Abstract Soil salinization represents a significant constraint on global agricultural development. Although several NAC gene families have been implicated in the response to salt stress, the mechanisms underlying salt stress tolerance remain unclear, particularly in perennial fruit trees. In this study, we identify MdJUB1, a NAC transcription factor, as a key regulator of salt stress response in apple. Under salt stress, MdJUB1 increases γ-aminobutyric acid (GABA) content, thereby positively regulating salt stress tolerance in apple. Further analysis using yeast one-hybrid, luciferase assays, and electrophoretic mobility shift assays demonstrated that MdJUB1 directly binds to the promoter of MdGAD1, promoting GABA synthesis. Additionally, under salt stress, overexpression of MdJUB1 increased the Na+/K+ ratio in apple. MdJUB1 also binds to the promoters of MdNHX1, MdHKT1, and MdSOS3, activating their expression. Using yeast two-hybrid screening, we identified MdDOF2.4 as an interactor of MdJUB1 and found that MdDOF2.4 positively regulates salt stress tolerance. Importantly, the interaction between MdDOF2.4 and MdJUB1 enhances the expression of downstream genes, including MdGAD1, MdNHX1, MdHKT1, and MdSOS3. Our results demonstrate that MdJUB1 is involved in the regulation of GABA levels and the maintenance of Na+/K+ homeostasis, which may contribute to enhanced salt tolerance in apple.
Abstract Litsea cubeba, an economically important woody species in the Lauraceae, is widely cultivated for spice and essential oil production. Core agronomic traits, particularly fruit morphology and yield per plant, directly determine its commercial value. However, genetic improvement of complex traits in this perennial species is hindered by intrinsic biological constraints, including a prolonged juvenile phase and an extended generation interval. Moreover, the genetic architecture and regulatory mechanisms underlying key agronomic traits remain poorly resolved. Conventional single nucleotide polymorphism (SNP)-based approaches, which depend on a single reference genome, often fail to capture large structural variants and non-reference sequences, thereby limiting the predictive performance of genomic selection (GS). To address these limitations, we performed SNP- and K-mer-based genome-wide association analyses to dissect the genetic basis of coordinated fruit morphological development and biomass accumulation. The results indicated that the K-mer strategy not only recapitulated most SNP-associated signals but also uniquely captured a novel locus associated with the fruit shape index. Additionally, we implemented a reference-free K-mer-based genomic prediction framework to overcome reference bias and incorporate additional genetic variation. Compared with SNP-based baseline models, the K-mer strategy improved prediction accuracy for key agronomic traits by 4.48%–7.71%. Collectively, this study elucidates the polygenic architecture and pleiotropic regulatory networks governing core agronomic traits in L. cubeba and demonstrates that reference-free K-mer-based strategies can enhance genomic prediction performance. These findings provide a conceptual and methodological framework for GS-assisted molecular breeding in highly heterozygous woody species.
Abstract Notoginsenosides are major bioactive ingredients that directly influence the quality of Panax notoginseng herbs. However, the current understanding of the interplay between environmental signaling and notoginsenoside biosynthesis is limited. Although jasmonates (JAs) are known to regulate the biosynthesis and accumulation of notoginsenosides, the associated signaling pathway remains largely unknown. Here, we identify PnMYC2, a central regulator of JA signaling, as a key node bridging JA signaling to notoginsenoside biosynthesis. Methyl jasmonate (MeJA) treatment markedly induced PnMYC2 expression, accompanied by enhanced notoginsenoside biosynthesis. Further assays revealed that PnMYC2 directly bound the promoters of the PnACAT, PnGGPS, and PnSE, which are involved in notoginsenoside biosynthesis, and activated their expression. Additionally, PnMYC2 also activated the expression of PnLOX5, a JA biosynthetic 13-lipoxygenase gene. Overexpression and interference of PnMYC2 in P. notoginseng calli and leaves indicated that PnMYC2 promoted notoginsenoside and JA biosynthesis by synergistically activating the expression of genes involved in both pathways. Furthermore, MeJA treatment reduced the protein abundance of PnJAZ2 and PnJAZ5, which serve as repressors of JA signaling and interact with PnMYC2 to repress its transactivation activity. Together, our results demonstrate that JAs promote notoginsenoside biosynthesis in P. notoginseng by enhancing the transcription and transactivation activity of PnMYC2. Our findings provide mechanistic insight into the transcriptional mechanism underlying JAs-mediated notoginsenoside biosynthesis.
Abstract Soil salinity triggers excessive reactive oxygen species (ROS) accumulation and oxidative damage in apple (Malus domestica), severely reducing fruit yield and quality. While melatonin confers salt tolerance by enhancing ROS scavenging, hydrogen sulfide (H2S), a vital gasotransmitter in plants, also plays a crucial role in stress responses, including salt stress. However, how H2S regulates melatonin-mediated salt tolerance remains largely unknown. Here we show that MdDES1, an L-cysteine desulfhydrase, positively regulates salt tolerance by increasing endogenous H₂S production. Biochemical and mutagenesis assays reveal that H2S directly modifies MdASMT10-the rate-limiting enzyme in melatonin biosynthesis-at Cys 12, Cys 55 and Cys158 via persulfidation, thereby improving its enzymatic activity and protein stability. Transgenic analysis confirms that persulfidation-deficient MdASMT10 no longer enhances salt tolerance. And exogenous NaHS most effectively improved salt tolerance in MdASMT10-overexpressing apple lines, while its effects on persulfidation-deficient MdASMT10 and wild type (WT) plants were comparable and weaker than those on MdASMT10-overexpressing lines. H2S thus promotes melatonin biosynthesis by persulfidating MdASMT10, which in turn strengthens ROS scavenging and alleviates salt stress. Collectively, our study elucidates the MdDES1-H2S-MdASMT10 regulatory cascade in apple salt tolerance, providing a promising molecular target for breeding salt-tolerant apple cultivars.
Abstract Fruit ripening and softening is primarily driven by fluctuations in hormone levels, which induce changes in cell turgor, adhesion, and cell wall remodeling. Although extensive research has examined the role of ethylene in promoting pear fruit ripening and softening, the molecular mechanisms underlying ethylene biosynthesis in pears (Pyrus L.) remain insufficiently understood, and the regulatory interactions between ethylene signal transduction and ethylene biosynthesis have yet to be fully elucidated. In this study, we characterize an ethylene-insensitive 3 (EIN3)/EIN3-Like transcription factor, PbrEIL1, which localizes to the nucleus and functions as a positive regulator of fruit ripening and softening by directly modulating ethylene biosynthesis. Transient transformation assays showed that overexpression of PbrEIL1 accelerated fruit ripening and softening in pear, whereas its silencing delayed these processes. Furthermore, PbrEIL1-overexpressing lines exhibited a significantly shorter ripening period and enhanced carotenoid accumulation compared with the wild type in the model plant tomato. The results of regulatory network analysis indicate that PbrEIL1 acts as a transcriptional activator that directly binds to the promoters of the ethylene biosynthesis genes PbrACS1 and PbrACS2, thereby activating their transcription. Moreover, overexpression of PbrACS1 and PbrACS2 also promoted ethylene production and accelerated pear fruit ripening and softening. In conclusion, the PbrEIL1–PbrACS1/2 module positively regulates pear fruit ripening and softening by controlling ethylene production, providing new insights into the regulatory mechanisms underlying these processes.
Abstract Trichomes are multicellular, non-glandular structures that play important roles in plant development, and provide protection against harmful ultraviolet radiation, insect damage and excessive water loss through transpiration. However, the regulation of trichome formation in cucumber remains incompletely understood. Although the phenotype of gl2 have been identified, no fine mapping and candidate gene for it has been determined so far. In this study, CsGL2 was mapped to a 72.5-kb region on chromosome 2, and via fine-mapping, an ethylene-responsive transcription factor ESR1, was identified as the candidate gene. We generated a CRISPR/Cas9 knockout mutant CsGL2 CRwhich was phenotypically resembled the natural gl2 mutant, demonstrating that CsESR1 is CsGL2. We further investigated whether CsGL2 interacts with CsGL1 and CsGL3, two known regulators of trichome development. Our results showed that CsGL2 directly binds to the promoter of CsGL1 and regulates its expression. Moreover, CsGL1 directly interacts with the CsGL3 protein. Genetic analysis demonstrated that CsGL3 is epistatic to CsGL1 and CsGL2, respectively. In conclusion, this study identified CsGL2 as a novel regulator of trichome development in cucumber, which is the first report of an ethylene-responsive transcription factor involved in cucumber trichome development. Furthermore, we elucidate the functional relationships among CsGL2, CsGL1 and CsGL3, providing new insights into the molecular network governing trichome development in cucumber.
Abstract Sweet cherry (Prunus avium) fruit is highly susceptible to postharvest softening, resulting in significant economic losses. This process is driven by the degradation of cell wall components, yet its transcriptional regulation remains unclear. Here, we explored the differential degradation of fruit cell walls in two sweet cherry cultivars with varying levels of fruit firmness. We identified the APETALA2/Ethylene Response Factor (AP2/ERF) family gene PavDREB2A, which is uniquely expressed during the ripening of the low-firmness cultivar. Phenotypic and transcriptomic analyses confirm its role in reducing cell wall integrity and fruit firmness. Through integrated transcriptomics, DAP-seq, and AlphaFold3 screening, we identified four cell wall-modifying genes (PG2, MAN7, EXP1, EXP4) as potential target genes of PavDREB2A. PavDREB2A directly activates these genes by binding to the DRE cis-element in their promoter regions, as shown by dual-luciferase, yeast one-hybrid, and EMSA assays. AlphaFold3 structural predictions indicate that six arginines in the AP2/ERF domain of PavDREB2A are essential for its binding and transcriptional activity. Our findings establish a PavDREB2A-mediated transcriptional pathway that promotes cell wall disassembly during ripening. This study provides both mechanistic insight into fruit texture control and potential molecular targets for breeding sweet cherry cultivars with enhanced firmness and shelf-life.
Abstract The tea plant (Camellia sinensis) possesses an array of specialized metabolites that contribute to its distinctive flavor and health-promoting properties. How they are regulated by long non-coding RNAs (lncRNAs) is not well elucidated. Here we systematically identify 65 675 high-confidence lncRNAs using the RNA sequencing dataset from the second-leaf samples of 133 accessions. Integrative co-expression analysis revealed 55 lncRNA-centered regulatory modules significantly associated with the accumulation of 57 specialized metabolites. We further show that a Gypsy-retrotransposon-derived lncRNA, CsLNC703, promotes CsMYB111-mediated regulation of the F3′H-associated flavan-3-ol biosynthetic branch, resulting in preferential accumulation of catechin (C), epicatechin (EC), and epicatechin gallate (ECG). These findings demonstrate that lncRNAs constitute an important regulatory layer controlling specialized metabolism in tea plants and reveal a transposon-derived lncRNA that modulates catechin biosynthesis. Our study provides new insights into the regulatory mechanisms underlying metabolic diversity in tea and highlights lncRNAs as potential targets for metabolic engineering and breeding of tea cultivars with improved quality traits.
Abstract Bacterial stem and root rot (BSRR), triggered by Dickeya dadantii, severely reduces sweetpotato productivity, yet the cellular mechanisms underlying root responses remain poorly understood. Here, we generated a single-cell transcriptomic landscape of 40 345 cells from infected and control sweetpotato root tips to uncover cell-type-specific defense patterns. Using canonical marker genes, we delineated seven major cell types and 16 transcriptionally distinct clusters. Cortex cells were identified as the principal responders to infection, characterized by extensive transcriptional reprogramming and enriched defense-related trajectories. Among the differentially expressed genes, IbWRKY40 emerged as a core regulator within the co-expression network of root immune responses. IbWRKY40 predominantly localized to the nucleus and exhibited strong induction approximately 9 h after pathogen exposure. Functional validation in sweet potato further suggested that IbWRKY40 negatively modulates resistance to D. dadantii. To our knowledge, this is the first report elucidating the cellular-level molecular mechanisms of sweet potato root tips in response to BSRR infection, providing valuable insights into plant defense strategies under pathogen stress.
Abstract Structural variations (SVs) represent an important source of genomic diversity and can contribute substantially to phenotypic variation in crops. However, the population-scale distribution and phenotypic effects of SVs in Theobroma cacao L. (cocoa) remain poorly understood. Here, we constructed a population-scale cocoa SV atlas using whole-genome resequencing data from 165 cocoa accessions representing ten previously defined genetic groups. Using a unified short-read-based SV detection strategy, we identified 11 271 high-confidence SVs, including deletions, duplications, and inversions. Using a framework based on term frequency-inverse document frequency (TF-IDF) algorithm, we identified 1078 fingerprint SVs for ten cocoa genetic groups of which 145 showed significant SV–trait associations. To further investigate integrated phenotypic divergence associated with functional SVs, we developed an analysis framework based on latent Dirichlet allocation (LDA). This analysis identified four latent phenotypic features showing significant divergence among cocoa genetic groups. Geographic populations from South America displayed extensive admixture of genetic groups, whereas populations outside South America showed reduced genetic diversity consistent with historical dispersal bottlenecks. Our study provides a population-scale SV resource for cocoa and demonstrates that SVs contribute to genetic differentiation, phenotypic divergence, and geographic adaptation in cocoa populations.
Abstract Among the traits that define the appeal of rose flowers, fragrance, which results from complex blends of volatile organic compounds (VOCs) holds a central cultural, sensory and economic role. These VOCs mainly include terpenoids, phenylpropanoid/benzenoid derivatives, phenolic methyl ethers and norisoprenoids, with strong variation among species and cultivars. In this review, we provide a broad, near-comprehensive and chronological synthesis of research on biosynthesis, regulation and emission of rose VOCs, with particular attention to the experimental approaches that have shaped the field, from classical biochemical and molecular studies to recent advances enabled by metabolic analyses, genomic resources and functional genomics. These developments have recently enabled to decipher major pathways leading to rose VOCs and highlighted the contribution of gene duplication, enzyme diversification and natural variation to scent diversity. Recent findings have been especially important for terpenoids. These includes the discovery of original cytosolic terpene biosynthetic pathways involving both terpene synthase-dependent and terpene synthase-independent routes, the cytosolic origin of the geraniol precursor GPP, the enzymatic conversion of geraniol to β-citronellol and the characterization of numerous terpene synthases involved in the production of major terpenes produced by rose flowers. We also discuss advances in the regulation of VOC biosynthesis, together with emerging questions regarding tissue specialization, intracellular trafficking, transport and emission of scent compounds. By integrating historical discoveries with recent findings, this review outlines how rose scent biology has been reshaped and discuss remaining challenges for understanding, breeding and engineering desirable floral fragrances.
Abstract Drought stress severely restricts the growth and yield of alfalfa (Medicago sativa L.), and ABA plays a vital role in plant drought tolerance. Here, we report that a transcription factor MsSPL12, induced by both drought and ABA treatment, mediates drought tolerance in alfalfa. Overexpression of MsSPL12 enhanced drought resistance and ABA sensitivity in alfalfa plants, while suppression of its expression led to the opposite phenotypes. Population genetic analysis across Medicago species revealed a SNP in the 5’UTR of MsSPL12 that converts the strong repressive Type I upstream open reading frame (uORF) into a weaker Type II. Disruption of the Type I uORF12 by using the CRISPR/Cas9 system markedly elevated the translation efficiency of MsSPL12, leading to enhanced ABA sensitivity and improved drought resistance in two alfalfa cultivars. Transcriptome analysis showed MsSPL12 modulates numerous drought-responsive genes, with ABA biosynthesis genes closely associated with altered ABA levels in transgenic plants. Biochemical assays verified that MsSPL12 directly activates the expression of MsBCH2 and MsCCD4, key genes in ABA biosynthesis, thereby promoting ABA accumulation. Overexpression of MsBCH2 and MsCCD4 significantly increased ABA content and drought tolerance in alfalfa plants and hairy roots, respectively. This study reveals the regulatory module of MsSPL12-mediated ABA biosynthesis in alfalfa drought tolerance, providing valuable molecular targets and uORF-based strategies for drought-tolerant alfalfa breeding.
Abstract Abscission is a fundamental developmental process in plants that can also be integrated into a defense strategy to restrict pathogen spread by shedding colonized organs. Members of Botryosphaeriaceae represent a major threat to global agriculture. These hemibiotrophic fungal pathogens colonize the vascular tissues of woody plants as endophytes, eliciting no visual symptoms. Under stress conditions, they switch to a necrotrophic lifestyle, causing destructive diseases such as stem dieback and fruit stem-end rot. Despite their economic significance, the role of the earliest, endophytic chronic colonization in triggering premature fruit drop, or abscission, remains largely unexplored. In this study, we uncover a previously overlooked phenomenon where Lasiodiplodia theobromae endophytically colonizes mango (Mangifera indica) flowers and fruitlets at the abscission zone (AZ), triggering premature abscission through activation of host ethylene-dependent defense responses. This causes the colonized fruitlets to drop prematurely, reducing the overall fruit yield. The presence of fungi initiates a cascade of events at the AZ, reprogramming the AZ cells through oxidative bursts and ethylene biosynthesis. Thereafter, the fungal secretion of ammonia leads to local cytoplasmic alkalization, ultimately accelerating callose deposition, accompanied by a depletion of auxin and cytokinin fluxes, and drives premature fruit detachment. The results elucidate two previously uncharacterized facets of the Botryosphaeriaceae endophytic/chronic colonization stage. We demonstrate that host damage and yield loss initiate before the necrotrophic transition and characterize a novel systemic defense response wherein the tree prematurely drops colonized fruits to limit pathogenic fungi colonization.
Abstract The basic leucine zipper (bZIP) transcription factor family plays crucial roles in plant development and stress responses, yet its evolutionary dynamics and functional diversification across green plants remain poorly understood. Here, we conducted a comprehensive analysis of the bZIP family across 114 green plant taxa, from algae to angiosperms, with emphasis on Rosaceae species and Malus accessions. Phylogenetic analysis showed that most bZIP subfamilies formed stable monophyletic clades across green plants, except for two atypical evolutionary patterns. An intertwined E-M-E-I complex suggested that the core domain of subfamily M originated from the group E lineage, whereas the highly divergent subclades, S1 and I1, highlighted the uneven evolutionary rates within the family. In Rosaceae, lineage-specific whole genome duplication (WGD) events, especially in Malinae subtribe and Potentilla, markedly expanded bZIP repertoires with asymmetric retention among subfamilies. Furthermore, lineage-specific pangenome in Malus identified 1251 core and 18 unique bZIP members across diverse accessions. Pan-transcriptome identified tissue-specific expression and stress-responsive co-expression modules. And functional characterization of MP_bZIP77, a gene derived from wild apple, provides a concrete example of this regulatory mechanism by demonstrating that its overexpression enhances salt tolerance through the activation of antioxidant enzyme activities. This study provides novel insights into the evolutionary origin, expansion pattern, and functional divergence of the bZIP family in green plants and Rosaceae, while laying a theoretical and genetic foundation for future molecular breeding aimed at improving stress resistance in fruit trees.
Abstract Grafting cucumber onto salt-tolerant pumpkin rootstocks is widely used to alleviate salt stress, yet the contribution of long-distance mRNA signaling to rootstock-conferred salt tolerance remains unclear. In this study, using reciprocal cucumber/pumpkin heterografts with SNP-resolved tracking, we demonstrate that salt stress rewires long-distance mRNA trafficking. Salinity imposes a pronounced directional bias, favouring mRNA export from the tolerant pumpkin to the sensitive cucumber while limiting reverse flux, irrespective of graft configuration. This identifies directional RNA mobility as an emergent property of salt stress adaptation. Among pumpkin-derived mobile transcripts, CmoHSP83 and CmoCAT2 were strongly enriched in cucumber scions and selected for functional validation. Their long-distance mobility was confirmed using transgenic hairy-root systems, species-specific RT–PCR, SNP-informed Hi-TOM sequencing, and GUS assays. Rootstock overexpression of either gene enhanced salt tolerance in grafted cucumber, accompanied by reduced ROS accumulation and improved Na +/K+ homeostasis. Collectively, these findings establish mobile mRNAs as functional long-distance signals in rootstock–scion communication under salinity and provide mechanistic insights into pumpkin rootstock-conferred systemic salt adaptation in grafted cucumber.
Abstract Ethylene is the key hormone controlling climacteric fruit ripening, a process that plays a crucial role in determining both fruit quality and shelf-life. Melon serves as an excellent model for studying ripening due to the presence of both climacteric and non-climacteric varieties. In this study, we fine-tuned ethylene production by combining the alleles of three non-climacteric QTLs, ETHQB3.5, ETHQV6.3, and ETHQV8.1, into a climacteric Cantaloupe background. This pyramiding strategy resulted in lines with reduced and delayed ethylene production, which in turn affected fruit flesh firmness and harvest timing. Regarding aroma, although overall volatile organic compounds (VOCs) production was reduced, the fruits retained the synthesis of specific ester-based compounds, preserving some of the characteristic sweet aroma of Cantaloupe. QTL pyramiding also revealed the impact of QTL-QTL interactions on ripening-related traits: ETHQV6.3 and ETHQV8.1 acted synergistically, while ETHQB3.5 showed an antagonistic effect on ETHQV6.3. This approach generated a range of climacteric behaviours, offering valuable genetic resources for breeding programs aimed at improving long-shelf life.
