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.

BackgroundThe molecular mechanisms contributing to the poor prognosis of Clonorchis sinensis (Cs)-infected hepatocellular carcinoma (HCC) remain poorly understood, especially when it comes to DNA methylation.MethodsUsing parallel whole-genome bisulfite sequencing (WGBS) and whole-genome oxidative bisulfite sequencing (oxWGBS), we characterized the profiles of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in Cs-infected HCC tumors and adjacent non-tumor tissues at single-nucleotide resolution. These profiles were compared with data from non-Cs-infected HCC available in the GEO database. Additionally, we investigated the impact of DNA methylation modifications on differential gene expression and prognosis in Cs-infected HCC by analyzing HCC related data from the TCGA database and RNA-seq data from previous studies.ResultsHere, we reported the methylation and hydroxymethylation landscapes of Cs-infected and Cs-non-infected HCC patients at single-nucleotide resolution by WGBS and oxWGBS, respectively. 29 differentially methylated regions (DMRs) and 13 differentially hydroxymethylated regions (DhMRs) were found in Cs-infected HCC tumor tissues compared to Cs-non-infected HCC tumor tissues. Following, we identified 28 differentially methylated/hydroxymethylated-associated genes (DAGs/DhAGs), two genes (DHDH and KCNQ3) of which were significantly correlated with the overall survival of HCC. Finally, we also revealed that four Cytosine-phosphate-Guanine sites in the promoter of DAGs/DhAGs certainly affect the survival outcomes of HCC.ConclusionsThis study provides the first comprehensive characterization of DNA methylome and hydroxymethylome landscapes in Cs-infected HCC, shedding light on the impact of on HCC methylation and offering new insights into the role of Cs in HCC progression. Our findings also highlight the key DMRs/DhMRs and DAGs/DhAGs in Cs-infected HCC tumors, which could serve as promising epigenetic biomarkers for predicting the prognosis and therapeutic targets of Cs-associated HCC.

Amid its dynamic roles within Polycomb group (PcG) complexes, a growing body of work shows that LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) interacts with diverse protein and long non-coding RNA (lncRNA) partners and integrates multiple epigenetic pathways to sculpt chromatin states and modulate gene activity. We survey expanding, integrative networks that position LHP1 as a regulatory hub in Arabidopsis thaliana and crops, ensuring developmental timing and fidelity, establishing bivalent chromatin domains and growth-stress/defense tradeoffs, and contributing to lineage-specific functional innovation. These multifaceted attributes qualify LHP1 as a Polycomb-beyond Composite Regulator 1 (PCR1), a designation that honors its PcG heritage while accommodating broader and evolving roles in gene regulation. This synthesis delineates conceptual and translational frontiers in chromatin biology, informing strategies to engineer resilient, high-performing crops.

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.

Cross-compartment communication is critical for maintaining cellular homeostasis, which is essential for cell function and survival under stressful conditions. However, the cellular cues that trigger interorganellar communication remain poorly understood. Mitochondrial Ca2+ (mtCa2+) homeostasis is fundamental to mitochondrial function; yet, how mitochondrial Ca2+ (mtCa2+) homeostasis modulates nuclear gene expression to establish and maintain cellular homeostasis remains unclear. Here, we first characterize the critical role of the mitochondrial Ca2+ uniporter (MCU) in control of mtCa2+ uptake and maintaining mtCa2+ homeostasis in planta. Using gain-of-function and sextuple MCU knockdown mutants, we then analyzed the effects of impaired MCU-controlled mtCa2+ homeostasis (iMUCH). We find that iMUCH elicits an interorganellar transcription program that activates multiple compartment-specific unfolded protein responses (UPRs) and genes critical for mitochondrial and cytosolic proteostasis. Additionally, iMUCH induces a post-transcriptional program that selectively represses the synthesis of ribosomes and RNA modification proteins. Furthermore, eukaryotic initiation factor α (eIFα and its phosphorylation likely serve as a protective mechanism under long-term mitochondrial proteotoxic stress induced by iMUCH. Collectively, the data demonstrate that MCU-controlled mtCa2+ homeostasis plays a pivotal role in sustaining mitochondrial and cytosolic proteostasis through an interconnected organelle quality control system, which ultimately determines cell growth and fitness.

This study introduces an intronic artificial microRNA (IamiRNA) strategy that combines CRISPR-Cas9-mediated knock-in with endogenous miRNA processing for targeted gene silencing in plants. By inserting amiRNA precursors into introns of endogenous genes, this approach enables effective, tissue-specific gene silencing without persistent transgene expression, offering a promising tool for functional genomics and crop improvement.

Papaya is a major tropical fruit crop with notable nutritional and economic value, yet its genetic improvement through modern breeding technologies faces substantial challenges. The traditional tissue culture process is both labor-intensive and time-consuming, causing gene-editing advancements in papaya to lag behind those in other crops. To overcome these obstacles, we developed a tissue culture-independent hairy root system in papaya, which enables efficient gene editing and significantly enhances the application and development of editing tools. This innovative platform allows for the pre-assessment of editing efficiency and supports the establishment of adenine base editor (ABE) and cytosine base editor (CBE) tools in papaya, thereby mitigating the high failure costs associated with the lengthy cycle of conventional genetic transformation. Utilizing this system, we pre-tested sgRNA activity and achieved high editing efficiency of CpWIP3 during stable transformation. Additionally, through promoter screening, we successfully developed ABE and CBE tools, marking the first precise single-nucleotide editing system in papaya. This gene-editing system provides a crucial platform for advancing functional genomics and accelerating precision breeding in papaya.

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.

The CBF transcription factors serve as key nodes that integrate cold signaling into the SVP-mediated flowering pathway, which fine-tunes seasonal flowering in response to ambient temperature.

DNA demethylation is essential for maintaining genome-wide DNA methylation balance. Despite the substantial risk to genome stability, the prevailing paradigm posits that the Arabidopsis demethylase ROS1 prevents genome-wide DNA hypermethylation in vivo mainly through its 5-methylcytosine DNA glycosylase/lyase activity. Here we challenge this paradigm by demonstrating that ROS1, through its occupancy, drives extensive passive demethylation independent of its glycosylase/lyase activity and maintains hypomethylation primarily by preventing de novo DNA methylation. This occupancy-based mechanism eliminates the need for genome-wide base excision for active demethylation, thereby minimizing threats to genomic fidelity and stability. Beyond its role in demethylation, ROS1 also functions as a key marker and regulator of chromatin accessibility. This regulation operates in both DNA methylation-dependent and -independent contexts, with ROS1 acting as either a reserve or active protector of accessible chromatin, depending on the functional state of DNA methylation systems. Our findings redefine the diverse roles of ROS1 in DNA methylation regulation and chromatin accessibility, highlighting their intricate interplay.

The therapeutic and research applications of CRISPR-Cas nucleases are constrained by their reliance on specific Protospacer Adjacent Motifs (PAMs), which limit the accessible sites in the genome. To overcome this critical barrier, we performed structure-guided engineering of SF01, a compact Cas12i nuclease. Using AlphaFold-predicted structural models, we identified and systematically mutagenized 38 residues at the PAM-interacting interface. This iterative engineering process yielded three superior variants-KR, IKRR, and STKRR-that exhibit dramatically relaxed PAM specificity, enabling efficient editing at a broad spectrum of 5'-NNTN-3' sites. Importantly, while the most broad-spectrum variant (STKRR) shows a trade-off at canonical sites, the IKRR variant retains high activity at canonical 5'-NTTN-3' PAMs while simultaneously enabling efficient editing at 5'-NNTN-3' sites. This near-PAMless activity expands the targetable portion of the genome to over 25%, a four-fold increase over the parental nuclease. Furthermore, adenine base editors (ABEs) constructed with these variants achieve high-efficiency editing (∼80%) at endogenous loci with expanded targeting scope. Comprehensive off-target analysis using GUIDE-tag and Digenome-seq revealed that the enhanced on-target activity of the SF01 variants is not accompanied by a loss of specificity. These engineered nucleases represent a powerful and versatile expansion of the genome editing toolkit, enabling applications previously inaccessible due to PAM constraints.

Reactive oxygen species (ROS) regulate plant growth, development, and responses to the environment. ROS production by the RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN D (RBOHD) protein is regulated by PHYTOCHROME B (phyB), and phyB is phosphorylated by FERONIA, highlighting the possibility that these 3 proteins interact to regulate ROS levels during stress. We used immunoprecipitation and proximity labeling, followed by split-luciferase and functional validation assays, to study interactions among FERONIA, phyB, and RBOHD under excess light (EL) stress in Arabidopsis (Arabidopsis thaliana). We found that phyB, RBOHD, and FERONIA interact, that phosphorylation of phyB by FERONIA, as well as the kinase activity of FERONIA, are required for RBOHD-driven ROS production in response to EL stress, and that CYSTEINE-RICH RECEPTOR LIKE KINASE 10 (CRK10) and PLASMA MEMBRANE INTRINSIC PROTEIN 2;6 (PIP2;6) interact with RBOHD and phyB and are also required for EL-driven RBOHD ROS production. Our findings uncover a putative plasma membrane complex among FERONIA, RBOHD, CRK10, and PIP2;6 that interacts with phyB to regulate ROS production in Arabidopsis in response to stress. This complex could play a canonical role in the integration and regulation of multiple signaling pathways in plants.

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.

Zero-shot mutation prediction is vital for low-resource protein engineering, yet existing protein language models (PLMs) often yield statistically confident results that ignore fundamental biophysical constraints. Currently, selecting candidates for wet-lab validation relies on manual expert auditing of PLM outputs, a process that is inefficient, subjective, and highly dependent on domain expertise. To address this, we propose Rank-and-Reason (VenusRAR), a two-stage agentic framework to automate this workflow and maximize expected wet-lab fitness. In the Rank-Stage, a Computational Expert and Virtual Biologist aggregate a context-aware multi-modal ensemble, establishing a new Spearman correlation record of 0.551 (vs. 0.518) on ProteinGym. In the Reason-Stage, an agentic Expert Panel employs chain-of-thought reasoning to audit candidates against geometric and structural constraints, improving the Top-5 Hit Rate by up to 367

RAF-like protein kinases constitute a major subclass of mitogen-activated protein kinase kinase kinases (MAPKKKs) in plants and function as critical regulators of stress and hormone signaling pathways. Unlike their animal counterparts, plant RAF kinases show extensive expansion and diversification, with distinct subgroups (B and C) exhibiting both conserved and specialized functions. Recent studies have unveiled their pivotal roles in sensing environmental stresses, such as hyperosmotic stress and elevated CO 2 , as well as in mediating hormonal responses, including those to abscisic acid (ABA), ethylene, and auxin. RAFs also participate in guard cell signaling, immune responses, and developmental processes, integrating diverse external and internal cues. This review summarizes the current knowledge of plant RAF kinases, emphasizing their functional diversity, mechanisms of activation, and physiological relevance in plant adaptation.

TnpB, a compact RNA-guided nuclease ancestral to Cas12, is optimized for plant genome editing. A 99-nt enhanced RNA and T5 exonuclease fusion generate TnpBe5, boosting rice editing 2.5-fold. Coupling with a single-strands annealing (SSA)-responsive HYYG surrogate (TnpBmax) enriches edited cells, achieving up to 81.5% efficiency and high homozygosity across rice and tomato.

Adenine and cytosine base editing using dCas-SF01 and the 35S-CmYLCV-U6 composite promoter successfully introduced targeted base substitutions at multiple loci in rice, with average editing efficiency of 33.3%. Using the protospacer adjacent motif (PAM)-relaxed SF01-IKRR variant enabled base editing using 5'-NTN PAMs in rice.

Transient gene expression in mesophyll protoplasts is a valuable approach for investigating gene function, plant physiological processes, and molecular mechanisms. Rubber dandelion (Taraxacum kok-saghyz, TKS) is an ideal model for studying rubber biosynthesis and serves as a promising source of natural rubber and inulin. However, developing efficient protoplast-based systems for TKS remains challenging. In this study, we established a robust method for isolating mesophyll protoplasts from TKS by optimizing enzymatic conditions for cell wall digestion. We subjected the protoplasts to PEG/calcium-mediated transfection and evaluated promoter activities, expressed and detected target proteins, confirmed the subcellular localization of the target proteins, and examined transcription factor-DNA interactions under physiological conditions. We also developed a rapid assessment strategy for genome-editing tools in TKS protoplasts using multiple reporter systems. We evaluated these optimized tools in a tissue-culture-free hairy root transformation system, establishing a dual-platform toolkit for functional genomics in TKS. This work provides an efficient approach for TKS protoplast preparation, facilitating studies of gene function and advancing biotechnological research in this rubber-producing crop.