Camptotheca acuminata, a tree native to southern China and cultivated for horticultural purposes, has attracted considerable attention due to its ability to synthesize camptothecin-type compounds with potent antitumor activity. Flavonoids and camptothecins share a conserved benzene ring scaffold, suggesting potential crosstalk in their biosynthetic pathways. Metabolic profiling of 1-year-old C. acuminata seedlings revealed tissue-specific accumulation of O-methylated quercetin and camptothecin in the stem phloem. Notably, CaOMT3, one of four clustered O-methyltransferase genes, showed expression levels that positively correlated with rhamnetin accumulation across tissues. Transient transformation in Nicotiana benthamiana, coupled with heterologous expression in vitro, demonstrated that CaOMT1, CaOMT2, and CaOMT3 efficiently methylated 9-hydroxycamptothecin and 10-hydroxycamptothecin. Regarding their flavonoid-methylation abilities, CaOMT1 and CaOMT3 were identified as bifunctional enzymes that targeted the 4′-hydroxyl and 7-hydroxyl groups of quercetin, respectively, whereas CaOMT2 exhibited strict specificity for the 4′-hydroxyl group of quercetin. Unfortunately, CaOMT4 showed no methylation activity toward the tested substrates. The catalytic dyad and key substrate-binding sites of the CaOMTs were identified via molecular docking and subsequently verified by site-directed mutagenesis. F166, M313, and L316 were proposed to be critical for activity toward both quercetin and hydroxycamptothecin. Notably, N261, S309, and T312 were suggested as key amino acids responsible for the lack of 7- O-methylation activity in CaOMT2. Taken together, our findings revealed a bifunctional OMT gene cluster and provided molecular insights into the substrate-binding pockets.
N6-methyladenosine (m6A) is a widespread RNA modification in eukaryotes, yet its contribution to growth and metabolic regulation in perennial leguminous forage crops remains poorly understood. Here, we identify MsMTA, a nuclear-localized m6A methyltransferase in alfalfa (Medicago sativa), as a key regulator of the global m6A landscape, plant architecture, and forage nutritional composition. Suppression of MsMTA expression reduced overall m6A levels and caused a pronounced dwarf phenotype. At the metabolic level, MsMTA knockdown lines showed a strong increase in soluble sugar content, accompanied by only a modest reduction in crude protein and decreased fiber, reflecting a reprogramming of carbon–nitrogen allocation that enhances agronomic quality in alfalfa. Integrated m6A sequencing and transcriptome profiling showed that m6A sites are preferentially enriched in the 3′ UTR and that the expression of 549 genes is significantly altered; these genes are mainly involved in oxidation–reduction processes, small-molecule metabolic processes, and organonitrogen compound biosynthesis. Representative metabolic genes, such as GS2 and SDR1, exhibited reduced m6A modification and decreased mRNA stability, directly linking MsMTA-dependent methylation to transcript turnover. Together, these findings establish MsMTA as a central node connecting the m6A epitranscriptome with carbon–nitrogen metabolic homeostasis in alfalfa, and indicate that targeted manipulation of MsMTA-dependent m6A methylation offers a potential route to develop alfalfa cultivars that maintain high protein content while substantially elevating soluble sugars, thereby improving forage yield and quality in leguminous crops.
Tomato serves as a globally important vegetable crop and a genetic model organism, and the improvement of its agronomic traits represents a central objective in breeding. While graph-based pangenomes comprehensively capture species-wide genetic diversity, those built from large cohorts of individuals frequently present considerable computational challenges. To address this, we constructed a graph-based pangenome that integrated only the spectrum of genetic variants between wild (Solanum pimpinellifolium) and cultivated tomato (Solanum lycopersicum). By balancing reduced resource demands with reliable variant calling accuracy, this resource establishes a practical foundation for high-throughput genetic analysis. Using the graph-based pangenome and an F7 recombinant inbred line (RIL) population, genome-wide association studies (GWAS) pinpointed known loci for trichome, vegetative form, and weight traits, as well as a novel fruit-weight locus, fw6.4. The candidate gene SlILL6 within this region likely affects fruit weight (FW) by modulating cell expansion. Metabolite profiling revealed micro-scale quality variation and identified 128 differentially accumulated metabolites. Among these, a GDSL lipase-like caffeoyltransferase (SlCGT) was found to function as a negative regulator of chlorogenic acid content, thereby affecting fruit resistance to pathogenic fungi. Integrating a graph-based pangenome with multi-omics data, this research deciphered the genetic basis of complex traits and supplies new genomic resources, analytical tools, and candidate genes for tomato improvement.
Strawberry aroma is a key component of fruit quality, influencing consumer preferences and playing important ecological roles, including plant defense. However, the genetic basis of volatile organic compound (VOC) biosynthesis remains only partially understood, particularly in the wild woodland strawberry Fragaria vesca, which has enormous potential to uncover genetic diversity within the genus for improving the commercial strawberries. Here, we performed Genome-Wide Association Studies (GWAS) across a diverse European collection of F. vesca accessions. We identified multiple novel candidate genes involved in the biosynthesis of diverse volatile esters, lactones, terpenoids, and methyl ketones. Among them, we characterized FvJMT2, a SABATH family methyltransferase, which we found associated with natural variation in benzenoid ester content. Transient expression in Nicotiana benthamiana and strawberry fruit confirmed its role in methyl benzoate biosynthesis, while enzymatic assays demonstrated that FvJMT2 encodes a promiscuous enzyme capable of methylating not only benzoic acid, but also cinnamic, salicylic, and jasmonic acids. Behavioral assays revealed that methyl benzoate at physiologically relevant concentrations significantly reduced the attraction of Drosophila suzukii flies, supporting a dual role of this VOC in both flavor and pest deterrence. Finally, natural variation analyses in wild Fragaria species and F. × ananassa cultivars showed that benzenoid esters have been largely lost in modern cultivars but retained in ancient and wild accessions. Altogether, this study provides novel insights into the genetics and ecological relevance of strawberry volatiles and identifies candidate loci and alleles for future studies.
Vernicia montana is a dioecious woody tree with a ZW sex determination system, yet the molecular basis of sex determination remains poorly understood. Here, we constructed a chromosome-level, haplotype-resolved female genome assembly using PacBio HiFi sequencing and Hi-C scaffolding, and integrated population-scale coverage-based genome-wide association analysis of 178 natural individuals to identify sex-associated loci. A highly localized sex-associated signal was detected on ChrB02, defining a ~61.4-kb W-specific sex-determining region (SDR) that is sharply bounded by conserved syntenic blocks but internally enriched in haplotype-specific insertions, duplications, inversions, and a complex inverted-repeat architecture. Polymerase chain reaction assays further validated multiple female-specific segments within this structurally heterogeneous region. Two conserved genes were identified at the SDR boundaries. Notably, VmBASS4.2 displayed pronounced developmental dynamics and broad reproductive expression and is positioned adjacent to major structural rearrangements within the SDR, making it a compelling candidate for functional validation. Heterologous overexpression in Arabidopsis impaired stigma receptivity and increased floral and silique abortion, supporting a dosage-sensitive role in reproductive development. Collectively, our findings suggest that sex determination in V. montana is not driven by the emergence of a novel sex-determining gene but instead is associated with local inverted-repeat-mediated structural remodeling that reshapes the regulatory landscape of pre-existing boundary genes such as VmBASS4.2. This study proposes an inverted-repeat-mediated SDR evolution model, providing a plausible framework linking local structural architecture to regulatory divergence during the early evolution of homomorphic sex chromosomes in plants.
R2R3-MYB transcription factors are central regulators of anthocyanin biosynthesis, but their role in coordinating the control of chlorophyll metabolism remains largely unexplored. Here, we identified a novel R2R3-MYB transcriptional repressor from pear, named PbrMYBL4. This nuclear-localized protein contains a canonical EAR repression motif and is predominantly expressed in the fruit pericarp. Functional analyses demonstrated that PbrMYBL4 could simultaneously inhibit anthocyanin accumulation and chlorophyll degradation. Mechanistically, it represses anthocyanin biosynthesis by directly downregulating key structural genes (PbrF3H and PbrANS) and by binding to the promoter of the master activator PbrMYB10b to suppress its transcription. Furthermore, PbrMYBL4 interacts with PbrbHLH3 to form a repressive complex that competitively disrupts the formation of the activating PbrMYB10b–PbrbHLH3 complex , thereby amplifying the repression of anthocyanin pathway genes. Concurrently, PbrMYBL4 could inhibit chlorophyll degradation by directly repressing the chlorophyll degradation gene PbrSGRL . Overall, these findings suggest that PbrMYBL4 could regulate pigment metabolism via distinct regulator y modules, PbrMYBL4-PbrMYB10b and PbrMYBL4-PbrSGRL. Importantly, our findings reveal a dual-function transcriptional regulator that fine -tunes fruit coloration, providing a strategic target for molecular breeding to improve pear fruit quality.
Root meristem growth factors (RGFs) are pivotal regulators of root growth in plants and are critically involved in the regulation of root development in fruit trees. In this study, we constructed a comprehensive small secreted peptide library from 64 plant species. Using a structure-based screening strategy, we identified a total of 495 RGF genes. Phylogenetic analysis revealed that the modern RGF gene family originated in ferns and exhibited an evolutionary pattern of initial contraction and subsequent expansion, consistent with the evolutionary emergence and diversification of plant root systems. Analysis of the Malus pan-genome indicated that RGF genes within the genus were classified into 12 clades, with some clades present exclusively in wild Malus species. Notably, the repertoire and copy number of RGF peptides vary significantly among Malus species with different ploidy levels, suggesting a potential association between RGF gene expansion and polyploidization. Functional analysis of a gene encoding the canonical RGF mature peptide ‘DYTPARKKPPIHN’ in Malus demonstrated a dose-dependent effect on primary root elongation in apple seedlings following exogenous application. Subsequent experiments confirmed that overexpression of MdglRGF1 negatively regulates adventitious root growth during regeneration-associated and hormone-induced adventitious rooting, a phenotype sustained throughout adventitious root development in soil-grown seedlings. Collectively, our research provides novel insights into the identification and functional characterization of small peptides and establishes a foundation for their potential application in apple rootstock breeding and the development of ideal root system architecture.
Plants face the challenge of balancing dynamic environmental stresses with internal developmental demands during growth, necessitating the precise regulation of gene expression networks to optimize resource allocation. Recent studies have shown that epigenetic regulatory systems, which serve as bridges between genotype and phenotype, enable plants to respond rapidly to environmental changes and facilitate adaptive evolution by establishing heritable epigenetic memory. This article systematically elucidates the molecular mechanisms by which core epigenetic processes coordinate plant growth and development with environmental adaptation, with particular emphasis on the transgenerational transmission of epigenetic information, such as DNA modification, RNA modification, histone modification, histone chaperones, chromatin remodeling, and non-coding RNA regulation. It further examines the interrelationships between these mechanisms and how they work in concert to achieve such coordination. It also explores key unresolved scientific questions in this field and proposes future perspectives for further research.
Theanine, a non-protein amino acid predominantly found in tea (Camellia sinensis), is a primary contributor to the characteristic umami and sweet taste of tea infusion and is associated with numerous health benefits. The biosynthesis of its direct precursor, ethylamine, is catalyzed by the enzyme CsAlaDC. The evolutionary origin of this enzyme and the molecular basis for its functional divergence from the serine decarboxylase CsSDC, however, remain poorly understood. In this study, through comprehensive genome-wide identification, phylogenetic, structural, and domain analyses, we demonstrate that CsAlaDC originated from CsSDC via gene duplication followed by functional specialization. Phe106 and Gly168 were identified as indispensable residues governing CsAlaDC enzymatic activity in vitro and in vivo through functional validation. Crucially, we pinpointed two specific codon substitutions-TAC(Tyr 112) to TTT(Phe 106) and TGT(Cys 174 ) to GGT(Gly 168 )-as key evolutionary mutations responsible for the functional shift from SDC to AlaDC activity. These findings elucidate the evolutionary trajectory of CsAlaDC and provide mechanistic insights into the molecular regulation of theanine biosynthesis in tea plants.
The olive tree (Olea europaea L.) is a key source of verbascoside, a potent phenylethanoid glycoside known for its antioxidant, anti-inflammatory, and neuroprotective properties. Traditional breeding for enhanced phenolic profiles is limited by the olive’s long juvenile period and complex genetics. This study dissected the unknown genetic architecture of verbascoside biosynthesis using a multi-omics strategy integrating metabolomics, Quantitative Trait Loci mapping, and transcriptomics across an F1 population and diverse cultivars. This approach identified a major genetic locus controlling verbascoside content on chromosome 21 (LOD [Logarithm of the Odds] = 17), which explained the wide metabolomic variation. The locus includes 193 genes, of which 29 were involved in verbascoside synthesis and 47 showed differential expression. A homology-based analysis, using data from five high-verbascoside Lamiales species, narrowed the search to a small set of high-confidence candidate genes, including BAHD acyltransferases (BEAT, AHCT, HCBT, DAT acyltransferase family) and various cytochrome P450s (CYPs). Digital PCR confirmed the differential expression of nine candidate CYP genes. The expression of both OeCYP71D55 (a premnaspirodiene oxygenase) and the precursor-producing OeACy3G peaked at the T5 stage, 165 days after flowering. This peak mirrored the maximum time of verbascoside accumulation, suggesting that the transcriptional regulation of these CYPs is a rate-limiting factor in the pathway. This study defines a critical genetic locus and a core set of candidate genes governing verbascoside production in olive cultivars. These discoveries provide valuable molecular tools to fast-track the breeding of new olive varieties with enhanced health benefits.
Plant regeneration is widely exploited in genetic transformation, genome editing, tissue culture, cuttage, and grafting, which are crucial means to improve crop productivity and resilience. However, plant regeneration is heavily restricted by their low regeneration efficiency and genotype recalcitrance, especially in many economically important plant species. To address these challenges, new approaches have been developed to greatly improve regeneration efficiency and genotype plasticity. Among them, small molecules represent attractive options that offer an effective, inexpensive, and convenient strategy to enhance regeneration efficiency in a stepwise and controllable manner. Here, we summarize current advances in the characterization of small molecules with superior efficacy and their molecular mechanisms in plant regeneration, highlighting their advantages for fundamental studies and practical applications. Finally, we conclude with a critical discussion on the remaining questions and future challenges in pinpointing small molecules with regeneration-enhancing potential.
Apple (Malus × domestica) is one of the world’s premier fruit crops. Apple Glomerella leaf spot (GLS) is caused by Colletotrichum spp., jeopardizing global apple supply and incurring millions of dollars in annual losses. The development of resistant apple varieties has evolved from conventional selection breeding to deliberate breeding strategies that incorporate statistical analysis and experimental design, and now increasingly integrate genetic and genomic data. Innovative methods like marker-assisted selection and genetic editing have facilitated the development of cultivars resistant to GLS. Novel breeding techniques (NBTs), particularly genome editing, are increasingly vital for the precise and rapid development of disease-resistant crops. The CRISPR/Cas9 system is a powerful tool for targeted mutagenesis, enabling the development of genetically edited crops that are not classified as transgenic. Consequently, researchers are working to identify genes associated with susceptibility and resistance. The article explores the established mechanisms of interaction between the GLS pathogen and its host, as well as the innate immune responses elicited in the apple plant, to expedite the development of resistant apple varieties. Recent advances in effector biology and host gene targeting offer new avenues to enhance immunity and engineer durable resistance traits against GLS.
During the domestication of Brassica campestris (syn. Brassica rapa), significant variations in bolting time have emerged, critically impacting crop yield and quality. To elucidate the genetic basis of bolting time regulation in B. campestris, we integrated selective-sweep analysis on 365 resequenced accessions with genome-wide association studies. This identified genes including BcLMI1-LF (LATE MERISTEM IDENTITY 1), an HD-ZIP I subfamily gene in the least fractionated subgenome of B. campestris. Further, gene ontology enrichment analysis indicated that BcLMI1-LF was a key candidate regulator of bolting. Haplotype analysis demonstrated the association of Hap3 of BcLMI1-LF with the late bolting phenotype. Functional validation confirmed that BcLMI1-LF-Hap3 overexpression significantly delays bolting. Mechanistically, yeast one-hybrid (Y1H), Dual-Luciferase Reporter Assay (DLRA), and chromatin immunoprecipitation followed by quantitative polymerase chain reaction (ChIP-qPCR) assays established that BcLMI1-LF-Hap3 directly represses BcTCP4-LF (TEOSINTE BRANCHED 1/ CYCLOIDEA/ PCF 4), a conserved bolting regulator. This study advances understanding of bolting regulation in B. campestris and provides molecular targets for breeding late-bolting crops.
Cannabis sativa produces a rich array of specialized metabolites, including cannabinoids, terpenoids, and flavonoids, which are of immense therapeutic interest. However, the genetic and regulatory complexity arising from its highly heterozygous genome has obscured a complete understanding of their biosynthesis. Here, we present a fully phased, chromosome-level genome assembly of the wild diploid cannabis accession CSLZ. Comparative genomics and transcriptomics reveal that extensive haplotype-specific variation is a major driver of metabolic diversification within this accession. We find that structural variants and transposable element insertions differentially shape the allelic architecture of key biosynthetic genes, leading to divergent expression and function between haplotypes. This is exemplified by haplotype-specific loss of germacrene D synthase activity, copy number variation in oxidosqualene cyclases, and silencing of a flavonoid glycosyltransferase allele via promoter motif loss. Furthermore, allele-specific expression contributes to the spatiotemporal regulation of metabolic pathways, from cuticle formation in leaves to cannabinoid production in glandular trichomes. Our haplotype-resolved resource uncovers the pervasive role of allelic divergence in generating the specialized metabolome of wild cannabis accession CSLZ, providing a foundation for genomics-guided breeding and metabolic engineering.
Flesh thickness (FT) is the edible portion of melon fruit and directly affects its utilization rate, with an important influence on commerciality and yield. In this study, the phenotype of F2 populations derived from PI 282448 (thin-fleshed, ssp. melo, wild type) and M4-135 (thick-fleshed, ssp. melo, cultivar) showed that flesh thickness is a quantitative trait. Graded Pool-seq (GPS) combined with genetic linkage analysis identified a stable major-effect quantitative trait locus (Cmqft4.1) related to flesh thickness on chromosome 4. Genetic mapping narrowed down the Cmqft4.1 locus to a 222-kb interval using the near-isogene line NIL_BC3 F2 population, which contained 13 candidate genes. Through gene expression and sequence alignment, MELO3C012896.2 (annotated as Ethylene-responsive transcription factor 1B-like) was predicted as the key candidate gene for flesh thickness in melon. A 286-bp deletion in the promoter region of MELO3C012896.2 was present in multiple thick-fleshed melon natural materials. These results provide a theoretical basis for further gene functional verification and breeding programs focused on flesh thickness in melon.
Wild populations of North American cranberry (Vaccinium macrocarpon Aiton) are reservoirs of genetic variation that may contribute to the improvement of breeding-relevant traits. However, the extent to which wild genetic variation is geographically structured and represented in elite germplasm remains unclear. We analysed 179 wild cranberry accessions from the upper Midwest and Eastern North America to estimate nucleotide diversity (π), population structure, and loci associated with genetic differentiation and environmental variables using a genome-informed targeted genotyping panel. Additionally, 14 demographic scenarios were evaluated using site-frequency-spectrum-based inference to identify historical events that could explain current genetic diversity. We observed extremely low nucleotide diversity within the targeted panel (π = 5 × 10−6). Rare allele distributions strongly influenced π and Tajima’s D values, suggesting constrained diversity in the genomic regions assayed that is not captured by heterozygosity-based estimates alone. However, we interpreted these results as conservative lower bounds on genome-wide neutral diversity because the targeted panel is enriched for genic and conserved regions. A clear separation between the Midwest and East populations was observed, with inbreeding coefficients ranging from −0.13 to 0.15. Furthermore, site frequency spectrum inference from the targeted panel supported a demographic scenario consistent with a significant population reduction ≈15–14 thousand years ago (kya), followed by a divergence between the two regions ≈12 kya, and an asymmetric gene flow ≈1.3 kya. We detected 254 candidate loci showing regional allele-frequency differentiation. Several of these loci colocalized with candidate genes linked to stress response, development, and metabolic processes. To evaluate the representation of geographically differentiated wild alleles in a breeding context, we analysed Rutgers breeding materials ( n = 484) and found that this panel is enriched for common alleles in Eastern wild populations. These findings indicate regionally structured allele-frequency variation in wild cranberry, with potential relevance to environmental response and breeding. This study extends prior wild cranberry population-genetic research by providing targeted-panel estimates of diversity, comparisons of demographic models, and breeding insights on geographically differentiated alleles, while highlighting the importance of conserving wild cranberry germplasm for use in modern breeding programs.
Rhizosphere microbiome critically influences plant growth and health, yet the genetic mechanisms underlying host regulation of microbiome composition remain unclear. Here, we analyzed whole-genome genotype and 16S rhizosphere microbiome data from 432 globally sourced eggplant accessions (Solanum melongena L.). Eggplant population structure corresponded to geographic origins, with rhizosphere microbiomes varying significantly among subpopulations. Host genetics explains 9%–39% of the variation in individual microbial taxa abundance, with core taxa more affected by host genetic variation. Microbial genome-wide association studies (mGWAS) identified 1235 significant genetic variants associated with 46 core microbial taxa, revealing key regulatory loci including chr10:7799021 near MYB113 (associated with Stenotrophomonas, P = 2.16 × 10−15) and chr10:19786889 near BLH9 (associated with Mycobacterium, P = 1.24 × 10−12), as well as a chromosome 5 locus with specific regulatory effects on Rhizobiales. These microbiome-associated genetic variants were enriched in secondary metabolic pathways, including anthocyanin biosynthesis, benzoxazinoid biosynthesis, and brassinosteroid biosynthesis, indicating that hosts regulate microbial communities through complex metabolic networks. Notably, genetic loci controlling microbial community structure underwent strong directional selection across eggplant subpopulations from different geographic origins, providing evidence for host–microbe coadaptive evolution. This study elucidates genetic regulatory patterns of eggplant rhizosphere microbiomes, enriching the theoretical framework of plant–microbe coevolution, with broad implications for microbiome-assisted crop improvement and sustainable agriculture.
Passion fruit (Passiflora edulis) production is significantly limited by sporadic cold stress events in unfavorable years. The existence of cultivars with varying cold tolerance offers opportunities for genetic improvement of this high-value crop. To elucidate the molecular basis of cold adaption in passion fruit, we conducted an integrated transcriptomic and metabolomic analysis comparing cold-tolerant and cold-sensitive passion fruit cultivars under low-temperature stress. Our multi-omics approach revealed that cold tolerance is associated with significant reprogramming of key pathways, particularly sphingolipid metabolism, phenylpropanoid metabolism, and terpene metabolism. We further validated that exogenous application of the key metabolites within these pathways, sphinganine, chlorogenic acid (CGA), rutin, and three terpenoids enhanced cold tolerance in cold-sensitive cultivars. Conversely, virus-induced gene silencing (VIGS) of core biosynthetic genes (e.g. PesSPT, PesKDSR, PesHCT, and PesFG2) in these pathways compromised cold resistance, leading to increased oxidative damage and more severe phenotypic injury. This study provides comprehensive insights into the molecular networks underlying cold tolerance in passion fruit, highlighting potential candidate genes and metabolites. Our findings establish a crucial foundation for future molecular breeding strategies aimed at improving cold resistance in this profitable horticultural crop.
Elucidating the genomic underpinnings of adaptive variation is highly important for the conservation, landscape application, and management of ornamental trees against the backdrop of global climate change. However, research on the genetic mechanisms underlying climate adaptation in Phoebe bournei-a near-threatened subtropical tree species endemic to China, which is endowed with exceptionally high ornamental and ecological value-remains scarce. Whole-genome resequencing was conducted on 362 individuals from 27 natural populations across the geographical range of the species. Genome-environment association analyses were employed to identify 1556 climate-associated variants and 167 candidate genes associated with temperature and precipitation variables. Through functional annotation and expression profiling, pivotal genes, including TRX-M4 and FBD1, were identified as integral to drought and heat stress responses, with adaptive alleles displaying distinct geographic frequency distributions and significant phenotypic differentiation. Divergent evolutionary trajectories were deduced among populations, with southeastern populations distinguished by elevated genetic diversity and strong signatures of local adaptation. Nevertheless, projections derived from the Risk of Non-Adaptedness and gradient forest models suggest that these southeastern populations will face substantial genomic offset under future climate scenarios, signaling heightened vulnerability and the need for prioritized conservation and management. This study provides the first genome-wide perspective into the adaptive evolution of P. bournei and offers a robust foundation for its conservation and climate-resilient management.
Pea (Pisum sativum L.) is one of the most important edible legumes in China, with both planting area and total yield ranking among the highest in the world. Fusarium wilt, caused by Fusarium oxysporum f. sp. pisi (Fop), is a severe factor limiting pea production. The deployment of resistant pea cultivars is the most effective and sustainable strategy for controlling this disease. In the present study, a novel resistance gene PsFwC9 , conferring resistance to Fop race 5, was identified in the resistant pure line Chengwan 9-8 (CW9-8), and its candidate gene Psat4g213640 was characterized and functionally validated to be associated with disease resistance. Genetic analysis of the F 2population derived from the cross between the resistant parent CW9-8 and the susceptible parent Chengwan 9-1 (CW9-1) revealed that PsFwC9 was controlled by a single dominant gene. Based on whole-genome resequencing, bulked segregant analysis sequencing (BSA-seq) and fine mapping, PsFwC9 was localized to an 817.06-kb region on chromosome 4 (i.e. linkage group IV, chr4LG4), flanked by KASP markers A016508 and A016511, and co-segregated with four markers. Haplotype analysis revealed that only the marker A016615 was significantly associated with Fusarium wilt resistance, and this marker was designated as a diagnostic marker for PsFwC9 . Marker A016615 was located at 425 699 725 bp on chr4LG4, corresponding to the 277 bp within Psat4g213640 , where a ‘A/G’ single-nucleotide polymorphism caused an amino acid substitution leading to an alteration in protein structure; therefore, Psat4g213640 was identified as the PsFwC9 candidate gene. Quantitative real-time PCR analysis showed no significant difference in the expression levels of Psat4g213640 between CW9-8 and CW9-1. Overexpression of the candidate gene Psat4g213640 CW9-8 in the hairy root system significantly enhanced the resistance of CW9-1 to Fusarium wilt, whereas RNA interference-mediated silencing of Psat4g213640 CW9-8 reduced the resistance of CW9-8, indicating that Psat4g213640 CW9-8 played a crucial role in pea resistance to Fusarium wilt. In addition, subcellular localization showed that the protein encoded by Psat4g213640 was targeted to the endoplasmic reticulum. Collectively, these findings not only enriched the gene resources for disease resistance in pea and provided an important foundation for elucidating the molecular mechanism of PsFwC9 -mediated resistance, but also provided important technical support for the practical application of molecular breeding for disease resistance in pea.
The flowers of Osmanthus fragrans are widely appreciated for their distinctive fragrance, predominantly derived from monoterpenes and apocarotenoid volatiles. However, the transcriptional mechanisms coordinating the production of these scent compounds remain poorly understood. In this study, integrated transcriptomic and metabolomic analyses of five cultivars with varying scent profiles identified OfWRKY48, a Group II-c WRKY TF. Functional studies demonstrated that transient overexpression and virus-induced gene silencing of OfWRKY48 significantly altered the accumulation of key scent terpenoids, including linalool, its oxides, and the apocarotenoids β-ionone, α-ionone, and dihydro- β-ionone. Mechanistic investigations revealed that OfWRKY48 activated key genes in the methylerythritol phosphate (MEP) pathway, including OfDXS, OfCMK, OfTPS1, and OfTPS2, thereby promoting linalool synthesis. Furthermore, OfWRKY48 upregulated upstream carotenogenic genes (OfPSY, OfPDS2, and OfLCYb2) and, in parallel, enhanced the expression of the carotenoid cleavage gene OfCCD4, collectively boosting the production of ionone derivatives. DNA-binding assays, including yeast one-hybrid (Y1H), luciferase reporter, and electrophoretic mobility shift assays, confirmed that OfWRKY48 bound to the promoters of OfTPS1, OfCMK, and OfLCYb2. These findings establish OfWRKY48 as a key regulator that orchestrates the biosynthesis of both monoterpene and apocarotenoid volatiles. Our work reveals a coordinated regulatory mechanism that synchronizes precursor supply in the MEP pathway with branching points in the carotenoid pathway, providing new insights into the complexity of floral fragrance regulation and suggesting a promising target for molecular breeding of aroma traits in horticultural crops.
Root elongation is a key adaptive trait that enables plants to respond to soil conditions and is jointly regulated by auxin signaling and very long-chain fatty acids (VLCFAs). However, the molecular mechanism underlying this pathway remains unclear in apple. In this study, we elucidate the roles and regulatory relationship between auxin and VLCFA biosynthesis in apple. VLCFA accumulation increases auxin levels in roots, reduces the expression of IAA-related genes, and promotes root morphogenesis. This interaction refines the conventional view of auxin as the sole regulator of root development and reveals a synergistic auxin–VLCFA regulatory network that integrates hormonal signaling with metabolic control.
Horticultural plants synthesize diverse flavonoids (e.g. anthocyanins, proanthocyanidins, and flavonols) that shape key agronomic traits, including flower pigmentation, fruit flavor, stress resilience, and nutritional value. MYB transcription factors (TFs), particularly R2R3-MYBs, serve as core regulators of flavonoid biosynthesis; their functional divergence and distinct regulatory mechanisms drive the interspecific diversity of flavonoid-related traits. This review systematically summarizes the evolutionary and functional divergence of flavonoid-regulatory R2R3-MYBs across monocot (including Poaceae and non-Poaceae) and eudicot horticultural plants. Phylogenetic analysis, integrating functionally validated R2R3-MYB sequences and Arabidopsis references, classifies the R2R3-MYB family into 37 subgroups. Flavonoid-regulatory members primarily cluster within key dedicated subgroups, which are first categorized into six functional groups and subsequently subdivided into 11 distinct subclades based on their monophyletic distribution in angiosperms across 44 horticultural species. Distinct lineage-specific evolutionary patterns emerge: some monocots (notably Poaceae) putatively lack Subgroup 6 (SG6) and rely on SG5 for anthocyanin biosynthesis, while eudicots expand SG6 via tandem duplications. Rosaceae exhibit adaptive expansion of SG5 associated with fruit trait evolution. We further discuss cross-species divergence within conserved flavonoid-regulatory subgroups, including variations in spatiotemporal expression patterns and dependency on the MYB–bHLH–WD40 (MBW) complex. Case studies of representative species (Arabidopsis, rice, strawberry, and freesia) demonstrate that paralogs derived from gene duplication achieve precise regulation of distinct flavonoid branch pathways through subfunctionalization or neofunctionalization. Ecological pressures, domestication, and structural determinants collectively drive MYB divergence in copy number and function. Finally, we outline the applications of these findings in crop improvement and propose future research directions.
Phoebe species are renowned for their highly valuable ‘golden thread’ timber; however, their protracted juvenile phase presents a significant obstacle to mechanistic investigations of floral induction. Phoebe chekiangensis, a rare early-flowering representative within this genus, provides a unique model system for dissecting the vegetative-to-reproductive phase transition. Nevertheless, the absence of a high-quality reference genome has severely hindered molecular insights into its developmental regulation. Here, we present the first telomere-to-telomere (T2T) genome assembly for P. chekiangensis, comprising two completely gap-free haplotypes with contig N50 values exceeding 65 Mb, base-level quality scores >36, and Long Terminal Repeat Assembly Index scores surpassing the gold standard threshold of 20. Approximately 29 000 genes were annotated per haplotype, supported by a BUSCO completeness score of >97%. Age-resolved transcriptomic landscapes identified two MADS-box transcription factors, PcMADS5 (AP1-like) and PcMADS19.1 (SOC1-like), as core activators of the floral transition. Both genes triggered precocious flowering when ectopically expressed in Arabidopsis thaliana. Whole-genome bisulfite sequencing revealed a progressive, age-dependent decline in CHG (where H is A, C, or T) DNA methylation, which was particularly pronounced at the PcMADS19.1 locus. Notably, DML1/2, which mediate active DNA demethylation, were coordinately upregulated during the onset of reproductive growth. Chemical demethylation using 5-azacytidine further diminished CHG methylation and selectively enhanced PcMADS19.1 expression, confirming a causal relationship between CHG hypomethylation and transcriptional activation. This work delivers the first chromosome-scale T2T genome within the genus Phoebe and uncovers CHG demethylation as a previously unrecognized epigenetic switch governing reproductive competence in woody perennials.
Tomatoes are highly nutritious and represent one of the important vegetable fruits worldwide. Both historically and moving forward, genetic decoding and precision breeding remain fundamental to tomato improvement. Here, we summarize pivotal advances in decoding tomato genomes across domestication, improvement and evolution processes and provide a perspective on future breeding through precision design. In-depth population genetic studies have revealed how artificial selection systematically prioritized yield-related alleles at the cost of narrowing genetic diversity, especially at flavor-related loci-highlighting the urgent need to reconcile these trade-offs. Comparative genomics across species, viewed through an evolutionary lens, has uncovered critical insights into functional genes, deepening our understanding of the genetic architecture and regulatory mechanisms underlying key traits. Collectively, these advances have enabled precise identification and functional characterization of key genetic elements, paving the way for systematic redomestication of tomato through precision genomic design. Looking ahead, more efficient and precise breeding strategies will be required to accelerate genetic gains in tomato in the coming decades. The integration of recent genomic advances, coupled with genomic selection and artificial intelligence, into genomic design breeding offers a transformative framework, unlocking unprecedented opportunities for developing highly flavorful and consumer-customized tomato varieties.
Ca2+-ATPase (CAP) is a key Ca2+ efflux protein in plants. Our previous research suggests that CAPs may play a crucial role in the adaptation of rhododendrons to high calcium environments. However, the evolution, variation, characteristic expression, and subfunctionalization of this gene family in Rhododendron remain unknown. Through the analysis of pan-genomes and pan-transcriptomes, we elucidated the systematic evolution of CAPs in plants, as well as their characteristic expression patterns in Rhododendron. During the evolutionary process from lower to higher plants, CAPs can be divided into six clades and exhibit structural conservation. CAPs have emerged and differentiated in lower plants such as algae, and they have undergone significant amplification in Eudicots plants like rhododendrons. Three Rhododendron species (Rhododendron bailiense, R. delavayi, and R. irroratum) located in the karst province of Guizhou in Southwest China exhibit the highest copies of CAPs, suggesting a strong association between CAP copy number variation and habitat, particularly in high calcium environments. Through multiple transcriptome analyses, we revealed that CAPs are induced under various environmental/developmental conditions (e.g. karst environments, high altitude, early flower development, hormones, etc.). Co-expression network analysis highlighted key members of calcineurin B-like protein (CBL) and CBL-interacting protein kinases (CIPK) that are associated with the high expression of CAPs. Experimental validation demonstrated that CAPb1 and CAPd1 significantly alleviate high calcium stress, and the CAPb1-CIPK1-CBL1 and CAPd1-CIPK2-CBL1 modules can further enhance the alleviation. These findings provide new insights into the evolution, characteristic expression, and function of CAPs, as well as new perspectives on the high calcium adaptability of rhododendrons.
The hardened endocarp/stone is a defining feature of drupe fruits and is essential for seed protection, yet it poses significant challenges for fruit processing. Stone development is not an isolated process; rather, it is closely coordinated with seed and fruit growth through complex hormonal and genetic signaling networks. This review synthesizes advances in elucidating how these networks guide the developmental trajectory from early cellular specification in the ovary to lignification and programmed cell death (PCD). Following fertilization, a conserved transcriptional cascade is initiated in which embryo-derived signals activate MADS-box and other regulatory factors to determine endocarp fate. Subsequent upregulation of additional transcription factors/genes, such as NAC, MYB, PODs, LACs, and others, drives cell division and specialization, ultimately leading to activation of the phenylpropanoid pathway and the biosynthesis and deposition of lignin in endocarp cells, which is central to stone hardening. As fruit ripens, the lignified tissue undergoes PCD, forming a rigid protective structure. This developmental process is highly coordinated, and disruptions-whether genetic, hormonal, or environmental-can produce phenotypes such as split-pit, incomplete lignification, or altered stone thickness. Despite research progress, the spatial and temporal regulation of genes and their associated biochemical pathways during development remains poorly understood. The mechanisms by which metabolic and hormonal fluxes in the endocarp are directed from embryonic tissues to stony precursor cells, and how these processes contribute to differential lignification between endocarp and mesocarp tissues, require further investigation. This can be achieved through the integration of pangenomics, single-cell spatial transcriptomics, and precision gene editing.
Artemisinin, a sesquiterpene lactone produced in Artemisia annua , is the most effective antimalarial compound, but its low natural abundance in A. annua remains a major bottleneck for large-scale production. Increasing its biosynthesis requires a better understanding of the regulatory networks controlling artemisinin biosynthesis genes. Several jasmonate (JA)-responsive transcription factors have been characterized, but the role of upstream kinases remains poorly understood. Here, we identify AaSnRK2.6, a SnRK2III-type protein kinase, as a novel positive regulator of artemisinin biosynthesis. AaSnRK2.6 expression was strongly induced by methyl jasmonate and abscisic acid, and the protein is localized to both the cytoplasm and nucleus. Protein interaction assays revealed that AaSnRK2.6 specifically interacts with the JA-responsive TF AaORA in the nucleus, and an in vitro kinase assay further showed that AaSnRK2.6 directly phosphorylates AaORA. Dual-luciferase assays showed that AaSnRK2.6 enhances AaORA-mediated activation of key artemisinin biosynthetic genes (amorpha-4,11-diene synthase, DBR2 , and ALDH1). Overexpression of AaSnRK2.6 alone or AaORA alone significantly increased artemisinin and dihydroartemisinic acid accumulation, while co-overexpression of AaSnRK2.6 and AaORA in transgenic plants produced the stronger effect. By contrast, RNA interference knockdown of AaSnRK2.6 showed no significant effect on artemisinin levels, likely due to functional redundancy of kinases. Collectively, this study supports a positive kinase-TF regulatory module involved in hormone-responsive artemisinin biosynthesis in A. annua and provides new insights into its regulatory mechanism.
The albino leaves of cold-sensitive tea plants exhibited a marked reduction in chlorophyll content. However, the underlying regulatory mechanism remains elusive. In this study, we determined lower chlorophyll levels in tender leaves of cold-sensitive ‘Baiye 1’ compared to green leaves during the spring. Comparative transcriptome analyses identified the chlorophyll biosynthesis gene CsPORA, which was significantly downregulated in albino leaves. Functional assays confirmed that CsPORA positively regulates chlorophyll content. Electrophoretic mobility shift assay, yeast one-hybrid, dual-luciferase, and GUS staining results demonstrated that CsNAC87 binds to the promoter of CsPORA and suppresses its expression. Additionally, CsZAT12 physically interacts with CsNAC87 to enhance the repression. Overexpression of either CsZAT12 or CsNAC87 negatively regulated chlorophyll accumulation in tea plants and tobacco leaves, while co-transformation of CsZAT12 and CsNAC87 intensified the albino phenotype in tobacco leaves. Low temperature treatment of ‘Baiye 1’ triggered the marked upregulation of CsNAC87 and CsZAT12, leading to dramatical downregulation of CsPORA and leaf albinism. Collectively, our study reveals a novel cold-responsive CsZAT12-CsNAC87 module that negatively regulates chlorophyll synthesis by repressing CsPORA, providing new insights into chlorophyll metabolism in albino leaves of cold-sensitive tea cultivars.