation through increases in the expression of genes involved in graft healing in both rootstock and scion tissues. We also observed that the ethylene biosynthetic gene ACS1 and the ethylene-responsive gene ERF5 were upregulated in both scions and rootstocks of the WT/ iaaM grafts. Furthermore, exogenous applications of the ethylene precursor ACC to the junction of WT/WT grafts promoted graft union formation, whereas application of the ethylene biosynthesis inhibitor AVG delayed graft healing in WT/WT grafts, and the observed delay was less pronounced in the WT/ iaaM grafts. These results demonstrated that elevated auxin levels in the iaaM rootstock in combination with the increased auxin levels in scions caused by upward transport/diffusion enhanced graft union formation and that ethylene was partially responsible for the effects of auxin on grafting. Our findings showed that grafting success can be enhanced by increasing the auxin levels in rootstocks using transgenic or gene-editing techniques.

Waxy apple cuticles predominantly accumulate ursane-type triterpenes, but the profile shifts with the induction of skin russeting towards lupane-type triterpenes. We previously characterised several key enzymes in the ursane-type and lupane-type triterpene pathways, but this switch in triterpene metabolism associated with loss of cuticle integrity is not fully understood. To analyse the relationship between triterpene biosynthesis and russeting, we used microscopy, RNA-sequencing and metabolite profiling during apple fruit development. We compared the skin of three genetically-close clones of ‘Golden Delicious’ (with waxy, partially russeted and fully russeted skin). We identified a unique molecular profile for the russet clone, including low transcript abundance of multiple cuticle-specific metabolic pathways in the early stages of fruit development. Using correlation analyses between gene transcription and metabolite concentration we found MYB transcription factors strongly associated with lupane-type triterpene biosynthesis. We showed how their transcription changed with the onset of cuticle cracking followed by russeting and that one factor, MYB66, was able to bind the promoter of the oxidosqualene cyclase OSC5, to drive the production of lupeol derivatives. These results provide insights into the breakdown of cuticle integrity leading to russet and how this drives MYB-regulated changes to triterpene biosynthesis.

Blooming seasonality is an important trait in ornamental plants and was selected by humans. Wild roses flower only in spring whereas most cultivated modern roses can flower continuously. This trait is explained by a mutation of a floral repressor gene, RoKSN, a TFL1 homologue. In this work, we studied the origin, the diversity and the selection of the RoKSN gene. We analyzed 270 accessions, including wild and old cultivated Asian and European roses as well as modern roses. By sequencing the RoKSN gene, we proposed that the allele responsible for continuous-flowering, RoKSN^copia, originated from Chinese wild roses (Indicae section), with a recent insertion of the copia element. Old cultivated Asian roses with the RoKSN^copia allele were introduced in Europe, and the RoKSN^copia allele was progressively selected during the 19th and 20th centuries, leading to continuous-flowering modern roses. Furthermore, we detected a new allele, RoKSN^A181, leading to a weak reblooming. This allele encodes a functional floral repressor and is responsible for a moderate accumulation of RoKSN transcripts. A transient selection of this RoKSN^A181 allele was observed during the 19th century. Our work highlights the selection of different alleles at the RoKSN locus for recurrent blooming in rose.

MYB transcription factors regulate diverse aspects of plant development and secondary metabolism, often by partnering in transcriptional regulatory complexes. Here, we harness genomic resources to identify novel MYBs, thereby producing an updated eudicot MYB phylogeny with revised relationships among subgroups as well as new information on sequence variation in the disordered C-terminus of anthocyanin-activating MYBs. BLAST® and hidden Markov model scans of gene annotations identified a total of 714 MYB transcription factors across the genomes of four crops that span the eudicots: apple, grape, kiwifruit and tomato. Codon model-based phylogenetic inference identified novel members of previously defined subgroups, and the function of specific anthocyanin-activating subgroup 6 members was assayed transiently in tobacco leaves. Sequence conservation within subgroup 6 highlighted one previously described and two novel short linear motifs in the disordered C-terminal region. The novel motifs have a mix of hydrophobic and acidic residues and are predicted to be relatively ordered compared with flanking protein sequences. Comparison of motifs with the Eukaryotic Linear Motif database suggests roles in protein–protein interaction. Engineering of motifs and their flanking regions from strong anthocyanin activators into weak activators, and vice versa, affected function. We conclude that, although the MYB C-terminal sequence diverges greatly even within MYB clades, variation within the C-terminus at and near relatively ordered regions offers opportunities for exploring MYB function and developing superior alleles for plant breeding.

Abscission, a cell separation process, is an important trait that influences grain and fruit yield. We previously reported that BEL1-LIKE HOMEODOMAIN 4 ( SlBL4) is involved in chloroplast development and cell wall metabolism in tomato fruit. In the present study, we showed that silencing SlBL4 resulted in the enlargement and pre-abscission of the tomato (Solanum lycopersicum cv. Micro-TOM) fruit pedicel. The anatomic analysis showed the presence of more epidermal cell layers and no obvious abscission zone (AZ) in the SlBL4 RNAi lines compared with the wild-type plants. RNA-seq analysis indicated that the regulation of abscission by SlBL4 was associated with the altered abundance of genes related to key meristems, auxin transporters, signaling components, and cell wall metabolism. Furthermore, SlBL4 positively affected the auxin concentration in the abscission zone. A dual-luciferase reporter assay revealed that SlBL4 activated the transcription of the JOINTLESS, OVATE, PIN1, and LAX3 genes. We reported a novel function of SlBL4, which plays key roles in fruit pedicel organogenesis and abscission in tomatoes.

The CmBBX8-CmFTL1 regulatory module is a key determinant in the transition from vegetative growth to reproductive development in summer-flowering chrysanthemum. However, the detailed regulatory mechanism of CmBBX8-mediated flowering remains elusive. In this study, we revealed that RADICAL-INDUCED CELL DEATH 1 (CmRCD1) physically associated with CmBBX8 through bimolecular fluorescence complementation (BiFC), pulldown and Coimmunoprecipitation (CoIP) assays. Furthermore, the RCD1-SRO1-TAF4 (RST) domain of CmRCD1 and the B-box of CmBBX8 mediated their interaction. In addition, Luciferase (LUC) assays and electrophoretic mobility shift assay (EMSAs) showed that CmRCD1 repressed the transcriptional activity of CmBBX8 and interfered with its binding to the CmFTL1 promoter, thereby leading to delayed flowering in the summer chrysanthemum ‘Yuuka’. These results provide insight into the molecular framework of CmRCD1-CmBBX8-mediated flowering in chrysanthemum.

The aluminum (Al) cation Al³⁺ in acidic soil shows severe rhizotoxicity that inhibits plant growth and development. Most woody plants adapted to acidic soils have evolved specific strategies against Al³⁺ toxicity, but the underlying mechanism remains elusive. The four-carbon amino acid gamma-aminobutyric acid (GABA) has been well studied in mammals as an inhibitory neurotransmitter; GABA also controls many physiological responses during environmental or biotic stress. The woody plant hybrid Liriodendron (L. chinense × tulipifera) is widely cultivated in China as a horticultural tree and provides high-quality timber; studying its adaptation to high Al stress is important for harnessing its ecological and economic potential. Here, we performed quantitative iTRAQ (isobaric tags for relative and absolute quantification) to study how protein expression is altered in hybrid Liriodendron leaves subjected to Al stress. Hybrid Liriodendron shows differential accumulation of several proteins related to cell wall biosynthesis, sugar and proline metabolism, antioxidant activity, cell autophagy, protein ubiquitination degradation, and anion transport in response to Al damage. We observed that Al stress upregulated glutamate decarboxylase (GAD) and its activity, leading to increased GABA biosynthesis. Additional GABA synergistically increased Al-induced antioxidant enzyme activity to efficiently scavenge ROS, enhanced proline biosynthesis, and upregulated the expression of MATE1/2, which subsequently promoted the efflux of citrate for chelation of Al³⁺. We also showed similar effects of GABA on enhanced Al³⁺ tolerance in Arabidopsis. Thus, our findings suggest a function of GABA signaling in enhancing hybrid Liriodendron tolerance to Al stress through promoting organic acid transport and sustaining the cellular redox and osmotic balance.

Water deficit is one of the major limiting factors for apple (Malus domestica) production on the Loess Plateau, a major apple cultivation area in China. The identification of genes related to the regulation of water use efficiency (WUE) is a crucial aspect of crop breeding programs. As a conserved degradation and recycling mechanism in eukaryotes, autophagy has been reported to participate in various stress responses. However, the relationship between autophagy and WUE regulation has not been explored. We have shown that a crucial autophagy protein in apple, MdATG8i, plays a role in improving salt tolerance. Here, we explored its biological function in response to long-term moderate drought stress. The results showed that MdATG8i-overexpressing (MdATG8i-OE) apple plants exhibited higher WUE than wild-type (WT) plants under long-term moderate drought conditions. Plant WUE can be increased by improving photosynthetic efficiency. Osmoregulation plays a critical role in plant stress resistance and adaptation. Under long-term drought conditions, the photosynthetic capacity and accumulation of sugar and amino acids were higher in MdATG8i-OE plants than in WT plants. The increased photosynthetic capacity in the OE plants could be attributed to their ability to maintain optimal stomatal aperture, organized chloroplasts, and strong antioxidant activity. MdATG8i overexpression also promoted autophagic activity, which was likely related to the changes described above. In summary, our results demonstrate that MdATG8i-OE apple lines exhibited higher WUE than WT under long-term moderate drought conditions because they maintained robust photosynthesis, effective osmotic adjustment processes, and strong autophagic activity.

Potato invertase inhibitor (StInvInh2) positively regulates cold-induced sweetening (CIS) resistance by inhibiting the activity of vacuolar invertase. The distinct expression patterns of StInvInh2 have been thoroughly characterized in different potato genotypes, but the related CIS ability has not been characterized. The understanding of the regulatory mechanisms that control StInvInh2 transcription is unclear. In this study, we identified an ERF‐VII transcription factor, StRAP2.3, that directly regulates StInvInh2 to positively modulate CIS resistance. Acting as a nuclear-localized transcriptional activator, StRAP2.3 directly binds the ACCGAC cis-element in the promoter region of StInvInh2, enabling promoter activity. Overexpression of StRAP2.3 in CIS-sensitive potato tubers induced StInvInh2 mRNA abundance and increased CIS resistance. In contrast, silencing StRAP2.3 in CIS-resistant potato tubers repressed the expression of StInvInh2 and decreased CIS resistance. We conclude that cold-responsive StInvInh2 is due to the binding of StRAP2.3 to the ACCGAC cis-element in the promoter region of StInvInh2. Overall, these findings indicate that StRAP2.3 directly regulates StInvInh2 to positively modulate CIS resistance, which may provide a strategy to improve the processing quality of potatoes.

Light plays a critical role in plant growth and development, but the mechanisms through which light regulates fruit ripening and nutritional quality in horticultural crops remain largely unknown. Here, we found that ELONGATED HYPOCOTYL 5 (HY5), a master regulator in the light signaling pathway, is required for normal fruit ripening in tomato (Solanum lycopersicum). Loss of function of tomato HY5 (SlHY5) impairs pigment accumulation and ethylene biosynthesis. Transcriptome profiling identified 2948 differentially expressed genes, which included 1424 downregulated and 1524 upregulated genes, in the Slhy5 mutants. In addition, genes involved in carotenoid and anthocyanin biosynthesis and ethylene signaling were revealed as direct targets of SlHY5 by chromatin immunoprecipitation. Surprisingly, the expression of a large proportion of genes encoding ribosomal proteins was downregulated in the Slhy5 mutants, and this downregulation pattern was accompanied by a decrease in the abundance of ribosomal proteins. Further analysis demonstrated that SlHY5 affected the translation efficiency of numerous ripening-related genes. These data indicate that SlHY5 regulates fruit ripening both at the transcriptional level by targeting specific molecular pathways and at the translational level by affecting the protein translation machinery. Our findings unravel the regulatory mechanisms of SlHY5 in controlling fruit ripening and nutritional quality and uncover the multifaceted regulation of gene expression by transcription factors.

Drought leads to reductions in plant growth and crop yields. Arbuscular mycorrhizal fungi (AMF), which form symbioses with the roots of the most important crop species, alleviate drought stress in plants. In the present work, we identified 14 GH3 genes in apple (Malus domestica) and provided evidence that MdGH3-2 and MdGH3-12 play important roles during AM symbiosis. The expression of both MdGH3-2 and MdGH3-12 was upregulated during mycorrhization, and the silencing of MdGH3-2/12 had a negative impact on AM colonization. MdGH3-2/12 silencing resulted in the downregulation of five genes involved in strigolactone synthesis, and there was a corresponding change in root strigolactone content. Furthermore, we observed lower root dry weights in RNAi lines under AM inoculation conditions. Mycorrhizal transgenic plants showed greater sensitivity to drought stress than WT, as indicated by their higher relative electrolytic leakage and lower relative water contents, osmotic adjustment ability, ROS scavenging ability, photosynthetic capacity, chlorophyll fluorescence values, and abscisic acid contents. Taken together, these data demonstrate that MdGH3-2/12 plays an important role in AM symbiosis and drought stress tolerance in apple.

The conserved histone variant H2A.Z is essential for transcriptional regulation; defense responses; and various biological processes in plants, such as growth, development, and flowering. However, little is known about how H2A.Z affects the developmental process and ripening of tomato fruits. Here, we utilized the CRISPR/Cas9 gene-editing system to generate a sl_hta9 sl_hta11 double-mutant, designated sl_h2a.z, and found that these two mutations led to a significant reduction in the fresh weight of tomato fruits. Subsequent messenger RNA (mRNA)-seq results showed that dysfunction of Sl_H2A.Z has profound effects on the reprogramming of genome-wide gene expression at different developmental stages of tomato fruits, indicating a ripening-dependent correlation between Sl_H2A.Z and gene expression regulation in tomato fruits. In addition, the expression of three genes, SlPSY1, SlPDS, and SlVDE, encoding the key enzymes in the biosynthesis pathway of carotenoids, was significantly upregulated in the later ripening stages, which was consistent with the increased contents of carotenoids in sl_h2a.z double-mutant fruits. Overall, our study reveals a role of Sl_H2A.Z in the regulation of carotenoids and provides a resource for the study of Sl_H2A.Z-dependent gene expression regulation. Hence, our results provide a link between epigenetic regulation via histone variants and fruit development, suggesting a conceptual framework to understand how histone variants regulate tomato fruit quality.

Comprehensive untargeted and targeted analysis of root exudate composition has advanced our understanding of rhizosphere processes. However, little is known about exudate spatial distribution and regulation. We studied the specific metabolite signatures of asparagus root exudates, root outer (epidermis and exodermis), and root inner tissues (cortex and vasculature). The greatest differences were found between exudates and root tissues. In total, 263 non-redundant metabolites were identified as significantly differentially abundant between the three root fractions, with the majority being enriched in the root exudate and/or outer tissue and annotated as ‘lipids and lipid-like molecules’ or ‘phenylpropanoids and polyketides’. Spatial distribution was verified for three selected compounds using MALDI-TOF mass spectrometry imaging. Tissue-specific proteome analysis related root tissue-specific metabolite distributions and rhizodeposition with underlying biosynthetic pathways and transport mechanisms. The proteomes of root outer and inner tissues were spatially very distinct, in agreement with the fundamental differences between their functions and structures. According to KEGG pathway analysis, the outer tissue proteome was characterized by a high abundance of proteins related to ‘lipid metabolism’, ‘biosynthesis of other secondary metabolites’ and ‘transport and catabolism’, reflecting its main functions of providing a hydrophobic barrier, secreting secondary metabolites, and mediating water and nutrient uptake. Proteins more abundant in the inner tissue related to ‘transcription’, ‘translation’ and ‘folding, sorting and degradation’, in accord with the high activity of cortical and vasculature cell layers in growth- and development-related processes. In summary, asparagus root fractions accumulate specific metabolites. This expands our knowledge of tissue-specific plant cell function.

Chrysanthemum (Chrysanthemum morifolium) is one of the four major cut-flower plants worldwide and possesses both high ornamental value and cultural connotation. As most chrysanthemum varieties flower in autumn, it is costly to achieve annual production. JAZ genes in the TIFY family are core components of the jasmonic acid (JA) signaling pathway; in addition to playing a pivotal role in plant responses to defense, they are also widely implicated in regulating plant development processes. Here, we characterized the TIFY family gene CmJAZ1-like from the chrysanthemum cultivar ‘Jinba’. CmJAZ1-like localizes in the nucleus and has no transcriptional activity in yeast. Tissue expression pattern analysis indicated that CmJAZ1-like was most active in the root and shoot apex. Overexpressing CmJAZ1-like with Jas domain deletion in chrysanthemum resulted in late flowering. RNA-Seq analysis of the overexpression lines revealed some differentially expressed genes (DEGs) involved in flowering, such as the homologs of the flowering integrators FT and SOC1, an FUL homolog involved in flower meristem identity, AP2 domain-containing transcription factors, MADS box genes, and autonomous pathway-related genes. Based on KEGG pathway enrichment analysis, the differentially transcribed genes were enriched in carbohydrate metabolic and fatty acid-related pathways, which are notable for their role in flowering in plants. This study preliminarily verified the function of CmJAZ1-like in chrysanthemum flowering, and the results can be used in molecular breeding programs aimed at flowering time regulation of chrysanthemum.

Drought severely limits plant development and growth; accordingly, plants have evolved strategies to prevent water loss and adapt to water deficit conditions. However, experimental cases that corroborate these evolutionary processes are limited. The LACCASEs (LACs) family is involved in various plant development and growth processes. Here, we performed an evolutionary analysis of LACs from Populus euphratica and characterized the functions of LACs in Arabidopsis and poplar. The results showed that in PeuLACs, multiple gene duplications led to apparent functional redundancy as the result of various selective pressures. Among them, PeuLAC2 underwent strong positive selection. Heterologous expression analyses showed that the overexpression of PeuLAC2 alters the xylem structure of plants, including thickening the secondary cell wall (SCW) and increasing the fiber cell length and stem tensile strength. Altogether, these changes improve the water transport capacity of plants. The analysis of the physiological experimental results showed that PeuLAC2-OE lines exhibited a stronger antioxidant response and greater drought tolerance than WT. Three genes screened by transcriptome analysis, NAC025, BG1, and UGT, that are associated with SCW synthesis and drought stress were all upregulated in the PeuLAC2-OE lines, implying that the overexpression of PeuLAC2 thickened the SCW, improved the water transport capacity of the plant, and further enhanced its drought tolerance. Our study highlights that genes that have undergone adaptive evolution may participate in the development of adaptive traits in P. euphratica and that PeuLAC2 could be a candidate gene for molecular genetic breeding in trees.

Hemerocallis citrina Borani (huang hua cai in Chinese) is an important horticultural crop whose flower buds are widely consumed as a delicious vegetable in Asia. Here we assembled a high-quality reference genome of H. citrina using single-molecule sequencing and Hi-C technologies. The genome assembly was 3.77 Gb and consisted of 3183 contigs with a contig N50 of 2.09 Mb, which were further clustered into 11 pseudochromosomes. A larger portion (3.25 Gb or 86.20%) was annotated as a repetitive content and 54,295 protein-coding genes were annotated in the genome. Genome evolution analysis showed that H. citrina experienced a recent whole-genome duplication (WGD) event at ~15.73 million years ago (Mya), which was the main factor leading to many multiple copies of orthologous genes. We used this reference genome to predict 20 genes involved in the rutin biosynthesis pathway. Moreover, our metabolomics data revealed neither colchicine nor its precursors in H. citrina, challenging the long-standing belief that this alkaloid causes poisoning by the plant. The results of our disruptive research are further substantiated by our genomic finding that H. citrina does not contain any genes involved in colchicine biosynthesis. The high-quality genome lays a solid foundation for genetic research and molecular breeding of H. citrina.

Leaf size influences plant development and biomass and is also an important agricultural trait in Brassica crops, in which leaves are the main organ produced for consumption. Leaf size is determined by the coordinated regulation of cell proliferation and cell expansion during leaf development, and these processes are strictly controlled by various integrated signals from the intrinsic regulatory network and the growth environment. Understanding the molecular mechanism of leaf size control is a prerequisite for molecular breeding for crop improvement purposes. Although research on leaf size control is just beginning in Brassica, recent studies have identified several genes and QTLs that are important in leaf size regulation. These genes have been proposed to influence leaf growth through different pathways and mechanisms, including phytohormone biosynthesis and signaling, transcription regulation, small RNAs, and others. In this review, we summarize the current findings regarding the genetic regulators of leaf size in Brassica and discuss future prospects for this research.

Endive (Cichorium endivia L.), a vegetable consumed as fresh or packaged salads, is mostly cultivated outdoors and known to be sensitive to waterlogging in terms of yield and quality. Phenotypic, metabolic and transcriptomic analyses were used to study variations in curly- (‘Domari’, ‘Myrna’) and smooth-leafed (‘Flester’, ‘Confiance’) cultivars grown in short-term waterlog due to rainfall excess before harvest. After recording loss of head weights in all cultivars (6-35%), which was minimal in ‘Flester’, NMR untargeted profiling revealed variations as influenced by genotype, environment and interactions, and included drop of total carbohydrates (6–50%) and polyols (3–37%), gain of organic acids (2–30%) and phenylpropanoids (98–560%), and cultivar-specific fluctuations of amino acids (−37 to +15%). The analysis of differentially expressed genes showed GO term enrichment consistent with waterlog stress and included the carbohydrate metabolic process. The loss of sucrose, kestose and inulin recurred in all cultivars and the sucrose-inulin route was investigated by covering over 50 genes of sucrose branch and key inulin synthesis (fructosyltransferases) and catabolism (fructan exohydrolases) genes. The lowered expression of a sucrose gene subset together with that of SUCROSE:SUCROSE-1-FRUCTOSYLTRANSFERASE (1-SST) may have accounted for sucrose and kestose contents drop in the leaves of waterlogged plants. Two anti-correlated modules harbouring candidate hub-genes, including 1-SST, were identified by weighted gene correlation network analysis, and proposed to control positively and negatively kestose levels. In silico analysis further pointed at transcription factors of GATA, DOF, WRKY types as putative regulators of 1-SST.

Cynodon species can be used for multiple purposes and have high economic and ecological significance. However, the genetic basis of the favorable agronomic traits of Cynodon species is poorly understood, partially due to the limited availability of genomic resources. In this study, we report a chromosome-scale genome assembly of a diploid Cynodon species, C. transvaalensis, obtained by combining Illumina and Nanopore sequencing, BioNano, and Hi-C. The assembly contains 282 scaffolds (~423.42 Mb, N50 = 5.37 Mb), which cover ~93.2% of the estimated genome of C. transvaalensis (~454.4 Mb). Furthermore, 90.48% of the scaffolds (~383.08 Mb) were anchored to nine pseudomolecules, of which the largest was 60.78 Mb in length. Evolutionary analysis along with transcriptome comparison provided a preliminary genomic basis for the adaptation of this species to tropical and/or subtropical climates, typically with dry summers. The genomic resources generated in this study will not only facilitate evolutionary studies of the Chloridoideae subfamily, in particular, the Cynodonteae tribe, but also facilitate functional genomic research and genetic breeding in Cynodon species for new leading turfgrass cultivars in the future.

Low temperature causes anther dysfunction, severe pollen sterility and, ultimately, major yield losses in crop plants. Previous studies have shown that the gibberellic acid (GA) metabolic pathway plays an important role in this process by regulating tapetum function and pollen development. However, the interaction mechanism of GA with other hormones mediating anther development is still unclear. Herein, we collected and analyzed almond (Amygdalus communis L.) anthers at the meiosis, tetrad, 1-nucleus, and mature 2-nucleus stages. The growth rate per 1000 anthers exhibited a significant positive correlation with the total bioactive GA compound content, and the levels of all bioactive GA compounds were highest in the 1-nucleus pollen stage. GA₃ treatment experiments indicated that exogenous GA₃ increased the levels of indole-3-acetic acid (IAA), trans-zeatin (tZ), and jasmonic acid (JA) and decreased the levels of salicylic acid (SA) and abscisic acid (ABA); moreover, GA₃ improved pollen viability and quantities under cold conditions, whereas PP₃₃₃ (paclobutrazol, an inhibitor of GA biosynthesis) was antagonistic with GA₃ in controlling anther development. RNA-seq and qRT-PCR results showed that GA played an important role in anther development by regulating the expression of other phytohormone pathway genes, dehydration-responsive element-binding/C-repeat binding factor (DREB1/CBF)-mediated signaling genes, and anther development pathway genes. Our results reveal the novel finding that GA interacts with other hormones to balance anther development under normal- and low-temperature conditions in almond.

In response to preharvest priming with exogenous methyl jasmonate (MeJA), tea plants adjust their physiological behavior at the molecular level. The whole-organism reconfiguration of aroma formation from the precursor to storage is poorly understood. In this study, we performed iTRAQ proteomic analysis and identified 337, 246, and 413 differentially expressed proteins in tea leaves primed with MeJA for 12 h, 24 h, and 48 h, respectively. Furthermore, a total of 266 nonvolatile and 100 volatile differential metabolites were identified by utilizing MS-based metabolomics. A novel approach that incorporated the integration of extended self-organizing map-based dimensionality was applied. The vivid time-scale changes tracing physiological responses in MeJA-primed tea leaves are marked in these maps. Jasmonates responded quickly to the activation of the jasmonic acid pathway in tea leaves, while hydroxyl and glycosyl jasmonates were biosynthesized simultaneously on a massive scale to compensate for the exhausted defense. The levels of α-linolenic acid, geranyl diphosphate, farnesyl diphosphate, geranylgeranyl diphosphate, and phenylalanine, which are crucial aroma precursors, were found to be significantly changed in MeJA-primed tea leaves. Green leaf volatiles, volatile terpenoids, and volatile phenylpropanoids/benzenoids were spontaneously biosynthesized from responding precursors and subsequently converted to their corresponding glycosidic forms, which can be stably stored in tea leaves. This study elucidated the physiological response of tea leaves primed with exogenous methyl jasmonate and revealed the molecular basis of source and sink changes on tea aroma biosynthesis and catabolism in response to exogenous stimuli. The results significantly enhance our comprehensive understanding of tea plant responses to exogenous treatment and will lead to the development of promising biotechnologies to improve fresh tea leaf quality.

Plants have evolved regulatory mechanisms at multiple levels to regulate gene expression in order to improve their cold adaptability. However, limited information is available regarding the stress response at the chromatin and translational levels. Here, we characterize the chromatin accessibility, transcriptional, and translational landscapes of tea plants in vivo under chilling stress for the first time. Chilling stress significantly affected both the transcription and translation levels as well as the translation efficiency of tea plants. A total of 3010 genes that underwent rapid and independent translation under chilling stress were observed, and they were significantly enriched in the photosynthesis-antenna protein and phenylpropanoid biosynthesis pathways. A set of genes that were significantly responsive to cold at the transcription and translation levels, including four (+)-neomenthol dehydrogenases (MNDs) and two (E)-nerolidol synthases (NESs) arranged in tandem on the chromosomes, were also found. We detected potential upstream open reading frames (uORFs) on 3082 genes and found that tea plants may inhibit the overall expression of genes by enhancing the translation of uORFs under chilling stress. In addition, we identified distal transposase hypersensitive sites (THSs) and proximal THSs and constructed a transcriptional regulatory network for tea plants under chilling stress. We also identified 13 high-confidence transcription factors (TFs) that may play a crucial role in cold regulation. These results provide valuable information regarding the potential transcriptional regulatory network in plants and help to clarify how plants exhibit flexible responses to chilling stress.

Beta-amylase (BAM) plays an important role in plant resistance to cold stress. However, the specific role of the BAM gene in freezing tolerance is poorly understood. In this study, we demonstrated that a cold-responsive gene module was involved in the freezing tolerance of kiwifruit. In this module, the expression of AaBAM3.1, which encodes a functional protein, was induced by cold stress. AaBAM3.1-overexpressing kiwifruit lines showed increased freezing tolerance, and the heterologous overexpression of AaBAM3.1 in Arabidopsis thaliana resulted in a similar phenotype. The results of promoter GUS activity and cis-element analyses predicted AaCBF4 to be an upstream transcription factor that could regulate AaBAM3.1 expression. Further investigation of protein-DNA interactions by using yeast one-hybrid, GUS coexpression, and dual luciferase reporter assays confirmed that AaCBF4 directly regulated AaBAM3.1 expression. In addition, the expression of both AaBAM3.1 and AaCBF4 in kiwifruit responded positively to cold stress. Hence, we conclude that the AaCBF-AaBAM module is involved in the positive regulation of the freezing tolerance of kiwifruit.

Osmanthus fragrans is a well-known ornamental plant that has been domesticated in China for 2500 years. More than 160 cultivars have been found during this long period of domestication, and they have subsequently been divided into four cultivar groups, including the Yingui, Jingui, Dangui, and Sijigui groups. These groups provide a set of materials to study genetic evolution and variability. Here, we constructed a reference genome of O. fragrans ‘Liuyejingui’ in the Jingui group and investigated its floral color traits and domestication history by resequencing a total of 122 samples, including 119 O. fragrans accessions and three other Osmanthus species, at an average sequencing depth of 15×. The population structure analysis showed that these 119 accessions formed an apparent regional cluster. The results of linkage disequilibrium (LD) decay analysis suggested that varieties with orange/red flower color in the Dangui group had undergone more artificial directional selection; these varieties had the highest LD values among the four groups, followed by the Sijigui, Jingui, and Yingui groups. Through a genome-wide association study, we further identified significant quantitative trait loci and genomic regions containing several genes, such as ethylene-responsive transcription factor 2 and Arabidopsis pseudoresponse regulator 2, that are positively associated with petal color. Moreover, we found a frameshift mutation with a 34-bp deletion in the first coding region of the carotenoid cleavage dioxygenase 4 gene. This frameshift mutation existed in at least one site on both alleles in all varieties of the Dangui group. The results from this study shed light on the genetic basis of domestication in woody plants, such as O. fragrans.

The most daunting issue of global climate change is the deleterious impact of extreme temperatures on tea productivity and quality, which has resulted in a quest among researchers and growers. The current study aims to unravel molecular programming underpinning thermotolerance by characterizing heat tolerance and sensitivity response in 20 tea cultivars. The significantly higher negative influence of heat stress was recorded in a sensitive cultivar with reduced water retention (47%), chlorophyll content (33.79%), oxidation potential (32.48%), and increase in membrane damage (76.4%). Transcriptional profiling of most tolerant and sensitive cultivars identified 78 differentially expressed unigenes with chaperon domains, including low and high molecular weight heat shock protein (HSP) and heat shock transcription factors (HSFs) involved in heat shock response (HSR). Further, predicted transcriptional interactome network revealed their key role in thermotolerance via well-co-ordinated transcriptional regulation of aquaporins, starch metabolism, chlorophyll biosynthesis, calcium, and ethylene mediated plant signaling system. The study identified the key role of HSPs (CsHSP90) in regulating HSR in tea, wherein, structure-based molecular docking revealed the inhibitory role of geldanamycin (GDA) on CsHSP90 by blocking ATP binding site at N-terminal domain of predicted structure. Subsequently, GDA mediated leaf disc inhibitor assay further affirmed enhanced HSR with higher expression of CsHSP17.6, CsHSP70, HSP101, and CsHSFA2 genes in tea. Through the current study, efforts were made to extrapolate a deeper understanding of chaperons mediated regulation of HSR attributing thermotolerance in tea.

Wild grapevines can show strong resistance to the downy mildew pathogen P. viticola, but the associated mechanisms are poorly described, especially at early stages of infection. Here, we performed comparative proteomic analyses of grapevine leaves from the resistant genotype V. davidii “LiuBa-8” (LB) and susceptible V. vinifera “Pinot Noir” (PN) 12 h after inoculation with P. viticola. By employing the iTRAQ technique, a total of 444 and 349 differentially expressed proteins (DEPs) were identified in LB and PN, respectively. The majority of these DEPs were related to photosynthesis, respiration, cell wall modification, protein metabolism, stress, and redox homeostasis. Compared with PN, LB showed fewer downregulated proteins associated with photosynthesis and more upregulated proteins associated with metabolism. At least a subset of PR proteins (PR10.2 and PR10.3) was upregulated upon inoculation in both genotypes, whereas HSP (HSP70.2 and HSP90.6) and cell wall-related XTH and BXL1 proteins were specifically upregulated in LB and PN, respectively. In the incompatible interaction, ROS signaling was evident by the accumulation of H₂O₂, and multiple APX and GST proteins were upregulated. These DEPs may play crucial roles in the grapevine response to downy mildew. Our results provide new insights into molecular events associated with downy mildew resistance in grapevine, which may be exploited to develop novel protection strategies against this disease.

The Mildew Resistance Locus O (MLO) gene family has been investigated in many species. However, there are few studies on chrysanthemum MLO genes. We report in this study that CmMLO17 in Chrysanthemum morifolium was upregulated after Alternaria alternata infection. Silencing of CmMLO17 by artificial microRNA resulted in reduced susceptibility of chrysanthemum to A. alternata infection. Genes in the abscisic acid (ABA) and Ca²⁺ signaling pathways were upregulated in the CmMLO17-silenced line R20 compared to the wild-type plants. We speculated that CmMLO17-silenced plants had a faster and stronger defense response that was mediated by the ABA and Ca²⁺ signaling pathways, resulting in reduced susceptibility of chrysanthemum to A. alternata infection. In addition, a candidate gene, CmKIC, that may interact with CmMLO17 was discovered by the yeast two-hybrid assay. The interaction between CmMLO17 and CmKIC was confirmed using the yeast two-hybrid assay and bimolecular fluorescence complementation (BiFC) analysis. CmMLO17 and CmKIC were both located on the plasma membrane, and CmKIC was also located on the nucleus. CmKIC overexpression increased the susceptibility of chrysanthemum to A. alternata, whereas CmKIC silencing resulted in reduced susceptibility. Therefore, CmMLO17 and CmKIC may work together in C. morifolium to support the growth of A. alternata. The results of this study will provide insight into the potential function of MLO and improve the understanding of plant defense responses to necrotrophic pathogens.

Trees in temperate regions exhibit evident seasonal patterns, which play vital roles in their growth and development. The activity of cambial stem cells is the basis for regulating the quantity and quality of wood, which has received considerable attention. However, the underlying mechanisms of these processes have not been fully elucidated. Here we performed a comprehensive analysis of morphological observations, transcriptome profiles, the DNA methylome, and miRNAs of the cambium in Populus tomentosa during the transition from dormancy to activation. Anatomical analysis showed that the active cambial zone exhibited a significant increase in the width and number of cell layers compared with those of the dormant and reactivating cambium. Furthermore, we found that differentially expressed genes associated with vascular development were mainly involved in plant hormone signal transduction, cell division and expansion, and cell wall biosynthesis. In addition, we identified 235 known miRNAs and 125 novel miRNAs. Differentially expressed miRNAs and target genes showed stronger negative correlations than other miRNA/target pairs. Moreover, global methylation and transcription analysis revealed that CG gene body methylation was positively correlated with gene expression, whereas CHG exhibited the opposite trend in the downstream region. Most importantly, we observed that the number of CHH differentially methylated region (DMR) changes was the greatest during cambium periodicity. Intriguingly, the genes with hypomethylated CHH DMRs in the promoter were involved in plant hormone signal transduction, phenylpropanoid biosynthesis, and plant–pathogen interactions during vascular cambium development. These findings improve our systems-level understanding of the epigenomic diversity that exists in the annual growth cycle of trees.

Botrytis cinerea is a major grapevine (Vitis spp.) pathogen, but some genotypes differ in their degree of resistance. For example, the Vitis vinifera cultivar Red Globe (RG) is highly susceptible, but V. amurensis Rupr Shuangyou (SY) is highly resistant. Here, we used RNA sequencing analysis to characterize the transcriptome responses of these two genotypes to B. cinerea inoculation at an early infection stage. Approximately a quarter of the genes in RG presented significant changes in transcript levels during infection, the number of which was greater than that in the SY leaves. The genes differentially expressed between infected leaves of SY and RG included those associated with cell surface structure, oxidation, cell death and C/N metabolism. We found evidence that an imbalance in the levels of reactive oxygen species (ROS) and redox homeostasis probably contributed to the susceptibility of RG to B. cinerea. SY leaves had strong antioxidant capacities and improved ROS homeostasis following infection. Regulatory network prediction suggested that WRKY and MYB transcription factors are associated with the abscisic acid pathway. Weighted gene correlation network analysis highlighted preinfection features of SY that might contribute to its increased resistance. Moreover, overexpression of VaWRKY10 in Arabidopsis thaliana and V. vinifera Thompson Seedless enhanced resistance to B. cinerea. Collectively, our study provides a high-resolution view of the transcriptional changes of grapevine in response to B. cinerea infection and novel insights into the underlying resistance mechanisms.

The growth of leaves and biosynthesis of characteristic secondary metabolites are critically important for tea production and quality control. However, little is known about the coordinated regulation of leaf development and catechin biosynthesis in tea plants. Here, we reported that TCP TFs are involved in both catechin biosynthesis and leaf development. An integrated analysis of catechin profiling and CsTCP expression in different tissues of plants under various environmental conditions at different developmental stages indicated significant correlations between the transcript levels of CIN-type TCPs and catechin production. CIN-type CsTCP3 and CsTCP4 and PCF-type CsTCP14 interacted with the MYB-bHLH-WD40 repeat (MBW) complex by forming a CsTCP3-CsTT8 heterodimer and modulating the transactivation activity of the promoters of anthocyanin synthase (CsANS1) and anthocyanidin reductase (CsANR1). Four types of microRNA/target modules, miR319b/CsTCP3-4, miR164b/CsCUC, miR396/CsGRF-GIF, and miR165b/HD-ZIPIII ones, were also identified and characterized for their functions in the regulation of the development of tea plant shoot tips and leaf shape. The results of these modules were reflected by their different expression patterns in developing buds and leaves that had distinctly different morphologies in three different tea plant varieties. Their roles in the regulation of catechin biosynthesis were also further verified by manipulation of microRNA319b (miR319b), which targets the transcripts of CsTCP3 and CsTCP4. Thus, CsTCPs represent at least one of these important groups of TFs that can integrate tea plant leaf development together with secondary metabolite biosynthesis. Our study provides new insight into shoot tip development and catechin production in tea plants and lays a foundation for further mechanistic understanding of the regulation of tea plant leaf development and secondary metabolism.

Abscission in plants is tightly controlled by multiple phytohormones and the expression of various genes. However, whether the plant hormone brassinosteroids (BRs) are involved in this process is largely unknown. Here, we found that exogenous application of BRs reduced the ethylene-induced fruitlet abscission of litchi due to lower ethylene (ET) production and suppressed the expression of the ethylene biosynthetic genes LcACS1/4 and LcACO2/3 in the fruitlet abscission zone (FAZ). Two genes that encode the BR core signaling components brassinazole resistant (BZR) proteins, namely, LcBZR1 and LcBZR2, were characterized. LcBZR1/2 were localized to the nucleus and acted as transcription repressors. Interestingly, the LcBZR1/2 transcript levels were not changed during ET-induced fruitlet abscission, while their expression levels were significantly increased after BR application. Moreover, gel shift and transient expression assays indicated that LcBZR1/2 could suppress the transcription of LcACS1/4 and LcACO2/3 by specifically binding to their promoters. Importantly, ectopic expression of LcBZR1/2 in Arabidopsis significantly delayed floral organ abscission and suppressed ethylene biosynthesis. Collectively, our results suggest that BRs suppress ET-induced fruitlet abscission through LcBZR1/2-controlled expression of genes related to ethylene biosynthesis in litchi. In addition, similar results were observed in the Arabidopsis gain-of-function mutant bzr1-1D, which showed delayed floral organ abscission in parallel with lower expression of ACS/ACO genes and reduced ethylene production, suggesting that the mechanism of BZR-controlled organ abscission via regulation of ethylene biosynthesis might be conserved in Arabidopsis.

The agricultural and consumer quality of Chinese cabbage is determined by its shape. The shape is defined by the folding of the heading leaves, which defines the head top shape (HTS). The overlapping HTS, in which the heading leaves curve inward and overlap at the top, is the shape preferred by consumers. To understand the genetic regulation of HTS, we generated a large segregating F₂ population from a cross between pak choi and Chinese cabbage, with phenotypes ranging from nonheading to heading with either outward curving or inward curving overlapping heading leaves. HTS was correlated with plant height, outer/rosette leaf length, and petiole length. A high-density genetic map was constructed. Quantitative trait locus (QTL) analysis resulted in the identification of 22 QTLs for leafy head-related traits, which included five HTS QTLs. Bulked segregant analysis (BSA) was used to confirm HTS QTLs and identify candidate genes based on informative single-nucleotide polymorphisms. Interestingly, the HTS QTLs colocalized with QTLs for plant height, outer/rosette leaf, and petiole length, consistent with the observed phenotypic correlations. Combined QTL analysis and BSA laid a foundation for molecular marker-assisted breeding of Chinese cabbage HTS and directions for further research on the genetic regulation of this trait.

Tea plants (Camellia sinensis) are commercially cultivated in >60 countries, and their fresh leaves are processed into tea, which is the most widely consumed beverage in the world. Although several chromosome-level tea plant genomes have been published, they collapsed the two haplotypes and ignored a large number of allelic variations that may underlie important biological functions in this species. Here, we present a phased chromosome-scale assembly for an elite oolong tea cultivar, “Huangdan”, that is well known for its high levels of aroma. Based on the two sets of haplotype genome data, we identified numerous genetic variations and a substantial proportion of allelic imbalance related to important traits, including aroma- and stress-related alleles. Comparative genomics revealed extensive structural variations as well as expansion of some gene families, such as terpene synthases (TPSs), that likely contribute to the high-aroma characteristics of the backbone parent, underlying the molecular basis for the biosynthesis of aroma-related chemicals in oolong tea. Our results uncovered the genetic basis of special features of this oolong tea cultivar, providing fundamental genomic resources to study evolution and domestication for the economically important tea crop.

Downy mildew (DM), caused by obligate parasitic oomycetes, is a destructive disease for a wide range of crops worldwide. Recent outbreaks of impatiens downy mildew (IDM) in many countries have caused huge economic losses. A system to reveal plant–pathogen interactions in the early stage of infection and quickly assess resistance/ susceptibility of plants to DM is desired. In this study, we established an early and rapid system to achieve these goals using impatiens as a model. Thirty-two cultivars of Impatiens walleriana and I. hawkeri were evaluated for their responses to IDM at cotyledon, first/second pair of true leaf, and mature plant stages. All I. walleriana cultivars were highly susceptible to IDM. While all I. hawkeri cultivars were resistant to IDM starting at the first true leaf stage, many (14/16) were susceptible to IDM at the cotyledon stage. Two cultivars showed resistance even at the cotyledon stage. Histological characterization showed that the resistance mechanism of the I. hawkeri cultivars resembles that in grapevine and type II resistance in sunflower. By integrating full-length transcriptome sequencing (Iso-Seq) and RNA- Seq, we constructed the first reference transcriptome for Impatiens comprised of 48,758 sequences with an N50 length of 2060 bp. Comparative transcriptome and qRT-PCR analyses revealed strong candidate genes for IDM resistance, including three resistance genes orthologous to the sunflower gene RGC203, a potential candidate associated with DM resistance. Our approach of integrating early disease-resistance phenotyping, histological characterization, and transcriptome analysis lay a solid foundation to improve DM resistance in impatiens and may provide a model for other crops.

Despite their economic importance and well-characterized domestication syndrome, the genomic impact of domestication and the identification of variants underlying the domestication traits in Cucurbita species (pumpkins and squashes) is currently lacking. Cucurbita argyrosperma, also known as cushaw pumpkin or silver-seed gourd, is a Mexican crop consumed primarily for its seeds rather than fruit flesh. This makes it a good model to study Cucurbita domestication, as seeds were an essential component of early Mesoamerican diet and likely the first targets of human-guided selection in pumpkins and squashes. We obtained population-level data using tunable Genotype by Sequencing libraries for 192 individuals of the wild and domesticated subspecies of C. argyrosperma across Mexico. We also assembled the first high-quality wild Cucurbita genome. Comparative genomic analyses revealed several structural variants and presence/absence of genes related to domestication. Our results indicate a monophyletic origin of this domesticated crop in the lowlands of Jalisco. We found evidence of gene flow between the domesticated and wild subspecies, which likely alleviated the effects of the domestication bottleneck. We uncovered candidate domestication genes that are involved in the regulation of growth hormones, plant defense mechanisms, seed development, and germination. The presence of shared selected alleles with the closely related species Cucurbita moschata suggests domestication-related introgression between both taxa.

Under high light conditions or UV radiation, tea plant leaves produce more flavonols, which contribute to the bitter taste of tea; however, neither the flavonol biosynthesis pathways nor the regulation of their production are well understood. Intriguingly, tea leaf flavonols are enhanced by UV-B but reduced by shading treatment. CsFLS, CsUGT78A14, CsMYB12, and CsbZIP1 were upregulated by UV-B radiation and downregulated by shading. CsMYB12 and CsbZIP1 bound to the promoters of CsFLS and CsUGT78A14, respectively, and activated their expression individually. CsbZIP1 positively regulated CsMYB12 and interacted with CsMYB12, which specifically activated flavonol biosynthesis. Meanwhile, CsPIF3 and two MYB repressor genes, CsMYB4 and CsMYB7, displayed expression patterns opposite to that of CsMYB12. CsMYB4 and CsMYB7 bound to CsFLS and CsUGT78A14 and repressed their CsMYB12-activated expression. While CsbZIP1 and CsMYB12 regulated neither CsMYB4 nor CsMYB7, CsMYB12 interacted with CsbZIP1, CsMYB4, and CsMYB7, but CsbZIP1 did not physically interact with CsMYB4 or CsMYB7. Finally, CsPIF3 bound to and activated CsMYB7 under shading to repress flavonol biosynthesis. These combined results suggest that UV activation and shading repression of flavonol biosynthesis in tea leaves are coordinated through a complex network involving CsbZIP1 and CsPIF3 as positive MYB activators and negative MYB repressors, respectively. The study thus provides insight into the regulatory mechanism underlying the production of bitter-tasting flavonols in tea plants.

Soil-borne plant pathogens represent a serious threat that undermines commercial walnut (Juglans regia) production worldwide. Crown gall, caused by Agrobacterium tumefaciens, and Phytophthora root and crown rots, caused by various Phytophthora spp., are among the most devastating walnut soil-borne diseases. A recognized strategy to combat soil-borne diseases is adoption of resistant rootstocks. Here, resistance to A. tumefaciens, P. cinnamomi, and P. pini is mapped in the genome of Juglans microcarpa, a North American wild relative of cultivated walnut. Half-sib J. microcarpa mother trees DJUG 31.01 and DJUG 31.09 were crossed with J. regia cv. Serr, producing 353 and 400 hybrids, respectively. Clonally propagated hybrids were genotyped by sequencing to construct genetic maps for the two populations and challenged with the three pathogens. Resistance to each of the three pathogens was mapped as a major QTL on the long arm of J. microcarpa chromosome 4D and was associated with the same haplotype, designated as haplotype b, raising the possibility that the two mother trees were heterozygous for a single Mendelian gene conferring resistance to all three pathogens. The deployment of this haplotype in rootstock breeding will facilitate breeding of a walnut rootstock resistant to both crown gall and Phytophthora root and crown rots.

Carotenoids, such as β-carotene, accumulate in chromoplasts of various fleshy fruits, awarding them with colors, aromas, and nutrients. The Orange (CmOr) gene controls β-carotene accumulation in melon fruit by posttranslationally enhancing carotenogenesis and repressing β-carotene turnover in chromoplasts. Carotenoid isomerase (CRTISO) isomerizes yellow prolycopene into red lycopene, a prerequisite for further metabolism into β-carotene. We comparatively analyzed the developing fruit transcriptomes of orange-colored melon and its two isogenic EMS-induced mutants, low-β (Cmor) and yofi (Cmcrtiso). The Cmor mutation in low-β caused a major transcriptomic change in the mature fruit. In contrast, the Cmcrtiso mutation in yofi significantly changed the transcriptome only in early fruit developmental stages. These findings indicate that melon fruit transcriptome is primarily altered by changes in carotenoid metabolic flux and plastid conversion, but minimally by carotenoid composition in the ripe fruit. Clustering of the differentially expressed genes into functional groups revealed an association between fruit carotenoid metabolic flux with the maintenance of the photosynthetic apparatus in fruit chloroplasts. Moreover, large numbers of thylakoid localized photosynthetic genes were differentially expressed in low-β. CmOR family proteins were found to physically interact with light-harvesting chlorophyll a–b binding proteins, suggesting a new role of CmOR for chloroplast maintenance in melon fruit. This study brings more insights into the cellular and metabolic processes associated with fruit carotenoid accumulation in melon fruit and reveals a new maintenance mechanism of the photosynthetic apparatus for plastid development.

Genetics of traits related to fruit cuticle deposition and composition was studied in two red-fruited tomato species. Two mapping populations derived from the cross between the cultivated tomato (Solanum lycopersicum L.) and its closest relative wild species Solanum pimpinellifolium L. were employed to conduct a QTL analysis. A combination of fruit cuticle deposition, components and anatomical traits were investigated and the individual effect of each QTL evaluated. A total of 70 QTLs were identified, indicating that all the cuticle traits analyzed have a complex polygenic nature. A combination of additive and epistatic interactions was observed for all the traits, with positive contribution of both parental lines to most of them. Colocalization of QTLs for various traits uncovered novel genomic regions producing extensive changes in the cuticle. Cuticle density emerges as an important trait since it can modulate cuticle thickness and invagination thus providing a strategy for sustaining mechanical strength without compromising palatability. Two genomic regions, located in chromosomes 1 and 12, are responsible for the negative interaction between cuticle waxes and phenolics identified in tomato fruit. Several candidate genes, including transcription factors and structural genes, are postulated and their expression analyzed throughout development.

The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) system is a powerful tool for targeted genome editing, with applications that include plant biotechnology and functional genomics research. However, the specificity of Cas9 targeting is poorly investigated in many plant species, including fruit trees. To assess the off-target mutation rate in grapevine (Vitis vinifera), we performed whole-genome sequencing (WGS) of seven Cas9-edited grapevine plants in which one of two genes was targeted by CRISPR/Cas9 and three wild-type (WT) plants. In total, we identified between 202,008 and 272,397 single nucleotide polymorphisms (SNPs) and between 26,391 and 55,414 insertions/deletions (indels) in the seven Cas9-edited grapevine plants compared with the three WT plants. Subsequently, 3272 potential off-target sites were selected for further analysis. Only one off-target indel mutation was identified from the WGS data and validated by Sanger sequencing. In addition, we found 243 newly generated off-target sites caused by genetic variants between the Thompson Seedless cultivar and the grape reference genome (PN40024) but no true off-target mutations. In conclusion, we observed high specificity of CRISPR/Cas9 for genome editing of grapevine.

Flowering time is known to be regulated by numerous pathways, such as the autonomous, gibberellin, aging, photoperiod-mediated, and vernalization pathways. These regulatory mechanisms involve both environmental triggers and endogenous hormonal cues. Additional flowering control mechanisms mediated by other phytohormones, such as auxin, are less well understood. We found that in cultivated strawberry (Fragaria × ananassa), the expression of auxin response factor4 (FaARF4) was higher in the flowering stage than in the vegetative stage. Overexpression of FaARF4 in Arabidopsis thaliana and woodland strawberry (Fragaria vesca) resulted in transgenic plants flowering earlier than control plants. In addition, FveARF4-silenced strawberry plants showed delayed flowering compared to control plants, indicating that FaARF4 and FveARF4 function similarly in regulating flowering. Further studies showed that ARF4 can bind to the promoters of the floral meristem identity genes APETALA1 (AP1) and FRUITFULL (FUL), inducing their expression and, consequently, flowering in woodland strawberry. Our studies reveal an auxin-mediated flowering pathway in strawberry involving the induction of ARF4 expression.

Venation is a common anthocyanin pattern displayed in flowers that confers important ornamental traits to plants. An anthocyanin-related R2R3-MYB transcription factor, DPL, has been proposed to regulate corolla tube venation in petunia plants. Here, however, we provide evidence redefining the role of DPL in petunia. A CRISPR/Cas9-mediated mutation of DPL resulted in the absence of the vein-associated anthocyanin pattern above the abaxial surface of the flower bud, but not corolla tube venation, thus indicating that DPL did not regulate the formation of corolla tube venation. Alternately, quantitative real-time PCR analysis demonstrated that the spatiotemporal expression pattern of another R2R3-MYB gene, AN4, coincided with the formation of corolla tube venation in petunia. Furthermore, overexpression of AN4 promoted anthocyanin accumulation by increasing the expression of anthocyanin biosynthesis genes. CRISPR/Cas9-mediated mutation of AN4 led to an absence of corolla tube venation, suggesting that this gene in fact determines this key plant trait. Taken together, the results presented here redefine the prime regulator of corolla tube venation, paving the way for further studies on the molecular mechanisms underlying the various venation patterns in petunia.

Mitogen-activated protein kinase (MAPK) cascades play important roles in plant immunity. Previously, we reported that the potato StMKK1 protein negatively regulates Nicotiana benthamiana resistance to Phytophthora infestans. However, the functions of StMKK1 in potato immunity are unknown. To investigate the roles of StMKK1 in potato resistance to different pathogens, such as the potato late-blight pathogen P. infestans, the bacterial wilt pathogen Ralstonia solanacearum, and the gray-mold fungal pathogen Botrytis cinerea, we generated StMKK1 transgenic lines and investigated the response of potato transformants to destructive oomycete, bacterial, and fungal pathogens. The results showed that overexpression and silencing of StMKK1 do not alter plant growth and development. Interestingly, we found that StMKK1 negatively regulated potato resistance to the hemibiotrophic/biotrophic pathogens P. infestans and R. solanacearum, while it positively regulated potato resistance to the necrotrophic pathogen B. cinerea. Further investigation showed that overexpression of StMKK1 suppressed potato pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and salicylic acid (SA)-related responses, while silencing of StMKK1 enhanced PTI and SA-related immune responses. Taken together, our results showed that StMKK1 plays dual roles in potato defense against different plant pathogens via negative regulation of PTI and SA-related signaling pathways.

Leaves provide energy for plants, and consequently for animals, through photosynthesis. Despite their important functions, plant leaf developmental processes and their underlying mechanisms have not been well characterized. Here, we provide a holistic description of leaf developmental processes that is centered on cytokinins and their signaling functions. Cytokinins maintain the growth potential (pluripotency) of shoot apical meristems, which provide stem cells for the generation of leaf primordia during the initial stage of leaf formation; cytokinins and auxins, as well as their interaction, determine the phyllotaxis pattern. The activities of cytokinins in various regions of the leaf, especially at the margins, collectively determine the final leaf morphology (e.g., simple or compound). The area of a leaf is generally determined by the number and size of the cells in the leaf. Cytokinins promote cell division and increase cell expansion during the proliferation and expansion stages of leaf cell development, respectively. During leaf senescence, cytokinins reduce sugar accumulation, increase chlorophyll synthesis, and prolong the leaf photosynthetic period. We also briefly describe the roles of other hormones, including auxin and ethylene, during the whole leaf developmental process. In this study, we review the regulatory roles of cytokinins in various leaf developmental stages, with a focus on cytokinin metabolism and signal transduction processes, in order to shed light on the molecular mechanisms underlying leaf development.

Fruit detection and counting are essential tasks for horticulture research. With computer vision technology development, fruit detection techniques based on deep learning have been widely used in modern orchards. However, most deep learning-based fruit detection models are generated based on fully supervised approaches, which means a model trained with one domain species may not be transferred to another. There is always a need to recreate and label the relevant training dataset, but such a procedure is time-consuming and labor-intensive. This paper proposed a domain adaptation method that can transfer an existing model trained from one domain to a new domain without extra manual labeling. The method includes three main steps: transform the source fruit image (with labeled information) into the target fruit image (without labeled information) through the CycleGAN network; Automatically label the target fruit image by a pseudo-label process; Improve the labeling accuracy by a pseudo-label self-learning approach. Use a labeled orange image dataset as the source domain, unlabeled apple and tomato image dataset as the target domain, the performance of the proposed method from the perspective of fruit detection has been evaluated. Without manual labeling for target domain image, the mean average precision reached 87.5% for apple detection and 76.9% for tomato detection, which shows that the proposed method can potentially fill the species gap in deep learning-based fruit detection.

Heterosis has historically been exploited in plants; however, its underlying genetic mechanisms and molecular basis remain elusive. In recent years, due to advances in molecular biotechnology at the genome, transcriptome, proteome, and epigenome levels, the study of heterosis in vegetables has made significant progress. Here, we present an extensive literature review on the genetic and epigenetic regulation of heterosis in vegetables. We summarize six hypotheses to explain the mechanism by which genes regulate heterosis, improve upon a possible model of heterosis that is triggered by epigenetics, and analyze previous studies on quantitative trait locus effects and gene actions related to heterosis based on analyses of differential gene expression in vegetables. We also discuss the contributions of yield-related traits, including flower, fruit, and plant architecture traits, during heterosis development in vegetables (e.g., cabbage, cucumber, and tomato). More importantly, we propose a comprehensive breeding strategy based on heterosis studies in vegetables and crop plants. The description of the strategy details how to obtain F₁ hybrids that exhibit heterosis based on heterosis prediction, how to obtain elite lines based on molecular biotechnology, and how to maintain heterosis by diploid seed breeding and the selection of hybrid simulation lines that are suitable for heterosis research and utilization in vegetables. Finally, we briefly provide suggestions and perspectives on the role of heterosis in the future of vegetable breeding.

Coptis chinensis Franch, a perennial herb, is mainly distributed in southeastern China. The rhizome of C. chinensis has been used as a traditional medicine for more than 2000 years in China and many other Asian countries. The pharmacological activities of C. chinensis have been validated by research. Here, we present a de novo high-quality genome of C. chinensis with a chromosome-level genome of ~958.20 Mb, a contig N50 of 1.58 Mb, and a scaffold N50 of 4.53 Mb. We found that the relatively large genome size of C. chinensis was caused by the amplification of long terminal repeat (LTR) retrotransposons. In addition, a whole-genome duplication event in ancestral Ranunculales was discovered. Comparative genomic analysis revealed that the tyrosine decarboxylase (TYDC) and (S)-norcoclaurine synthase (NCS) genes were expanded and that the aspartate aminotransferase gene (ASP5) was positively selected in the berberine metabolic pathway. Expression level and HPLC analyses showed that the berberine content was highest in the roots of C. chinensis in the third and fourth years. The chromosome-level reference genome of C. chinensis provides important genomic data for molecular-assisted breeding and active ingredient biosynthesis.

Simple sequence repeats (SSRs) are one of the most important genetic markers and widely exist in most species. Here, we identified 249,822 SSRs from 3,951,919 genes in 112 plants. Then, we conducted a comprehensive analysis of these SSRs and constructed a plant SSR database (PSSRD). Interestingly, more SSRs were found in lower plants than in higher plants, showing that lower plants needed to adapt to early extreme environments. Four specific enriched functional terms in the lower plant Chlamydomonas reinhardtii were detected when it was compared with seven other higher plants. In addition, Guanylate_cyc existed in more genes of lower plants than of higher plants. In our PSSRD, we constructed an interactive plotting function in the chart interface, and users can easily view the detailed information of SSRs. All SSR information, including sequences, primers, and annotations, can be downloaded from our database. Moreover, we developed Web SSR Finder and Batch SSR Finder tools, which can be easily used for identifying SSRs. Our database was developed using PHP, HTML, JavaScript, and MySQL, which are freely available at http://www.pssrd.info/. We conducted an analysis of the Myb gene families and flowering genes as two applications of the PSSRD. Further analysis indicated that whole-genome duplication and whole-genome triplication played a major role in the expansion of the Myb gene families. These SSR markers in our database will greatly facilitate comparative genomics and functional genomics studies in the future.

Deep learning is known as a promising multifunctional tool for processing images and other big data. By assimilating large amounts of heterogeneous data, deep-learning technology provides reliable prediction results for complex and uncertain phenomena. Recently, it has been increasingly used by horticultural researchers to make sense of the large datasets produced during planting and postharvest processes. In this paper, we provided a brief introduction to deep-learning approaches and reviewed 71 recent research works in which deep-learning technologies were applied in the horticultural domain for variety recognition, yield estimation, quality detection, stress phenotyping detection, growth monitoring, and other tasks. We described in detail the application scenarios reported in the relevant literature, along with the applied models and frameworks, the used data, and the overall performance results. Finally, we discussed the current challenges and future trends of deep learning in horticultural research. The aim of this review is to assist researchers and provide guidance for them to fully understand the strengths and possible weaknesses when applying deep learning in horticultural sectors. We also hope that this review will encourage researchers to explore some significant examples of deep learning in horticultural science and will promote the advancement of intelligent horticulture.

Melatonin (MT) is a tryptophan-derived natural product that plays a vital role in plant response to abiotic stresses, including heavy metals (HMs). However, it remains elusive how exogenous MT mediates lead (Pb) accumulation and detoxification at the methylation and transcriptional levels in radish. In this study, decreased Pb accumulation and increased antioxidant enzyme activity were detected under MT treatment in radish. Single-base resolution maps of DNA methylation under Pb stress (Pb200) and Pb plus MT treatment (Pb_50MT) were first generated. The genome-wide methylation level was increased under Pb stress, while an overall loss of DNA methylation was observed under MT treatment. The differentially methylated region (DMR)-associated genes between Pb_50MT and Pb200 were uniquely enriched in ion binding terms, including cation binding, iron ion binding, and transition metal ion binding. Hyper-DMRs between Pb200 and Control exhibited a decreasing trend of methylation under Pb_50MT treatment. A few critical upregulated antioxidant genes (e.g., RsAPX2, RsPOD52 and RsGST) exhibited decreased methylation levels under MT treatment, which enabled the radish plants to scavenge lead-induced reactive oxygen species (ROS) and decrease oxidative stress. Notably, several MT-induced HM transporter genes with low methylation (e.g., RsABCF5, RsYSL7 and RsHMT) and transcription factors (e.g., RsWRKY41 and RsMYB2) were involved in reducing Pb accumulation in radish roots. These findings could facilitate comprehensive elucidation of the molecular mechanism underlying MT-mediated Pb accumulation and detoxification in radish and other root vegetable crops.

Dioecious species evolved from species with monomorphic sex systems in order to achieve overall fitness gains by separating male and female functions. As reproductive organs, unisexual flowers have different reproductive roles and exhibit conspicuous sexual dimorphism. To date, little is known about the temporal variations in and molecular mechanisms underlying the morphology and reproductive costs of dioecious flowers. We investigated male and female flowers of Salix paraplesia in three flowering stages before pollination (the early, blooming and late stages) via transcriptional sequencing as well as metabolite content and phenotypic analysis. We found that a large number of sex-biased genes, rather than sex-limited genes, were responsible for sexual dimorphism in S. paraplesia flowers and that the variation in gene expression in male flowers intensified this situation throughout flower development. The temporal dynamics of sex-biased genes derived from changes in reproductive function during the different flowering stages. Sexually differentiated metabolites related to respiration and flavonoid biosynthesis exhibited the same bias directions as the sex-biased genes. These sex-biased genes were involved mainly in signal transduction, photosynthesis, respiration, cell proliferation, phytochrome biosynthesis, and phenol metabolism; therefore, they resulted in more biomass accumulation and higher energy consumption in male catkins. Our results indicated that sex-biased gene expression in S. paraplesia flowers is associated with different reproductive investments in unisexual flowers; male flowers require a greater reproductive investment to meet their higher biomass accumulation and energy consumption needs.

Light is required for initiating chloroplast biogenesis and photosynthesis; however, the photosystem II reaction center (PSII RC) can be photodamaged. In this study, we characterized pvsl1, a seedling-lethal mutant of Phaseolus vulgaris. This mutant showed lethality when exposed to sunlight irradiation and a yellow-green leaf phenotype when grown in a growth chamber under low-light conditions. We developed 124 insertion/deletion (INDEL) markers based on resequencing data of Dalong1 and PI60234, two local Chinese common bean cultivars, for genetic mapping. We identified Phvul.002G190900, which encodes the PvFtsH2 protein, as the candidate gene for this pvsl1 mutation through fine-mapping and functional analysis. A single-base deletion occurred in the coding region of Phvul.002G190900 in the pvsl1 mutant, resulting in a frameshift mutation and a truncated protein lacking the Zn²⁺ metalloprotease domain. Suppressed expression of Phvul.002G190900 at the transcriptional level was detected, while no change in the subcellular localization signal was observed. The seedlings of pvsl1 exhibited hypersensitivity to photoinhibition stress. In the pvsl1 mutant, abnormal accumulation of the D1 protein indicated a failure to rapidly degrade damaged D1 protein in the PSII RC. The results of this study demonstrated that PvFtsH2 is critically required for survival and maintaining photosynthetic activity by degrading photodamaged PSII RC D1 protein in common bean.

The proliferation and development of chloroplasts are important for maintaining the normal chloroplast population in plant tissues. Most studies have focused on chloroplast maintenance in leaves. In this study, we identified a spontaneous mutation in a tomato mutant named suffulta (su), in which the stems appeared albinic while the leaves remained normal. Map-based cloning showed that Su encodes a DnaJ heat shock protein that is a homolog of the Arabidopsis gene AtARC6, which is involved in chloroplast division. Knockdown and knockout of SlARC6 in wild-type tomato inhibit chloroplast division, indicating the conserved function of SlARC6. In su mutants, most mesophyll cells contain only one or two giant chloroplasts, while no chloroplasts are visible in 60% of stem cells, resulting in the albinic phenotype. Compared with mature tissues, the meristem of su mutants suggested that chloroplasts could partially divide in meristematic cells, suggesting the existence of an alternative mechanism in those dividing cells. Interestingly, the adaxial petiole cells of su mutants contain more chloroplasts than the abaxial cells. In addition, prolonged lighting can partially rescue the albinic phenotypes in su mutants, implying that light may promote SlACR6-independent chloroplast development. Our results verify the role of SlACR6 in chloroplast division in tomato and uncover the tissue-specific regulation of chloroplast development.

In plants, chloroplasts are the sites at which photosynthesis occurs, and an increased abundance of chloroplasts increases the nutritional quality of plants and the resultant color of fruits. However, the molecular mechanisms underlying chlorophyll synthesis and chloroplast development in tomato fruits remain unknown. In this study, we isolated a chlorophyll-deficient mutant, reduced chlorophyll mutant 1 (rcm1), by ethylmethanesulfonate mutagenesis; this mutant produced yellowish fruits with altered chloroplast development. MutMap revealed that Solyc08g005010 is the causal gene underlying the rcm1 mutant phenotype. A single-nucleotide base substitution in the second exon of SlRCM1 results in premature termination of its translated protein. SlRCM1 encodes a chloroplast-targeted metalloendopeptidase that is orthologous to the BCM1 protein of Arabidopsis and the stay-green G protein of soybean (Glycine max L. Merr.). Notably, the yellowish phenotype of the lutescent1 mutant can be restored with the allele of SlRCM1 from wild-type tomato. In contrast, knockout of SlRCM1 by the CRISPR/Cas9 system in Alisa Craig yielded yellowish fruits at the mature green stage, as was the case for lutescent1. Amino acid sequence alignment and functional complementation assays showed that SlRCM1 is indeed Lutescent1. These findings provide new insights into the regulation of chloroplast development in tomato fruits.

Stevia (Stevia rebaudiana Bertoni) is well known for its very sweet steviol glycosides (SGs) consisting of a common tetracyclic diterpenoid steviol backbone and a variable glycone. Steviol glycosides are 150–300 times sweeter than sucrose and are used as natural zero-calorie sweeteners. However, the most promising compounds are biosynthesized in small amounts. Based on Illumina, PacBio, and Hi-C sequencing, we constructed a chromosome-level assembly of Stevia covering 1416 Mb with a contig N50 value of 616.85 kb and a scaffold N50 value of 106.55 Mb. More than four-fifths of the Stevia genome consisted of repetitive elements. We annotated 44,143 high-confidence protein-coding genes in the high-quality genome. Genome evolution analysis suggested that Stevia and sunflower diverged ~29.4 million years ago (Mya), shortly after the whole-genome duplication (WGD) event (WGD-2, ~32.1 Mya) that occurred in their common ancestor. Comparative genomic analysis revealed that the expanded genes in Stevia were mainly enriched for biosynthesis of specialized metabolites, especially biosynthesis of terpenoid backbones, and for further oxidation and glycosylation of these compounds. We further identified all candidate genes involved in SG biosynthesis. Collectively, our current findings on the Stevia reference genome will be very helpful for dissecting the evolutionary history of Stevia and for discovering novel genes contributing to SG biosynthesis and other important agronomic traits in future breeding programs.

Small secreted proteins (SSPs) are less than 250 amino acids in length and are actively transported out of cells through conventional protein secretion pathways or unconventional protein secretion pathways. In plants, SSPs have been found to play important roles in various processes, including plant growth and development, plant response to abiotic and biotic stresses, and beneficial plant–microbe interactions. Over the past 10 years, substantial progress has been made in the identification and functional characterization of SSPs in several plant species relevant to agriculture, bioenergy, and horticulture. Yet, there are potentially a lot of SSPs that have not been discovered in plant genomes, which is largely due to limitations of existing computational algorithms. Recent advances in genomics, transcriptomics, and proteomics research, as well as the development of new computational algorithms based on machine learning, provide unprecedented capabilities for genome-wide discovery of novel SSPs in plants. In this review, we summarize known SSPs and their functions in various plant species. Then we provide an update on the computational and experimental approaches that can be used to discover new SSPs. Finally, we discuss strategies for elucidating the biological functions of SSPs in plants.

Weeping Prunus mume (mei) has long been cultivated in East Asia for its specific ornamental value. However, little is known about the regulatory mechanism of the weeping trait in mei, which limits molecular breeding for the improvement of weeping-type cultivars. Here, we quantified the weeping trait in mei using nested phenotyping of 214 accessions and 342 F₁ hybrids. Two major associated loci were identified from the genome-wide association study (GWAS), which was conducted using 3,014,409 single nucleotide polymorphisms (SNPs) derived from resequencing, and 8 QTLs and 55 epistatic loci were identified from QTL mapping using 7,545 specific lengths amplified fragment (SLAF) markers. Notably, an overlapping PmWEEP major QTL was fine mapped within a 0.29 Mb region on chromosome 7 (Pa7), and a core SNP locus closely associated with the weeping trait was screened and validated. Furthermore, a total of 22 genes in the PmWEEP QTL region were expressed in weeping or upright mei based on RNA-seq analysis. Among them, only a novel gene (Pm024213) containing a thioredoxin (Trx) domain was found to be close to the core SNP and specifically expressed in buds and branches of weeping mei. Co-expression analysis of Pm024213 showed that most of the related genes were involved in auxin and lignin biosynthesis. These findings provide insights into the regulatory mechanism of the weeping trait and effective molecular markers for molecular-assisted breeding in Prunus mume.

Since ancient times, humans have bred several plants that we rely on today. However, little is known about the divergence of most of these plants. In the present study, we investigated the divergence of Mibuna (Brassica rapa L. subsp. nipposinica L. H. Bailey), a traditional leafy vegetable in Kyoto (Japan), by combining genetic analysis and a survey of ancient literature. Mibuna is considered to have been bred 200 years ago from Mizuna, another traditional leafy vegetable in Kyoto. Mibuna has simple spatulate leaves, whereas Mizuna has characteristic serrated leaves. The quantitative trait loci (QTL) and gene expression analyses suggested that the downregulation of BrTCP15 expression contributed to the change in the leaf shape from serrated to simple spatulate. Interestingly, the SNP analysis indicated that the genomic region containing the BrTCP15 locus was transferred to Mibuna by introgression. Furthermore, we conducted a survey of ancient literature to reveal the divergence of Mibuna and found that hybridization between Mizuna and a simple-leaved turnip might have occurred in the past. Indeed, the genomic analysis of multiple turnip cultivars showed that one of the cultivars, Murasakihime, has almost the same sequence in the BrTCP15 region as Mibuna. These results suggest that the hybridization between Mizuna and turnip has resulted in the establishment of Mibuna.

Bamboo is known for its edible shoots and beautiful texture and has considerable economic and ornamental value. Unique among traditional flowering plants, many bamboo plants undergo extensive synchronized flowering followed by large-scale death, seriously affecting the productivity and application of bamboo forests. To date, the molecular mechanism of bamboo flowering characteristics has remained unknown. In this study, a SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1)-like gene, BoMADS50, was identified from Bambusa oldhamii. BoMADS50 was highly expressed in mature leaves and the floral primordium formation period during B. oldhamii flowering and overexpression of BoMADS50 caused early flowering in transgenic rice. Moreover, BoMADS50 could interact with APETALA1/FRUITFULL (AP1/FUL)-like proteins (BoMADS14-1/2, BoMADS15-1/2) in vivo, and the expression of BoMADS50 was significantly promoted by BoMADS14-1, further indicating a synergistic effect between BoMADS50 and BoAP1/FUL-like proteins in regulating B. oldhamii flowering. We also identified four additional transcripts of BoMADS50 (BoMADS50-1/2/3/4) with different nucleotide variations. Although the protein-CDS were polymorphic, they had flowering activation functions similar to those of BoMADS50. Yeast one-hybrid and transient expression assays subsequently showed that both BoMADS50 and BoMADS50-1 bind to the promoter fragment of itself and the SHORT VEGETATIVE PHASE (SVP)-like gene BoSVP, but only BoMADS50-1 can positively induce their transcription. Therefore, nucleotide variations likely endow BoMADS50-1 with strong regulatory activity. Thus, BoMADS50 and BoMADS50-1/2/3/4 are probably important positive flowering regulators in B. oldhamii. Moreover, the functional conservatism and specificity of BoMADS50 and BoMADS50-1 might be related to the synchronized and sporadic flowering characteristics of B. oldhamii.

Plantations and production of yellowhorn, one of the most important woody oil and urban greening trees widely cultivated in northern China, have gradually become limited by drought stress. The epicuticular wax layer plays a key role in the protection of yellowhorn trees from drought and other stresses. However, there is no research on the mechanism of wax loading in yellowhorn trees. In this study, we investigated the anatomical and physiological characteristics of leaves from different germplasm resources and different parts of the same tree and compared their cuticle properties. In addition, the different expression patterns of genes involved in wax accumulation were analyzed, and a coexpression network was built based on transcriptome sequencing data. Morphological and physiological comparisons found that the sun leaves from the outer part of the crown had thicker epicuticular wax, which altered the permeability and improved the drought resistance of leaves, than did shade leaves. Based on transcriptome data, a total of 3008 and 1324 differentially expressed genes (DEGs) were identified between the sun leaves and shade leaves in glossy- and non-glossy-type germplasm resources, respectively. We identified 138 DEGs involved in wax biosynthesis and transport, including structural genes (such as LACS8, ECH1, and ns-LTP) and transcription factors (such as MYB, WRKY, and bHLH transcription factor family proteins). The coexpression network showed a strong correlation between these DEGs. The differences in gene expression patterns between G- and NG-type germplasm resources under different light conditions were very clear. These results not only provide a theoretical basis for screening and developing drought-resistant yellowhorn germplasm resources but also provide a data platform to reveal the wax accumulation process of yellowhorn leaves.

Morinda officinalis is a well-known medicinal and edible plant that is widely cultivated in the Lingnan region of southern China. Its dried roots (called bajitian in traditional Chinese medicine) are broadly used to treat various diseases, such as impotence and rheumatism. Here, we report a high-quality chromosome-scale genome assembly of M. officinalis using Nanopore single-molecule sequencing and Hi-C technology. The assembled genome size was 484.85 Mb with a scaffold N50 of 40.97 Mb, and 90.77% of the assembled sequences were anchored on eleven pseudochromosomes. The genome includes 27,698 protein-coding genes, and most of the assemblies are repetitive sequences. Genome evolution analysis revealed that M. officinalis underwent core eudicot γ genome triplication events but no recent whole-genome duplication (WGD). Likewise, comparative genomic analysis showed no large-scale structural variation after species divergence between M. officinalis and Coffea canephora. Moreover, gene family analysis indicated that gene families associated with plant–pathogen interactions and sugar metabolism were significantly expanded in M. officinalis. Furthermore, we identified many candidate genes involved in the biosynthesis of major active components such as anthraquinones, iridoids and polysaccharides. In addition, we also found that the DHQS, GGPPS, TPS-Clin, TPS04, sacA, and UGDH gene families-which include the critical genes for active component biosynthesis-were expanded in M. officinalis. This study provides a valuable resource for understanding M. officinalis genome evolution and active component biosynthesis. This work will facilitate genetic improvement and molecular breeding of this commercially important plant.

Rain-induced fruit cracking is a major problem in sweet cherry cultivation. Basic research has been conducted to disentangle the physiological and mechanistic bases of this complex phenomenon, whereas genetic studies have lagged behind. The objective of this work was to disentangle the genetic determinism of rain-induced fruit cracking. We hypothesized that a large genetic variation would be revealed, by visual field observations conducted on mapping populations derived from well-contrasted cultivars for cracking tolerance. Three populations were evaluated over 7–8 years by estimating the proportion of cracked fruits for each genotype at maturity, at three different areas of the sweet cherry fruit: pistillar end, stem end, and fruit side. An original approach was adopted to integrate, within simple linear models, covariates potentially related to cracking, such as rainfall accumulation before harvest, fruit weight, and firmness. We found the first stable quantitative trait loci (QTLs) for cherry fruit cracking, explaining percentages of phenotypic variance above 20%, for each of these three types of cracking tolerance, in different linkage groups, confirming the high complexity of this trait. For these and other QTLs, further analyses suggested the existence of at least two-linked QTLs in each linkage group, some of which showed confidence intervals close to 5 cM. These promising results open the possibility of developing marker-assisted selection strategies to select cracking-tolerant sweet cherry cultivars. Further studies are needed to confirm the stability of the reported QTLs over different genetic backgrounds and environments and to narrow down the QTL confidence intervals, allowing the exploration of underlying candidate genes.

Transfer RNAs (tRNA) are crucial adaptor molecules between messenger RNA (mRNA) and amino acids. Recent evidence in plants suggests that dicistronic tRNA-like structures also act as mobile signals for mRNA transcripts to move between distant tissues. Co-transcription is not a common feature in the plant nuclear genome and, in the few cases where polycistronic transcripts have been found, they include non-coding RNA species, such as small nucleolar RNAs and microRNAs. It is not known, however, the extent to which dicistronic transcripts of tRNA and mRNAs are expressed in field-grown plants, or the factors contributing to their expression. We analysed tRNA–mRNA dicistronic transcripts in the major horticultural crop grapevine (Vitis vinifera) using a novel pipeline developed to identify dicistronic transcripts from high-throughput RNA-sequencing data. We identified dicistronic tRNA–mRNA in leaf and berry samples from 22 commercial vineyards. Of the 124 tRNA genes that were expressed in both tissues, 18 tRNA were expressed forming part of 19 dicistronic tRNA–mRNAs. The presence and abundance of dicistronic molecules was tissue and geographic sub-region specific. In leaves, the expression patterns of dicistronic tRNA–mRNAs significantly correlated with tRNA expression, suggesting that their transcriptional regulation might be linked. We also found evidence of syntenic genomic arrangements of tRNAs and protein-coding genes between grapevine and Arabidopsis thaliana, and widespread prevalence of dicistronic tRNA–mRNA transcripts among vascular land plants but no evidence of these transcripts in non-vascular lineages. This suggests that the appearance of plant vasculature and tRNA–mRNA occurred concurrently during the evolution of land plants.

Within large-fruited germplasm, fruit size is influenced by flat and globe shapes. Whereas flat fruits are smaller and retain better marketability, globe fruits are larger and more prone to cuticle disorders. Commercial hybrids are often developed from crosses between flat and globe shaped parents because flat shape is thought to be dominant and fruit size intermediate. The objectives of this study were to determine the genetic basis of flat/globe fruit shape in large-fruited fresh-market tomato germplasm and to characterize its effects on several fruit traits. Twenty-three advanced single plant selections from the Fla. 8000 × Fla. 8111B cross were selectively genotyped using a genome-wide SNP array, and inclusive composite interval mapping identified a single locus on the upper arm of chromosome 12 associated with shape, which we termed globe. A 238-plant F₂ population and 69 recombinant inbred lines for this region from the same parents delimited globe to approximately 392-kilobases. A germplasm survey representing materials from multiple breeding programs demonstrated that the locus explains the flat/globe shape broadly. A single base insertion in an exon of Solyc12g006860, a gene annotated as a brassinosteroid hydroxylase, segregated completely with shape in all populations tested. CRISPR/Cas9 knock out plants confirmed this gene as underlying the globe locus. In silico analysis of the mutant allele of GLOBE among 595 wild and domesticated accessions suggested that the allele arose very late in the domestication process. Fruit measurements in three genetic backgrounds evidenced that globe impacts fruit size and several fruit shape attributes, pedicel length/width, and susceptibility of fruit to weather check. The mutant allele of GLOBE appears mostly recessive for all traits except fruit size where it acts additively.

Bud endodormancy is a complex physiological process that is indispensable for the survival, growth, and development of deciduous perennial plants. The timely release of endodormancy is essential for flowering and fruit production of deciduous fruit trees. A better understanding of the mechanism of endodormancy will be of great help in the artificial regulation of endodormancy to cope with climate change and in creating new cultivars with different chilling requirements. Studies in poplar have clarified the mechanism of vegetative bud endodormancy, but the endodormancy of floral buds in fruit trees needs further study. In this review, we focus on the molecular regulation of endodormancy induction, maintenance and release in floral buds of deciduous fruit trees. We also describe recent advances in quantitative trait loci analysis of chilling requirements in fruit trees. We discuss phytohormones, epigenetic regulation, and the detailed molecular network controlling endodormancy, centered on SHORT VEGETATIVE PHASE (SVP) and Dormancy-associated MADS-box (DAM) genes during endodormancy maintenance and release. Combining previous studies and our observations, we propose a regulatory model for bud endodormancy and offer some perspectives for the future.

Auxin is a key phytohormone that modulates fruit formation in many fleshy fruits through the regulation of cell division and expansion. Auxin content rapidly increases after pollination and the manipulation in its levels may lead to the parthenocarpic development. ln Vitis vinifera L., little is known about the early fruit development that encompasses from pollination to fruit set. Pollination/fertilization events trigger fruit formation, and auxin treatment mimics their effect in grape berry set. However, the role of auxin in this process at the molecular level is not well understood. To elucidate the participation of auxin in grapevine fruit formation, morphological, reproductive, and molecular events from anthesis to fruit set were described in sequential days after pollination. Exploratory RNA-seq analysis at four time points from anthesis to fruit set revealed that the highest percentage of genes induced/repressed within the hormone-related gene category were auxin-related genes. Transcript profiling showed significant transcript variations in auxin signaling and homeostasis-related genes during the early fruit development. Indole acetic acid and several auxin metabolites were present during this period. Finally, application of an inhibitor of auxin action reduced cell number and the mesocarp diameter, similarly to unpollinated berries, further confirming the key role of auxin during early berry development. This work sheds light into the molecular features of the initial fruit development and highlights the auxin participation during this stage in grapevine.

Rosa rugosa, commonly known as rugged rose, is a perennial ornamental shrub. It produces beautiful flowers with a mild fragrance and colorful seed pods. Unlike many other cultivated roses, R. rugosa adapts to a wide range of habitat types and harsh environmental conditions such as salinity, alkaline, shade, drought, high humidity, and frigid temperatures. Here, we produced and analyzed a high-quality genome sequence for R. rugosa to understand its ecology, floral characteristics and evolution. PacBio HiFi reads were initially used to construct the draft genome of R. rugosa, and then Hi-C sequencing was applied to assemble the contigs into 7 chromosomes. We obtained a 382.6 Mb genome encoding 39,704 protein-coding genes. The genome of R. rugosa appears to be conserved with no additional whole-genome duplication after the gamma whole-genome triplication (WGT), which occurred ~100 million years ago in the ancestor of core eudicots. Based on a comparative analysis of the high-quality genome assembly of R. rugosa and other high-quality Rosaceae genomes, we found a unique large inverted segment in the Chinese rose R. chinensis and a retroposition in strawberry caused by post-WGT events. We also found that floral development- and stress response signaling-related gene modules were retained after the WGT. Two MADS-box genes involved in floral development and the stress-related transcription factors DREB2A-INTERACTING PROTEIN 2 (DRIP2) and PEPTIDE TRANSPORTER 3 (PTR3) were found to be positively selected in evolution, which may have contributed to the unique ability of this plant to adapt to harsh environments. In summary, the high-quality genome sequence of R. rugosa provides a map for genetic studies and molecular breeding of this plant and enables comparative genomic studies of Rosa in the near future.

Polyploidization plays a crucial role in the evolution of angiosperm species. Almost all newly formed polyploids encounter genetic or epigenetic instabilities. However, the molecular mechanisms contributing to genomic instability in synthetic polyploids have not been clearly elucidated. Here, we performed a comprehensive transcriptomic and methylomic analysis of natural and synthetic polyploid rapeseeds (Brassica napus). Our results showed that the CHG methylation levels of synthetic rapeseed in different genomic contexts (genes, transposon regions, and repeat regions) were significantly lower than those of natural rapeseed. The total number and length of CHG-DMRs between natural and synthetic polyploids were much greater than those of CG-DMRs and CHH-DMRs, and the genes overlapping with these CHG-DMRs were significantly enriched in DNA damage repair and nucleotide metabolism pathways. These results indicated that CHG methylation may be more sensitive than CG and CHH methylation in regulating the stability of the polyploid genome of B. napus. In addition, many genes involved in DNA damage repair, nucleotide metabolism, and cell cycle control were significantly differentially expressed between natural and synthetic rapeseeds. Our results highlight that the genes related to DNA repair and nucleotide metabolism display differential CHG methylation patterns between natural and synthetic polyploids and reveal the potential connection between the genomic instability of polyploid plants with DNA methylation defects and dysregulation of the DNA repair system. In addition, it was found that the maintenance of CHG methylation in B. napus might be partially regulated by MET1. Our study provides novel insights into the establishment and evolution of polyploid plants and offers a potential idea for improving the genomic stability of newly formed Brassica polyploids.

Cucumber (Cucumis sativus L.) is an important vegetable crop species with great economic value. Shoot architecture determines the visual appearance of plants and has a strong impact on crop management and yield. Unlike most model plant species, cucumber undergoes vegetative growth and reproductive growth simultaneously, in which leaves are produced from the shoot apical meristem and flowers are generated from leaf axils, during the majority of its life, a feature representative of the Cucurbitaceae family. Despite substantial advances achieved in understanding the regulation of plant form in Arabidopsis thaliana, rice, and maize, our understanding of the mechanisms controlling shoot architecture in Cucurbitaceae crop species is still limited. In this review, we focus on recent progress on elucidating the genetic regulatory pathways underlying the determinant/indeterminant growth habit, leaf shape, branch outgrowth, tendril identity, and vine length determination in cucumber. We also discuss the potential of applying biotechnology tools and resources for the generation of ideal plant types with desired architectural features to improve cucumber productivity and cultivation efficiency.

Papaya (Carica papaya L.) is regarded as an excellent model for genomic studies of tropical trees because of its short generation time and its small genome that has been sequenced. However, functional genomic studies in papaya depend on laborious genetic transformations because no rapid tools exist for this species. Here, we developed a highly efficient virus-induced gene silencing (VIGS) vector for use in papaya by modifying an artificially attenuated infectious clone of papaya leaf distortion mosaic virus (PLDMV; genus: Potyvirus), PLDMV-E, into a stable Nimble Cloning (NC)-based PLDMV vector, pPLDMV-NC, in Escherichia coli. The target fragments for gene silencing can easily be cloned into pPLDMV-NC without multiple digestion and ligation steps. Using this PLDMV VIGS system, we silenced and characterized five endogenous genes in papaya, including two common VIGS marker genes, namely, phytoene desaturase, Mg-chelatase H subunit, putative GIBBERELLIN (GA)-INSENSITIVE DWARF1A and 1B encoding GA receptors; and the cytochrome P450 gene CYP83B1, which encodes a key enzyme involved in benzylglucosinolate biosynthesis. The results demonstrate that our newly developed PLDMV VIGS vector is a rapid and convenient tool for functional genomic studies in papaya.

Mature pollen germinates rapidly on the stigma, extending its pollen tube to deliver sperm cells to the ovule for fertilization. The success of this process is an important factor that limits output. The flavonoid content increased significantly during pollen germination and pollen tube growth, which suggests it may play an important role in these processes. However, the specific mechanism of this involvement has been little researched. Our previous research found that hyperoside can prolong the flowering period of Abelmoschus esculentus (okra), but its specific mechanism is still unclear. Therefore, in this study, we focused on the effect of hyperoside in regulating the actin-depolymerizing factor (ADF), which further affects the germination and growth of pollen. We found that hyperoside can prolong the effective pollination period of okra by 2–3-fold and promote the growth of pollen tubes in the style. Then, we used Nicotiana benthamiana cells as a research system and found that hyperoside accelerates the depolymerization of intercellular microfilaments. Hyperoside can promote pollen germination and pollen tube elongation in vitro. Moreover, AeADF1 was identified out of all AeADF genes as being highly expressed in pollen tubes in response to hyperoside. In addition, hyperoside promoted AeADF1-mediated microfilament dissipation according to microfilament severing experiments in vitro. In the pollen tube, the gene expression of AeADF1 was reduced to 1/5 by oligonucleotide transfection. The decrease in the expression level of AeADF1 partially reduced the promoting effect of hyperoside on pollen germination and pollen tube growth. This research provides new research directions for flavonoids in reproductive development.

The use of heterografts is widely applied for the production of several important commercial crops, but the molecular mechanism of graft union formation remains poorly understood. Here, cucumber grafted onto pumpkin was used to study graft union development, and genome-wide tempo-spatial gene expression at the graft interface was comprehensively investigated. Histological analysis suggested that resumption of the rootstock growth occurred after both phloem and xylem reconnection, and the scion showed evident callus production compared with the rootstock 3 days after grafting. Consistently, transcriptome data revealed specific responses between the scion and rootstock in the expression of genes related to cambium development, the cell cycle, and sugar metabolism during both vascular reconnection and healing, indicating distinct mechanisms. Additionally, lower levels of sugars and significantly changed sugar enzyme activities at the graft junction were observed during vascular reconnection. Next, we found that the healing process of grafted etiolated seedlings was significantly delayed, and graft success, xylem reconnection, and the growth of grafted plants were enhanced by exogenous glucose. This demonstrates that graft union formation requires the correct sugar content. Furthermore, we also found that graft union formation was delayed with a lower energy charge by the target of rapamycin (TOR) inhibitor AZD-8055, and xylem reconnection and the growth of grafted plants were enhanced under AZD-8055 with exogenous glucose treatment. Taken together, our results reveal that sugars play a positive role in graft union formation by promoting the growth of cucumber/pumpkin and provide useful information for understanding graft union healing and the application of heterografting in the future.

Sex types of papaya are controlled by a pair of nascent sex chromosomes, but molecular genetic mechanisms of sex determination and sex differentiation in papaya are still unclear. We performed comparative analysis of transcriptomic profiles of male and female floral buds at the early development stage before the initiation of reproductive organ primordia at which there is no morphological difference between male and female flowers. A total of 1734 differentially expressed genes (DEGs) were identified, of which 923 showed female-biased expression and 811 showed male-biased expression. Functional annotation revealed that genes related to plant hormone biosynthesis and signaling pathways, especially in abscisic acid and auxin pathways, were overrepresented in the DEGs. Transcription factor binding motifs, such as MYB2, GAMYB, and AP2/EREBP, were enriched in the promoters of the hormone-related DEGs, and transcription factors with those motifs also exhibited differential expression between sex types. Among these DEGs, we also identified 11 genes in the non-recombining region of the papaya sex chromosomes and 9 genes involved in stamen and carpel development. Our results suggested that sex differentiation in papaya may be regulated by multiple layers of regulation and coordination and involved transcriptional, epigenetic, and phytohormone regulation. Hormones, especially ABA and auxin, transcription factors, and genes in the non-recombination region of the sex chromosome could be involved in this process. Our findings may facilitate the elucidation of signal transduction and gene interaction in sex differentiation of unisexual flowers in papaya.

There is a critical need to identify germplasm resources and genes that promote cold tolerance of tomato because global tomato production is threatened by cold stress. We found that the expression of an F-box gene family member named ShPP2-1 from Solanum habrochaites is cold inducible and studied its contribution to cold tolerance. Overexpression of ShPP2-1 in cultivated tomato (AC) reduced cold tolerance by intensifying damage to cell membranes. To explore the underlying molecular mechanism, we conducted a yeast two-hybrid library screen and found that a protein containing ACT domain repeats named ACR11A interacts with PP2-1. Overexpression of SlACR11A in AC enhanced the cold tolerance of seedlings and germinating seeds. Cold tolerance decreased in tomato plants that overexpressed both of these genes. Additionally, we performed seed germination experiments in the cold with 177 tomato accessions and identified two alleles of SlACR11A that differ in one single-nucleotide polymorphism. We found that one of these alleles, SlACR11A^G, is significantly enriched in cold-tolerant tomato plants. Taken together, our findings indicate that the combination of low expression levels of PP2-1 and high expression levels of ACR11A can promote cold tolerance. These genes may therefore serve as direct targets for both genetic engineering and improvement projects that aim to enhance the cold tolerance of tomato.

Common bean (Phaseolus vulgaris L.), one of the most consumed food legumes worldwide, is threatened by two main constraints that are found frequently together in nature, water deficit (WD) and fusarium wilt (Fop). To understand the shared and unique responses of common bean to Fop and WD, we analyzed the transcriptomic changes and phenotypic responses in two accessions, one resistant and one susceptible to both stresses, exposed to single and combined stresses. Physiological responses (photosynthetic performance and pigments quantification) and disease progression were also assessed. The combined FopWD imposition negatively affected the photosynthetic performance and increased the susceptible accession disease symptoms. The susceptible accession revealed a higher level of transcriptional changes than the resistant one, and WD single stress triggered the highest transcriptional changes. While 89 differentially expressed genes were identified exclusively in combined stresses for the susceptible accession, 35 were identified in the resistant one. These genes belong mainly to “stress”, “signaling”, “cell wall”, “hormone metabolism”, and “secondary metabolism” functional categories. Among the up-regulated genes with higher expression in the resistant accession, the cysteine-rich secretory, antigen 5 and Pr-1 (CAP) superfamily protein, a ribulose bisphosphate carboxylase family protein, and a chitinase A seem promising targets for multiple stress breeding.

Mango (Mangifera indica L., Anacardiaceae), the fifth most consumed fruit worldwide, is one of the most important fruit crops in tropical regions, but its vascular anatomy is quite unexplored. Previous studies examined the xylem structure in the stems of mango, but the anatomy of the phloem has remained elusive, leaving the long-distance transport of photoassimilates understudied. We combined fluorescence and electron microscopy to evaluate the structure of the phloem tissue in the tapering branches of mango trees, and used this information to describe the hydraulic conductivity of its sieve tube elements following current models of fluid transport in trees. We revealed that the anatomy of the phloem changes from current year branches, where it was protected by pericyclic fibres, to older ones, where the lack of fibres was concomitant with laticiferous canals embedded in the phloem tissue. Callose was present in the sieve plates, but also in the walls of the phloem sieve cells, making them discernible from other phloem cells. A scaling geometry of the sieve tube elements-including the number of sieve areas and the pore size across tapering branches-resulted in an exponential conductivity towards the base of the tree. These evaluations in mango fit with previous measurements of the phloem architecture in the stems of forest trees, suggesting that, despite agronomic management, the phloem sieve cells scale with the tapering branches. The pipe model theory applied to the continuous tubing system of the phloem appears as a good approach to understand the hydraulic transport of photoassimilates in fruit trees.

Resveratrol plays a crucial phytoalexin role in the grapevine and is beneficial to human health. However, the molecular mechanism of resveratrol accumulation in the enhancement of disease resistance is unclear. Here, we report that the transcription factor VqMYB154 from Vitis quinquangularis accession Danfeng-2 is strongly expressed under artificial inoculation with Uncinula necator and regulates resveratrol accumulation. Unlike its homolog, VqMYB154 has a pathogen-induced promoter and responds to stimulation by U. necator, Pseudomonas syringae, and other treatments. Yeast one-hybrid and GUS activity assays confirmed that VqMYB154 can activate the stilbene synthase genes VqSTS9, VqSTS32, and VqSTS42 by directly binding to their promoters. Overexpression of VqMYB154 in grape leaves resulted in activation of the stilbene pathway, upregulation of STS genes, and accumulation of stilbenoids. In addition, heterologous overexpression of VqMYB154 in Arabidopsis activated resistance-related genes and resulted in greater programmed cell death and accumulation of reactive oxygen species, which led to resistance against P. syringae. These results suggest that the transcription factor VqMYB154 from V. quinquangularis accession Danfeng-2 participates in the regulatory mechanism that improves the biosynthesis and accumulation of stilbenes and enhances resistance to disease in grapevine.

Loquat (Eriobotrya japonica) is a subtropical tree that bears fruit that ripens during late spring. Fruit size is one of the dominant factors inhibiting the large-scale production of this fruit crop. To date, little is known about fruit size regulation. In this study, we first discovered that cell size is more important to fruit size than cell number in loquat and that the expression of the EjBZR1 gene is negatively correlated with cell and fruit size. Virus-induced gene silencing (VIGS) of EjBZR1 led to larger cells and fruits in loquat, while its overexpression reduced cell and plant size in Arabidopsis. Moreover, both the suppression and overexpression of EjBZR1 inhibited the expression of brassinosteroid (BR) biosynthesis genes, especially that of EjCYP90A. Further experiments indicated that EjCYP90A, a cytochrome P450 gene, is a fruit growth activator, while EjBZR1 binds to the BRRE (CGTGTG) motif of the EjCYP90A promoter to repress its expression and fruit cell enlargement. Overall, our results demonstrate a possible pathway by which EjBZR1 directly targets EjCYP90A and thereby affects BR biosynthesis, which influences cell expansion and, consequently, fruit size. These findings help to elucidate the molecular functions of BZR1 in fruit growth and thus highlight a useful genetic improvement that can lead to increased crop yields by repressing gene expression.

Powdery mildew (PM) caused by Podosphaera aphanis is a major fungal disease of cultivated strawberry. Mildew Resistance Locus O (MLO) is a gene family described for having conserved seven-transmembrane domains. Induced loss-of-function in specific MLO genes can confer durable and broad resistance against PM pathogens. However, the genomic structure and potential role of MLO genes for PM resistance have not been characterized yet in the octoploid cultivated strawberry. In the present study, MLO gene families were characterized in four diploid progenitor species (Fragaria vesca, F. iinumae, F. viridis, and F. nipponica) and octoploid cultivated (Fragaria ×ananassa) strawberry, and potential sources of MLO-mediated susceptibility were identified. Twenty MLO sequences were identified in F. vesca and 68 identified in F. ×ananassa. Phylogenetic analysis divided diploid and octoploid strawberry MLO genes into eight different clades, in which three FveMLO (MLO10, MLO17, and MLO20) and their twelve orthologs of FaMLO were grouped together with functionally characterized MLO genes conferring PM susceptibility. Copy number variations revealed differences in MLO composition among homoeologous chromosomes, supporting the distinct origin of each subgenome during the evolution of octoploid strawberry. Dissecting genomic sequence and structural variations in candidate FaMLO genes revealed their potential role associated with genetic controls and functionality in strawberry against PM pathogen. Furthermore, the gene expression profiling and RNAi silencing of putative FaMLO genes in response to the pathogen indicate the function in PM resistance. These results are a critical first step in understanding the function of strawberry MLO genes and will facilitate further genetic studies of PM resistance in cultivated strawberry.

DNA methylation has been proposed to regulate plant stress resistance. However, the dynamic changes in DNA methylation in woody plants and their correlations with pathogenic responses are not fully understood. Here, we present single-base maps of the DNA methylomes of mulberry (Morus notabilis) leaves that were subjected to a mock treatment or inoculation with Botrytis cinerea. Compared with the former, the latter showed decreased mCG and mCHG levels and increased mCHH levels. DNA methylation inhibitors reduced resistance gene methylation levels and enhanced mulberry resistance, suggesting that the hypomethylation of resistance genes affects mulberry resistance to B. cinerea. Virus-induced gene silencing of MnMET1 enhanced the expression of mulberry-resistance genes, thereby increasing the plant’s resistance to B. cinerea. We also found that MITEs play a dominant role in controlling DNA methylation levels. MITEs appear to be the main sources of 24-nt siRNAs that regulate gene expression through the RNA-directed DNA methylation pathway.

Understanding corm development in flower bulbs is of importance for securing the quality of cut flowers and propagation of commercial stocks. Gladiolus is one of the most popular bulb plants worldwide. Its corm development is characterized by starch accumulation. Previous research has shown that phytohormones (especially gibberellin (GA)) are involved in tuber development. However, the relationship between abscisic acid (ABA)/GA and starch during corm development remains unclear. To gain deeper insights into the biological process of corm development, we performed a detailed anatomical characterization of different stages of corm development and analyzed phytohormone levels. Our study showed that corm development is linked to hormones (ABA and GA) and carbohydrates (sucrose and starch). Exogenous hormone treatment and silencing of endogenous hormone biosynthesis genes indicated that ABA positively regulates corm development, while GA acts as an antagonist of ABA function. A sucrose synthase gene (GhSUS2) was shown to be involved in the antagonism between ABA and GA. GhSUS2 was upregulated by ABA and downregulated by GA. The increase in the transcript level of GhSUS2 coincided with the development of corm/cormels. Silencing of GhSUS2 repressed corm development and starch accumulation. In conclusion, we propose that GhSUS2, an essential enzyme in sucrose degradation, is differentially regulated by ABA and GA and controls corm development in Gladiolus.

The anthocyanin content in apple skin determines its red coloration, as seen in a Fuji apple mutant. Comparative RNA-seq analysis was performed to determine differentially expressed genes at different fruit development stages between the wild-type and the skin color mutant. A novel R2R3-MYB transcription factor, MdMYB90-like, was uncovered as the key regulatory gene for enhanced coloration in the mutant. The expression of MdMYB90-like was 21.3 times higher in the mutant. MdMYB90-like regulates anthocyanin biosynthesis directly through the activation of anthocyanin biosynthesis genes and indirectly through the activation of other transcription factors that activate anthocyanin biosynthesis. MdMYB90-like bound to the promoters of both structural genes (MdCHS and MdUFGT) and other transcription factor genes (MdMYB1 and MdbHLH3) in the yeast one-hybrid system, electrophoretic mobility shift assay, and dual-luciferase assay. Transgenic analysis showed that MdMYB90-like was localized in the nucleus, and its overexpression induced the expression of other anthocyanin-related genes, including MdCHS, MdCHI, MdANS, MdUFGT, MdbHLH3, and MdMYB1. The mutant had reduced levels of DNA methylation in two regions (−1183 to −988 and −2018 to −1778) of the MdMYB90-like gene promoter, which might explain the enhanced expression of the gene and the increased anthocyanin content in the mutant apple skin.

Camellia oleifera (C. oleifera) is one of the four major woody oil-bearing crops in the world and has relatively high ecological, economic, and medicinal value. Its seeds undergo a series of complex physiological and biochemical changes during ripening, which is mainly manifested as the accumulation and transformation of certain metabolites closely related to oil quality, especially flavonoids and fatty acids. To obtain new insights into the underlying molecular mechanisms, a parallel analysis of the transcriptome and proteome profiles of C. oleifera seeds at different maturity levels was conducted using RNA sequencing (RNA-seq) and isobaric tags for relative and absolute quantification (iTRAQ) complemented with gas chromatography-mass spectrometry (GC-MS) data. A total of 16,530 transcripts and 1228 proteins were recognized with significant differential abundances in pairwise comparisons of samples at various developmental stages. Among these, 317 were coexpressed with a poor correlation, and most were involved in metabolic processes, including fatty acid metabolism, α-linolenic acid metabolism, and glutathione metabolism. In addition, the content of total flavonoids decreased gradually with seed maturity, and the levels of fatty acids generally peaked at the fat accumulation stage; these results basically agreed with the regulation patterns of genes or proteins in the corresponding pathways. The expression levels of proteins annotated as upstream candidates of phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) as well as their cognate transcripts were positively correlated with the variation in the flavonoid content, while shikimate O-hydroxycinnamoyltransferase (HCT)-encoding genes had the opposite pattern. The increase in the abundance of proteins and mRNAs corresponding to alcohol dehydrogenase (ADH) was associated with a reduction in linoleic acid synthesis. Using weighted gene coexpression network analysis (WGCNA), we further identified six unique modules related to flavonoid, oil, and fatty acid anabolism that contained hub genes or proteins similar to transcription factors (TFs), such as MADS intervening keratin-like and C-terminal (MIKC_MADS), type-B authentic response regulator (ARR-B), and basic helix-loop-helix (bHLH). Finally, based on the known metabolic pathways and WGCNA combined with the correlation analysis, five coexpressed transcripts and proteins composed of cinnamyl-alcohol dehydrogenases (CADs), caffeic acid 3-O-methyltransferase (COMT), flavonol synthase (FLS), and 4-coumarate: CoA ligase (4CL) were screened out. With this exploratory multiomics dataset, our results presented a dynamic picture regarding the maturation process of C. oleifera seeds on Hainan Island, not only revealing the temporal specific expression of key candidate genes and proteins but also providing a scientific basis for the genetic improvement of this tree species.

Plant glandular trichomes (GTs) are epidermal outgrowths with the capacity to biosynthesize and secrete specialized metabolites, that are of great scientific and practical significance. Our understanding of the developmental process of GTs is limited, and no single plant species serves as a unique model. Here, we review the genetic mechanisms of GT initiation and development and provide a summary of the biosynthetic pathways of GT-specialized metabolites in nonmodel plant species, especially horticultural crops. We discuss the morphology and classification of GT types. Moreover, we highlight technological advancements in methods employed for investigating GTs. Understanding the molecular basis of GT development and specialized metabolites not only offers useful avenues for research in plant breeding that will lead to the improved production of desirable metabolites, but also provides insights for plant epidermal development research.

Jasmonic acid (JA) plays an important role in regulating leaf senescence. However, the molecular mechanisms of leaf senescence in apple (Malus domestica) remain elusive. In this study, we found that MdZAT10, a C2H2-type zinc finger transcription factor (TF) in apple, markedly accelerates leaf senescence and increases the expression of senescence-related genes. To explore how MdZAT10 promotes leaf senescence, we carried out liquid chromatography/mass spectrometry screening. We found that MdABI5 physically interacts with MdZAT10. MdABI5, an important positive regulator of leaf senescence, significantly accelerated leaf senescence in apple. MdZAT10 was found to enhance the transcriptional activity of MdABI5 for MdNYC1 and MdNYE1, thus accelerating leaf senescence. In addition, we found that MdZAT10 expression was induced by methyl jasmonate (MeJA), which accelerated JA-induced leaf senescence. We also found that the JA-responsive protein MdBT2 directly interacts with MdZAT10 and reduces its protein stability through ubiquitination and degradation, thereby delaying MdZAT10-mediated leaf senescence. Taken together, our results provide new insight into the mechanisms by which MdZAT10 positively regulates JA-induced leaf senescence in apple.

Horticulturists are interested in evaluating how cultivar, environment, or production system inputs can affect postharvest quality. Ambient mass spectrometry approaches enable analysis of minimally processed samples under ambient conditions and offer an attractive high-throughput alternative for assessing quality characteristics in plant products. Here, we evaluate direct analysis in real time (DART-MS) mass spectrometry and rapid evaporative ionization-mass spectrometry (REIMS) to assess quality characteristics in various pepper (Capsicum annuum L.) cultivars. DART-MS exhibited the ability to discriminate between pod colors and pungency based on chemical fingerprints, while REIMS could distinguish pepper market class (e.g., bell, lunchbox, and popper). Furthermore, DART-MS analysis resulted in the putative detection of important bioactive compounds in human diet such as vitamin C, p-coumaric acid, and capsaicin. The results of this study demonstrate the potential for these approaches as accessible and reliable tools for high throughput screening of pepper quality.

Grapevine downy mildew (DM) is a destructive oomycete disease of viticulture worldwide. MrRPV1 is a typical TIR-NBS-LRR type DM disease resistance gene cloned from the wild North American grapevine species Muscadinia rotundifolia. However, the molecular basis of resistance mediated by MrRPV1 remains poorly understood. Downy mildew-susceptible Vitis vinifera cv. Shiraz was transformed with a genomic fragment containing MrRPV1 to produce DM-resistant transgenic Shiraz lines. Comparative transcriptome analysis was used to compare the transcriptome profiles of the resistant and susceptible genotypes after DM infection. Transcriptome modulation during the response to P. viticola infection was more rapid, and more genes were induced in MrRPV1-transgenic Shiraz than in wild-type plants. In DM-infected MrRPV1-transgenic plants, activation of genes associated with Ca²⁺ release and ROS production was the earliest transcriptional response. Functional analysis of differentially expressed genes revealed that key genes related to multiple phytohormone signaling pathways and secondary metabolism were highly induced during infection. Coexpression network and motif enrichment analysis showed that WRKY and MYB transcription factors strongly coexpress with stilbene synthase (VvSTS) genes during defense against P. viticola in MrRPV1-transgenic plants. Taken together, these findings indicate that multiple pathways play important roles in MrRPV1-mediated resistance to downy mildew.

Plants respond differently to the identity of their neighbors, such as their sex and kinship, showing plasticity in their traits. However, how the functional traits of dioecious trees are shaped by the recognition of neighbors with different sex and kinship remains unknown. In this study, we set up an experiment with different kin/nonkin and inter/intrasexual combinations for a dioecious tree species, Diospyros morrisiana. The results showed that plants grew better with nonkin and intrasexual neighbors than with kin and intersexual neighbors. Kin combinations had significantly shorter root length in the resource-overlapping zone than nonkin combinations, suggesting that kin tended to reduce competition by adjusting their root distribution, especially among female siblings. Our study suggested that the seedling growth of D. morrisiana was affected by both the relatedness and sexual identity of neighboring plants. Further analysis by gas chromatography-mass spectrometry showed that the root exudate composition of female seedlings differed from that of male seedlings. Root exudates may play important roles in sex competition in dioecious plants. This study indicates that sex-specific competition and kin recognition interact and co-shape the traits of D. morrisiana seedlings, while intrasexual and nonkin neighbors facilitate the growth of seedlings. Our study implies that kin- and sex-related interactions depend on different mechanisms, kin selection, and niche partitioning, respectively. These results are critical for understanding how species coexist and how traits are shaped in nature.

Anthocyanins play vital roles in plant stress tolerance and growth regulation. Previously, we reported that the photomorphogenesis-related transcription factor SlBBX20 regulates anthocyanin accumulation in tomato. However, the underlying mechanism remains unclear. Here, we showed that SlBBX20 promotes anthocyanin biosynthesis by binding the promoter of the anthocyanin biosynthesis gene SlDFR, suggesting that SlBBX20 directly activates anthocyanin biosynthesis genes. Furthermore, we found by yeast two-hybrid screening that SlBBX20 interacts with the COP9 signalosome subunit SlCSN5-2, and the interaction was confirmed by bimolecular fluorescence complementation and coimmunoprecipitation assays. SlCSN5 gene silencing led to anthocyanin hyperaccumulation in the transgenic tomato calli and shoots, and SlCSN5-2 overexpression decreased anthocyanin accumulation, suggesting th SlCSN5-2 enhanced the ubiquitination of SlBBX20 and promoted the degradation of SlBBX20 in vivo. Consistently, silencing the SlCSN5-2 homolog in tobacco significantly increased the accumulation of the SlBBX20 protein. Since SlBBX20 is a vital regulator of photomorphogenesis, the SlBBX20- SlCSN5-2 module may represent a novel regulatory pathway in light-induced anthocyanin biosynthesis.

Pitaya (Hylocereus) is the most economically important fleshy-fruited tree of the Cactaceae family that is grown worldwide, and it has attracted significant attention because of its betalain-abundant fruits. Nonetheless, the lack of a pitaya reference genome significantly hinders studies focused on its evolution, as well as the potential for genetic improvement of this crop. Herein, we employed various sequencing approaches, namely, PacBio-SMRT, Illumina HiSeq paired-end, 10× Genomics, and Hi-C (high-throughput chromosome conformation capture) to provide a chromosome-level genomic assembly of ‘GHB’ pitaya (H. undatus, 2n = 2x = 22 chromosomes). The size of the assembled pitaya genome was 1.41 Gb, with a scaffold N50 of ~127.15 Mb. In total, 27,753 protein-coding genes and 896.31 Mb of repetitive sequences in the H. undatus genome were annotated. Pitaya has undergone a WGT (whole-genome triplication), and a recent WGD (whole-genome duplication) occurred after the gamma event, which is common to the other species in Cactaceae. A total of 29,328 intact LTR-RTs (~696.45 Mb) were obtained in H. undatus, of which two significantly expanded lineages, Ty1/copia and Ty3/gypsy, were the main drivers of the expanded genome. A high-density genetic map of F1 hybrid populations of ‘GHB’ × ‘Dahong’ pitayas (H. monacanthus) and their parents were constructed, and a total of 20,872 bin markers were identified (56,380 SNPs) for 11 linkage groups. More importantly, through transcriptomic and WGCNA (weighted gene coexpression network analysis), a global view of the gene regulatory network, including structural genes and the transcription factors involved in pitaya fruit betalain biosynthesis, was presented. Our data present a valuable resource for facilitating molecular breeding programs of pitaya and shed novel light on its genomic evolution, as well as the modulation of betalain biosynthesis in edible fruits.

CCCH is a subfamily of zinc finger proteins involved in plant growth, development, and stresses response. The function of CCCH in regulating leaf senescence, especially its roles in abscisic acid (ABA)-mediated leaf senescence is largely unknown. The objective of this study was to determine functions and mechanisms of CCCH gene in regulating leaf senescence in switchgrass (Panicum virgatum). A CCCH gene, PvCCCH69 (PvC3H69), was cloned from switchgrass. Overexpressing PvC3H69 in rice suppressed both natural senescence with leaf aging and dark-induced leaf senescence. Endogenous ABA content, ABA biosynthesis genes (NCED3, NCED5, and AAO3), and ABA signaling-related genes (SnRKs, ABI5, and ABF2/3/4) exhibited significantly lower levels in senescencing leaves of PvC3H69-OE plants than those in WT plants. PvC3H69-suppression of leaf senescence was associated with transcriptional upregulation of genes mainly involved in the light-dependent process of photosynthesis, including light-harvesting complex proteins, PSI proteins, and PSII proteins and downregulation of ABA biosynthesis and signaling genes and senescence-associated genes. PvC3H69 could act as a repressor for leaf senescence via upregulating photosynthetic proteins and repressing ABA synthesis and ABA signaling pathways.

Breeding has been used successfully for many years in the fruit industry, giving rise to most of today’s commercial fruit cultivars. More recently, new molecular breeding techniques have addressed some of the constraints of conventional breeding. However, the development and commercial introduction of such novel fruits has been slow and limited with only five genetically engineered fruits currently produced as commercial varieties-virus-resistant papaya and squash were commercialized 25 years ago, whereas insect-resistant eggplant, non-browning apple, and pink-fleshed pineapple have been approved for commercialization within the last 6 years and production continues to increase every year. Advances in molecular genetics, particularly the new wave of genome editing technologies, provide opportunities to develop new fruit cultivars more rapidly. Our review, emphasizes the socioeconomic impact of current commercial fruit cultivars developed by genetic engineering and the potential impact of genome editing on the development of improved cultivars at an accelerated rate.

The spread of transgenes and exotic germplasm from planted crops into wild or feral species is a difficult problem for public and regulatory acceptance of genetically engineered plants, particularly for wind-pollinated trees such as poplar. We report that overexpression of a poplar homolog of the floral repressor SHORT VEGETATIVE PHASE-LIKE (SVL), a homolog of the Arabidopsis MADS-box repressor SHORT VEGETATIVE PHASE (SVP), delayed the onset of flowering several years in three genotypes of field-grown transgenic poplars. Higher expression of SVL correlated with a delay in flowering onset and lower floral abundance, and did not cause morphologically obvious or statistically significant effects on leaf characteristics, tree form, or stem volume. Overexpression effects on reproductive and vegetative phenology in spring was modest and genotype-specific. Our results suggest that use of SVL and related floral repressors can be useful tools to enable a high level of containment for vegetatively propagated short-rotation woody energy or pulp crops.

The skin protects a fruit from environmental stresses and supports the fruit’s structure. Failure of the skin leads to fruit splitting and may compromise commercial production for fruit growers. The mechanical properties of the cuticle and skin cell walls might influence the splitting susceptibility of fleshy fruits. Thin shell theory and fracture mechanics were utilized in this study to target the potential factors contributing to splitting susceptibility. The study analyzed the structure of the cuticle and epidermis in ripening grape berries and examined the temporal dynamics of berry splitting. Cuticular waxes were partially removed, and skin cell walls were manipulated using wall stiffening and loosening solutions that altered reactions involving hydrogen peroxide. A more than twofold difference in cuticle thickness among grape cultivars did not account for their differences in splitting resistance. However, while removing predominantly epicuticular wax did not alter the berries’ splitting resistance, their surface appearance and increasing yield strength following partial wax removal support the notion that cuticular waxes contribute to berry mechanical properties. Immersing berries in H₂O₂-based cell wall loosening solutions increased the splitting probability and accelerated berry splitting, whereas cell wall stiffening solutions decreased the splitting probability and delayed berry splitting. These results showed that both cuticle and skin cell walls contribute to the mechanical properties of grape berries and to their splitting resistance. The results also suggest that the two current explanations for fruit splitting, the critical turgor model and the zipper model, should be viewed as complementary rather than incompatible.

Fruit quality traits play a significant role in consumer preferences and consumption in blueberry (Vaccinium corymbosum L). The objectives of this study were to construct a high-density linkage map and to identify the underlying genetic basis of fruit quality traits in blueberry. A total of 287 F₁ individuals derived from a cross between two southern highbush blueberry cultivars, ‘Reveille’ and ‘Arlen’, were phenotyped over three years (2016–2018) for fruit quality-related traits, including titratable acidity, pH, total soluble solids, and fruit weight. A high-density linkage map was constructed using 17k single nucleotide polymorphisms markers. The linkage map spanned a total of 1397 cM with an average inter-loci distance of 0.08 cM. The quantitative trait loci interval mapping based on the hidden Markov model identified 18 loci for fruit quality traits, including seven loci for fruit weight, three loci for titratable acidity, five loci for pH, and three loci for total soluble solids. Ten of these loci were detected in more than one year. These loci explained phenotypic variance ranging from 7 to 28% for titratable acidity and total soluble solid, and 8–13% for pH. However, the loci identified for fruit weight did not explain more than 10% of the phenotypic variance. We also reported the association between fruit quality traits and metabolites detected by Proton nuclear magnetic resonance analysis directly responsible for these fruit quality traits. Organic acids, citric acid, and quinic acid were significantly (P &lt; 0.05) and positively correlated with titratable acidity. Sugar molecules showed a strong and positive correlation with total soluble solids. Overall, the study dissected the genetic basis of fruit quality traits and established an association between these fruit quality traits and metabolites.

Sex dimorphism and gene expression were studied in developing catkins in 159 F₂ individuals from the bioenergy crop Salix purpurea, and potential mechanisms and pathways for regulating sex development were explored. Differential expression, eQTL, bisulfite sequencing, and network analysis were used to characterize sex dimorphism, detect candidate master regulator genes, and identify pathways through which the sex determination region (SDR) may mediate sex dimorphism. Eleven genes are presented as candidates for master regulators of sex, supported by gene expression and network analyses. These include genes putatively involved in hormone signaling, epigenetic modification, and regulation of transcription. eQTL analysis revealed a suite of transcription factors and genes involved in secondary metabolism and floral development that were predicted to be under direct control of the sex determination region. Furthermore, data from bisulfite sequencing and small RNA sequencing revealed strong differences in expression between males and females that would implicate both of these processes in sex dimorphism pathways. These data indicate that the mechanism of sex determination in Salix purpurea is likely different from that observed in the related genus Populus. This further demonstrates the dynamic nature of SDRs in plants, which involves a multitude of mechanisms of sex determination and a high rate of turnover.

Tomato (Solanum lycopersicum) is one of the most economically important vegetables throughout the world. It is one of the best studied cultivated dicotyledonous plants, often used as a model system for plant research into classical genetics, cytogenetics, molecular genetics, and molecular biology. Tomato plants are affected by different pathogens such as viruses, viroids, fungi, oomycetes, bacteria, and nematodes, that reduce yield and affect product quality. The study of tomato as a plant-pathogen system helps to accelerate the discovery and understanding of the molecular mechanisms underlying disease resistance and offers the opportunity of improving the yield and quality of their edible products. The use of functional genomics has contributed to this purpose through both traditional and recently developed techniques, that allow the identification of plant key functional genes in susceptible and resistant responses, and the understanding of the molecular basis of compatible interactions during pathogen attack. Next-generation sequencing technologies (NGS), which produce massive quantities of sequencing data, have greatly accelerated research in biological sciences and offer great opportunities to better understand the molecular networks of plant–pathogen interactions. In this review, we summarize important research that used high-throughput RNA-seq technology to obtain transcriptome changes in tomato plants in response to a wide range of pathogens such as viruses, fungi, bacteria, oomycetes, and nematodes. These findings will facilitate genetic engineering efforts to incorporate new sources of resistance in tomato for protection against pathogens and are of major importance for sustainable plant-disease management, namely the ones relying on the plant’s innate immune mechanisms in view of plant breeding.

Recognizing plant cultivars reliably and efficiently can benefit plant breeders in terms of property rights protection and innovation of germplasm resources. Although leaf image-based methods have been widely adopted in plant species identification, they seldom have been applied in cultivar identification due to the high similarity of leaves among cultivars. Here, we propose an automatic leaf image-based cultivar identification pipeline called MFCIS (Multi-feature Combined Cultivar Identification System), which combines multiple leaf morphological features collected by persistent homology and a convolutional neural network (CNN). Persistent homology, a multiscale and robust method, was employed to extract the topological signatures of leaf shape, texture, and venation details. A CNN-based algorithm, the Xception network, was fine-tuned for extracting high-level leaf image features. For fruit species, we benchmarked the MFCIS pipeline on a sweet cherry ( Prunus avium L.) leaf dataset with &gt;5000 leaf images from 88 varieties or unreleased selections and achieved a mean accuracy of 83.52%. For annual crop species, we applied the MFCIS pipeline to a soybean ( Glycine max L. Merr.) leaf dataset with 5000 leaf images of 100 cultivars or elite breeding lines collected at five growth periods. The identification models for each growth period were trained independently, and their results were combined using a score-level fusion strategy. The classification accuracy after score-level fusion was 91.4%, which is much higher than the accuracy when utilizing each growth period independently or mixing all growth periods. To facilitate the adoption of the proposed pipelines, we constructed a user-friendly web service, which is freely available at http://www.mfcis.online.

Flavonoids play important roles in regulating plant growth and development. In this study, three kaempferol 3-O-glycosides were identified and mainly accumulated in flowers but not in leaves or fruits of Malus. In Malus, flower petal color is normally white, but some genotypes have red flowers containing anthocyanin. Anthocyanin biosynthesis appears to be in competition with kaempferol 3-O-glycosides production and controlled by the biosynthetic genes. The white flower Malus genotypes had better-developed seeds than the red flower genotypes. In flowers, the overexpression of MYB10 in Malus domestica enhanced the accumulation of anthocyanin, but decreased that of kaempferol 3-O-glycosides. After pollination the transgenic plants showed slower pollen tube growth and fewer developed seeds. Exogenous application of different flavonoid compounds suggested that kaempferol 3-O-glycosides, especially kaempferol 3-O-rhamnoside, regulated pollen tube growth and seed set rather than cyanidin or quercetin 3-O-glycosides. It was found that kaempferol 3-O-rhamnoside might regulate pollen tube growth through effects on auxin, the Rho of plants (ROP) GTPases, calcium and the phosphoinositides signaling pathway. With the inhibition of auxin transport, the transcription levels of Heat Shock Proteins (HSPs) and ROP GTPases were downregulated while the levels were not changed or even enhanced when blocking calcium signaling, suggesting that HSPs and ROP GTPases were downstream of auxin signaling, but upstream of calcium signaling. In summary, kaempferol glycoside concentrations in pistils correlated with auxin transport, the transcription of HSPs and ROP GTPases, and calcium signaling in pollen tubes, culminating in changes to pollen tube growth and seed set.

Flowering is crucial to plant reproduction and controlled by multiple factors. However, the mechanisms underlying the regulation of flowering in perennial plants are still largely unknown. Here, we first report a super long blooming 1 (slb1) mutant of the relict tree Liriodendron chinense possessing a prolonged blooming period of more than 5 months, in contrast to the 1 month blooming period in the wild type (WT). Phenotypic characterization showed that earlier maturation of lateral shoots was caused by accelerated axillary bud fate, leading to the phenotype of continuous flowering in slb1 mutants. The transcriptional activity of genes related to hormone signaling (auxin, cytokinin, and strigolactone), nutrient availability, and oxidative stress relief further indicated active outgrowth of lateral buds in slb1 mutants. Interestingly, we discovered a unique FT splicing variant with intron retention specific to slb1 mutants, representing a potential causal mutation in the slb1 mutants. Surprisingly, most slb1 inbred offspring flowered precociously with shorter juvenility (~4 months) than that (usually 8–10 years) required in WT plants, indicating heritable variation underlying continuous flowering in slb1 mutants. This study reports an example of a perennial tree mutant that flowers continuously, providing a rare resource for both breeding and genetic research.

Although multiple microscopic techniques have been applied to horticultural research, few studies of individual organelles in living fruit cells have been reported to date. In this paper, we established an efficient system for the transient transformation of citrus fruits using an Agrobacterium-mediated method. Kumquat (Fortunella crassifolia Swingle) was used; it exhibits higher transformation efficiency than all citrus fruits that have been tested and a prolonged-expression window. Fruits were transformed with fluorescent reporters, and confocal microscopy and live-cell imaging were used to study their localization and dynamics. Moreover, various pH sensors targeting different subcellular compartments were expressed, and the local pH environments in cells from different plant tissues were compared. The results indicated that vacuoles are most likely the main organelles that contribute to the low pH of citrus fruits. In summary, our method is effective for studying various membrane trafficking events, protein localization, and cell physiology in fruit and can provide new insight into fruit biology research.

Tea, coffee, and cocoa are the three most popular nonalcoholic beverages in the world and have extremely high economic and cultural value. The genomes of four tea plant varieties have recently been sequenced, but there is some debate regarding the characterization of a whole-genome duplication (WGD) event in tea plants. Whether the WGD in the tea plant is shared with other plants in order Ericales and how it contributed to tea plant evolution remained unanswered. Here we re-analyzed the tea plant genome and provided evidence that tea experienced only WGD event after the core-eudicot whole-genome triplication (WGT) event. This WGD was shared by the Polemonioids-Primuloids-Core Ericales (PPC) sections, encompassing at least 17 families in the order Ericales. In addition, our study identified eight pairs of duplicated genes in the catechins biosynthesis pathway, four pairs of duplicated genes in the theanine biosynthesis pathway, and one pair of genes in the caffeine biosynthesis pathway, which were expanded and retained following this WGD. Nearly all these gene pairs were expressed in tea plants, implying the contribution of the WGD. This study shows that in addition to the role of the recent tandem gene duplication in the accumulation of tea flavor-related genes, the WGD may have been another main factor driving the evolution of tea flavor.

Polyploidization plays a key role in plant evolution, but the forces driving the fate of homoeologs in polyploid genomes, i.e., paralogs resulting from a whole-genome duplication (WGD) event, remain to be elucidated. Here, we present a chromosome-scale genome assembly of tetraploid scarlet sage (Salvia splendens), one of the most diverse ornamental plants. We found evidence for three WGD events following an older WGD event shared by most eudicots (the γ event). A comprehensive, spatiotemporal, genome-wide analysis of homoeologs from the most recent WGD unveiled expression asymmetries, which could be associated with genomic rearrangements, transposable element proximity discrepancies, coding sequence variation, selection pressure, and transcription factor binding site differences. The observed differences between homoeologs may reflect the first step toward sub- and/or neofunctionalization. This assembly provides a powerful tool for understanding WGD and gene and genome evolution and is useful in developing functional genomics and genetic engineering strategies for scarlet sage and other Lamiaceae species.

Organic tea is more popular than conventional tea that originates from fertilized plants. Amino acids in organic soils constitute a substantial pool nitrogen (N) available for plants. However, the amino-acid contents in soils of tea plantations and how tea plants take up these amino acids remain largely unknown. In this study, we show that the amino-acid content in the soil of an organic tea plantation is significantly higher than that of a conventional tea plantation. Glutamate, alanine, valine, and leucine were the most abundant amino acids in the soil of this tea plantation. When¹⁵N-glutamate was fed to tea plants, it was efficiently absorbed and significantly increased the contents of other amino acids in the roots. We cloned seven CsLHT genes encoding amino-acid transporters and found that the expression of CsLHT1, CsLHT2, and CsLHT6 in the roots significantly increased upon glutamate feeding. Moreover, the expression of CsLHT1 or CsLHT6 in a yeast amino-acid uptake-defective mutant, 22Δ10α, enabled growth on media with amino acids constituting the sole N source. Amino-acid uptake assays indicated that CsLHT1 and CsLHT6 are H⁺-dependent high- and low-affinity amino-acid transporters, respectively. We further demonstrated that CsLHT1 and CsLHT6 are highly expressed in the roots and are localized to the plasma membrane. Moreover, overexpression of CsLHT1 and CsLHT6 in Arabidopsis significantly improved the uptake of exogenously supplied¹⁵N-glutamate and¹⁵N-glutamine. Taken together, our findings are consistent with the involvement of CsLHT1 and CsLHT6 in amino-acid uptake from the soil, which is particularly important for tea plants grown in organic tea plantations.

Computational tool-assisted primer design for real-time reverse transcription (RT) PCR (qPCR) analysis largely ignores the sequence similarities between sequences of homologous genes in a plant genome. It can lead to false confidence in the quality of the designed primers, which sometimes results in skipping the optimization steps for qPCR. However, the optimization of qPCR parameters plays an essential role in the efficiency, specificity, and sensitivity of each gene’s primers. Here, we proposed an optimized approach to sequentially optimizing primer sequences, annealing temperatures, primer concentrations, and cDNA concentration range for each reference (and target) gene. Our approach started with a sequence-specific primer design that should be based on the single-nucleotide polymorphisms (SNPs) present in all the homologous sequences for each of the reference (and target) genes under study. By combining the efficiency calibrated and standard curve methods with the 2^−ΔΔCt method, the standard cDNA concentration curve with a logarithmic scale was obtained for each primer pair for each gene. As a result, an R² ≥ 0.9999 and the efficiency (E) = 100 ± 5% should be achieved for the best primer pair of each gene, which serve as the prerequisite for using the 2^−ΔΔCt method for data analysis. We applied our newly developed approach to identify the best reference genes in different tissues and at various inflorescence developmental stages of Tripidium ravennae, an ornamental and biomass grass, and validated their utility under varying abiotic stress conditions. We also applied this approach to test the expression stability of six reference genes in soybean under biotic stress treatment with Xanthomonas axonopodis pv. glycines (Xag). Thus, these case studies demonstrated the effectiveness of our optimized protocol for qPCR analysis.

Tomato seedlings were used as experimental materials and treated with 1.0, 2.0, 3.0, and 4.0 mg/L ozone water irrigation and 0.2, 0.4, 0.6, and 0.8 mg/L ozone water spray treatments. Indexes including the malondialdehyde (MDA) content, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), activities, soil and plant analysis development (SPAD) value, and nitrogen content of leaves were measured. Furthermore, the expression of antioxidant enzyme, chlorophyll synthesis and nitrogen absorption genes was analyzed after optimal ozone water treatment. The results showed that the activities of antioxidant enzymes in tomato leaves were significantly increased, and the MDA content in tomato leaves was significantly reduced by ozone water irrigation and spray treatment, which indicated that ozone water treatment can significantly improve the stress tolerance of tomato seedlings. Ozone water irrigation and spraying could also significantly increase the leaf SPAD value and nitrogen content of tomato seedlings, and the optimal concentrations of ozone water irrigation and spraying were 3.0 mg/L and 0.6 mg/L, respectively. The effect of ozone water irrigation on improving the physiological characteristics of tomato seedlings was better than that of spraying. After treatment with the optimal concentration of ozone water, the relative expression of antioxidant enzyme, chlorophyll synthesis, and nitrogen absorption genes was significantly increased, and the maximum expression level was reached at 12 h. In addition, ozone water irrigation could promote the expression of genes more than ozone water spraying, which was consistent with the improvements in the physiological characteristics of the tomato seedlings.

Overcoming short-day-dependent tuberization to adapt to long-day conditions is critical for the widespread geographical success of potato. The genetic pathways of photoperiodic tuberization are similar to those of photoperiodic flowering. DNA methylation plays an important role in photoperiodic flowering. However, little is known about how DNA methylation affects photoperiodic tuberization in potato. Here, we verified the effect of a DNA methylation inhibitor on photoperiodic tuberization and compared the DNA methylation levels and differentially methylated genes (DMGs) in the photoperiodic tuberization process between photoperiod-sensitive and photoperiod-insensitive genotypes, aiming to dissect the role of DNA methylation in the photoperiodic tuberization of potato. We found that a DNA methylation inhibitor could promote tuber initiation in strict short-day genotypes. Whole-genome DNA methylation sequencing showed that the photoperiod-sensitive and photoperiod-insensitive genotypes had distinct DNA methylation modes in which few differentially methylated genes were shared. Transcriptome analysis confirmed that the DNA methylation inhibitor regulated the expression of the key genes involved in the photoperiod and GA pathways to promote tuber initiation in the photoperiod-sensitive genotype. Comparison of the DNA methylation levels and transcriptome levels identified 52 candidate genes regulated by DNA methylation that were predicted to be involved in photoperiodic tuberization. Our findings provide a new perspective for understanding the relationship between photoperiod-dependent and GA-regulated tuberization. Uncovering the epigenomic signatures of these pathways will greatly enhance potato breeding for adaptation to a wide range of environments.

Temperature changes affect apple development and production. Phenylpropanoid metabolism and hormone signaling play a crucial role in regulating apple growth and development in response to temperature changes. Here, we found that McMYB4 is induced by treatment at 28 °C and 18 °C, and McMYB4 overexpression results in flavonol and lignin accumulation in apple leaves. Yeast one-hybrid (Y1H) assays and electrophoretic mobility shift assays (EMSAs) further revealed that McMYB4 targets the promoters of the flavonol biosynthesis genes CHS and FLS and the lignin biosynthesis genes CAD and F5H. McMYB4 expression resulted in higher levels of flavonol and lignin biosynthesis in apple during growth at 28 °C and 18 °C than during growth at 23 °C. At 28 °C and 18 °C, McMYB4 also binds to the AUX/ARF and BRI/BIN promoters to activate gene expression, resulting in acceleration of the auxin and brassinolide signaling pathways. Taken together, our results demonstrate that McMYB4 promotes flavonol biosynthesis and brassinolide signaling, which decreases ROS contents to improve plant resistance and promotes lignin biosynthesis and auxin signaling to regulate plant growth. This study suggests that McMYB4 participates in the abiotic resistance and growth of apple in response to temperature changes by regulating phenylpropanoid metabolism and hormone signaling.

As one of the largest families of angiosperms, the Orchidaceae family is diverse. Dendrobium represents the second largest genus of the Orchidaceae. However, an assembled high-quality genome of species in this genus is lacking. Here, we report a chromosome-scale reference genome of Dendrobium chrysotoxum, an important ornamental and medicinal orchid species. The assembled genome size of D. chrysotoxum was 1.37 Gb, with a contig N50 value of 1.54 Mb. Of the sequences, 95.75% were anchored to 19 pseudochromosomes. There were 30,044 genes predicted in the D. chrysotoxum genome. Two whole-genome polyploidization events occurred in D. chrysotoxum. In terms of the second event, whole-genome duplication (WGD) was also found to have occurred in other Orchidaceae members, which diverged mainly via gene loss immediately after the WGD event occurred; the first duplication was found to have occurred in most monocots (tau event). We identified sugar transporter (SWEET) gene family expansion, which might be related to the abundant medicinal compounds and fleshy stems of D. chrysotoxum. MADS-box genes were identified in D. chrysotoxum, as well as members of TPS and Hsp90 gene families, which are associated with resistance, which may contribute to the adaptive evolution of orchids. We also investigated the interplay among carotenoid, ABA, and ethylene biosynthesis in D. chrysotoxum to elucidate the regulatory mechanisms of the short flowering period of orchids with yellow flowers. The reference D. chrysotoxum genome will provide important insights for further research on medicinal active ingredients and breeding and enhances the understanding of orchid evolution.

Leaf size and flatness directly affect photosynthesis and are closely related to agricultural yield. The final leaf size and shape are coordinately determined by cell proliferation, differentiation, and expansion during leaf development. Lettuce (Lactuca sativa L.) is one of the most important leafy vegetables worldwide, and lettuce leaves vary in shape and size. However, the molecular mechanisms of leaf development in lettuce are largely unknown. In this study, we showed that the lettuce APETALA2 (LsAP2) gene regulates leaf morphology. LsAP2 encodes a transcriptional repressor that contains the conserved EAR motif, which mediates interactions with the TOPLESS/TOPLESS-RELATED (TPL/TPR) corepressors. Overexpression of LsAP2 led to small and crinkly leaves, and many bulges were seen on the surface of the leaf blade. LsAP2 physically interacted with the CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors and inhibited their transcriptional activation activity. RNA sequencing analysis showed that LsAP2 affected the expression of auxin- and polarity-related genes. In addition, LsAP2 directly repressed the abaxial identity gene KANADI2 (LsKAN2). Together, these results indicate that LsAP2 regulates leaf morphology by inhibiting CIN-like TCP transcription factors and repressing LsKAN2, and our work provides insights into the regulatory mechanisms of leaf development in lettuce.

Strawberry (Fragaria spp.) is a member of the Rosoideae subfamily in the family Rosaceae. The self-incompatibility (SI) of some diploid species is a key agronomic trait that acts as a basic pollination barrier; however, the genetic mechanism underlying SI control in strawberry remains unclear. Two candidate S-RNases (S_a- and S_b-RNase) identified in the transcriptome of the styles of the self-incompatible Fragaria viridis 42 were confirmed to be SI determinants at the S locus following genotype identification and intraspecific hybridization using selfing progenies. Whole-genome collinearity and RNase T2 family analysis revealed that only an S locus exists in Fragaria; however, none of the compatible species contained S-RNase. Although the results of interspecific hybridization experiments showed that F. viridis (SI) styles could accept pollen from F. mandshurica (self-compatible), the reciprocal cross was incompatible. S_a and S_b-RNase contain large introns, and their noncoding sequences (promotors and introns) can be transcribed into long noncoding RNAs (lncRNAs). Overall, the genus Fragaria exhibits S-RNase-based gametophytic SI, and S-RNase loss occurs at the S locus of compatible germplasms. In addition, a type of SI-independent unilateral incompatibility exists between compatible and incompatible Fragaria species. Furthermore, the large introns and neighboring lncRNAs in S-RNase in Fragaria could offer clues about S-RNase expression strategies.

Sugars, especially glucose and fructose, contribute to the taste and quality of tomato fruits. These compounds are translocated from the leaves to the fruits and then unloaded into the fruits by various sugar transporters at the plasma membrane. SWEETs, are sugar transporters that regulate sugar efflux independently of energy or pH. To date, the role of SWEETs in tomato has received very little attention. In this study, we performed functional analysis of SlSWEET7a and SlSWEET14 to gain insight into the regulation of sugar transport and storage in tomato fruits. SlSWEET7a and SlSWEET14 were mainly expressed in peduncles, vascular bundles, and seeds. Both SlSWEET7a and SlSWEET14 are plasma membrane-localized proteins that transport fructose, glucose, and sucrose. Apart from the resulting increase in mature fruit sugar content, silencing SlSWEET7a or SlSWEET14 resulted in taller plants and larger fruits (in SlSWEET7a-silenced lines). We also found that invertase activity and gene expression of some SlSWEET members increased, which was consistent with the increased availability of sucrose and hexose in the fruits. Overall, our results demonstrate that suppressing SlSWEET7a and SlSWEET14 could be a potential strategy for enhancing the sugar content of tomato fruits.

Watercore is a physiological disorder in apple (Malus × domestica Borkh.) fruits that appears as water-soaked tissues adjacent to the vascular core, although there is little information on what exactly occurs at cell level in the watercored apples, particularly from the viewpoint of cell water relations. By combining picolitre pressure-probe electrospray-ionization mass spectrometry (picoPPESI-MS) with freezing point osmometry and vapor pressure osmometry, changes in cell water status and metabolisms were spatially assayed in the same fruit. In the watercored fruit, total soluble solid was lower in the watercore region than the normal outer parenchyma region, but there was no spatial difference in the osmotic potentials determined with freezing point osmometry. Importantly, a disagreement between the osmotic potentials determined with two methods has been observed in the watercore region, indicating the presence of significant volatile compounds in the cellular fluids collected. In the watercored fruit, cell turgor varied across flesh, and a steeper water potential gradient has been established from the normal outer parenchyma region to the watercore region, retaining the potential to transport water to the watercore region. Site-specific analysis using picoPPESI-MS revealed that together with a reduction in turgor, remarkable metabolic modifications through fermentation have occurred at the border, inducing greater production of watercore-related volatile compounds, such as alcohols and esters, compared with other regions. Because alcohols including ethanol have low reflection coefficients, it is very likely that these molecules would have rapidly penetrated membranes to accumulate in apoplast to fill. In addition to the water potential gradient detected here, this would physically contribute to the appearance with high tissue transparency and changes in colour differences. Therefore, it is concluded that these spatial changes in cell water relations are closely associated with watercore symptoms as well as with metabolic alterations.

Ginger (Zingiber officinale) is one of the most valued spice plants worldwide; it is prized for its culinary and folk medicinal applications and is therefore of high economic and cultural importance. Here, we present a haplotype-resolved, chromosome-scale assembly for diploid ginger anchored to 11 pseudochromosome pairs with a total length of 3.1 Gb. Remarkable structural variation was identified between haplotypes, and two inversions larger than 15 Mb on chromosome 4 may be associated with ginger infertility. We performed a comprehensive, spatiotemporal, genome-wide analysis of allelic expression patterns, revealing that most alleles are coordinately expressed. The alleles that exhibited the largest differences in expression showed closer proximity to transposable elements, greater coding sequence divergence, more relaxed selection pressure, and more transcription factor binding site differences. We also predicted the transcription factors potentially regulating 6-gingerol biosynthesis. Our allele-aware assembly provides a powerful platform for future functional genomics, molecular breeding, and genome editing in ginger.

Ginger (Zingiber officinale), the type species of Zingiberaceae, is one of the most widespread medicinal plants and spices. Here, we report a high-quality, chromosome-scale reference genome of ginger ‘Zhugen’, a traditionally cultivated ginger in Southwest China used as a fresh vegetable, assembled from PacBio long reads, Illumina short reads, and high-throughput chromosome conformation capture (Hi-C) reads. The ginger genome was phased into two haplotypes, haplotype 1 (1.53 Gb with a contig N50 of 4.68 M) and haplotype 0 (1.51 Gb with a contig N50 of 5.28 M). Homologous ginger chromosomes maintained excellent gene pair collinearity. In 17,226 pairs of allelic genes, 11.9% exhibited differential expression between alleles. Based on the results of ginger genome sequencing, transcriptome analysis, and metabolomic analysis, we proposed a backbone biosynthetic pathway of gingerol analogs, which consists of 12 enzymatic gene families, PAL, C4H, 4CL, CST, C3’H, C3OMT, CCOMT, CSE, PKS, AOR, DHN, and DHT. These analyses also identified the likely transcription factor networks that regulate the synthesis of gingerol analogs. Overall, this study serves as an excellent resource for further research on ginger biology and breeding, lays a foundation for a better understanding of ginger evolution, and presents an intact biosynthetic pathway for species-specific gingerol biosynthesis.

The ancient tea plant, as a precious natural resource and source of tea plant genetic diversity, is of great value for studying the evolutionary mechanism, diversification, and domestication of plants. The overall genetic diversity among ancient tea plants and the genetic changes that occurred during natural selection remain poorly understood. Here, we report the genome resequencing of eight different groups consisting of 120 ancient tea plants: six groups from Guizhou Province and two groups from Yunnan Province. Based on the 8,082,370 identified high-quality SNPs, we constructed phylogenetic relationships, assessed population structure, and performed genome-wide association studies (GWAS). Our phylogenetic analysis showed that the 120 ancient tea plants were mainly clustered into three groups and five single branches, which is consistent with the results of principal component analysis (PCA). Ancient tea plants were further divided into seven subpopulations based on genetic structure analysis. Moreover, it was found that the variation in ancient tea plants was not reduced by pressure from the external natural environment or artificial breeding (nonsynonymous/synonymous = 1.05). By integrating GWAS, selection signals, and gene function prediction, four candidate genes were significantly associated with three leaf traits, and two candidate genes were significantly associated with plant type. These candidate genes can be used for further functional characterization and genetic improvement of tea plants.

Distant hybridization is widely used to develop crop cultivars, whereas the hybridization process of embryo abortion often severely reduces the sought-after breeding effect. The LEAFY COTYLEDON1 (LEC1) gene has been extensively investigated as a central regulator of seed development, but it is far less studied in crop hybridization breeding. Here we investigated the function and regulation mechanism of CmLEC1 from Chrysanthemum morifolium during its seed development in chrysanthemum hybridization. CmLEC1 encodes a nucleic protein and is specifically expressed in embryos. CmLEC1’s overexpression significantly promoted the seed-setting rate of the cross, while the rate was significantly decreased in the amiR-CmLEC1 transgenic chrysanthemum. The RNA-Seq analysis of the developing hybrid embryos revealed that regulatory genes involved in seed development, namely, CmLEA (late embryogenesis abundant protein), CmOLE (oleosin), CmSSP (seed storage protein), and CmEM (embryonic protein), were upregulated in the OE (overexpressing) lines but downregulated in the amiR lines vs. wild-type lines. Future analysis demonstrated that CmLEC1 directly activated CmLEA expression and interacted with CmC3H, and this CmLEC1–CmC3H interaction could enhance the transactivation ability of CmLEC1 for the expression of CmLEA. Further, CmLEC1 was able to induce several other key genes related to embryo development. Taken together, our results show that CmLEC1 plays a positive role in the hybrid embryo development of chrysanthemum plants, which might involve activating CmLEA’s expression and interacting with CmC3H. This may be a new pathway in the LEC1 regulatory network to promote seed development, one perhaps leading to a novel strategy to not only overcome embryo abortion during crop breeding but also increase the seed yield.

Auxin plays a central role in control of organ abscission, and it is thought that changes in the auxin gradient across the abscission zone are the primary determinant of the onset of abscission. The nature of this gradient, whether in concentration, flow, or perhaps in the response system has not conclusively been determined. We employed a DR5::GUS auxin response reporter system to examine the temporal and spatial distribution of the auxin response activity in response to developmental and environmental cues during pedicel abscission in tomato. In pedicels of young and fully open flowers, auxin response, as indicated by GUS activity, was predominantly detected in the vascular tissues and was almost entirely confined to the abscission zone (AZ) and to the distal portion of the pedicel, with a striking reduction in the proximal tissues below the AZ-a ‘step’, rather than a gradient. Following pollination and during early fruit development, auxin response increased substantially throughout the pedicel. Changes in GUS activity following treatments that caused pedicel abscission (flower removal, high temperature, darkness, ethylene, or N-1-naphthylphthalamic acid (NPA) treatment) were relatively minor, with reduced auxin response in the AZ and some reduction above and below it. Expression of genes encoding some auxin efflux carriers (PIN) and influx carriers (AUX⁄LAX) was substantially reduced in the abscission zone of NPA-treated pedicels, and in pedicels stimulated to abscise by flower removal. Our results suggest that changes in auxin flow distribution through the abscission zone are likely more important than the auxin response system in the regulation of abscission.

Transcriptomic changes at the cessation of sugar accumulation in the pericarp of Vitis vinifera were addressed on single berries re-synchronised according to their individual growth patterns. The net rates of water, sugars and K⁺ accumulation inferred from individual growth and solute concentration confirmed that these inflows stopped simultaneously in the ripe berry, while the small amount of malic acid remaining at this stage was still being oxidised at low rate. Re-synchronised individual berries displayed negligible variations in gene expression among triplicates. RNA-seq studies revealed sharp reprogramming of cell-wall enzymes and structural proteins at the stop of phloem unloading, associated with an 80% repression of multiple sugar transporters and aquaporins on the plasma or tonoplast membranes, with the noticeable exception of H⁺/sugar symporters, which were rather weakly and constitutively expressed. This was verified in three genotypes placed in contrasted thermo-hydric conditions. The prevalence of SWEET suggests that electrogenic transporters would play a minor role on the plasma membranes of SE/CC complex and the one of the flesh, while sucrose/H⁺ exchangers dominate on its tonoplast. Cis-regulatory elements present in their promoters allowed to sort these transporters in different groups, also including specific TIPs and PIPs paralogs, and cohorts of cell wall-related genes. Together with simple thermodynamic considerations, these results lead to propose that H⁺/sugar exchangers at the tonoplast, associated with a considerably acidic vacuolar pH, may exhaust cytosolic sugars in the flesh and alleviate the need for supplementary energisation of sugar transport at the plasma membrane.

Ultraviolet-B radiation (280–315 nm), perceived by the plant photoreceptor UVR8, is a key environmental signal that influences plant growth and development and can reduce disease and pest incidence. The positive effect of UV-B on disease resistance and incidence in various plant species supports the implementation of supplemental UV-B radiation in sustainable crop production. However, despite many studies focusing on UV-B light, there is no consensus on the best mode of application. This review aims to analyze, evaluate, and organize the different application strategies of UV-B radiation in crop production with a focus on disease resistance. We summarize the physiological effects of UV-B light on plants and discuss how plants perceive and transduce UV-B light by the UVR8 photoreceptor as well as how this perception alters plant specialized metabolite production. Next, we bring together conclusions of various studies with respect to different UV-B application methods to improve plant resistance. In general, supplemental UV-B light has a positive effect on disease resistance in many plant–pathogen combinations, mainly through the induction of the production of specialized metabolites. However, many variables (UV-B light source, plant species, dose and intensity, timing during the day, duration, background light, etc.) make it difficult to compare and draw general conclusions. We compiled the information of recent studies on UV-B light applications, including e.g., details on the UV-B light source, experimental set-up, calculated UV-B light dose, intensity, and duration. This review provides practical insights and facilitates future research on UV-B radiation as a promising tool to reduce disease and pest incidence.

Polyphenols are the main active components of the anti-inflammatory compounds in dandelion, and chlorogenic acid (CGA) is one of the primary polyphenols. However, the molecular mechanism underlying the transcriptional regulation of CGA biosynthesis remains unclear. Hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase (HQT2) is the last rate-limiting enzyme in chlorogenic acid biosynthesis in Taraxacum antungense. Therefore, using the TaHQT2 gene promoter as a probe, a yeast one-hybrid library was performed, and a basic helix-loop-helix (bHLH) transcription factor, TabHLH1, was identified that shared substantial homology with Gynura bicolor DC bHLH1. The TabHLH1 transcript was highly induced by salt stress, and the TabHLH1 protein was localized in the nucleus. CGA and luteolin concentrations in TabHLH1-overexpression transgenic lines were significantly higher than those in the wild type, while CGA and luteolin concentrations in TabHLH1-RNA interference (RNAi) transgenic lines were significantly lower. Quantitative real-time polymerase chain reaction demonstrated that overexpression and RNAi of TabHLH1 in T. antungense significantly affected CGA and luteolin concentrations by upregulating or downregulating CGA and luteolin biosynthesis pathway genes, especially TaHQT2, 4-coumarate-CoA ligase (Ta4CL), chalcone isomerase (TaCHI), and flavonoid-3′-hydroxylase (TaF3′H). Dual-luciferase, yeast one-hybrid, and electrophoretic mobility shift assays indicated that TabHLH1 directly bound to the bHLH-binding motifs of pro TaHQT2 and pro Ta4CL. This study suggests that TabHLH1 participates in the regulatory network of CGA and luteolin biosynthesis in T. antungense and might be useful for metabolic engineering to promote plant polyphenol biosynthesis.

Studies have shown that melatonin regulates the expression of various elements in the biosynthesis and catabolism of plant hormones. In contrast, the effects of these different plant hormones on the biosynthesis and metabolism of melatonin and their underlying molecular mechanisms are still unclear. In this study, the melatonin biosynthesis pathway was proposed from constructed metabolomic and transcriptomic libraries from hickory (Carya cathayensis Sarg.) nuts. The candidate pathway genes were further identified by phylogenetic analysis, amino-acid sequence alignment, and subcellular localization. Notably, most of the transcription factor-related genes coexpressed with melatonin pathway genes were hormone-responsive genes. Furthermore, dual-luciferase and yeast one-hybrid assays revealed that CcEIN3 (response to ethylene) and CcAZF2 (response to abscisic acid) could activate melatonin biosynthesis pathway genes, a tryptophan decarboxylase coding gene (CcTDC1) and an N-acetylserotonin methyltransferase coding gene (CcASMT1), by directly binding to their promoters, respectively. Our results provide a molecular basis for the characterization of novel melatonin biosynthesis regulatory mechanisms and demonstrate for the first time that abscisic acid and ethylene can regulate melatonin biosynthesis.

Dormancy-associated MADS-box (DAM) genes serve as crucial regulators of the endodormancy cycle in rosaceous plants. Although pear DAM genes have been identified previously, the lack of a high-quality reference genome and techniques to study gene function have prevented accurate genome-wide analysis and functional verification of such genes. Additionally, the contribution of other genes to the regulation of endodormancy release remains poorly understood. In this study, a high-quality genome assembly for 'Cuiguan' pear (Pyrus pyrifolia), which is a leading cultivar with a low chilling requirement cultivated in China, was constructed using PacBio and Hi-C technologies. Using this genome sequence, we revealed that pear DAM genes were tandemly clustered on Chr8 and Chr15 and were differentially expressed in the buds between 'Cuiguan' and the high-chilling-requirement cultivar 'Suli' during the dormancy cycle. Using a virus-induced gene silencing system, we determined the repressive effects of DAM genes on bud break. Several novel genes potentially involved in the regulation of endodormancy release were identified by RNA sequencing and H3K4me3 chromatin immunoprecipitation sequencing analyses of 'Suli' buds during artificial chilling using the new reference genome. Our findings enrich the knowledge of the regulatory mechanism underlying endodormancy release and chilling requirements and provide a foundation for the practical regulation of dormancy release in fruit trees as an adaptation to climate change.

DNA methylation is a crucial regulatory mechanism in many biological processes. However, limited studies have dissected the contribution of DNA methylation to sexual differentiation in dioecious plants. In this study, we investigated the variances in methylation and transcriptional patterns of male and female flowers of garden asparagus. Compared with male flowers, female flowers at the same stages showed higher levels of DNA methylation. Both male and female flowers gained DNA methylation globally from the premeiotic to meiotic stages. Detailed analysis revealed that the increased DNA methylation was largely due to increased CHH methylation. Correlation analysis of differentially expressed genes and differentially methylated regions suggested that DNA methylation might not have contributed to the expression variation of the sex-determining genes SOFF and TDF1 but probably played important roles in sexual differentiation and flower development of garden asparagus. The upregulated genes AoMS1, AoLAP3, AoAMS, and AoLAP5 with varied methylated CHH regions might have been involved in sexual differentiation and flower development of garden asparagus. Plant hormone signaling genes and transcription factor genes also participated in sexual differentiation and flower development with potential epigenetic regulation. In addition, the CG and CHG methylation levels in the Y chromosome were notably higher than those in the X chromosome, implying that DNA methylation might have been involved in Y chromosome evolution. These data provide insights into the epigenetic modification of sexual differentiation and flower development and improve our understanding of sex chromosome evolution in garden asparagus.

The comprehension of molecular processes underlying the development and progression of flowering in plants is a hot topic, not only because that often the products of interest for human and animal nutrition are linked to the development of fruits or seeds, but also because the processes of gametes formation occurring in sexual organs are at the basis of recombination and genetic variability which constitutes the matter on which evolution acts, whether understood as natural or human driven. In the present study, we used an NGS approach to produce a grapevine flower transcriptome snapshot in different whorls and tissues including calyx, calyptra, filament, anther, stigma, ovary, and embryo in both pre- and post-anthesis phases. Our investigation aimed at identifying hub genes that unequivocally distinguish the different tissues providing insights into the molecular mechanisms that are at the basis of floral whorls and tissue development. To this end we have used different analytical approaches, some now consolidated in transcriptomic studies on plants, such as pairwise comparison and weighted-gene coexpression network analysis, others used mainly in studies on animals or human’s genomics, such as the tau (τ) analysis aimed at isolating highly and absolutely tissue-specific genes. The intersection of data obtained by these analyses allowed us to gradually narrow the field, providing evidence about the molecular mechanisms occurring in those whorls directly involved in reproductive processes, such as anther and stigma, and giving insights into the role of other whorls not directly related to reproduction, such as calyptra and calyx. We believe this work could represent an important genomic resource for functional analyses of grapevine floral organ growth and fruit development shading light on molecular networks underlying grapevine reproductive organ determination.

Centipedegrass [ Eremochloa ophiuroides (Munro) Hack.], a member of the Panicoideae subfamily, is one of the most important warm-season turfgrasses originating from China. This grass has an extremely developed prostrate growth habit and has been widely used in transitional and warm climatic regions. To better understand the genetic basis of important biological characteristics, such as prostrate growth and seed yield, in warm-season turfgrasses, we present a high-quality reference genome for centipedegrass and use PacBio, BioNano, and Hi-C technologies to anchor the 867.43 Mb genome assembly into nine pseudochromosomes, with a scaffold N50 of 86.05 Mb and 36,572 annotated genes. Centipedegrass was most closely related to sorghum and diverged from their common ancestor ~16.8 Mya. We detected a novel chromosome reshuffling event in centipedegrass, namely, the nest chromosome fusion event in which fusion of chromosomes 8 and 10 of sorghum into chromosome 3 of centipedegrass likely occurred after the divergence of centipedegrass from sorghum. The typical prostrate growth trait in centipedegrass may be linked to the expansion of candidate PROSTRATE GROWTH 1 (PROG1) genes on chromosome 2. Two orthologous genes of OsPROG1, EoPROG1, and EoPROG2, were confirmed to increase the stem number and decrease the stem angle in Arabidopsis. Collectively, our assembled reference genome of centipedegrass offers new knowledge and resources to dissect the genome evolution of Panicoideae and accelerate genome-assisted breeding and improvement of plant architecture in turf plants.

Pedigree information is of fundamental importance in breeding programs and related genetics efforts. However, many individuals have unknown pedigrees. While methods to identify and confirm direct parent–offspring relationships are routine, those for other types of close relationships have yet to be effectively and widely implemented with plants, due to complications such as asexual propagation and extensive inbreeding. The objective of this study was to develop and demonstrate methods that support complex pedigree reconstruction via the total length of identical by state haplotypes (referred to in this study as “summed potential lengths of shared haplotypes”, SPLoSH). A custom Python script, HapShared, was developed to generate SPLoSH data in apple and sweet cherry. HapShared was used to establish empirical distributions of SPLoSH data for known relationships in these crops. These distributions were then used to estimate previously unknown relationships. Case studies in each crop demonstrated various pedigree reconstruction scenarios using SPLoSH data. For cherry, a full-sib relationship was deduced for ‘Emperor Francis, and ‘Schmidt’, a half-sib relationship for ‘Van’ and ‘Windsor’, and the paternal grandparents of ‘Stella’ were confirmed. For apple, 29 cultivars were found to share an unknown parent, the pedigree of the unknown parent of ‘Cox’s Pomona’ was reconstructed, and ‘Fameuse’ was deduced to be a likely grandparent of ‘McIntosh’. Key genetic resources that enabled this empirical study were large genome-wide SNP array datasets, integrated genetic maps, and previously identified pedigree relationships. Crops with similar resources are also expected to benefit from using HapShared for empowering pedigree reconstruction.

Genome-wide association study (GWAS) is effective in identifying favorable alleles for traits of interest with high mapping resolution in crop species. In this study, we conducted GWAS to explore quantitative trait loci (QTL) for eight fruit traits using 162 tomato accessions with diverse genetic backgrounds. The eight traits included fruit weight, fruit width, fruit height, fruit shape index, pericarp thickness, locule number, fruit firmness, and brix. Phenotypic variations of these traits in the tomato collection were evaluated with three replicates in field trials over three years. We filtered 34,550 confident SNPs from the 51 K Axiom^® tomato array based on < 10% of missing data and > 5% of minor allele frequency for association analysis. The 162 tomato accessions were divided into seven clusters and their membership coefficients were used to account for population structure along with a kinship matrix. To identify marker-trait associations (MTAs), four phenotypic data sets representing each of three years and combined were independently analyzed in the multilocus mixed model (MLMM). A total of 30 significant MTAs was detected over data sets for eight fruit traits at P < 0.0005. The number of MTA per trait ranged from one (brix) to seven (fruit weight and fruit width). Two SNP markers on chromosomes 1 and 2 were significantly associated with multiple traits, suggesting pleiotropic effects of QTL. Furthermore, 16 of 30 MTAs suggest potential novel QTL for eight fruit traits. These results facilitate genetic dissection of tomato fruit traits and provide a useful resource to develop molecular tools for improving fruit traits via marker-assisted selection and genomic selection in tomato breeding programs.

Fire blight disease, caused by the bacterium Erwinia amylovora (E. amylovora), is responsible for substantial losses in cultivated apples worldwide. An important mechanism of plant immunity is based on the recognition of conserved microbial molecules, named pathogen-associated or microbe-associated molecular patterns (PAMPs or MAMPs), through pattern recognition receptors (PRRs), leading to pattern-triggered immunity (PTI). The interspecies transfer of PRRs represents a promising strategy to engineer broad-spectrum and durable disease resistance in crops. EFR, the Arabidopsis thaliana PRR for the PAMP elf18 derived from the elongation factor thermal unstable (EF-Tu) proved to be effective in improving bacterial resistance when expressed into Solanaceae and other plant species. In this study, we tested whether EFR can affect the interaction of apple with E. amylovora by its ectopic expression in the susceptible apple rootstock M.26. Stable EFR expression led to the activation of PAMP-triggered immune response in apple leaves upon treatment with supernatant of E. amylovora, as measured by the production of reactive oxygen species and the induction of known defense genes. The amount of tissue necrosis associated with E. amylovora infection was significantly reduced in the EFR transgenic rootstock compared to the wild-type. Our results show that the expression of EFR in apple rootstock may be a valuable biotechnology strategy to improve the resistance of apple to fire blight.

Zanthoxylum bungeanum is an important spice and medicinal plant that is unique for its accumulation of abundant secondary metabolites, which create a characteristic aroma and tingling sensation in the mouth. Owing to the high proportion of repetitive sequences, high heterozygosity, and increased chromosome number of Z. bungeanum, the assembly of its chromosomal pseudomolecules is extremely challenging. Here, we present a genome sequence for Z. bungeanum, with a dramatically expanded size of 4.23 Gb, assembled into 68 chromosomes. This genome is approximately tenfold larger than that of its close relative Citrus sinensis. After the divergence of Zanthoxylum and Citrus, the lineage-specific whole-genome duplication event η-WGD approximately 26.8 million years ago (MYA) and the recent transposable element (TE) burst ~6.41 MYA account for the substantial genome expansion in Z. bungeanum. The independent Zanthoxylum-specific WGD event was followed by numerous fusion/fission events that shaped the genomic architecture. Integrative genomic and transcriptomic analyses suggested that prominent species-specific gene family expansions and changes in gene expression have shaped the biosynthesis of sanshools, terpenoids, and anthocyanins, which contribute to the special flavor and appearance of Z. bungeanum. In summary, the reference genome provides a valuable model for studying the impact of WGDs with recent TE activity on gene gain and loss and genome reconstruction and provides resources to accelerate Zanthoxylum improvement.

Sclareol, an antifungal specialized metabolite produced by clary sage, Salvia sclarea, is the starting plant natural molecule used for the hemisynthesis of the perfume ingredient ambroxide. Sclareol is mainly produced in clary sage flower calyces; however, the cellular localization of the sclareol biosynthesis remains unknown. To elucidate the site of sclareol biosynthesis, we analyzed its spatial distribution in the clary sage calyx epidermis using laser desorption/ ionization mass spectrometry imaging (LDI–FTICR-MSI) and investigated the expression profile of sclareol biosynthesis genes in isolated glandular trichomes (GTs). We showed that sclareol specifically accumulates in GTs’ gland cells in which sclareol biosynthesis genes are strongly expressed. We next isolated a glabrous beardless mutant and demonstrate that more than 90% of the sclareol is produced by the large capitate GTs. Feeding experiments, using 1-¹³C-glucose, and specific enzyme inhibitors further revealed that the methylerythritol-phosphate (MEP) biosynthetic pathway is the main source of isopentenyl diphosphate (IPP) precursor used for the biosynthesis of sclareol. Our findings demonstrate that sclareol is an MEP-derived diterpene produced by large capitate GTs in clary sage emphasing the role of GTs as biofactories dedicated to the production of specialized metabolites.

The structurally robust biopolymer sporopollenin is the major constituent of the exine layer of pollen wall and plays a vital role in plant reproductive success. The sporopollenin precursors are synthesized through an ancient polyketide biosynthetic pathway consisting of a series of anther-specific enzymes that are widely present in all land plant lineages. Tetraketide α-pyrone reductase 1 (TKPR1) and TKPR2 are two reductases catalyzing the final reduction of the carbonyl group of the polyketide synthase-synthesized tetraketide intermediates to hydroxylated α-pyrone compounds, important precursors of sporopollenin. In contrast to the functional conservation of many sporopollenin biosynthesis associated genes confirmed in diverse plant species, TKPR2’s role has been addressed only in Arabidopsis, where it plays a minor role in sporopollenin biosynthesis. We identified in gerbera two non-anther-specific orthologues of AtTKPR2, Gerbera reductase 1 (GRED1) and GRED2. Their dramatically expanded expression pattern implies involvement in pathways outside of the sporopollenin pathway. In this study, we show that GRED1 and GRED2 are still involved in sporopollenin biosynthesis with a similar secondary role as AtTKPR2 in Arabidopsis. We further show that this secondary role does not relate to the promoter of the gene, AtTKPR2 cannot rescue pollen development in Arabidopsis even when controlled by the AtTKPR1 promoter. We also identified the gerbera orthologue of AtTKPR1, GTKPR1, and characterized its crucial role in gerbera pollen development. GTKPR1 is the predominant TKPR in gerbera pollen wall formation, in contrast to the minor roles GRED1 and GRED2. GTKPR1 is in fact an excellent target for engineering male-sterile gerbera cultivars in horticultural plant breeding.

Flowering time is an important agronomic trait that contributes to fitness in plants. However, the genetic basis of flowering time has not been extensively studied in pepper. To understand the genetics underlying flowering time, we constructed an F₂ population by crossing a spontaneous early flowering mutant and a late-flowering pepper line. Using bulked segregant RNA-seq, a major locus controlling flowering time in this population was mapped to the end of chromosome 2. An APETALA2 (AP2) homolog (CaFFN) cosegregated with flowering time in 297 individuals of the F₂ population. A comparison between the parents revealed a naturally occurring rare SNP (SNP2T &gt; C) that resulted in the loss of a start codon in CaFFN in the early flowering mutant. Transgenic Nicotiana benthamiana plants with high CaFFN expression exhibited a delay in flowering time and floral patterning defects. On the other hand, pepper plants with CaFFN silencing flowered early. Therefore, the CaFFN gene acts as a flowering repressor in pepper. CaFFN may function as a transcriptional activator to activate the expression of CaAGL15 and miR156e and as a transcriptional repressor to repress the expression of CaAG, CaAP1, CaSEP3, CaSOC1, and miR172b based on a qRT-PCR assay. Direct activation of CaAGL15 by CaFFN was detected using yeast one-hybrid and dual-luciferase reporter assays, consistent with the hypothesis that CaFFN regulates flowering time. Moreover, the CaFFN gene association analysis revealed a significant association with flowering time in a natural pepper population, indicating that the CaFFN gene has a broad effect on flowering time in pepper. Finally, the phylogeny, evolutionary expansion and expression patterns of CaFFN/AP2 homologs were analyzed to provide valuable insight into CaFFN. This study increases our understanding of the involvement of CaFFN in controlling flowering time in pepper, thus making CaFFN a target gene for breeding early maturing pepper.

Peach is a typical climacteric fruit that releases ethylene during fruit ripening. Several studies have been conducted on the transcriptional regulation of ethylene biosynthesis in peach fruit. Herein, an ethylene response factor, PpERF.A16, which was induced by exogenous ethylene, could enhance ethylene biosynthesis by directly inducing the expression of 1-aminocyclopropane-1-carboxylic acid synthase (PpACS1) and 1-aminocyclopropane-1-carboxylic acid oxidase (PpACO1) genes. Moreover, the NAM/ATAF1/2/CUC2 (NAC) transcription factor (TF) PpNAC.A59 was coexpressed with PpERF.A16 in all tested peach cultivars. Interestingly, PpNAC.A59 can directly interact with the promoter of PpERF.A16 to induce its expression but not enhance LUC activity driven by any promoter of PpACS1 or PpACO1. Thus, PpNAC.A59 can indirectly mediate ethylene biosynthesis via the NAC-ERF signaling cascade to induce the expression of both PpACS1 and PpACO1. These results enrich the genetic network of fruit ripening in peach and provide new insight into the ripening mechanism of other perennial fruits.

Melatonin is a pleiotropic signaling molecule that regulates plant growth and responses to various abiotic stresses. The last step of melatonin synthesis in plants can be catalyzed by caffeic acid O-methyltransferase (COMT), a multifunctional enzyme reported to have N-acetylserotonin O-methyltransferase (ASMT) activity; however, the ASMT activity of COMT has not yet been characterized in nonmodel plants such as watermelon (Citrullus lanatus). Here, a total of 16 putative O-methyltransferase (ClOMT) genes were identified in watermelon. Among them, ClOMT03 (Cla97C07G144540) was considered a potential COMT gene (renamed ClCOMT1) based on its high identities (60.00–74.93%) to known COMT genes involved in melatonin biosynthesis, expression in almost all tissues, and upregulation under abiotic stresses. The ClCOMT1 protein was localized in the cytoplasm. Overexpression of ClCOMT1 significantly increased melatonin contents, while ClCOMT1 knockout using the CRISPR/Cas-9 system decreased melatonin contents in watermelon calli. These results suggest that ClCOMT1 plays an essential role in melatonin biosynthesis in watermelon. In addition, ClCOMT1 expression in watermelon was upregulated by cold, drought, and salt stress, accompanied by increases in melatonin contents. Overexpression of ClCOMT1 enhanced transgenic Arabidopsis tolerance against such abiotic stresses, indicating that ClCOMT1 is a positive regulator of plant tolerance to abiotic stresses.

Protoplast transient expression is a powerful strategy for gene functional characterization, especially in biochemical mechanism studies. We herein developed a highly efficient transient expression system for apple protoplasts. The abilities of the Arabidopsis thaliana and Malus domestica ubiquitin-10 (AtUBQ10 and MdUBQ10) promoters to drive the expression of multiple genes were compared with that of the CaMV 35S promoter, and the results revealed that the AtUBQ10 and MdUBQ10 promoters were more efficient in apple protoplasts. With this system, we demonstrated that active AtMKK7ac could activate MAPK6/3/4 signaling cascades, which further regulated MdWRKY33 phosphorylation and stability in apple. Furthermore, the ligand-induced interaction between the immune receptor AtFLS2 and the coreceptor AtBAK1 was reconstituted in apple protoplasts. We also found that the stability of the bacterial effector AvrRpt2 was regulated by feedback involving auxin and the immune regulator RIN4. The system established herein will serve as a useful tool for the molecular and biochemical analyses of apple genes.

Many studies showed that few degrees above tomato optimum growth temperature threshold can lead to serious loss in production. Therefore, the development of innovative strategies to obtain tomato cultivars with improved yield under high temperature conditions is a main goal both for basic genetic studies and breeding activities. In this paper, a F4 segregating population was phenotypically evaluated for quantitative and qualitative traits under heat stress conditions. Moreover, a genotyping by sequencing (GBS) approach has been employed for building up genomic selection (GS) models both for yield and soluble solid content (SCC). Several parameters, including training population size, composition and marker quality were tested to predict genotype performance under heat stress conditions. A good prediction accuracy for the two analyzed traits (0.729 for yield production and 0.715 for SCC) was obtained. The predicted models improved the genetic gain of selection in the next breeding cycles, suggesting that GS approach is a promising strategy to accelerate breeding for heat tolerance in tomato. Finally, the annotation of SNPs located in gene body regions combined with QTL analysis allowed the identification of five candidates putatively involved in high temperatures response, and the building up of a GS model based on calibrated panel of SNP markers.

Prunus species include many important perennial fruit crops, such as peach, plum, apricot, and related wild species. Here, we report de novo genome assemblies for five species, including the cultivated species peach (Prunus persica), plum (Prunus salicina), and apricot (Prunus armeniaca), and the wild peach species Tibetan peach (Prunus mira) and Chinese wild peach (Prunus davidiana). The genomes ranged from 240 to 276 Mb in size, with contig N50 values of 2.27−8.30 Mb and 25,333−27,826 protein-coding gene models. As the phylogenetic tree shows, plum diverged from its common ancestor with peach, wild peach species, and apricot ~7 million years ago (MYA). We analyzed whole-genome resequencing data of 417 peach accessions, called 3,749,618 high-quality SNPs, 577,154 small indels, 31,800 deletions, duplications, and inversions, and 32,338 insertions, and performed a structural variant-based genome-wide association study (GWAS) of key agricultural traits. From our GWAS data, we identified a locus associated with a fruit shape corresponding to the OVATE transcription factor, where a large inversion event correlates with higher OVATE expression in flat-shaped accessions. Furthermore, a GWAS revealed a NAC transcription factor associated with fruit developmental timing that is linked to a tandem repeat variant and elevated NAC expression in early-ripening accessions. We also identified a locus encoding microRNA172d, where insertion of a transposable element into its promoter was found in double-flower accessions. Thus, our efforts have suggested roles for OVATE, a NAC transcription factor, and microRNA172d in fruit shape, fruit development period, and floral morphology, respectively, that can be connected to traits in other crops, thereby demonstrating the importance of parallel evolution in the diversification of several commercially important domesticated species. In general, these genomic resources will facilitate functional genomics, evolutionary research, and agronomic improvement of these five and other Prunus species. We believe that structural variant-based GWASs can also be used in other plants, animal species, and humans and be combined with deep sequencing GWASs to precisely identify candidate genes and genetic architecture components.

Drought stress is a major agricultural problem restricting the growth, development, and productivity of plants. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) significantly influence the plant response to different stresses. However, the molecular mechanisms of CBL–CIPK in the drought stress response of pepper are still unknown. Here, the function of CaCIPK3 in the regulation of drought stress in pepper (Capsicum annuum L.) was explored. Transcriptomic data and quantitative real-time PCR (qRT-PCR) analysis revealed that CaCIPK3 participates in the response to multiple stresses. Knockdown of CaCIPK3 in pepper increased the sensitivity to mannitol and methyl jasmonate (MeJA). Transient overexpression of CaCIPK3 improved drought tolerance by enhancing the activities of the antioxidant system and positively regulating jasmonate (JA)-related genes. Ectopic expression of CaCIPK3 in tomato also improved drought and MeJA resistance. As the CaCIPK3-interacting partner, CaCBL2 positively influenced drought resistance. Additionally, CaWRKY1 and CaWRKY41 directly bound the CaCIPK3 promoter to influence its expression. This study shows that CaCIPK3 acts as a positive regulator in drought stress resistance via the CBL–CIPK network to regulate MeJA signaling and the antioxidant defense system.

The genetic diversity of germplasm is critical for exploring genetic and phenotypic resources and has important implications for crop-breeding sustainability and improvement. However, little is known about the factors that shape and maintain genetic diversity. Here, we assembled a high-quality chromosome-level reference of the Chinese common apricot ‘Yinxiangbai’, and we resequenced 180 apricot accessions that cover four major ecogeographical groups in China and other accessions from occidental countries. We concluded that Chinese-cultivated common apricot germplasms possessed much higher genetic diversity than those cultivated in Western countries. We also detected seven migration events among different apricot groups, where 27% of the genome was identified as being introgressed. Remarkably, we demonstrated that these introgressed regions drove the current high level of germplasm diversity in Chinese-cultivated common apricots by introducing different genes related to distinct phenotypes from different cultivated groups. Our results highlight the consideration that introgressed regions may provide an important reservoir of genetic resources that can be used to sustain modern breeding programs.

Drought stress is a major agricultural problem restricting the growth, development, and productivity of plants. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) significantly influence the plant response to different stresses. However, the molecular mechanisms of CBL–CIPK in the drought stress response of pepper are still unknown. Here, the function of CaCIPK3 in the regulation of drought stress in pepper (Capsicum annuum L.) was explored. Transcriptomic data and quantitative real-time PCR (qRT-PCR) analysis revealed that CaCIPK3 participates in the response to multiple stresses. Knockdown of CaCIPK3 in pepper increased the sensitivity to mannitol and methyl jasmonate (MeJA). Transient overexpression of CaCIPK3 improved drought tolerance by enhancing the activities of the antioxidant system and positively regulating jasmonate (JA)-related genes. Ectopic expression of CaCIPK3 in tomato also improved drought and MeJA resistance. As the CaCIPK3-interacting partner, CaCBL2 positively influenced drought resistance. Additionally, CaWRKY1 and CaWRKY41 directly bound the CaCIPK3 promoter to influence its expression. This study shows that CaCIPK3 acts as a positive regulator in drought stress resistance via the CBL–CIPK network to regulate MeJA signaling and the antioxidant defense system.

Artemisia annua, a traditional Chinese medicinal plant, remains the only plant source for artemisinin production, yet few genes have been identified to be involved in both the response to biotic stresses, such as pathogens, and artemisinin biosynthesis. Here, we isolated and identified the WRKY transcription factor (TF) AaWRKY17, which could significantly increase the artemisinin content and resistance to Pseudomonas syringae in A. annua. Yeast one-hybrid (Y1H), dual-luciferase (dual-LUC), and electrophoretic mobility shift assay (EMSA) results showed that AaWRKY17 directly bound to the W-box motifs in the promoter region of the artemisinin biosynthetic pathway gene amorpha-4,11-diene synthase (ADS) and promoted its expression. Real-time quantitative PCR (RT-qPCR) analysis revealed that the transcript levels of two defense marker genes, Pathogenesis-Related 5 (PR5) and NDR1/HIN1-LIKE 10 (NHL10), were greatly increased in AaWRKY17-overexpressing transgenic A. annua plants. Additionally, overexpression of AaWRKY17 in A. annua resulted in decreased susceptibility to P. syringae. These results indicated that AaWRKY17 acted as a positive regulator in response to P. syringae infection. Together, our findings demonstrated that the novel WRKY transcription factor AaWRKY17 could potentially be used in transgenic breeding to improve the content of artemisinin and pathogen tolerance in A. annua.

Valencia orange (Citrus sinensis Osbeck) (VO) is a type of late-ripening sweet orange whose ripening occurs 4 to 5 months later than that of the mid-ripening common sweet orange (CO). Notably, the mastication trait of VO fruit is inferior to that of CO fruit. To date, how inferior pulp mastication trait forms in VO has not been determined. In this study, 13 VO varieties and 12 CO varieties were subjected to whole-genome resequencing. A total of 2.98 million SNPs were identified from 25 varieties, and a SNP molecular marker was developed to distinguish VO and CO. Moreover, 144 and 141 genes identified by selective sweep analysis were selected during VO and CO evolution, respectively. Based on gene functional enrichment analysis, most of the selected VO genes were related to the stress response and lignin biosynthesis. Simultaneously, we comparatively analyzed the transcriptome profiles of peel and pulp tissues among three VO varieties and three CO varieties, and the results demonstrated differences in lignin biosynthesis between VO and CO fruits. Furthermore, coexpression network analysis was performed to identify hub genes of lignin-related and variety-specific networks, which included CsERF74, CsNAC25, CsHSFB3, CsSPL4/13, etc. Overall, this study provides important insights into the mastication trait formation of Valencia orange fruit.

Alternaria leaf spot in apple (Malus x domestica), caused by the fungal pathogen Alternaria alternata f. sp. mali (also called A. mali), is a devastating disease resulting in substantial economic losses. We previously established that the resistance (R) protein MdRNL2, containing a coiled-coil, nucleotide-binding, and leucine-rich repeat (CC_R-NB-LRR) domain, interacts with another CC_R-NB-LRR protein, MdRNL6, to form a MdRNL2–MdRNL6 complex that confers resistance to A. mali. Here, to investigate the function of the MdRNL2–MdRNL6 complex, we identified two novel pathogenesis-related (PR) proteins, MdPR10-1 and MdPR10-2, that interact with MdRNL2. Yeast two-hybrid (Y2H) assays and bimolecular fluorescence complementation (BiFC) assays confirmed that MdPR10-1 and MdPR10-2 interact with MdRNL2 and MdRNL6 at the leucine-rich repeat domain. Transient expression assays demonstrated that accumulation of MdPR10-1 and MdPR10-2 enhanced the resistance of apple to four strains of A. mali that we tested: ALT1, GBYB2, BXSB5, and BXSB7. In vitro antifungal activity assays demonstrated that both the proteins contribute to Alternaria leaf spot resistance by inhibiting fungal growth. Our data provide evidence for a novel regulatory mechanism in which MdRNL2 and MdRNL6 interact with MdPR10-1 and MdPR10-2 to inhibit fungal growth, thereby contributing to Alternaria leaf spot resistance in apple. The identification of these two novel PR proteins will facilitate breeding for fungal disease resistance in apple.

High temperature (HT) is one of the most important environmental stress factors and seriously threatens plant growth, development, and production. VQ motif-containing proteins are transcriptional regulators that have been reported to regulate plant growth and developmental processes, including responses to biotic and abiotic stresses. However, the relationships between VQ motif-containing proteins and HT stress have not been studied in depth in plants. In this study, transgenic apple (Malus domestica) plants overexpressing the apple VQ motif-containing protein-coding gene (MdVQ37) were exposed to HT stress, and the transgenic lines exhibited a heat-sensitive phenotype. In addition, physiological and biochemical studies revealed that, compared with WT plants, transgenic lines had lower enzymatic activity and photosynthetic capacity and lower amounts of nonenzymatic antioxidant system metabolites under HT stress. Transcriptome analysis revealed 1379 genes whose expression differed between the transgenic lines and WT plants. GO and KEGG pathway analyses showed that transcription factor activity and plant hormone signaling pathways were differentially influenced and enriched in the transgenic lines. Salicylic acid (SA) content analysis indicated that overexpression of MdVQ37 reduced the content of endogenous SA by regulating the expression of SA catabolism-related genes, which ultimately resulted in disruption of the SA-dependent signaling pathway under HT stress. The application of SA slightly increased the survival rate of the transgenic lines under HT stress. Taken together, our results indicate that apple MdVQ37 has a regulatory function in basal thermotolerance by modulating the activity of transcription factors and SA homeostasis. Overall, this study provides novel insights that improve our understanding of the various functions of VQ motif-containing proteins.

Prickles are common structures in plants that play a key role in defense against herbivores. In the Rosa genus, prickles are widely present with great diversity in terms of form and density. For cut rose production, prickles represent an important issue, as they can damage the flower and injure workers. Our objectives were to precisely describe the types of prickles that exist in roses, their tissues of origin and their development. We performed a detailed histological analysis of prickle initiation and development in a rose F₁ population. Based on the prickle investigation of 110 roses, we proposed the first categorization of prickles in the Rosa genus. They are mainly divided into two categories, nonglandular prickles (NGPs) and glandular prickles (GPs), and subcategories were defined based on the presence/ absence of hairs and branches. We demonstrated that NGPs and GPs both originate from multiple cells of the ground meristem beneath the protoderm. For GPs, the gland cells originate from the protoderm of the GP at the early developmental stage. Our findings clearly demonstrate that prickles are not modified trichomes (which originate from the protoderm). These conclusions are different from the current mainstream hypothesis. These results provide a foundation for further studies on prickle initiation and development in plants.

Rapid alkalinization factors (RALFs) are cysteine-rich peptides that play important roles in a variety of biological processes, such as cell elongation and immune signaling. Recent studies in Arabidopsis have shown that RALFs regulate pollen tube growth via plasma membrane receptor-like kinases (RLKs). However, the downstream signal transduction mechanisms of RLKs in pollen tubes are unknown. Here, we identified PbrRALF2, a pear (Pyrus bretschneideri) pollen RALF peptide that inhibits pollen tube growth. We found that PbrRALF2 interacts with a malectin-like domain-containing RLK, PbrCrRLK1L13. The relative affinity between PbrRALF2 and PbrCrRLK1L13 was at the submicromolar level, which is consistent with the values of ligand–receptor kinase pairs and the physiological concentration for PbrRALF2-mediated inhibition of pollen tube growth. After binding to its extracellular domain, PbrRALF2 activated the phosphorylation of PbrCrRLK1L13 in a dose-dependent manner. We further showed that the MAP kinase PbrMPK18 is a downstream target of PbrCrRLK1L13 that mediates PbrRALF2-elicited reactive oxygen species (ROS) production. The excessive accumulation of ROS inhibits pollen tube growth. We show that MPK acts as a mediator for CrRLK1L to stimulate ROS production, which might represent a general mechanism by which RALF and CrRLK1L function in signaling pathways.

Color is an important trait for horticultural crops. Carotenoids are one of the main pigments for coloration and have important implications for photosynthesis in plants and benefits for human health. Here, we identified an APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) transcription factor named MdAP2-34 in apple (Malus domestica Borkh.). MdAP2-34 expression exhibited a close correlation with carotenoid content in ‘Benin Shogun’ and ‘Yanfu 3’ fruit flesh. MdAP2-34 promotes carotenoid accumulation in MdAP2-34-OVX transgenic apple calli and fruits by participating in the carotenoid biosynthesis pathway. The major carotenoid contents of phytoene and β-carotene were much higher in overexpressing MdAP2-34 transgenic calli and fruit skin, yet the predominant compound of lutein showed no obvious difference, indicating that MdAP2-34 regulates phytoene and β-carotene accumulation but not lutein. MdPSY2-1 (phytoene synthase 2) is a major gene in the carotenoid biosynthesis pathway in apple fruit, and the MdPSY2-1 gene is directly bound and transcriptionally activated by MdAP2-34. In addition, overexpressing MdPSY2-1 in apple calli mainly increases phytoene and total carotenoid contents. Our findings will advance and extend our understanding of the complex molecular mechanisms of carotenoid biosynthesis in apple, and this research is valuable for accelerating the apple breeding process.

The Alternaria alternata apple pathotype adversely affects apple (Malus domestica Borkh.) cultivation. However, the molecular mechanisms underlying enhanced resistance to this pathogen in apple remain poorly understood. We have previously reported that MdWRKY75 expression is upregulated by A. alternata infection in ‘Sushuai’ apples. In this study, we discovered that overexpression of MdWRKY75e increased the resistance of transgenic apple lines to A. alternata infection, whereas silencing this gene enhanced susceptibility to A. alternata infection. Furthermore, we found that MdWRKY75e directly binds to the MdLAC7 promoter to regulate the biosynthesis of laccase and increase the biosynthesis of lignin during A. alternata infection. Moreover, the thickening of the cell wall enhanced the mechanical defense capabilities of apple. In addition, we found that jasmonic acid remarkably induced MdWRKY75e expression, and its levels in transgenic apple lines were elevated. These results indicate that MdWRKY75e confers resistance to the A. alternata apple pathotype mainly via the jasmonic acid pathway and that pathogenesis-related genes and antioxidant-related enzyme activity are involved in the disease resistance of MdWRKY75e transgenic plants. In conclusion, our findings provide insights into the importance of MdWRKY75e for resistance to A. alternata infection in apples.

Crops face increased risk from heat stress due to climate change. Potato (Solanum tuberosum L.) tubers grown in hot summers often have defects including pre-harvest sprouting (“heat sprouts”). We have used 18 potato cultivars to investigate whether heat stress (HS) conditions alone could cause heat sprouting and dormancy changes in tubers. We also examined transcriptomic responses of potato to HS and whether these responses are like those induced by postharvest sprouting. We demonstrated that HS alone caused heat sprouts and shortened postharvest dormancy period, heat-sprouted tubers became dormant after harvest, and cultivars varied substantially for producing heat spouts but there was no clear association with cultivar maturity earliness. Cultivar Innovator did not show any heat sprouts and still had long dormancy. Dormancy-associated genes (DOG1 and SLP) were downregulated in HS tubers like in postharvest sprouting tubers. We have identified 1201 differentially expressed genes, 14 enriched GO terms and 12 enriched KEGG pathways in response to HS in growing tubers of ‘Russet Burbank’. Transcriptomic response of ‘Russet Burbank’ to HS showed significant similarities to that of postharvest non-HS sprouted tubers. Gibberellin biosynthesis pathway was enriched in heat-stressed tubers and was likely involved in heat sprouting and dormancy release. Heat sprouting and postharvest sprouting shared common candidate genes and had significant similarity in gene expression. Our study has significance for selecting potato cultivars for farming, planning storage and utilization of heat-stressed tubers, identifying sprouting-related genes, understanding heat-stress biology, and breeding heat-tolerant potato cultivars, especially for sustainable potato production under climate change.

In fleshy fruits, organic acids are the main source of fruit acidity and play an important role in regulating osmotic pressure, pH homeostasis, stress resistance, and fruit quality. The transport of organic acids from the cytosol to the vacuole and their storage are complex processes. A large number of transporters carry organic acids from the cytosol to the vacuole with the assistance of various proton pumps and enzymes. However, much remains to be explored regarding the vacuolar transport mechanism of organic acids as well as the substances involved and their association. In this review, recent advances in the vacuolar transport mechanism of organic acids in plants are summarized from the perspectives of transporters, channels, proton pumps, and upstream regulators to better understand the complex regulatory networks involved in fruit acid formation.

Vaccinium darrowii Camp (2n = 2x = 24) is a native North American blueberry species and an important source of traits such as low chill requirement in commercial southern highbush blueberry breeding ( Vaccinium corymbosum, 2n = 4x = 48). We present a chromosomal-scale genome of V. darrowii generated by the combination of PacBio sequencing and high throughput chromatin conformation capture (Hi–C) scaffolding technologies, yielding a total length of 1.06 Gigabases (Gb). Over 97.8% of the genome sequences are scaffolded into 24 chromosomes representing the two haplotypes. The primary haplotype assembly of V. darrowii contains 34,809 protein-coding genes. Comparison to a V. corymbosum haplotype assembly reveals high collinearity between the two genomes with small intrachromosomal rearrangements in eight chromosome pairs. With small RNA sequencing, the annotation was further expanded to include more than 200,000 small RNA loci and 638 microRNAs expressed in berry tissues. Transcriptome analysis across fruit development stages indicates that genes involved in photosynthesis are downregulated, while genes involved in flavonoid and anthocyanin biosynthesis are significantly increased at the late stage of berry ripening. A high-quality reference genome and accompanying annotation of V. darrowii is a significant new resource for assessing the evergreen blueberry contribution to the breeding of southern highbush blueberries.

As auxins are among the most important phytohormones, the regulation of auxin homeostasis is complex. Generally, auxin conjugates, especially IAA glucosides, are predominant at high auxin levels. Previous research on terminal glucosylation focused mainly on the O-position, while IAA-N-glucoside and IAA-Asp-N-glucoside have been neglected since their discovery in 2001. In our study, IAA-Asp-N-glucoside was found to be specifically abundant (as high as 4.13 mg/g) in the seeds of 58 ginkgo cultivars. Furthermore, a novel N-glucosyltransferase, termed GbNGT1, was identified via differential transcriptome analysis and in vitro enzymatic testing. It was found that GbNGT1 could catalyze IAA-Asp and IAA to form their corresponding N-glucosides. The enzyme was demonstrated to possess a specific catalytic capacity toward the N-position of the IAA-amino acid or IAA from 52 substrates. Docking and site-directed mutagenesis of this enzyme confirmed that the E15G mutant could almost completely abolish its N-glucosylation ability toward IAA-Asp and IAA in vitro and in vivo. The IAA modification of GbNGT1 and GbGH3.5 was verified by transient expression assay in Nicotiana benthamiana. The effect of GbNGT1 on IAA distribution promotes root growth in Arabidopsis thaliana.

Leaf veins play an important role in plant growth and development, and the bundle sheath (BS) is believed to greatly improve the photosynthetic efficiency of C₄ plants. The OBV mutation in tomato (Solanum lycopersicum) results in dark veins and has been used widely in processing tomato varieties. However, physiological performance has difficulty explaining fitness in production. In this study, we confirmed that this mutation was caused by both the increased chlorophyll content and the absence of bundle sheath extension (BSE) in the veins. Using genome-wide association analysis and map-based cloning, we revealed that OBV encoded a C₂H₂L domain class transcription factor. It was localized in the nucleus and presented cell type-specific gene expression in the leaf veins. Furthermore, we verified the gene function by generating CRISPR/Cas9 knockout and overexpression mutants of the tomato gene. RNA sequencing analysis revealed that OBV was involved in regulating chloroplast development and photosynthesis, which greatly supported the change in chlorophyll content by mutation. Taken together, these findings demonstrated that OBV affected the growth and development of tomato by regulating chloroplast development in leaf veins. This study also provides a solid foundation to further decipher the mechanism of BSEs and to understand the evolution of photosynthesis in land plants.

Loquat (Eriobotrya japonica) is a popular fruit and medicinal plant. Here, a high-quality draft genome of the E. japonica ‘Big Five-pointed Star’ cultivar that covers ~98% (733.32 Mb) of the estimated genome size (749.25 Mb) and contains a total of 45,492 protein-coding genes is reported. Comparative genomic analysis suggests that the loquat genome has evolved a unique genetic mechanism of chromosome repair. Resequencing data from 52 loquat cultivars, including 16 white-fleshed and 36 yellow-fleshed variants, were analyzed, and the flower, leaf, and root metabolomes of ‘Big Five-pointed Star’ were determined using a UPLC-ESI-MS/M system. A genome-wide association study identified several candidate genes associated with flesh color in E. japonica, linking these phenotypes to sugar metabolism. A total of 577 metabolites, including 98 phenolic acids, 95 flavonoids, and 28 terpenoids, were found, and 191 metabolites, including 46 phenolic acids, 33 flavonoids, and 7 terpenoids, showed no differences in concentration among the leaves, roots, and flowers. Candidate genes related to the biosynthesis of various medicinal ingredients, such as phenolics, flavonoids, terpenoids, and polysaccharides, were identified. Some of these genes were confirmed to be members of expanding gene families, suggesting that the high concentrations of beneficial metabolites in loquat may be associated with the number of biosynthetic genes in this plant. In summary, this study provides fundamental molecular insights into the nutritional and medical properties of E. japonica.

Fruit size and shape are critical agronomical and pomological attributes and prime targets in peach breeding programs. Apart from the flat peach type, a Mendelian trait well-characterized at the genetic level, ample diversity of fruit size and shapes is present across peach germplasms. Nevertheless, knowledge of the underlying genomic loci remains limited. In this work, fruit size and shape were assessed in a collection of non-flat peach accessions and selections, under controlled fruit load conditions. The architecture of these traits was then dissected by combining association and linkage mapping, revealing a major locus on the proximal end of chromosome 6 (qSHL/Fs6.1) explaining a large proportion of phenotypic variability for longitudinal shape and also affecting fruit size. A second major locus for fruit longitudinal shape (qSHL5.1), probably also affecting fruit size, was found co-localizing at locus G, suggesting pleiotropic effects of peach/nectarine traits. An additional QTL for fruit longitudinal shape (qSHL6.2) was identified in the distal end of chromosome 6 in a cross with an ornamental double-flower peach and co-localized with the Di2 locus, controlling flower morphology. Besides assisting breeding activities, knowledge of loci controlling fruit size and shape paves the way for more in-depth studies aimed at the identification of underlying genetic variant(s).

The Rosaceae family has striking phenotypic diversity and high syntenic conservation. Gillenia trifoliata is sister species to the Maleae tribe of apple and ~1000 other species. Gillenia has many putative ancestral features, such as herb/sub-shrub habit, dry fruit-bearing and nine base chromosomes. This coalescence of ancestral characters in a phylogenetically important species, positions Gillenia as a ‘rosetta stone’ for translational science within Rosaceae. We present genomic and phenological resources to facilitate the use of Gillenia for this purpose. The Gillenia genome is the first fully annotated chromosome-level assembly with an ancestral genome complement (x = 9), and with it we developed an improved model of the Rosaceae ancestral genome. MADS and NAC gene family analyses revealed genome dynamics correlated with growth and reproduction and we demonstrate how Gillenia can be a negative control for studying fleshy fruit development in Rosaceae.

Green fluorescent protein (GFP) has been widely used for monitoring gene expression and protein localization in diverse organisms. However, highly sensitive imaging equipment, like fluorescence microscope, is usually required for the visualization of GFP, limitings its application to fixed locations in samples. A reporter that can be visualized in real-time regardless the shape, size and location of the target samples will increase the flexibility and efficiency of research work. Here, we report the application of a GFP-like protein, called eYGFPuv, in both transient expression and stable transformation, in two herbaceous plant species (Arabidopsis and tobacco) and two woody plant species (poplar and citrus). We observed bright fluorescence under UV light in all of the four plant species without any effects on plant growth or development. eYGFPuv was shown to be effective for imaging transient expression in leaf and root tissues. With a focus on in vitro transformation, we demonstrated that the transgenic events expressing 1x eYGFPuv could be easily identified visually during the callus stage and the shoot stage, enabling early and efficient selection of transformants. Furthermore, whole-plant level visualization of eYGFPuv revealed its ubiquitous stability in transgenic plants. In addition, our transformation experiments showed that eYGFPuv can also be used to select transgenic plants without antibiotics. This work demonstrates the feasibility of utilizing 1x eYGFPuv in studies of gene expression and plant transformation in diverse plants.

Tree peony (Paeonia suffruticosa Andr.) is a popular ornamental plant in China due to its showy and colorful flowers. However, yellow-colored flowers are rare in both wild species and domesticated cultivars. The molecular mechanisms underlying yellow pigmentation remain poorly understood. Here, petal tissues of two tree peony cultivars, “High Noon” (yellow flowers) and “Roufurong” (purple–red flowers), were sampled at five developmental stages (S1–S5) from early flower buds to full blooms. Five petal color indices (brightness, redness, yellowness, chroma, and hue angle) and the contents of ten different flavonoids were determined. Compared to “Roufurong,” which accumulated abundant anthocyanins at S3–S5, the yellow-colored “High Noon” displayed relatively higher contents of tetrahydroxychalcone (THC), flavones, and flavonols but no anthocyanin production. The contents of THC, flavones, and flavonols in “High Noon” peaked at S3 and dropped gradually as the flower bloomed, consistent with the color index patterns. Furthermore, RNA-seq analyses at S3 showed that structural genes such as PsC4Hs, PsDFRs, and PsUFGTs in the flavonoid biosynthesis pathway were downregulated in “High Noon,” whereas most PsFLSs, PsF3Hs, and PsF3’Hs were upregulated. Five transcription factor (TF) genes related to flavonoid biosynthesis were also upregulated in “High Noon.” One of these TFs, PsMYB111, was overexpressed in tobacco, which led to increased flavonols but decreased anthocyanins. Dual-luciferase assays further confirmed that PsMYB111 upregulated PsFLS. These results improve our understanding of yellow pigmentation in tree peony and provide a guide for future molecular-assisted breeding experiments in tree peony with novel flower colors.

Nitrate is a major nitrogen resource for plant growth and development and acts as both a crucial nutrient and a signaling molecule for plants; hence, understanding nitrate signaling is important for crop production. Abscisic acid (ABA) has been demonstrated to be involved in nitrate signaling, but the underlying mechanism is largely unknown in apple. In this study, we found that exogenous ABA inhibited the transport of nitrate from roots to shoots in apple, and the transcription of the nitrate transporter MdNRT1.5/MdNPF7.3 was noticeably reduced at the transcriptional level by ABA, which inhibited the transport of nitrate from roots to shoots. Then, it was found that the ABA-responsive transcription factor MdABI5 bound directly to the ABRE recognition site of the MdNRT1.5 promoter and suppressed its expression. Overexpression of MdABI5 inhibited ABA-mediated transport of nitrate from roots to shoots. Overall, these results demonstrate that MdABI5 regulates the transport of nitrate from roots to shoots partially by mediating the expression of MdNRT1.5, illuminating the molecular mechanism by which ABA regulates nitrate transport in apple.

Strigolactones are carotenoid-derived phytohormones that impact plant growth and development in diverse ways. However, the roles of strigolactones in the responses to temperature stresses are largely unknown. Here, we demonstrated that strigolactone biosynthesis is induced in tomato (Solanum lycopersicum) by heat and cold stresses. Compromised strigolactone biosynthesis or signaling negatively affected heat and cold tolerance, while application of the synthetic strigolactone analog GR24^5DS enhanced heat and cold tolerance. Strigolactone-mediated heat and cold tolerance was associated with the induction of abscisic acid (ABA), heat shock protein 70 (HSP70) accumulation, C-REPEAT BINDING FACTOR 1 (CBF1) transcription, and antioxidant enzyme activity. Importantly, a deficiency in ABA biosynthesis compromised the GR24^5DS effects on heat and cold stresses and abolished the GR24^5DS-induced transcription of HSP70, CBF1, and antioxidant-related genes. These results support that strigolactones positively regulate tomato heat and cold tolerance and that they do so at least partially by the induction of CBFs and HSPs and the antioxidant response in an ABA-dependent manner.

Fruit lignification is due to lignin deposition in the cell wall during cell development. However, there are few studies on the regulation of cell wall lignification and lignin biosynthesis during fruit pigmentation. In this study, we investigated the regulation of cell wall lignification and lignin biosynthesis during pigmentation of winter jujube. The cellulose content decreased, while the lignin content increased in the winter jujube pericarp during pigmentation. Safranin O-fast green staining showed that the cellulose content was higher in the cell wall of winter jujube prior to pigmentation, whereas the lignin in the cell wall increased after pigmentation. The thickness of the epidermal cells decreased with pericarp pigmentation. A combined metabolomics and transcriptomics analysis showed that guaiacyl-syringyl (G-S) lignin was the main lignin type in the pericarp of winter jujube, and F5H (LOC107424406) and CCR (LOC107420974) were preliminarily identified as the key genes modulating lignin biosynthesis in winter jujube. Seventeen MYB and six NAC transcription factors (TFs) with potential regulation of lignin biosynthesis were screened out based on phylogenetic analysis. Three MYB and two NAC TFs were selected as candidate genes and further studied in detail. Arabidopsis ectopic expression and winter jujube pericarp injection of the candidate genes indicated that the MYB activator (LOC107425254) and the MYB repressor (LOC107415078) control lignin biosynthesis by regulating CCR and F5H, while the NAC (LOC107435239) TF promotes F5H expression and positively regulates lignin biosynthesis. These findings revealed the lignin biosynthetic pathway and associated genes during pigmentation of winter jujube pericarp and provide a basis for further research on lignin regulation.

Understanding germline specification in plants could be advantageous for agricultural applications. In recent decades, substantial efforts have been made to understand germline specification in several plant species, including Arabidopsis, rice, and maize. However, our knowledge of germline specification in many agronomically important plant species remains obscure. Here, we characterized the female germline specification and subsequent female gametophyte development in pineapple using callose staining, cytological, and whole-mount immunolocalization analyses. We also determined the male germline specification and gametophyte developmental timeline and observed male meiotic behavior using chromosome spreading assays. Furthermore, we identified 229 genes that are preferentially expressed at the megaspore mother cell (MMC) stage during ovule development and 478 genes that are preferentially expressed at the pollen mother cell (PMC) stage of anther development using comparative transcriptomic analysis. The biological functions, associated regulatory pathways and expression patterns of these genes were also analyzed. Our study provides a convenient cytological reference for exploring pineapple germline development and a molecular basis for the future functional analysis of germline specification in related plant species.

Fusarium oxysporum, a global soil-borne pathogen, causes severe disease in various cultivated plants. The mechanism underlying infection and resistance remains largely elusive. Vernicia fordii, known as the tung tree, suffers from disease caused by F. oxysporum f. sp. fordiis (Fof-1), while its sister species V. montana displays high resistance to Fof-1. To investigate the process of infection and resistance ability, we demonstrated that Fof-1 can penetrate the epidermis of root hairs and then centripetally invade the cortex and phloem in both species. Furthermore, Fof-1 spread upwards through the root xylem in susceptible V. fordii trees, whereas it failed to infect the root xylem in resistant V. montana trees. We found that D6 PROTEIN KINASE LIKE 2 (VmD6PKL2) was specifically expressed in the lateral root xylem and was induced after Fof-1 infection in resistant trees. Transgenic analysis in Arabidopsis and tomato revealed that VmD6PKL2 significantly enhanced resistance in both species, whereas the d6pkl2 mutant displayed reduced resistance against Fof-1. Additionally, VmD6PKL2 was identified to interact directly with synaptotagmin (VmSYT3), which is specifically expressed in the root xylem and mediates the negative regulation responding to Fof-1. Our data suggested that VmD6PKL2 could act as a resistance gene against Fof-1 through suppression of VmSYT3-mediated negative regulation in the lateral root xylem of the resistant species. These findings provide novel insight into Fusarium wilt resistance in plants.

In horticulture, grafting is a popular technique used to combine positive traits from two different plants. This is achieved by joining the plant top part (scion) onto a rootstock which contains the stem and roots. Rootstocks can provide resistance to stress and increase plant production, but despite their wide use, the biological mechanisms driving rootstock-induced alterations of the scion phenotype remain largely unknown. Given that epigenetics plays a relevant role during distance signalling in plants, we studied the genome-wide DNA methylation changes induced in eggplant (Solanum melongena) scion using two interspecific rootstocks to increase vigour. We found that vigour was associated with a change in scion gene expression and a genome-wide hypomethylation in the CHH context. Interestingly, this hypomethylation correlated with the downregulation of younger and potentially more active long terminal repeat retrotransposable elements (LTR-TEs), suggesting that graft-induced epigenetic modifications are associated with both physiological and molecular phenotypes in grafted plants. Our results indicate that the enhanced vigour induced by heterografting in eggplant is associated with epigenetic modifications, as also observed in some heterotic hybrids.

Plants have developed sophisticated mechanisms to survive in dynamic environments. Plants can communicate via volatile organic compounds (VOCs) to warn neighboring plants of threats. In most cases, VOCs act as positive regulators of plant defense. However, the communication and role of volatiles in response to drought stress are poorly understood. Here, we showed that tea plants release numerous VOCs. Among them, methyl salicylate (MeSA), benzyl alcohol, and phenethyl alcohol markedly increased under drought stress. Interestingly, further experiments revealed that drought-induced MeSA lowered the abscisic acid (ABA) content in neighboring plants by reducing 9-cis-epoxycarotenoid dioxygenase (NCED) gene expression, resulting in inhibition of stomatal closure and ultimately decreasing early drought tolerance in neighboring plants. Exogenous application of ABA reduced the wilting of tea plants caused by MeSA exposure. Exposure of Nicotiana benthamiana to MeSA also led to severe wilting, indicating that the ability of drought-induced MeSA to reduce early drought tolerance in neighboring plants may be conserved in other plant species. Taken together, these results provide evidence that drought-induced volatiles can reduce early drought tolerance in neighboring plants and lay a novel theoretical foundation for optimizing plant density and spacing.

The fruits of Physalis (Solanaceae) have a unique structure, a lantern-like fruiting calyx known as inflated calyx syndrome (ICS) or the Chinese lantern, and are rich in steroid-related compounds. However, the genetic variations underlying the origin of these characteristic traits and diversity in Physalis remain largely unknown. Here, we present a high-quality chromosome-level reference genome assembly of Physalis floridana (~1.40 Gb in size) with a contig N50 of ~4.87 Mb. Through evolutionary genomics and experimental approaches, we found that the loss of the SEP-like MADS-box gene MBP21 subclade is likely a key mutation that, together with the previously revealed mutation affecting floral MPF2 expression, might have contributed to the origination of ICS in Physaleae, suggesting that the origination of a morphological novelty may have resulted from an evolutionary scenario in which one mutation compensated for another deleterious mutation. Moreover, the significant expansion of squalene epoxidase genes is potentially associated with the natural variation of steroid-related compounds in Physalis fruits. The results reveal the importance of gene gains (duplication) and/or subsequent losses as genetic bases of the evolution of distinct fruit traits, and the data serve as a valuable resource for the evolutionary genetics and breeding of solanaceous crops.

Arbuscular mycorrhizal (AM) fungi may help protect plants against herbivores; however, their use for the pest control of woody plants requires further study. Here, we investigated the effect of Glomus mosseae colonization on the interactions between gypsy moth larvae and Populus alba × P. berolinensis seedlings and deciphered the regulatory mechanisms underlying the mycorrhizal-induced resistance in the leaves of mycorrhizal poplar using RNA-seq and nontargeted metabolomics. The resistance assay showed that AM fungus inoculation protected poplar seedlings against gypsy moth larvae, as evidenced by the decreased larval growth and reduced larval survival. A transcriptome analysis revealed that differentially expressed genes (DEGs) were involved in jasmonic acid biosynthesis (lipoxygenase, hydroperoxide dehydratase, and allene oxide cyclase) and signal transduction (jasmonate-ZIM domain and transcription factor MYC2) and identified the genes that were upregulated in mycorrhizal seedlings. Except for chalcone synthase and anthocyanidin synthase, which were downregulated in mycorrhizal seedlings, all DEGs related to flavonoid biosynthesis were upregulated, including 4-coumarate-CoA ligase, chalcone isomerase, flavanone 3-hydroxylase, flavonol synthase, and leucoanthocyanidin reductase. The metabolome analysis showed that several metabolites with insecticidal properties, including coumarin, stachydrine, artocarpin, norizalpinin, abietic acid, 6-formylumbelliferone, and vanillic acid, were significantly accumulated in the mycorrhizal seedlings. These findings suggest the potential of mycorrhiza-induced resistance for use in pest management of woody plants and demonstrate that the priming of JA-dependent responses in poplar seedlings contributes to mycorrhiza-induced resistance to insect pests.

Raphanobrassica (R^rR^rC^rC^r, 2n = 4x = 36), which is generated by distant hybridization between the maternal parent Raphanus sativus (R^sR^s, 2n = 2x = 18) and the paternal parent Brassica oleracea (C°C°, 2n = 2x = 18), displays intermediate silique phenotypes compared to diploid progenitors. However, the hybrid shares much more similarities in silique phenotypes with those of B. oleracea than those of R. sativus. Strikingly, the silique of Raphanobrassica is obviously split into two parts. To investigate the gene expression patterns behind these phenomena, transcriptome analysis was performed on the upper, middle, and lower sections of pods (RCsiu, RCsim, and RCsil), seeds in the upper and lower sections of siliques (RCseu and RCsel) from Raphanobrassica, whole pods (Rsi and Csi) and all seeds in the siliques (Rse and Cse) from R. sativus and B. oleracea. Transcriptome shock was observed in all five aforementioned tissues of Raphanobrassica. Genome-wide unbalanced biased expression and expression level dominance were also discovered, and both of them were toward B. oleracea in Raphanobrassica, which is consistent with the observed phenotypes. The present results reveal the global gene expression patterns of different sections of siliques of Raphanobrassica, pods, and seeds of B. oleracea and R. sativus, unraveling the tight correlation between global gene expression patterns and phenotypes of the hybrid and its parents.

Flower and fruit development are two key steps for plant reproduction. The ABCE model for flower development has been well established in model plant species; however, the functions of ABCE genes in fruit crops are less understood. In this work, we identified an EMS mutant named R27 in woodland strawberry (Fragaria vesca), showing the conversion of petals, stamens, and carpels to sepaloid organs in a semidominant inheritance fashion. Mapping by sequencing revealed that the class E gene homolog FveSEP3 (FvH4_4g23530) possessed the causative mutation in R27 due to a G to E amino acid change in the conserved MADS domain. Additional fvesep3^CR mutants generated by CRISPR/Cas9 displayed similar phenotypes to fvesep3-R27. Overexpressing wild-type or mutated FveSEP3 in Arabidopsis suggested that the mutation in R27 might cause a dominant-negative effect. Further analyses indicated that FveSEP3 physically interacted with each of the ABCE proteins in strawberry. Moreover, both R27 and fvesep3^CR mutants exhibited parthenocarpic fruit growth and delayed fruit ripening. Transcriptome analysis revealed that both common and specific differentially expressed genes were identified in young fruit at 6–7 days post anthesis (DPA) of fvesep3 and pollinated wild type when compared to unpollinated wild type, especially those in the auxin pathway, a key hormone regulating fruit set in strawberry. Together, we provided compelling evidence that FveSEP3 plays predominant E functions compared to other E gene homologs in flower development and that FveSEP3 represses fruit growth in the absence of pollination and promotes fruit ripening in strawberry.

Flavones are among the major colorless pigments synthesized through branches of the flavonoid pathway in plants. However, due to the absence of a gene encoding flavone synthase (FNS) in the model plant Arabidopsis thaliana species, the regulatory mechanism of FNS-catalyzed flavone biosynthesis has rarely been studied in plants. Here, it was found that flavones play a predominant role in the elimination of excess reactive oxygen species (ROS) at high temperatures in colorless plant organs. A novel atypical subgroup 7 (SG7) R2R3-MYB transcription factor, CmMYB012, was found to be induced in response to prolonged high temperatures and to inhibit flavone biosynthesis by directly regulating CmFNS. Moreover, CmMYB012 was also found to inhibit anthocyanin biosynthesis by suppressing the expression of CmCHS, CmDFR, CmANS, and CmUFGT. CmMYB012 overexpression exerted a negative influence on plant fitness and pink flower color formation, while CmMYB012 suppression had the opposite effect in response to high temperatures. Our findings provide new insights into the mechanisms by which high temperatures regulate the metabolism of flavones and anthocyanins to affect plant fitness and flower color formation.

The production of reactive oxygen species (ROS) by NADPH oxidase, which is also referred to as respiratory burst oxidase homolog (RBOH), affects several processes in plants. However, the role of RBOHs in cell wall lignification is not well understood. In this study, we show that PuRBOHF, an RBOH isoform, plays an important role in secondary wall formation in pear stone cells. ROS were closely associated with lignin deposition and stone cell formation according to microscopy data. In addition, according to the results of an in situ hybridization analysis, the stage-specific expression of PuRBOHF was higher in stone cells than in cells of other flesh tissues. Inhibitors of RBOH activity suppressed ROS accumulation and stone cell lignification in pear fruit. Moreover, transient overexpression of PuRBOHF caused significant changes in the amount of ROS and lignin that accumulated in pear fruit and flesh calli. We further showed that PuMYB169 regulates PuRBOHF expression, while PuRBOHF-derived ROS induces the transcription of PuPOD2 and PuLAC2. The findings of this study indicate that PuRBOHF-mediated ROS production, which is regulated by a lignin-related transcriptional network, is essential for monolignol polymerization and stone cell formation in pear fruit.

To identify cytoplasmic male sterility (CMS)-associated genes in tomato, we determined the genome sequences of mitochondria and chloroplasts in three CMS tomato lines derived from independent asymmetric cell fusions, their nuclear and cytoplasmic donors, and male fertile weedy cultivated tomato and wild relatives. The structures of the CMS mitochondrial genomes were highly divergent from those of the nuclear and cytoplasmic donors, and genes of the donors were mixed up in these genomes. On the other hand, the structures of CMS chloroplast genomes were moderately conserved across the donors, but CMS chloroplast genes were unexpectedly likely derived from the nuclear donors. Comparative analysis of the structures and contents of organelle genes and transcriptome analysis identified three genes that were uniquely present in the CMS lines, but not in the donor or fertile lines. RNA-sequencing analysis indicated that these three genes transcriptionally expressed in anther, and identified different RNA editing levels in one gene, orf265, that was partially similar to ATP synthase subunit 8, between fertile and sterile lines. The orf265 was a highly potential candidate for CMS-associated gene. This study suggests that organelle reorganization mechanisms after cell fusion events differ between mitochondria and chloroplasts, and provides insight into the development of new F1 hybrid breeding programs employing the CMS system in tomato.

Selection for favorable inflorescence architecture to improve yield is one of the crucial targets in crop breeding. Different tomato varieties require distinct inflorescence-branching structures to enhance productivity. While a few important genes for tomato inflorescence-branching development have been identified, the regulatory mechanism underlying inflorescence branching is still unclear. Here, we confirmed that SISTER OF TM3 (STM3), a homolog of Arabidopsis SOC1, is a major positive regulatory factor of tomato inflorescence architecture by map-based cloning. High expression levels of STM3 underlie the highly inflorescence-branching phenotype in ST024. STM3 is expressed in both vegetative and reproductive meristematic tissues and in leaf primordia and leaves, indicative of its function in flowering time and inflorescence-branching development. Transcriptome analysis shows that several floral development-related genes are affected by STM3 mutation. Among them, FRUITFULL1 (FUL1) is downregulated in stm3cr mutants, and its promoter is bound by STM3 by ChIP-qPCR analysis. EMSA and dual-luciferase reporter assays further confirmed that STM3 could directly bind the promoter region to activate FUL1 expression. Mutation of FUL1 could partially restore inflorescence-branching phenotypes caused by high STM3 expression in ST024. Our findings provide insights into the molecular and genetic mechanisms underlying inflorescence development in tomato.

Switchgrass (Panicum virgatum L.) is an important perennial, noninvasive, tall ornamental grass that adds color and texture to gardens and landscapes. Moreover, switchgrass has been considered a forage and bioenergy crop because of its vigorous growth, low-input requirements, and broad geography. Here, we identified PvWOX3a from switchgrass, which encodes a WUSCHEL-related homeobox transcription factor. Transgenic overexpression of PvWOX3a in switchgrass increased stem length, internode diameter, and leaf blade length and width, all of which contributed to a 95% average increase in dry weight biomass compared with control plants. Yeast one-hybrid and transient dual-luciferase assays showed that PvWOX3a can repress the expression of gibberellin 2-oxidase and cytokinin oxidase/dehydrogenase through apparently direct interaction with their promoter sequences. These results suggested that overexpression of PvWOX3a could increase gibberellin and cytokinin levels in transgenic switchgrass plants, which promotes cell division, elongation, and vascular bundle development. We also overexpressed PvWOX3a in a transgenic miR156-overexpressing switchgrass line that characteristically exhibited more tillers, thinner internodes, and narrower leaf blades. Double transgenic switchgrass plants displayed significant increases in internode length and diameter, leaf blade width, and plant height but retained a tiller number comparable to that of plants expressing miR156 alone. Ultimately, the double transgenic switchgrass plants produced 174% more dry-weight biomass and 162% more solubilized sugars on average than control plants. These findings indicated that PvWOX3a is a viable potential genetic target for engineering improved shoot architecture and biomass yield of horticulture, fodder, and biofuel crops.

Tea plants are subjected to multiple stresses during growth, development, and postharvest processing, which affects levels of secondary metabolites in leaves and influences tea functional properties and quality. Most studies on secondary metabolism in tea have focused on gene, protein, and metabolite levels, whereas upstream regulatory mechanisms remain unclear. In this review, we exemplify DNA methylation and histone acetylation, summarize the important regulatory effects that epigenetic modifications have on plant secondary metabolism, and discuss feasible research strategies to elucidate the underlying specific epigenetic mechanisms of secondary metabolism regulation in tea. This information will help researchers investigate the epigenetic regulation of secondary metabolism in tea, providing key epigenetic data that can be used for future tea genetic breeding.

Silicon (Si), the second most abundant element in Earth’s crust, exerts beneficial effects on the growth and productivity of a variety of plant species under various environmental conditions. However, the benefits of Si and its importance to plants are controversial due to differences among the species, genotypes, and the environmental conditions. Although Si has been widely reported to alleviate plant drought stress in both the Si-accumulating and nonaccumulating plants, the underlying mechanisms through which Si improves plant water status and maintains water balance remain unclear. The aim of this review is to summarize the morphoanatomical, physiological, biochemical, and molecular processes that are involved in plant water status that are regulated by Si in response to drought stress, especially the integrated modulation of Si-triggered drought stress responses in Si accumulators and intermediate- and excluder-type plants. The key mechanisms influencing the ability of Si to mitigate the effects of drought stress include enhancing water uptake and transport, regulating stomatal behavior and transpirational water loss, accumulating solutes and osmoregulatory substances, and inducing plant defense-associated with signaling events, consequently maintaining whole-plant water balance. This study evaluates the ability of Si to maintain water balance under drought stress conditions and suggests future research that is needed to implement the use of Si in agriculture. Considering the complex relationships between Si and different plant species, genotypes, and the environment, detailed studies are needed to understand the interactions between Si and plant responses under stress conditions.

The marvelously diverse Orchidaceae constitutes the largest family of angiosperms. The genus Cymbidium in Orchidaceae is well known for its unique vegetation, floral morphology, and flower scent traits. Here, a chromosome-scale assembly of the genome of Cymbidium ensifolium (Jianlan) is presented. Comparative genomic analysis showed that C. ensifolium has experienced two whole-genome duplication (WGD) events, the most recent of which was shared by all orchids, while the older event was the τ event shared by most monocots. The results of MADS-box genes analysis provided support for establishing a unique gene model of orchid flower development regulation, and flower shape mutations in C. ensifolium were shown to be associated with the abnormal expression of MADS-box genes. The most abundant floral scent components identified included methyl jasmonate, acacia alcohol and linalool, and the genes involved in the floral scent component network of C. ensifolium were determined. Furthermore, the decreased expression of photosynthesis-antennae and photosynthesis metabolic pathway genes in leaves was shown to result in colorful striped leaves, while the increased expression of MADS-box genes in leaves led to perianth-like leaves. Our results provide fundamental insights into orchid evolution and diversification.

Seedlessness is a valuable agronomic trait in watermelon (Citrullus lanatus) breeding. Conventional less seed watermelons are mainly triploid, which has many disadvantages due to unbalanced genome content. Less seed watermelon can be achieved at the diploid level when certain reproductive genes are mutated or by chromosome translocation, which leads to defects during meiosis. However, the formation mechanism of diploid less seed watermelons remains largely unknown. Here, we identified a spontaneous mutant line, watermelon line “148”, which can set seeds normally when self-pollinated. A total of 148 × JM F₁ hybrid plants exhibited seed number reductions to 50.3% and 47.3% of those of the two parental lines, respectively, which are considered to be less seed. Examination of pollen viability and hybridization experiments revealed that F₁ hybrids produce semisterile pollen and ovules. Further cytological observations indicated that semisterility was a result of a reciprocal translocation of chromosomes, which exhibited one quadrivalent ring of four chromosomes at prometaphase I during meiosis. RT-qPCR analysis indirectly confirmed that the semisterile phenotype is caused by chromosome translocation rather than disruption of specific meiotic gene expression. F₂ population genetic analysis indicated that the “148” watermelon line is a homozygous translocation and that the less seed phenotype of the F₁ hybrid is prompted by one chromosome fragment translocation. The translocated fragment was further fine mapped to a 2.09 Mb region on chromosome 6 by whole-genome resequencing and genetic map cloning procedures. Our work revealed that a 2.09 Mb chromosome fragment translocation on chromosome 6, causing meiotic defects at metaphase I during meiosis, leads to diploid less seed watermelon. Our findings provide a new promising method for less seed watermelon breeding at the diploid level, as well as a fragment size reference for breeding less seed watermelon through artificially induced chromosome translocation.

Artemisia annua is a medicinal plant rich in terpenes and flavonoids with useful biological activities such as antioxidant, anticancer, and antimalarial activities. The transcriptional regulation of flavonoid biosynthesis in A. annua has not been well-studied. In this study, we identified a YABBY family transcription factor, AaYABBY5, as a positive regulator of anthocyanin and total flavonoid contents in A. annua. AaYABBY5 was selected based on its similar expression pattern to the phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and flavonol synthase (FLS) genes. A transient dual-luciferase assay in Nicotiana bethamiana with the AaYABBY5 effector showed a significant increase in the activity of the downstream LUC gene, with reporters AaPAL, AaCHS, AaCHI, and AaUFGT. The yeast one-hybrid system further confirmed the direct activation of these promoters by AaYABBY5. Gene expression analysis of stably transformed AaYABBY5 overexpression, AaYABBY5 antisense, and control plants revealed a significant increase in the expression of AaPAL, AaCHS, AaCHI, AaFLS, AaFSII, AaLDOX, and AaUFGT in AaYABBY5 overexpression plants. Moreover, their total flavonoid content and anthocyanin content were also found to increase. AaYABBY5 antisense plants showed a significant decrease in the expression of flavonoid biosynthetic genes, as well as a decrease in anthocyanin and total flavonoid contents. In addition, phenotypic analysis revealed deep purple-pigmented stems, an increase in the leaf lamina size, and higher trichome densities in AaYABBY5 overexpression plants. Together, these data proved that AaYABBY5 is a positive regulator of flavonoid biosynthesis in A. annua. Our study provides candidate transcription factors for the improvement of flavonoid concentrations in A. annua and can be further extended to elucidate its mechanism of regulating trichome development.

Although male sterility has been identified as a useful trait for hybrid vigor utilization and hybrid seed production, its underlying molecular mechanisms in Cucurbitaceae species are still largely unclear. Here, a spontaneous male-sterile watermelon mutant, Se18, was reported to have abnormal tapetum development, which resulted in completely aborted pollen grains. Map-based cloning demonstrated that the causal gene Citrullus lanatus Abnormal Tapetum 1 (ClATM1) encodes a basic helix-loop-helix (bHLH) transcription factor with a 10-bp deletion and produces a truncated protein without the bHLH interaction and functional (BIF) domain in Se18 plants. qRT–PCR and RNA in situ hybridization showed that ClATM1 is specifically expressed in the tapetum layer and in microsporocytes during stages 6–8a of anther development. The genetic function of ClATM1 in regulating anther development was verified by CRISPR/Cas9-mediated mutagenesis. Moreover, ClATM1 was significantly downregulated in the Se18 mutant, displaying a clear dose effect at the transcriptional level. Subsequent dual-luciferase reporter, β-glucuronidase (GUS) activity, and yeast one-hybrid assays indicated that ClATM1 could activate its own transcriptional expression through promoter binding. Collectively, ClATM1 is the first male sterility gene cloned from watermelon, and its self-regulatory activity provides new insights into the molecular mechanism underlying anther development in plants.

Chlorophylls and carotenoids are essential and beneficial substances for both plant and human health. Identifying the regulatory network of these pigments is necessary for improving fruit quality. In a previous study, we identified an R2R3-MYB transcription factor, SlMYB72, that plays an important role in chlorophyll and carotenoid metabolism in tomato fruit. Here, we demonstrated that the SlMYB72-interacting protein SlZHD17, which belongs to the zinc-finger homeodomain transcription factor family, also functions in chlorophyll and carotenoid metabolism. Silencing SlZHD17 in tomato improved multiple beneficial agronomic traits, including dwarfism, accelerated flowering, and earlier fruit harvest. More importantly, downregulating SlZHD17 in fruits resulted in larger chloroplasts and a higher chlorophyll content. Dual-luciferase, yeast one-hybrid and electrophoretic mobility shift assays clarified that SlZHD17 regulates the chlorophyll biosynthesis gene SlPOR-B and chloroplast developmental regulator SlTKN2 in a direct manner. Chlorophyll degradation and plastid transformation were also retarded after suppression of SlZHD17 in fruits, which was caused by the inhibition of SlSGR1, a crucial factor in chlorophyll degradation. On the other hand, the expression of the carotenoid biosynthesis genes SlPSY1 and SlZISO was also suppressed and directly regulated by SlZHD17, which induced uneven pigmentation and decreased the lycopene content in fruits with SlZHD17 suppression at the ripe stage. Furthermore, the protein–protein interactions between SlZHD17 and other pigment regulators, including SlARF4, SlBEL11, and SlTAGL1, were also presented. This study provides new insight into the complex pigment regulatory network and provides new options for breeding strategies aiming to improve fruit quality.

Specific populations of plant microtubules cooperate with the plasma membrane to sense and process abiotic stress signals, such as cold stress. The current study derived from the question, to what extent this perception system is active in biotic stress signalling. The experimental system consisted of grapevine cell lines, where microtubules or actin filaments are visualised by GFP, such that their response became visible in vivo. We used the bacterial elicitors harpin (inducing cell-death related defence), or flg22 (inducing basal immunity) in combination with modulators of membrane fluidity, or microtubules. We show that DMSO, a membrane rigidifier, can cause microtubule bundling and trigger defence responses, including activation of phytoalexin transcripts. However, DMSO inhibited the gene expression in response to harpin, while promoting the gene expression in response to flg22. Treatment with DMSO also rendered microtubules more persistent to harpin. Paradoxically, Benzylalcohol (BA), a membrane fluidiser, acted in the same way as DMSO. Neither GdCl₃, nor diphenylene iodonium were able to block the inhibitory effect of membrane rigidification on harpin-induced gene expression. Treatment with taxol stabilised microtubule against harpin but amplified the response of PAL transcripts. Therefore, the data support implications of a model that deploys specific responses to pathogen-derived signals.

DORMANCY-ASSOCIATED MADS-BOX (DAM) genes have recently emerged as key potential regulators of the dormancy cycle and climate adaptation in perennial species. Particularly, PpeDAM6 has been proposed to act as a major repressor of bud dormancy release and bud break in peach (Prunus persica). PpeDAM6 expression is downregulated concomitantly with the perception of a given genotype-dependent accumulation of winter chilling time, and the coincident enrichment in H3K27me3 chromatin modification at a specific genomic region. We have identified three peach BASIC PENTACYSTEINE PROTEINs (PpeBPCs) interacting with two GA-repeat motifs present in this H3K27me3-enriched region. Moreover, PpeBPC1 represses PpeDAM6 promoter activity by transient expression experiments. On the other hand, the heterologous overexpression of PpeDAM6 in European plum (Prunus domestica) alters plant vegetative growth, resulting in dwarf plants tending toward shoot meristem collapse. These alterations in vegetative growth of transgenic lines associate with impaired hormone homeostasis due to the modulation of genes involved in jasmonic acid, cytokinin, abscisic acid, and gibberellin pathways, and the downregulation of shoot meristem factors, specifically in transgenic leaf and apical tissues. The expression of many of these genes is also modified in flower buds of peach concomitantly with PpeDAM6 downregulation, which suggests a role of hormone homeostasis mechanisms in PpeDAM6-dependent maintenance of floral bud dormancy and growth repression.

Water dropwort (Liyang Baiqin, Oenanthe javanica (BI.) DC.) is an aquatic perennial plant from the Apiaceae family with abundant protein, dietary fiber, vitamins, and minerals. It usually grows in wet soils and can even grow in water. Here, whole-genome sequencing of O. javanica via HiSeq 2000 sequencing technology was reported for the first time. The genome size was 1.28 Gb, including 42,270 genes, of which 93.92% could be functionally annotated. An online database of the whole-genome sequences of water dropwort, Water dropwortDB, was established to share the results and facilitate further research on O. javanica (database homepage: http://apiaceae.njau.edu.cn/waterdropwortdb). Water dropwortDB offers whole-genome and transcriptome sequences and a Basic Local Alignment Search Tool. Comparative analysis with other species showed that the evolutionary relationship between O. javanica and Daucus carota was the closest. Twenty-five gene families of O. javanica were found to be expanded, and some genetic factors (such as genes and miRNAs) related to phenotypic and anatomic differentiation in O. javanica under different water conditions were further investigated. Two miRNA and target gene pairs (miR408 and Oja15472, miR171 and Oja47040) were remarkably regulated by water stress. The obtained reference genome of O. javanica provides important information for future work, thus making in-depth genetic breeding and gene editing possible. The present study also provides a foundation for the understanding of the O. javanica response to water stress, including morphological, anatomical, and genetic differentiation.