Carrot taproots exhibit a wide range of colors due to variations in carotenoid and anthocyanin contents. TouXinHong4 (TXH4), a Chinese red carrot landrace from western China, is appreciated for its storability, stress tolerance, and good flavor. In this study, we generated a high-quality, telomere-to-telomere (T2T), gap-free genome assembly of TXH4, with a total size of 449.92 Mb. Repetitive sequences accounted for 48.6% of the genome. A total of 34 225 genes were identified, with 34 016 genes associated with at least one functional annotation. Comparison with two previously assembled carrot genomes, Daucus carota T2T (DcT2T) and D. carota v2.0 (DcRef), revealed 2 466 422 and 2 037 986 single nucleotide polymorphisms and 500 579 and 474 704 insertions/deletions in DcT2T and DcRef, respectively. Carotenoid analysis showed that the lycopene content in TXH4 roots was 1965-fold higher than that in the leaves, while α-carotene and β-carotene levels in the roots were only 2.7% and 3.5% of those in the leaves, respectively. This finding was consistent with the lack of transcription of lycopene β-cyclase 1 (LCYB1) and lycopene ε-cyclase (LCYE) in TXH4 roots. Furthermore, overexpression of DcLCYB1 and DcLCYE resulted in reduced lycopene levels, while their knockout led to elevated lycopene accumulation. Downregulation of DcLCYB1 and DcLCYE was identified as a critical factor contributing to lycopene accumulation, resulting in the red root phenotype of TXH4 roots. The gapless genome assembly of TXH4 offers important insights into the red carrot genome and expands the genomic resources for breeding, facilitating more efficient genome-assisted breeding strategies for crop improvement.
The precise manipulation of genome sequences through gene targeting (GT) is beneficial; however, the low efficiency of homology-directed repair (HDR) in seed plants has made GT difficult to achieve. Generation of double-strand breaks (DSBs) at the target DNA site of interest represents a promising approach to facilitate HDR-mediated GT in organisms. Despite recent advances, GT remains a significant challenge in seed plants. To address these challenges, we propose that the efficiency of CRISPR/Cas9-mediated GT could be enhanced by the exclusive selection of plants that exhibit high levels of HDR activity. To test this hypothesis, a surrogate screening system was developed, which consists of a nonfunctional split-selection marker gene. In this system, DSBs generated by CRISPR/Cas9 at the linker sequence of the tandem repeat will be repaired via single-strand annealing (SSA), a subtype of HDR, resulting in the achievement of antibiotic resistance in plants. This approach allows for a 2- to 23-fold increase in precise and heritable GT efficiency in Arabidopsis and rice. The results indicate that screening with SSA-mediated surrogate system can enrich cells and plants with high HDR activity as well as DSB activity, thus facilitating the establishment of highly efficient GTs at target loci in these plants.
Certain specialist herbivorous insects have evolved elegant mechanisms to manipulate the physiology of their host plants, including the ability to redirect the fate of plant cells toward the creation of a novel, tumor-like organ, called ‘galls’. While some plants have evolved resistance to gall-inducing insects, the underlying genetic mechanisms remain poorly understood. In this study, we focused on the chalcid gall-inducing wasp, Hemadas nubilipennis (Ormyridae) and its host plant, highbush blueberry Vaccinium corymbosum (Ericaceae). To identify the genetic basis of resistance to gall induction in blueberry, we developed a genetic mapping population derived from the susceptible ‘Liberty’ and resistant ‘Draper’ cultivars. We identified four quantitative trait loci (QTLs) associated with galling resistance, with candidate genes in these regions associated with plant defense, biotic stress response, and phytohormone metabolism. Furthermore, we analyzed gene expression on days one through seven post-oviposition in both susceptible and resistant genotypes, compared to controls, to identify genes and pathways that may contribute to galling resistance. Gene expression analyses, including genes within the four identified QTL regions, revealed a robust early defense response in the resistant genotype, marked by upregulation of defense, stress, and immunity genes following oviposition, ultimately leading to insect death. Conversely, the susceptible genotype exhibited a delayed and weaker response, allowing gall development and insect survival. We expect these results to serve as a resource that will enable breeding programs to employ molecular approaches for selection of resistant cultivars, while also guiding future research aimed at studying the evolution of galling resistance.
Cultivated strawberry is a globally important fruit crop with high economic value. Fruit shape is an important aspect of fruit quality and diversity, and a key target in breeding programs; however, few regulatory genes governing fruit shape are known in strawberry. Here, we identified an ethyl methanesulfonate (EMS) ‘round fruit’ (rf) mutant that produces round or flat fruits in woodland strawberry. The causal point mutation is located in the second exon of FvH4_2g22810, resulting in a premature termination at amino acid 266. The encoded protein shares a high sequence similarity with TON1 RECRUITING MOTIF 5 (TRM5) in different plant species and was therefore named FveTRM5. Consistently, transforming the rf mutant with FveTRM5pro:FveTRM5 restored the wild-type fruit phenotype. FveTRM5 is ubiquitously expressed in various organs, and the protein localized to microtubules. Overexpression of FveTRM5 produced elongated organs in both Arabidopsis and woodland strawberry, suggesting a conserved role in different species. Observation of cell shape showed that FveTRM5 promotes cell elongation and inhibits cell division in the medial-lateral direction in the receptacle. Transcriptome analysis revealed 183 differentially expressed genes (DEGs) in the young receptacles of rf and 2976 DEGs in those of FveTRM5-OE, including several involved in the auxin and gibberellic acid pathways. In conclusion, our results suggest that FveTRM5 plays an essential role in regulating strawberry fruit shape by influencing cell elongation and cell division, providing an excellent target gene for breeding new fruit shape cultivars.
WRKY transcription factors are essential for mediating many developmental processes, such as fruit ripening, a highly controlled and intricate physiological phenomenon. In the current research, a novel transcription factor, MdWRKY9, was identified and categorized, which significantly promotes apple fruit ripening. Its role was validated through a combination of transient injection and stable overexpression-transformed tomato experiments. Notably, MdWRKY9 interacts with the fruit ripening suppressor MdERF5L at protein and DNA levels. This interaction counteracts MdERF5L-mediated MdACS1 repression. MdACS1 is an important ethylene biosynthesis enzyme; the mentioned process eventually allows fruit ripening. Furthermore, phosphorylation, a post-translational modification, regulates maturation and ethylene synthesis. Through liquid chromatography-tandem mass spectrometry, we identified phosphorylation sites within the MdWRKY9-GFP protein. MdMAPK6-MdWRKY9 interaction and MdMAPK6-mediated phosphorylation of MdWRKY9 were confirmed through protein-protein interaction assays, such as bimolecular fluorescence complementation (BIFC), yeast two-hybrid (Y2H), protein phosphorylation, luciferase complementation imaging (LCI), and pull-down assays. Specifically, MdMAPK6 phosphorylates MdWRKY9 at Tyr394 site, enhancing the stability and activity of MdWRKY9 and thus modulating its regulatory role in fruit maturation. These findings provide new directions and insights into the intricate regulatory network governing the ripening of apples.
Root shape is a defining feature of marketability and breeding strategies in the Beta vulgaris crop complex encompassing sugar beet, fodder beet, table beet, and Swiss chard. This study leverages the Wisconsin Beta Diversity Panel of 234 accessions to understand the genetic architecture underlying root shape traits, utilizing field trials, genome-wide association, and population structure analyses. High heritability estimates for many root shape traits (H2 > 0.9) suggest genetic control as the primary determinant of root shape with minimal genotype-by-environment interactions across locations and years. Digital biomass was not correlated with length-width ratio, a key shape descriptor. Key quantitative trait loci (QTL) on Chromosomes 4, 7, and 8 associated with traits such as length, width, and length-to-width ratio, collectively explained up to 55% of phenotypic variance. Several loci co-localize with predicted gene families known to influence organ shape in other plant species. Candidate genes near shape QTL were significantly enriched for microtubule organization and auxin response. Genomic estimated breeding values for shape traits showed high predictive accuracy, particularly for length-to-width ratio. Admixture analyses revealed eight genetic populations, suggesting distinct domestication and breeding histories of crop types in the complex. Swiss chard and wild germplasm showed unique ancestry, while sugar and fodder beet shared genetic proximity. Our analysis identifies candidate loci and molecular markers for root shape, providing resources for molecular breeding strategies in B. vulgaris. The findings add to and clarify the current knowledge on root shape inheritance, advancing the genetic improvement of these crops of economic, nutritional, and cultural significance.
Root tips, which represent the initial stage of taproot development, serve as an ideal model for investigating plant growth and secondary metabolism. However, studies of root tips in Panax species have been limited, restricting our understanding of cell fate transitions during early root development and the cellular heterogeneity associated with ginsenosides biosynthesis. To address this gap, we conducted single-cell RNA sequencing (scRNA-seq) and spatial metabolomics analyses on the root tips of three Panax species: Panax notoginseng, Panax ginseng, and Panax quinquefolium. Our research reconstructed the developmental trajectory of the early endodermis and revealed epidermis-specific expression patterns of key enzyme genes involved in ginsenosides biosynthesis. We identified several novel transcription factors (TFs): IAA29 (which positively regulates endodermis suberization) and MYB2/MYB78 (positive regulators of ginsenosides biosynthesis), validated by dual-LUC reporter and electrophoretic mobility shift assay (EMSA). Conserved and divergent ligand-receptor interaction patterns across the three Panax species were discovered, with the FAD gene family exhibiting tissue-and species-specific expression. Cell-specific genes expression was confirmed by RNA in situ hybridization. Mass spectrometry imaging (MSI) mapped ginsenosides spatial distribution, while LC-MS/MS verified species-specific biosynthesis. This study presents a single-cell transcriptional landscape of early differentiation and cell type-specific ginsenosides accumulation in the Panax genus.
UDP-dependent glycosyltransferases (UGTs) play a critical role in producing glycosylated metabolites that mediate plant-environment interactions. Recent studies have examined the role of UGT genes across various plant genomes. However, the evolutionary history and functional divergence of the UGT pan-gene family in the genus Solanum have not yet been explored. This study integrated data from 61 tomatoes and 9 representative genomes, ranging from algae to angiosperms, to identify 12 073 genes. The phylogeny of the UGT gene family reveals a clear evolutionary trajectory from unicellular algae to ferns, mosses, gymnosperms, and angiosperms. The study identified a significant number of tomato-specific UGT genes and explored the expansions of UGT73 and UGT85 subfamilies. The entire UGT genes (10 769) in tomato were classified into 118 orthologous gene groups, including 58 core, 31 softcore, 10 dispensable, 19 private orthologous gene groups, and the core groups contained 7811 genes, representing 72.53% of the total UGT genes. Analysis of gene family expansion revealed that whole-genome triplication and tandem duplication events play significant roles in the expansion of the UGT gene family. Selection pressure analysis revealed that the UGT genes experienced purifying selection in the genus Solanum. Additionally, expression profiles of some UGT genes in different tissues demonstrated expression divergence of multicopy genes across different UGT subfamilies due to the increase in gene dosage. Subcellular localization prediction revealed that most genes are localized in the chloroplast. These findings provide critical insights into the evolution and function of the UGT genes in tomato, laying a foundation for further exploration in adaptive evolution.
High temperatures impair pollen viability and reduce fruit set, ultimately affecting the yield of crops. Understanding the genetic components involved in the heat stress (HS) response is essential for developing climate-resilient crop varieties. However, the regulatory mechanisms governing HS responses during pollen development in tomato (Solanum lycopersicum) remain unexplored. In this study, we identified the microspore mother cell stage as the most heat-sensitive phase in tomato pollen development. Furthermore, we generated a comprehensive RNA expression profile of tomato flower buds under HS, encompassing 8051 mRNAs, 5738 lncRNAs, 62 circRNAs, and 24 miRNAs. Comparative analysis of these RNAs revealed three distinct response phases, early, late, and dual, and enabled the identification of coexpression modules comprising both coding and noncoding transcripts. Among these, SlERF162 was identified as a key regulatory gene that promotes pollen thermotolerance. We further identified the lncRNA TCONS_00023929 (designated SllncERF162) as a positive regulator of SlERF162 expression. Both SlERF162 and SllncERF162 contributed to maintaining pollen viability under HS. Additional experiments demonstrated that the SllncERF162-SlERF162 regulatory module enhances basal thermotolerance by directly targeting and activating the heat-responsive genes SlHsfB1 and SlsHSP. Overall, this study provides a high-resolution expression atlas of RNAs under HS and uncovers a novel noncoding RNA-mediated regulatory network that promotes thermotolerance during tomato pollen development.
BAK1 was initially identified as a coreceptor of BRI1 in regulating the brassinosteroid-triggered signaling pathway in Arabidopsis. Over the past two decades, increasing pieces of evidence have demonstrated that BAK1 and its close paralogs form receptor-coreceptor complexes with distinct ligand-binding receptors. Through ligand-induced heterodimerization with receptor-like protein kinases or receptor-like proteins, BAK1 thereby regulates a variety of physiological events such as plant development, immunity, and stress responses. Thus, BAK1 plays a central role in directly governing the trade-offs of multiple signaling pathways. Deciphering the molecular mechanisms underlying how BAK1 coordinates plant growth and defense, with specific emphasis on its coreceptor functions, holds significant potential for future advancements in crop breeding. This review seeks to explore the latest insights into how BAK1 impacts the intricate equilibrium between plant development and immunity, as well as its roles in coordinating the activation of pattern-triggered immunity and effector-triggered immunity or programmed cell death. Furthermore, it offers significant perspectives on why BAK1 has been chosen as a shared component in determining plant growth and defense mechanisms across model plants to horticultural crops.
High-quality reference genomes at the population scale are fundamental for advancing pan-genomic research. However, high-quality genome assembly at the population scale is costly and time-consuming. To overcome these limitations, we developed Reference-Assisted Genome Assembly (RAGA), a hybrid computational tool that combines de novo and reference-based assembly approaches. RAGA efficiently employs existing reference genomes from the same or closely related species in combination with PacBio HiFi reads to produce high-quality alternative long sequences. These sequences can be integrated with de novo assemblies to improve assembly quality across population-scale datasets. The performance of RAGA across various plant genomes demonstrated its ability to reduce the number of contigs, decrease gaps, and correct genome assembly errors. The implementation of RAGA (available at https://github.com/wzxie/RAGA) significantly streamlines population-scale genome assembly workflows, providing a robust foundation for comprehensive pan-genomic investigations. This tool represents a substantial advancement in making large-scale genomic studies more accessible and efficient.
Clubroot is a devastating soil-borne disease that parasitizes cruciferous crops, posing a severe threat to rapeseed production. To date, no clubroot-resistant (CR) genes have been successfully cloned in cabbage (Brassica oleracea). This study aimed to identify CR genes and elucidate the molecular mechanisms underlying clubroot resistance in B. oleracea. A BC1 mapping population was developed from a cross between CR cabbage W12 and clubroot-susceptible cabbage Z5. A major CR locus, Bol.CR7.1, was identified on chromosome C07 by Bulk Segregant Analysis. Subsequently, the Bol.CR7.1 was fine-mapped to a 170.2-kb interval using linkage analysis. Two candidate genes, Bol.TNL.2 and Bol.TNL.3, exhibiting sequence variations between the parents were induced upon Plasmodiophora brassicae infection. Overexpression of Bol.TNL.2W (CR cabbage W12) in Arabidopsis and rapeseed significantly reduced the disease index compared to the wild type (WT) after P. brassicae inoculation. In contrast, plants overexpressing Bol.TNL.2Z (the susceptible cabbage Z5), Bol.TNL.3W, and Bol.TNL.3Z exhibited symptoms comparable to those of WT, indicating that Bol.TNL.2 is a CR gene. RNA-seq analysis revealed that Bol.TNL.2 may mediate resistance to P. brassicae by modulating pathways related to reactive oxygen species, cell wall metabolism and modification, as well as secondary metabolite synthesis. In addition, long noncoding RNAs were found to play a significant role in regulating gene expression associated with P. brassicae interaction. This study broadens the repertoire of CR genes, offering a solid foundation for breeding CR cruciferous crops. Additionally, it provides novel insights into resistance mechanisms in response to P. brassicae infection in B. oleracea.
Aquaporins (AQPs) are integral membrane channel proteins that facilitate water transport and contribute significantly to plant adaptation under drought stress. However, the evolutionary origins and mechanisms of functional diversity of this gene family remain to be elucidated. A comprehensive genome-wide analysis was therefore performed on 104 representative species spanning the green plant lineage, from algae to angiosperms. This study used two datasets: Taxon I (algae to eudicots) and Taxon II (angiosperms including drought-tolerant and drought-sensitive plants). By systematically optimizing the gene structure, codon preferences, motifs, and cis-elements of these two datasets, the molecular mechanisms of AQP genes in plant adaptation evolution and drought-tolerance evolution were revealed. The results of phylogenetic analysis indicate that the AQP gene family is divided into five main subfamilies: PIPs, NIPs, TIPs, SIPs, and XIPs. Through in-depth analysis of the evolution characteristics of each subfamily, it was found that the emergence and loss of different subclusters are related to the ecological adaptation needs of specific species. By systematically analyzing the evolutionary history of the members of PIPs and TIPs subfamilies and subclusters, and combining their gene expression patterns, it was confirmed that PIP2, TIP1, and TIP4 subcluster members exhibit more significant expression response characteristics under drought stress. This study is the first to analyze the evolutionary patterns and drought-tolerance mechanisms of the AQP gene family at a multidimensional scale, providing important molecular targets for crop drought resistance breeding.
The MYB transcription factor (TF) family, which is involved in plant growth and development, is large and diverse. Previous studies on MYB family in Cucurbitaceae were mostly based on a single genome or focused on the R2R3 subfamily. Here, we analyzed 91 genomes of Cucurbitaceae and identified a total of 15 858 MYB genes. According to phylogenetic relationships, these genes were divided into 27 subgroups. The identified MYB genes were further classified into 121 MYB orthologous gene groups (OGGs), including 25 core, 57 softcore, 19 shell and 20 line-specific/cloud groups. Whole-genome duplication was the most common mechanism of MYB genes expansion. In core group, the higher proportions of MYB genes were found to be in the coexpression network constructed by the RNA-seq data. Through the comprehensive analysis including phylogeny and gene expression profile of cucumber MYB genes, as well as genetic variations in 103 cucumber germplasms, we identified a MYB gene CsRAX5, which may be related to cucumber plant height. We used gene editing technology to knockout and overexpress CsRAX5. In the knockout lines, Csrax5, the height was significantly increased compared with wild type (WT), whereas after overexpression the height of CsRAX5-OE plants was significantly decreased compared with WT. These results indicated that MYB gene CsRAX5 negatively regulated cucumber plant height. The large-scale analysis of MYB genes in Cucurbitaceae in this study provides insights for further investigating the evolution and function of MYB genes in Cucurbitaceae crops.
Plant LATERAL ORGAN BOUNDARIES DOMAIN (LBD) family is crucial for defining organ boundaries and participates in various developmental processes, but its role in fruit weight has rarely been elucidated. Here, we characterized an LBD gene, Physalis organ size 3 (POS3), in Physalis floridana, designated as PfPOS3. This gene exhibited high expression levels in floral meristems, carpels, and developing seeds and fruits when compared to Solanum pimpinellifolium and Solanum lycopersicum. The floral organ size, seed weight, and mature fruit weight were significantly reduced in PfPOS3 knockdown and knockout plants. Consistent with overexpression analyses, PfPOS3 promoted cell size and inhibited cell division during berry development. Moreover, overexpression of PfPOS3 and SlPOS3 shared identical phenotypic variation in transgenic Physalis plants. Both PfPOS3 and SlPOS3 interacted with Teosinte branched1/Cycloidea/Proliferating cell factor 15 (TCP15) and TCP18, and the POS3-TCP modules directly regulated the expression of Cyclin D1;1 (CYCD1;1) and CYCB1;1. Overall, POS3 may have the capability to orchestrate cell number and cell size, thus regulating fruit weight variation within Solanaceae. However, a significant reduction in the expression of SlPOS3 may result in a pronounced weakening or complete loss of this function within Solanum. Our findings shed new light on the reproductive organ size control, the developmental evolution of fruit morphology, and the breeding of Physalis crops.
Tea plant [Camellia sinensis (L.) O. Kuntze] is a globally important crop but is severely threatened by Toxoptera aurantia infestations, which impact yield and safety. However, the response of tea plants to aphid feeding remains largely unexplored. This study investigates the feeding behavior of T. aurantia on different cultivars and identifies ‘Huangjinya’ and ‘Qiancha 1’ as susceptible and resistant cultivars, respectively. Transcriptome analysis revealed that CsUGT89A2 was significantly upregulated in response to T. aurantia infestation. In vitro biochemical assays demonstrated that CsUGT89A2 encodes a flavonoid 7-glycosyltransferase that catalyzes the conversion of flavonoids and UDP-glucose into flavonoid 7-O-glucosides. In vivo, silencing CsUGT89A2 significantly reduced flavonoid glycoside accumulation. To further clarify the role of CsUGT89A2 in tea plant resistance to T. aurantia, we used tobacco and tea flowers to evaluate aphid feeding and reproduction under chemical treatment, gene silencing, and gene overexpression conditions. Statistical analysis showed that, compared with flavonoids, the application of flavonoid 7-O-glycosides significantly reduced T. aurantia reproductive capacity. Furthermore, compared with the control, overexpression of CsUGT89A2 significantly reduced the reproductive ability of aphids, while its silencing increased reproductive rates. Overall, our findings demonstrate that CsUGT89A2 mediates flavonoid glycosylation and enhances insect resistance in tea plants by increasing flavonoid glycoside levels, offering new insights into the role of flavonoid glycosides in the insect resistance of C. sinensis.
Epigenetic modifications, such as DNA methylation, histone modifications, chromatin remodeling, and RNA-associated silencing, play critical roles in regulating gene expression without altering the DNA sequence. In horticultural crops, these mechanisms control key biological processes, including fruit development and ripening, flowering time, stress adaptation, and phenotypic plasticity. Driven by high-throughput sequencing and multi-omics technologies, researchers have begun to uncover the dynamic landscape of plant epigenomes. Notably, the Encyclopedia of DNA Elements (ENCODE) project was developed to systematically map functional elements within the genome. Inspired by this initiative, similar strategies have been increasingly applied to plants to identify regulatory elements, chromatin states, and transcriptional networks. This review integrates recent findings on epigenetic regulation in model and horticultural species, emphasizing the role of epigenomic tools and ENCODE-like approaches in annotating cis-regulatory elements, epigenetic markers, and long non-coding RNAs (lncRNAs). We discuss how epigenetic modifications mediate developmental transitions and responses to environmental cues. Finally, we propose a framework for integrating ENCODE-derived insights with precision breeding to improve yield, quality, and stress resilience in horticultural crops. These advancements offer exciting opportunities for translating epigenomic knowledge into practical crop improvement strategies.
Hydrogen sulfide (H2S), a gasotransmitter molecule, plays critical roles in stomatal closure and cellular bioenergetics. Alternative splicing (AS) is a key regulatory mechanism during plant development and stress responses; however, the interplay between H2S signaling and AS in drought tolerance remains unexplored in Chinese cabbage. In this study, we found that the mitochondrial inner membrane enzyme succinate dehydrogenase (SDH) responds to H2S signaling during stomatal closure. Silencing of BrSDH1-1 impaired the effects of H2S on stomatal closure, SDH activity, and ATP production. RNA-Seq analysis revealed that H2S modulates the AS of BrSDH1-1, resulting in transcript variants with differential expression. Overexpression of BrSDH1-1A and BrSDH1-1C in Arabidopsis enhanced drought resistance, whereas BrSDH1-1B had no significant effect. H2S enhanced SDH activity and ATP production, promoted stomatal closure, and reduced excess reactive oxygen species (ROS) in OE-BrSDH1-1A and OE-BrSDH1-1C lines but not in OE-BrSDH1-1B. Furthermore, biotin-switch assays demonstrated that H2S induced persulfidation of BrSDH1-1A and BrSDH1-1C, with no effect on variant BrSDH1-1B. These findings reveal a novel regulatory mechanism by which H2S modulates BrSDH1-1 splicing to mediate stomatal closure and improve drought tolerance, offering valuable molecular insights for enhancing stress resilience in horticultural crops.
Kiwifruit bacterial canker, caused by Pseudomonas syringae pv. actinidiae, poses a critical threat to global kiwifruit production. Previous studies implicated jasmonic acid (JA) signaling in kiwifruit responses to this pathogen; however, the molecular mechanisms underlying JA-mediated regulation remain largely unclear. Here, we identified and characterized AcJAZ2L2, a pivotal jasmonate-signaling regulator that confers substantial resistance against P. syringae pv. actinidiae. Transcriptomic profiling coupled with consensus co-expression network analysis revealed that AcJAZ2L2 expression is uniquely up-regulated in resistant kiwifruit cultivars after pathogen infection. Functional validation through genome editing with the clustered regularly interspaced short palindromic repeat-associated protein 9 nuclease and, through transgenic overexpression, confirmed the essential role of AcJAZ2L2 in resistance. Specifically, lines overexpressing AcJAZ2L2 displayed markedly reduced disease symptoms, lower pathogen colonization, and decreased stomatal density, whereas knockout lines exhibited increased susceptibility. Mechanistically, AcJAZ2L2 directly interacts with AcMYC2-like transcription factors, repressing downstream JA-responsive genes (AcVSP2L1 and AcVSP2L2) and maintaining stomatal closure to prevent pathogen entry. Promoter analysis further revealed cultivar-specific allelic divergence that drives differential AcJAZ2L2 transcriptional activation, explaining genotype-dependent resistance levels. Our findings establish a novel JAZ-MYC regulatory module that links JA signaling to stomatal immunity in kiwifruit and provide precise genetic targets for breeding cultivars with enhanced resistance to bacterial canker.
Impatiens noli-tangere accumulates abundant α-linolenic acid (ALA) and its metabolic volatiles, which hold significant potential for applications in healthcare and agriculture. However, the genetic basis underlying their biosynthesis has not been systematically investigated. Here, we present a high-quality genome assembly for I. noli-tangere (614.46 Mb). Despite a high repetitive sequence content (70.46%), it avoided excessive expansion due to the efficient elimination of long terminal repeat retrotransposons. Phylogenomic analyses revealed that I. noli-tangere experienced two whole-genome duplication (WGD) events, with WGD-derived genes predominating in oil biosynthesis. Notably, IntFAD3, a WGD-duplicated fatty acid desaturase, was identified as a key seed-specific gene for ALA biosynthesis. Its regulation by the transcription factor IntbZIP38 was functionally validated through yeast one-hybrid, luciferase, β-glucuronidase, and transgenic functional assays. Furthermore, (E)-2-hexenal, the predominant ALA-derived volatile in leaves, exhibited potent antifungal activity against Botrytis cinerea (minimum inhibitory concentration: 0.188 ml/l), with its biosynthesis linked to Int13-HPL. These findings provide genomic and functional insights into ALA biosynthesis and metabolic volatiles in I. noli-tangere, supporting its potential in sustainable agriculture and bioactive compound development.
Apple replant disease (ARD) poses a major threat to global orchard productivity, yet its biological causes remain poorly understood. Although microbial dysbiosis in replant soils has been recognized as a major contributing factor, the specific pathogenic agents involved and the efficacy of synthetic microbial communities in mitigating ARD remain unclear. In this study, we integrated physiological, transcriptomic, metabolomic, and microbiome analyses to investigate the effects of replant soils on the growth of Malus domestica rootstock M26. Absolute quantification amplicon sequencing of 16S rRNA and ITS regions revealed a marked decline in rhizospheric microbial diversity in replant soils compared to fallow controls, accompanied by an enrichment of fungal genera such as Fusarium, Aspergillus, and Acremonium. Pathogenicity assays and seedling colonization experiments verified strong pathogenicity for five isolates—Acremonium sp., Aspergillus niger, Fusarium solani, Macrophomina phaseolina, and Aspergillus stellatus—implicating them as potential causal agents of ARD. High-throughput culturing and confrontation assays were used to isolate and screen candidate microbial antagonists. A synthetic microbiota (SynMs) composed of 12 bacterial strains and Trichoderma sp. was developed. Inoculation with SynMs significantly inproved plant height by 133% (P < 0.05) and total root length by 186% (P < 0.01), and effectively suppressed pathogen proliferation of the five pathogenic isolates in replant soils. Collectively, these findings identify key fungal pathogens underlying ARD and propose a sustainable microbiota-based strategy for its effective mitigation, offering both mechanistic insights and practical solutions for microbiome-informed orchard management.
Cytokinins play crucial roles in regulating the flower bud differentiation in fruit trees. However, the molecular mechanisms by which cytokinins promote flowering in plants are largely unknown. The litchi (Litchi chinensis Sonn.) is a typical subtropical fruit tree that suffers from severe alternate fruiting due to unstable flowering. Here, we developed a novel alternate-fruiting management, which can ensure 100% flowering of the on-year trees, while the off-year trees hardly flower at all. The abundance of two types of cytokinins (tZR, iPR) and LcFT1 expression in the leaves of on-year trees were continuously increased throughout the period of floral bud physiological differentiation. In contrast, these corresponding indicators in off-year trees were maintained at a significantly lower level. Exogenous application of 40 mg/kg 6-BA significantly promoted flowering and increased LcFT1 expression level in the leaves of the off-year trees. LcIPT3, encoding a pivotal rate-limiting enzyme in cytokinin biosynthesis, was identified as the key gene determining the differences in cytokinin levels between on-year trees and off-year trees. Interestingly, we discovered that both LcIPT3 and LcFT1 are directly activated by LcARR11, a type-B cytokinin response factor, as demonstrated through both in vitro and in vivo assays. Furthermore, ectopic expression of LcARR11 in Arabidopsis resulted in elevated IPT expression and cytokinin content, alongside increased FT expression and earlier flowering. Collectively, our findings suggest that cytokinin-mediated promotion of flowering in litchi might be orchestrated by LcARR11, which could promote floral bud physiological differentiation by activating both LcIPT3 and LcFT1.
EIN3 binding F-box (EBF) proteins have been reported to play important roles in ethylene signaling pathway by mediating the ubiquitin-dependent degradation of EIN3-Like (EIL) proteins, but little is known about their roles in postharvest disease resistance. Here, we showed that SlEBF3 confers resistance against Botrytis cinerea by ubiquitin-mediated degradation of SlBBX20. Overexpression of SlEBF3 enhanced resistance to B. cinerea and increased the expression levels of genes related to PR (pathogenesis-related) and JA (jasmonic acid) in tomato, while knockdown of SlEBF3 does not affect tomato resistance to B. cinerea. Further study demonstrated that SlEBF3 interacts with SlBBX20 the interaction between SlEBF3 and SlBBX20 promotes SlBBX20 degradation via the 26S proteasome, which confers enhanced resistance to B. cinerea through the JA signaling pathway mediated by the SlBBX20-SlMYC2-SlMED25 module. Meanwhile, SlEBF3 extends fruit shelf life by remodeling cell wall composition and promoting cuticular accumulation. Additionally, SlEBF3 is involved in carotenoid metabolism regulation by interacting with SlBBX20, SlRIN, SlFUL1, and SlTAGL1, which is independent of the degradation of EIL proteins. Overall, this study revealed the molecular mechanism by which SlEBF3 responds to JA signaling to regulate B. cinerea resistance, enriched the roles of SlEBF3 in the regulatory network of carotenoids metabolism, and provided new insights into the extension of fruit shelf life.
Fusarium wilt caused by Fusarium oxysporum (Foc) is one of the most destructive diseases in global banana production. The response of root system to Foc infection through gene expression in multiple cell types is crucial for understanding the disease resistance mechanism in banana. Here, we reported a single-cell transcriptional landscape of banana root tips in response to Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) infection. We characterized 10 major cell types from 19 cell clusters. We explored differentiation trajectories of meristematic cells, root cap cells, and pericycle cells through pseudotime analysis, and identified pericycle cell as the dominant root cell type under Foc TR4 infection. Moreover, we identified 11 co-expression regulatory networks, of which eight were significantly associated with Foc TR4 response. Specifically, MaKAN4 was co-expressed with two Zn 2+-dependent genes (MaACA7 and MaADH3) in M5 module, which was associated with pericycle cell type and responded to Foc TR4 infection. Further analysis demonstrated that MaKAN4 protein could interact with the promoters of MaACA7 and MaADH3 to promote their expression levels, highlighting a crucial role of MaKAN4 in banana disease resistance by regulating the Zn 2+-dependent MaACA7/MaADH3 module. These findings provide a comprehensive view of cell fate determination in banana root tips and highlight novel insights into the regulatory mechanisms of major cell types in response to Foc TR4 infection, laying a useful foundation for developing disease-resistant banana cultivars.
The domestication of ornamental plants is primarily driven by aesthetic values and usually involves frequent hybridization events. Camellia spp., a globally famous woody flower, exemplifies the complex origins and extensive phenotypic variation. Here, based on the whole genome resequencing 220 germplasms, we developed Camellia21K, a high-density SNP array enabling cost-effective genome-wide genotyping. We demonstrated that Camellia21K accurately resolves 69 cultivars with complex hybridization histories. For molecular identification of closely related varieties, we developed a set of fingerprinting SNPs to support variety discrimination. To dissect the genomic basis of ornamental traits, we performed a genome-wide association study (GWAS) analysis of five leaf shape traits using the Camellia21K array and screened 31 SNP loci significantly associated with the traits. Further, by analyzing the genotypes of the SNP loci and the haplotypes of the surrounding segments, we identified potential genes regulating leaf tip length, thus demonstrating the versatility of the array. To enhance breeding efficiency, we evaluated and optimized four genomic selection (GS) models for leaf trait prediction. We found that the number of SNPs and model selection significantly affected prediction performance, with optimal predictive accuracy (PC) from 0.362 to 0.542, which was positively correlated with heritability. Finally, we integrated fixed-effects SNPs from GWAS and found significant enhancement of PC (24.7%-64.7%), indicating that the combination of GWAS and GS is indispensable for precision breeding applications. We demonstrated that Camellia21K is effective in discriminating the origin of varieties, in genetic analysis of traits and in genomic prediction, and thus informative for crop breeding.
Plant-parasitic root-knot nematodes (Meloidogyne species) are highly polyphagous parasites that alter cellular identity of terminally differentiated root cells to induce the formation of giant cells and knot-like structures known as galls, whose ontogeny remains largely unknown. In this study, we generated single-nucleus RNA-seq data of galls and neighboring root tissues at two distinct stages of Meloidogyne incognita infection of tomato (Solanum lycopersicum) plants. Analysis of 35 393 high-quality nuclei resulted in the identification of three stele-associated cell clusters that captured young and more differentiated giant cells, where 772 genes were preferentially expressed. Giant cell-specific expression patterns of a set of these genes were validated using promoter activity assays. We used pseudotime analysis to trace how gene activity changes as giant cells develop. Developmental trajectory analysis revealed a gradual activation of more complex gene regulatory networks as young giant cells adopt specific fates and become more differentiated. Functional assays using gene silencing confirmed the functional importance of giant cell-expressed genes in mediating plant susceptibility to M. incognita. Cell type-specific gene expression analysis revealed that xylem, phloem, stele, endodermal, and protophloem cells undergo extensive transcriptome reprograming, which facilitates coordinated cellular responses to nematode infection, including immune signaling, structural support, and metabolic adjustments. Together, our analyses represent the first single-nucleus transcriptomic map of nematode-induced giant cells and provide novel insights into the molecular events leading to the formation of a new plant organ and feeding cells orchestrated by an animal parasite.
Bee pollination enhances crop productivity and food security globally. However, its impact on pollen performance within pistil tissues and the underlying regulatory mechanisms remain unclear. In this study, artificial self-pollination yielded the highest pollen deposition on stigmas (119879.33 ± 43037.92 grains), followed by bee pollination (95464.60 ± 3985.01 grains). Conversely, bee pollination achieved the highest seed set rate (55.21% ± 1.84%), significantly exceeding the artificial self-pollination rate (7.27% ± 1.87%). A positive correlation was observed between pollen load on the stigmatic pollination band and seed set rate. Bee pollination delivers ample high-quality pollen to the stigmas of oil tree peony, enhancing seed production. Moreover, a trend high correlation was observed between pollen deposition on the stigmatic pollination band and seed set rate. Fluorescence microscopy and endogenous hormone analyses revealed that bee pollination stimulated a rapid increase in ZR, IAA, and GA3 levels in the pistil tissues, promoting pollen germination and pollen tube growth. Transcriptome analysis identified PoFAR2, a key candidate gene involved in pollen development, in the pistil tissues after bee pollination. This gene exhibits high homology with genes found in other crops. The PoFAR2 gene localizes to the cell membrane, validating earlier predictions, and exhibits strong transcriptional activity. Silencing PoFAR2 disrupts pollen development in Paeonia ostii ‘Fengdan’ manifesting as structural defects in pollen walls and significantly reduces pollen viability. In conclusion, bees enhance fertilization in oil tree peony by delivering high-quality pollen that promotes germination and pollen tube growth. Crucially, we identified PoFAR2, a membrane-localized key gene regulating pollen development. This study establishes a crucial foundation for deciphering the molecular mechanisms by which bee pollination and phytohormone signaling mediate pollen development.
Centromeres are essential for centromere-specific histone H3 (CENH3) recruitment and kinetochore assembly, ensuring accurate chromosome segregation and maintaining genome stability in plants. Although extensively studied in model species, the structural organization of centromeres in nonmodel plants, such as fruit trees, remains poorly explored. Our previous study revealed that jujube centromeres lack the typical tandem repeat (TR)-rich structure, complicating their precise identification. In this study, we updated the genome assembly of jujube (Ziziphus jujuba Mill. ‘Dongzao’) to a haplotype-resolved T2T version, enabling accurate mapping and comparison of centromeres between haplotypes using CENH3 ChIP-seq. These centromeres, ranging from 0.75 to 1.40 Mb, are largely conserved between haplotypes, except for a localized inversion on chromosome 10. Unlike the TR-rich centromeres found in many plant species, jujube centromeres are predominantly composed of Gypsy-type long-terminal repeat retrotransposons (LTR-RTs). Among these, we identified a centromere-enriched LTR family, centromeric retrotransposons of jujube (CRJ), which is particularly abundant in terminal LTRs compared to the internal transposon regions. Comparative analysis across plant species revealed that centromeric retrotransposons primarily fall into three subfamilies—CRM, Tekay, and Athila—highlighting strong subfamily specificity. Notably, early insertions of CRJ-derived LTR segments contributed to the formation of TR-like structures, suggesting a mechanistic link between transposable elements and the evolution of centromeric tandem repeats. This work provides the first in-depth characterization of a TE-dominated centromere architecture in a fruit tree, offering new insights into the diversity and evolution of plant centromeres.