DNA methylation is a key epigenetic regulator in plant development. However, the changes in methylation patterns between the early and late stages of grape berry development, the two phases with the most pronounced morphological differences, and the respective roles of methylation at these stages remain largely unexplored. To investigate the dynamic DNA methylation changes during this stage and their regulatory role in fruit development, we constructed genome-wide methylation maps of grape at two key time points: the early development stage (7 days after flowering, 7DAF; hereafter referred to as S1) and the late development stages (78 days after flowering, 78DAF; hereafter referred to as S2). Global cytosine methylation increased from 12.57% (S1) to 14.16% (S2), driven primarily by a substantial increase in CHH methylation (from 5.88% to 7.92%; p < 0.001), whereas CG and CHG methylation showed no statistically significant change. Most differentially methylated regions (DMRs) were hypermethylated in S2, predominantly in the CHH context. Integrative methylome and transcriptome analysis revealed that CHH hypermethylation was associated with the downregulation of YABBY5 (a berry size repressor) and upregulation of UGPase (a cell wall biosynthesis gene), suggesting a potential regulatory role in fruit expansion. Because our study compares only two time points, it cannot distinguish between gradual and stage-specific methylation changes, and functional validation of the identified genes is required. Nevertheless, these findings provides a valuable resource for understanding stage-specific DNA methylation dynamics and their association with gene expression during grape berry development.

Abstract Reduction of DNA methylation has traditionally been associated with gene activation. Here, we show that DNA hypomethylation permits the binding of a transcriptional repressor, leading to gene silencing. In tomato, the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE TF SlSPL-CNR exhibits methylation-sensitive DNA binding and preferentially occupies unmethylated GTACGG motifs. During fruit ripening, DEMETER-LIKE 2 (SlDML2)-mediated DNA demethylation at the alcohol acyltransferase 1 ( SlAAT1 ) promoter allows SlSPL-CNR binding, which in turn represses SlAAT1 expression and thereby modulates the biosynthesis of ester metabolites—key components of fruit flavor. Structural analysis reveals that cytosine methylation introduces a steric clash with Gln94 in the SBP domain of SlSPL-CNR, explaining its methylation sensitivity. CRISPR knockout of SlSPL-CNR de-represses SlAAT1 and increases ester accumulation, confirming its inhibitory role. Importantly, this methylation-sensitive binding is conserved across SBP domain proteins from rice, maize, and tomato. Our findings reveal a mechanism in which DNA hypomethylation facilitates repressor recruitment, establishing a regulatory logic linking epigenetic dynamics to metabolic control in plants.

Dietary deficiencies in essential micronutrients and other phytonutrients represent a global health and economic burden, contributing to "hidden hunger" and chronic diseases. While genome editing has been employed to improve individual nutritional traits in crops, multibiofortification through simultaneous modification of multiple distinct metabolic pathways is more challenging. Here, we designed a multiplex CRISPR-Cas strategy to edit five key genes in tomato: Sl7-DR2, SlGAD3, SlSGR1, SlGGP1, and SlGGP2. This approach successfully generated quintuple mutant (5m) tomato lines simultaneously biofortified with seven health-promoting compounds: vitamin D3 (from 0 to 0.70 μg/g dry weight), vitamin C (up to 2.53-fold), provitamin A/β-carotene (up to 3.86-fold), α-carotene (up to 2.47-fold), lutein (up to 3.26-fold), lycopene (up to 7.07-fold), and γ-aminobutyric acid (GABA, up to 5.26-fold). Notably, these multibiofortified tomatoes exhibited no significant trade-offs in plant growth or fruit quality. Extracts from 5m tomatoes showed enhanced suppression of colorectal cancer cell proliferation in vitro. This antiproliferative effect was validated in vivo, where dietary supplementation with 5m tomato powder significantly inhibited tumor growth in a mouse xenograft model. Our work demonstrates an effective strategy for developing a next generation of "functional foods" through multibiofortification, creating a single, nutrient-dense crop that combats both micronutrient malnutrition and chronic diseases.

Hyperosmolarity caused by drought, high salinity, or cold stress inhibits plant growth and crop productivity. A conserved protein-kinase cascade of cytosolic B-RAFs and SnRK2s is rapidly activated upon osmotic stresses to initiate downstream adaptive responses, which represents one of the fastest known responses to osmotic stress in plants. How the kinase cascade is activated by osmotic stress is unknown. Here, we show that Arabidopsis B4 subgroup RAFs have intrinsically disordered regions and directly sense both ionic and nonionic hyperosmolarity by reversible condensation. B4-RAFs recruit and cocondense with subclass-I SnRK2s to phosphorylate and turn on SnRK2s, evading the noncondensable inhibitory A-clade PP2C phosphatases. This straightforward osmosensing and relaying module can be fully reconstituted in Escherichia coli by coexpressing three components or in solution in a test tube using recombinant proteins. Our findings identify B-RAFs as the chief cellular osmosensors that detect low water potential by cocondensation, forming a signal hub with SnRK2s to orchestrate adaptive responses in plants, and represent an evolutionarily conserved osmosensing mechanism across kingdoms.

Many dicotyledonous plants exhibit considerable developmental plasticity and are capable of regenerating new organs upon wounding. Leveraging this wound-induced cellular pluripotency, a simple, efficient, and genotype-independent "cut-dip-budding" transformation system has been developed in recent years. Although genetic transformation in dicotyledonous crops like tomato is still largely based on tissue culture methods, and research into their molecular regulatory mechanisms and regeneration factors is extensive, the mechanisms underlying cut-induced shoot regeneration (cut-budding) remain poorly understood. This study investigated the molecular basis of cut-budding in tomato, focusing on the role of GROWTH-REGULATING FACTOR1 (GRF1) and its association with gibberellin (GA) signaling. By combining single-cell RNA sequencing, time-course transcriptome analysis, and genetic validation, we elucidated the key stages of shoot regeneration and identified SlGRF1 as a critical regulator. SlGRF1 was revealed to be essential for shoot initiation, with its expression significantly upregulated during cut-budding. Functional characterization using CRISPR/Cas9 knockout mutants (grf1-cr) demonstrated that SlGRF1 is required for pluripotency acquisition and shoot formation. Additionally, GA signaling negatively regulated shoot initiation by repressing SlGRF1 expression. An exogenous GA treatment inhibited shoot regeneration, while a paclobutrazol (GA biosynthesis inhibitor) treatment had the opposite effect. Moreover, grf1-cr mutants were similar to GA1-treated samples in terms of transcriptional changes and phenotypes, further indicating that GA signaling represses SlGRF1 expression. A ChIP-seq analysis showed that SlGRF1 controls cut-budding by activating the expression of shoot apical meristem regulator-encoding genes, including NAM1, EPF4, and ER2. NAM1 overexpression rescued the defective regeneration of grf1-cr1 mutants, highlighting the role of NAM1 as a downstream effector of SlGRF1. The study findings further clarify the molecular mechanisms governing cut-budding in tomato.

The regulation of serotonin metabolism during fruit development and ripening remains poorly understood, despite its potential roles in plant defence and human nutrition. Here, we demonstrated that the MADS-box transcription factor FUL2 acts as a key repressor of serotonin accumulation in tomato by forming a functional module with MADS1. CRISPR-Cas9-generated ful2-cr mutants exhibited delayed ripening, reduced fruit size and a striking 10-fold increase in serotonin levels, suggesting a previously unrecognised link between FUL2 and secondary metabolism. Immunoprecipitation-mass spectrometry (IP-MS) revealed that FUL2 physically interacts with MADS1, and genetic analyses showed that mads1-cr mutants phenocopied both the developmental and serotonin hyperaccumulation phenotypes of ful2-cr mutants. Furthermore, ChIP-seq and transcriptomic profiling demonstrated that the FUL2-MADS1 complex directly binds CArG-box motifs in the promoter of ASMT5 (a key enzyme in serotonin-to-melatonin conversion), activating its expression while repressing TDC1 (tryptophan decarboxylase). Electrophoretic mobility shift assays (EMSA) and dual-luciferase reporter assays confirmed their cooperative DNA binding and synergistic transcriptional regulation. Our work establishes a MADS-box transcriptional module that gates serotonin flux by coordinately regulating biosynthetic and metabolic genes. These findings provided a framework for engineering serotonin content in crops and deepen understanding of how developmental transcription factors govern specialised metabolism during ripening.

Supplementation of Driver and Kuniyuki Walnut Medium with phloroglucinol enhanced regeneration efficiency in tomato tissue culture. Heterologous expression of an Arabidopsis growth-regulating factor gene, GROWTH-REGULATING FACTOR5 (GRF5), in tomato improved regeneration and transformation efficiency, suggesting a synergistic effect between phloroglucinol treatment and GRF-mediated pathways.

Seed germination is a crucial transition in spermatophytes, regulated by gibberellins (GAs). GA levels are controlled by GA2-oxidases (GA2ox), but how DNA methylation participates in this regulation remains unclear. Here, we identified the tomato methyl-CpG-binding domain (MBD) protein SlMBD5 as a regulator of seed germination. The slmbd5 mutant exhibits delayed germination and reduced GA4/GA7 levels, which can be rescued by exogenous GA4+7 application. Transcriptomic and biochemical analyses revealed that SlMBD5 represses the GA catabolism gene SlGA2ox4 by directly binding to its hypermethylated promoter. Furthermore, we show that SlMBD5 interacts with the histone methylation reader SlEBS, forming a functional complex that promotes the transcriptional repression of SlGA2ox4. Consistent with the model that SIMBD5 promotes seed germination through its repression of SlGA2ox4, the slmbd5/slga2ox4 double mutant shows partially restored germination. This study thus reveals an SlMBD5-SlEBS module that regulates GA homeostasis to modulate seed germination in tomato.

The transcription factors (TFs) RIPENING-INHIBITOR (RIN) and NONRIPENING (NOR) are key regulators of fruit ripening in tomato (Solanum lycopersicum) fruit ripening. However, the spontaneous rin and nor alleles that were first described were demonstrated to be gain-of-function mutants, prompting a reevaluation of the roles of RIN and NOR in tomato fruit ripening. Here, we show that the slnor slrin double mutant (a double homozygous loss-of-function mutant of NOR and RIN) exhibits a complete cessation of fruit ripening, revealing that NOR and RIN redundantly but differentially regulate fruit ripening. Besides serving as activators, NOR and RIN are essential in suppressing genes related to photosynthesis. Additionally, at the initiation of ripening, NOR activates RIN expression by binding to its promoter. Following climacteric ethylene production, RIN represses NOR expression. This temporal interaction is crucial for the regulation of abscisic acid (ABA) and ethylene biosynthesis during fruit ripening. Interestingly, NOR and RIN do not form a transcriptional complex. Collectively, our findings provide insights into the regulatory network involving NOR and RIN in fruit ripening and uncover their roles in the crosstalk between the ripening hormones ethylene and ABA.

RNA-based immunity plays a central role in host defense against pathogens, with both hosts and pathogens continually evolving antagonistic strategies in their ongoing arms race. Although the presence of N6-methyl-adenosine (m6A) in viruses has been recognized for decades, its functional significance in plant antiviral defenses has only recently been revealed. Moreover, viral counterstrategies targeting m6A-mediated defenses remain largely unexplored. Here, we uncover a mutually antagonistic mechanism between m6A-mediated antiviral defense and a countermeasure employed by the RNA virus Cucumber mosaic virus (CMV). The deposition of m6A modification on CMV genomic RNAs was validated through m6A antibody-mediated MeRIP and nanopore-based direct RNA sequencing (DRS). During infection, plant m6A methyltransferases are translocated to the cytoplasm through their interaction with the viral coat protein (CP), facilitating viral m6A deposition. The plant EVOLUTIONARILY CONSERVED C-TERMINAL REGION 8 (ECT8) protein acts as a reader of viral m6A, destabilizing viral RNAs and mediating antiviral activity. Conversely, the CMV-2b protein, known as a viral suppressor of RNA silencing (VSR), antagonizes this defense by inhibiting viral m6A deposition. This occurs via direct interactions between 2b and the m6A methyltransferase components MTB and HAKAI, disrupting the methyltransferase complex's functionality. Furthermore, CMV-2b also downregulates global plant m6A levels, leading to the misexpression of defense-related transcripts. Collectively, our findings elucidate a previously unrecognized layer of host-virus interaction in which m6A modification serves as a regulatory battleground, positioning m6A dynamics as a new frontier in plant-virus coevolution.

Epigenetic mechanisms are integral to plant growth, development, and adaptation to environmental stimuli. Over the past two decades, our comprehension of these complex regulatory processes has expanded remarkably, producing a substantial body of knowledge on both locus-specific mechanisms and genome-wide regulatory patterns. Studies initially grounded in the model plant Arabidopsis have been broadened to encompass a diverse array of crop species, revealing the multifaceted roles of epigenetics in physiological and agronomic traits. With recent technological advancements, epigenetic regulations at the single-cell level and at the large-scale population level are emerging as new focuses. This review offers an in-depth synthesis of the diverse epigenetic regulations, detailing the catalytic machinery and regulatory functions. It delves into the intricate interplay among various epigenetic elements and their collective influence on the modulation of crop traits. Furthermore, it examines recent breakthroughs in technologies for epigenetic modifications and their integration into strategies for crop improvement. The review underscores the transformative potential of epigenetic strategies in bolstering crop performance, advocating for the development of efficient tools to fully exploit the agricultural benefits of epigenetic insights.

DNA methylation regulates fruit ripening in tomato, and disruption of the DNA demethylase DEMETER-LIKE 2 (DML2) results in genome-wide DNA hypermethylation and impaired ripening. We report here that the transcription factors Ripening Inhibitor (RIN) and FRUITFULL 1 (FUL1) play critical roles in mediating the effect of DNA methylation on tomato fruit ripening. RIN and FUL1 are silenced in dml2 mutant plants, and the defective ripening phenotype of dml2 is mimicked by the rin/ful1 double mutant. Restoration of RIN expression in dml2 partially rescues its ripening defects. DNA methylation controls ripening not only by regulating the expression of RIN and FUL1 but also by interfering with the genomic binding of RIN. In dml2 mutant plants, RIN cannot bind to some of its targets in vivo even though DNA methylation does not interfere with RIN binding in vitro; this inhibited binding in vivo is correlated with increased DNA methylation and histone H3 enrichment within 100 bp of the binding site. Our work uncovers the molecular mechanisms underlying DNA methylation control of fruit ripening in tomato.

Epigenome and cis-regulome, comprising cis-regulatory elements (CREs) and modules (CRMs), jointly define the architecture of gene regulation. However, the causal mechanisms by which epigenetic marks influence CRM function remain elusive. To address this, modular epigenome editing frameworks, exemplified by dead Cas9-coupled DNA demethylation (dCd) and DNA methylation (dCm) platforms, are developed for programmable dissection and engineering of CRM activity. The dCd system modulates methylation levels and transcriptional output at CRMs in situ or ex situ, in accordance with CRM-specific methylation responsiveness, and alters co-transcriptional RNA processing to yield predictable phenotypic outcomes in plants. These findings underscore the reliability of targeted DNA demethylation. In parallel, the dCm system reconstitutes methylation-dependent and -sensitive CRMs of diverse origins in Saccharomyces cerevisiae, a species devoid of native DNA methylation, enabling causal dissection of epigenetic regulation and revealing cross-species portability. This system further uncovers crosstalk between DNA methylation and chromatin modifications, and enables logic-gated control of endogenous genes through CRM engineering. Incorporation of optogenetic and temperature-sensitive anti-CRISPR inhibitors confers tunable, reversible regulation, proposing dCm as a foundation for input-responsive synthetic epigenome editors. Together, these frameworks provide a versatile platform to decode and reprogram cis-regulatory epigenetic logic, with broad applications in trait design and synthetic biology.

Plants, as sessile organisms, must adapt to a range of abiotic stresses, including drought, salinity, heat, and cold, which are increasingly exacerbated by climate change. These stresses significantly impact crop productivity, posing challenges for sustainable agriculture and food security. Recent advances in omics studies and genetics have shed light on molecular mechanisms underlying plant stress responses, including the role of calcium (Ca2⁺) signaling, liquid–liquid phase separation (LLPS), and cell wall-associated sensors in detecting and responding to environmental changes. However, gaps remain in understanding how rapid stress signaling is integrated with slower, adaptive processes. Emerging evidence also highlights crosstalk between abiotic stress responses, plant immunity, and growth regulation, mediated by key components such as RAF-SnRK2 kinase cascades, DELLA proteins, etc. Strategies to enhance crop stress resistance without compromising yield include introducing beneficial alleles, spatiotemporal optimization of stress responses, and decoupling stress signaling from growth inhibition. This review emphasizes the importance of interdisciplinary approaches and innovative technologies to bridge fundamental research and practical agricultural applications, aiming to develop resilient crops for sustainable food production in an era of escalating environmental challenges.

Papaya (Carica papaya) is a promising model system for genetic and genomic studies of fruit traits and sex determination in tropical trees and fruits. However, the genomic basis of the artificial selection for key fruit traits and commercially crucial hermaphroditism remains poorly understood. In this study, we assembled the genomes for two phenotypically divergent hermaphroditic cultivars, including their haplotype-phased sex-determining regions (SDRs). Population genomic analyses of wild, common-type (for vegetable use), and fruit-type (for fresh consumption) papayas revealed a clear domestication history and geographic spread model. By combining genome-wide association study (GWAS), selection scan, and functional validation, we revealed a stepwise selection targeting CpPUP11 and CpICMT during domestication and improvement to reshape fruit size, and artificial selection on CpMAPK1, CpCOX, CpCIN, and CpUBE3 during the improvement process to increase fruit sweetness and vitamin C content. Furthermore, we demonstrated the independent origins of two hermaphroditic lineages through polyphyletic selection from wild male populations, resulting in two distinct hermaphrodite-specific Yh regions (HSY1 and HSY3). This transition was driven by selection targeting male-biased genes, which exhibited stable dominance in their methylation and transcription. Notably, we identified a hermaphrodite-specific, selectively fixed 13-bp insertion in the male-biased gene CpPGLP1A (HSY3-TR-13bp), which is strongly associated with the male-to-hermaphrodite transition. Collectively, our study provides novel insights into the genomic architecture of papaya domestication, revealing a trajectory of stepwise selection reshaping fruit traits and male-biased selection driving the emergence of a novel hermaphroditic sexual system.

Tools to edit DNA methylation in a targeted manner are vital for establishing causal relationships between DNA methylation and its function, as well as for plant breeding and gene therapy. Here, by constructing dCas9 fusions to a panel of effectors and cofactors, we develop a range of highly effective tools for editing DNA methylation in Arabidopsis, including five tools for DNA methylation and six tools for DNA demethylation. Our tools show a diversity of performance features in terms of specificity and efficiency, offering either the capacity to edit DNA methylation in a target-specific manner or the ability to edit DNA methylation genome-wide due to potent off-target effect. Importantly, DNA methylation edited by these tools is inherited in the absence of transgene. These versatile tools pave the way for diverse applications of DNA methylation editing in not only research but also epigenetic breeding of crops.

GABA, a non-proteinogenic amino acid with anti-hypertensive properties, holds health-beneficial potential when enriched in crops. Previous studies have established that targeted disruption of the calmodulin-binding domain (CaMBD) of the tomato glutamate decarboxylase 3 (SlGAD3) enhances GABA biosynthesis. In this study, we used CRISPR/Cas9-mediated gene editing to precisely modify the CaMBD coding sequence of SlGAD3 in three elite tomato varieties (SFT1, SFT2, and SFT3). Under our experimental conditions, targeted editing of SlGAD3 led to substantial accumulation of GABA in all three varieties without compromising key agronomic traits such as fruit size and number. Although flowering was delayed in SFT2 and SFT3 mutants, SFT1 mutants had higher GABA levels but also maintained a wild-type flowering time. This result highlights the critical importance of selecting specific varieties, such as SFT1, to minimize pleiotropic effects. By identifying varieties that can accumulate high levels of GABA without major reductions in growth and yield potential, this work bridges a critical gap between plant metabolic-engineering research and practical applications in commercial crop-improvement programs.

Protein kinases in the mitogen-activated protein kinase kinase kinase (MAPKKK) superfamily are known to be central actors in orchestrating cell differentiation, growth, and responses to extracellular stimuli in eukaryotes. However, the rice (Oryza sativa L.) MAPKKKs, especially the rapidly accelerated fibrosarcoma (RAF) subgroup ones, have rarely been studied yet. In this study, we generate high-order mutants of B1, B2, and B3 RAF subgroup MAPKKKs. The resulting raf-b2, raf-b3q, and raf-b2/3q mutants are hyposensitive to abscisic acid (ABA) but show dramatic sensitivity to various osmotic stresses. The rice B2 and B3 RAFs can phosphorylate stress-activated protein kinases (SAPKs), and in the rice raf-b2/3q, the ABA-triggered activation of SAPKs is largely impaired, and the expression of stress-response genes is decreased compared to that in the wild type. Our study reveals a crucial role of B2 and B3 subgroup RAFs in ABA signaling and osmotic stresses and sheds light on the functional divergence of the RAF-SAPK cascade in different plant species.