Search Results (86)

Climate change is increasing the frequency and severity of abiotic stresses, including drought, salinity, waterlogging, and temperature extremes, thereby threatening the productivity and quality of Brassica crops. This review synthesizes recent progress in abiotic stress tolerance from physiological, genetic, epigenetic, and multi-omics perspectives, with an emphasis on how mechanistic discoveries can be translated into breeding decisions. We first outline the signaling hierarchy that links stress perception at the plasma membrane and cell wall interface to Ca²⁺ signaling, MAPK cascades, hormone crosstalk, osmotic adjustment, ROS homeostasis, and metabolic reprogramming. We then examine the genetic architecture of stress tolerance through QTL mapping, GWAS, and functional genomics, highlighting how allopolyploidy, subgenome specialization, homoeologous gene divergence, and alternative splicing create both opportunities and complications for Brassica improvement. We further evaluate how transcriptomic, epigenomic, metabolomic, and microbiome-related data are revealing regulatory complexity but remain underused for prediction and causal inference. Major bottlenecks include the inefficient conversion of association signals into validated functional markers, the descriptive rather than predictive use of multi-omics datasets, limited mechanistic understanding of combined stresses, and insufficient field validation across genetic backgrounds. Finally, we discuss integrated breeding strategies, including marker-assisted selection, genomic selection, genome editing, wild germplasm utilization, microbiome-assisted approaches, and synthetic biology. By connecting stress biology with translational breeding, this review provides a framework for developing climate-resilient Brassica cultivars. Full article

DNA methylation has long been regarded as a stable, maintenance-based epigenetic marker. However, this classical binary model struggles to fully explain the dynamic and situational dependence of methylation regulation at the multi-biological level. This review defines DNA methylation as a programmable information layer that systematically integrates the latest advances in three interrelated dimensions of molecular coding, disease status indication, and epigenomic engineering. At the molecular level, this paper describes how the chemical diversity of cytosine modification, the writing–erasing enzyme network, and the three-dimensional structure of chromatin jointly construct a methylated polymorphic coding system and evaluates the performance of emerging sequencing technologies in DNA integrity, reading length, modification resolution, and analytical complexity through a multidimensional scoring framework. At the cellular and clinical levels, this paper comprehensively demonstrates methylation as a quantifiable indicator of cell identity, biological aging and disease status, covering circulating free DNA biomarkers and spatial heterogeneity analysis. Critically, this paper evaluates how the clustered regularly interspaced short palindromic repeats (CRISPR)-based epigenome editing platform achieves causal inference and promotes the transformation of methylation from related biomarkers to functional therapeutic targets. At the same time, persistent challenges such as off-target specificity, in vivo delivery, and spatiotemporal regulation encountered in epigenetic gene editing are discussed. This review reveals the paradigm shift of DNA methylation from passive observation markers to actively engineered regulatory parameters, which has direct therapeutic application prospects. Full article

Cereal crops including wheat, rice, maize, barley, and sorghum collectively supply most global caloric and protein requirements, yet their productivity is increasingly constrained by combined abiotic stresses that co-occur under field conditions. Simultaneous drought, heat, salinity, and cold impose yield losses that consistently exceed those caused by individual stresses and elicit molecular responses that are qualitatively distinct from single-stress reactions and cannot be inferred from them. Despite this agronomic reality, the molecular mechanisms governing combined stress responses in cereals remain poorly resolved, and no integrated framework connecting the transcriptional, epigenetic, and genome-editing dimensions of combined stress tolerance has previously been articulated for this crop group. This review proposes a three-tier integrated framework for understanding and engineering combined abiotic stress tolerance in major cereals. The first tier encompasses transcription factor networks, including bZIP, WRKY, NAC, AP2/ERF, DREB, MYB, and HSF families, that translate combined stress signals into transcriptional reprogramming through ABA-dependent and ABA-independent pathways, hormonal crosstalk, and osmoprotectant and antioxidant defence systems. The second tier addresses the epigenetic regulatory layer, encompassing DNA methylation, histone modifications, and non-coding RNA pathways that gate TF binding site accessibility and encode stress memory in cereals. The third tier examines CRISPR-based tools, including multiplexed Cas9 editing and dCas9-based epigenome editing, that engineer validated targets from both tiers, while confronting polyploid off-target effects, growth penalties, and a laboratory-to-field validation gap. The three tiers are mechanistically coupled, with TF activity shaping epigenetic landscapes, epigenetic states gating TF access, and both providing precision engineering targets. Critical gaps include the absence of combined-stress epigenomic datasets, limited characterisation in barley and sorghum, and early-stage combined-stress-specific strategies. Full article

Rett syndrome (RTT) is a severe X-linked neurodevelopmental disorder that is caused in most cases by pathogenic variants in MECP2, the gene encoding methyl-CpG-binding protein 2 (MeCP2). Despite substantial progress in the development of gene therapy, restoring MECP2 expression remains challenging because MeCP2 is highly dosage-sensitive. Both deficiency and excessive expression of this protein are associated with severe neurological abnormalities. This makes simple viral vector-mediated replacement of MECP2 potentially unsafe and underscores the need for multilayered systems that control transgene expression. This review discusses current and emerging strategies for regulating MeCP2 expression in RTT, with an emphasis on non-coding RNA-based and epigenetic mechanisms. Particular attention is given to the limitations of conventional AAV-mediated gene therapy, the use of cell-specific and endogenous promoters, miRNA-regulated elements, autoregulatory systems, and post-transcriptional control of MECP2 expression. Strategies for reactivating the inactive X chromosome are also discussed, including XIST-dependent regulation and epigenome editing. In addition, the review considers CRISPR-mediated regulation, selective epigenetic activation, and combined therapeutic platforms that integrate viral delivery, RNA-dependent post-transcriptional control, and endogenous gene regulation. Overall, clinically applicable gene therapy for RTT will likely need to move beyond simple MECP2 replacement and instead rely on precise cell- and dose-dependent regulation of its expression. Non-coding RNA and epigenetic mechanisms represent important layers of such control and may contribute to the development of safer gene therapy strategies for RTT. Full article

Reversible genetic medicines are emerging as controllable alternatives to permanent genome editing by enabling programmable modulation of RNA sequence, transcript abundance, chromatin state, and gene expression without irreversible genomic alteration. However, reversibility is not a single binary property: transient editor exposure, decay of the molecular effect, recovery of cellular function, and clinical capacity to stop, redose, or counteract treatment may diverge. This review therefore distinguishes mechanistic, functional, and clinical reversibility while examining targeted delivery systems for RNA-editing and epigenome-editing therapeutics. Key payloads include ADAR-recruiting oligonucleotides, CRISPR-Cas13 RNA editors, guide RNAs, chemically modified RNAs, editor-encoding mRNAs, dCas9 transcriptional regulators, DNA methylation editors, histone-modifying systems, and CRISPRoff-like platforms. We evaluate extracellular and intracellular delivery barriers, including nuclease degradation, immune recognition, renal clearance, liver uptake, cellular entry, endosomal escape, cytoplasmic release, nuclear localization, chromatin access, editing-window duration, off-target activity, immunogenicity, repeat-dosing feasibility, manufacturing, quality control, potency assays, and regulatory translation. Overall, delivery systems for reversible genetic medicines should be judged by tissue selectivity, functional editing, duration of action, reversibility after treatment withdrawal, safety, manufacturability, and clinical controllability. Full article

Aims: Treatment of higher-risk myelodysplastic syndromes (HR-MDS) with azacytidine (AZA) exerts significant effects on the epigenome, primarily through DNA demethylation and reactivation of epigenetically silenced genes. Beyond this established mechanism, molecular AZA-linked effects are increasingly being recognized. Materials and methods: Liquid chromatography combined with mass spectrometry (LC-MS/MS) was employed for the accurate assessment of various RNA and DNA modifications pre- and post-AZA treatment of an HR-MDS cohort (N = 8). Mapping of the AZA treatment-responsive regulatory pathways was performed by miRNA-next generation sequencing (NGS), followed by a multi-layered bioinformatic pipeline, integrating miRNA differential expression, gene set enrichment, and network analyses. The precise number of mitochondrial (mt)DNA copies pre- and post-AZA was evaluated by a digital PCR assay. Results: Cell pathways affected by miRNA differential expression patterns pre- and post-AZA treatment discriminated the clinical phenotypes of Responders against Non-Responders to therapy. Intracellular RNA modifications: N6-methyladenosine (m6A), 5-methylcytidine (m5C), N1-methyladenosine (m1A), 2′-O-methylguanosine (Gm) and adenosine-to-inosine (A → I) editing were evaluated for their potential impact in treatment response. Nuclear DNA/mtDNA methylation profiles and mtDNA copy number reduction manifested the mitochondrial features affected by AZA. Our results suggest that neoplastic HSPCs in HR-MDS Responders to AZA adapt by normalizing glycolytic metabolism and enhancing ribosomal activity. The observed reduction of mtDNA content can be associated with improved survival and suppression of malignant progression. Non-Responders, despite experiencing mtDNA depletion, seem unable to coordinate such metabolic reprogramming and remain disadvantaged to AZA therapy. Full article

Cannabinoid content can vary several-fold across Cannabis sativa L. cultivars that carry functional, closely related CBDA synthase (CBDAS) and THCA synthase (THCAS) alleles, a difference that coding-sequence variation among functional alleles does not fully explain. Structural and copy-number variation at the synthase loci, the functional or pseudogenised state of synthase alleles, and linkage at the B locus account for much of the qualitative drug-versus-hemp chemotype; however, these genetic factors do not fully explain the quantitative, several-fold variation in cannabinoid content among cultivars that carry functional, near-identical synthase alleles. Three independent lines of evidence now indicate that chromatin-level regulation contributes to this variation. H3K4me3 and H3K56ac co-occupy the promoters and gene bodies of THCAS, CBDAS, OLS, and OAC exclusively in glandular trichome tissue while H3K27me3 marks the identical loci in vegetative tissues, indicating that a Polycomb-to-Trithorax chromatin switch is associated with trichome-specific cannabinoid gene expression, potentially independently of transcription factor availability. Progressive DNA hypomethylation accumulates during micropropagation in a cultivar-specific manner, with promoter-region differentially methylated positions reaching up to 22% by twenty subcultures, identifying DNA methylation maintenance as a candidate determinant of epigenetic stability in clonally propagated material, although the functional consequences for cannabinoid pathway gene expression and yield remain to be quantitatively established. In Cannabis indica cell suspension cultures, UV irradiation increases genome-wide DNA methylation (detected by MSAP, which does not resolve locus-specific changes) and elevates CBDAS transcript levels approximately 4-fold relative to non-irradiated controls. This coupling of an environmentally triggered methylation change to cannabinoid pathway gene expression in the Cannabis genus is suggestive, but the genome-wide MSAP signal and the use of C. indica cell cultures mean the locus-specific methylation change at the CBDAS promoter remains to be demonstrated. These findings identify specific, experimentally documented chromatin states as candidate targets for dCas9-effector intervention, including the H3K27me3 repressive state at cannabinoid loci that could be addressed by dCas9-KDM6A and the propagation-induced CG hypomethylation that could be addressed by dCas9-DNMT3A; these editing strategies remain hypotheses that have not yet been tested in Cannabis. We synthesize functional evidence from Catharanthus roseus, Papaver somniferum, and Artemisia annua demonstrating that equivalent chromatin switches control secondary metabolite yield in medicinal plants, evaluate which dCas9-effector architectures are most appropriate for each Cannabis chromatin target, and identify the critical mechanistic gaps that must be closed before epigenome editing can be rationally deployed for cannabinoid yield enhancement in Cannabis. Full article

Plant tissue culture is a crucial part of biotechnology that supports crop improvement, plant conservation, and other related fields. Although tissue culture has been successfully used in many plants, low regeneration and transformation rates still exist in some species. Agrobacterium-mediated transformation is commonly used in genetic engineering, but its effectiveness depends heavily on establishing a reliable in vitro regeneration system through organogenesis or somatic embryogenesis. Over the past decade, substantial progress has been made in understanding the molecular basis of regeneration; however, most reviews have focused on individual aspects such as hormone regulation or transcription factor networks in isolation. In contrast, this review provides a comprehensive and integrated framework that systematically links four critical layers—wound signaling, hormonal regulation, developmental regulators, and epigenetic modifications—into a unified regulatory network governing plant regeneration. Furthermore, we highlight recent cutting-edge advances, including artificial intelligence-assisted prediction, single-cell and spatial transcriptomics, epigenome editing, and CRISPR-based activation systems, and we discuss their transformative potential in overcoming genotype-dependent recalcitrance. By synthesizing classical regulatory mechanisms with emerging technologies, this review offers a forward-looking perspective that distinguishes it from earlier publications and provides both theoretical foundations and practical strategies for improving plant regeneration and genetic transformation. Full article

Factors regulating the genesis and expansion of basal progenitor cell sub-populations, including intermediate progenitor cells, are critical determinants of cortical neurogenesis and brain growth. Epigenetic (chromatin) marks have emerged as notable regulators of cortical development. However, it is unclear how these factors specifically interact in the epigenome to orchestrate brain development. Here, we combined in vivo electroporation and a CRISPR-dead (d)Cas9 system to probe the contribution of a specific histone modification mark, H3K9ac (H3K9 acetylation), in the epigenome of isolated intermediate progenitor cells in developing mouse cortices. CRISPR-dCas9-mediated addition of H3K9ac at the promoter region of lncRNA Sox1ot resulted in Sox1ot upregulation, with attendant increase in the intermediate progenitor pool and augmented neurogenesis. Thus, we found an interplay between H3K9ac and Sox1ot, which drives the amplification of cortical intermediate progenitor cell population. By targeting H3K9ac to the Sox1ot promoter, we were able to stimulate the proliferation of intermediate progenitor cells leading to enhanced cortical neurogenesis. In essence, we have identified H3K9ac as a key epigenetic regulator of Sox1ot expression, which may dynamically be involved in intermediate progenitor cell pool expansion during brain development and evolution. Full article

Epigenetic regulation has played a fundamental role in the evolution of plant reproduction. Across more than a billion years, ancestral genome-defense mechanisms in early eukaryotes were progressively expanded, diversified, and repurposed throughout the green lineage. Streptophyte algae assembled the first plant-specific methylation and small RNA systems, providing pre-adaptations for terrestrial reproduction. In bryophytes and early vascular plants, these systems became integrated into gametophyte development, sporogenesis, and meiotic genome protection. Seed plants experienced substantial diversification and expansion of chromatin regulators and small RNA machinery, enabling increasingly sophisticated control of cone, ovule, and embryo development. Angiosperms underwent the most dramatic rewiring of epigenetic pathways, including gene-family diversification, subfunctionalization, and the emergence of genomic imprinting, endosperm-specific demethylation, and lineage-specific reproductive small RNAs such as phasiRNAs. Convergent solutions, including imprinting, meiotic transposable element (TE) silencing, and TE-derived regulatory elements, arose independently across lineages. Rather than reflecting the emergence of entirely new molecular machinery, these innovations illustrate repeated functional co-option and regulatory rewiring of deeply conserved epigenetic modules. Ecological and life-history pressures further shaped epigenetic diversification, linking environmental stress, mating systems, and domestication to reproductive epigenetic plasticity. Recent evidence further demonstrates that epigenetic plasticity underlies the recurrent evolution of alternative reproductive strategies such as apomixis and contributes to reproductive responses to environmental stress. Advances in comparative epigenomics, single-cell technologies, and epigenome editing are now providing unprecedented opportunities to reconstruct the evolutionary history of reproductive epigenetic pathways and to harness them for crop improvement. Together, these findings reveal epigenetic regulation as a dynamic, modular, and deeply evolvable framework that has repeatedly enabled reproductive innovation throughout plant evolution. Full article

Soybean is a strategic crop for global protein and vegetable oil supply chains; however, genetic improvement remains constrained by genotype-dependent regeneration, variable transformation efficiency, and regulatory concerns regarding stable transgene integration. This review synthesizes emerging DNA-free and genotype-independent genome-editing frameworks for soybean, where genotype independence is defined as the ability to recover fertile, non-chimeric edited plants across elite germplasm. We critically examine the soybean genome-editing toolbox, including CRISPR-Cas9, Cas12a, multiplex editing systems, base editing, and prime editing, and discuss persistent bottlenecks associated with target selection, off-target assessment, editability, and plant recovery. Particular emphasis is placed on artificial intelligence (AI)-assisted approaches that integrate genomic, epigenomic, chromatin-accessibility, and multi-omics datasets to improve target prioritization, guide RNA design, off-target prediction, and locus- and genotype-specific editability assessment. We further evaluate DNA-free genome-editing technologies, including CRISPR-Cas ribonucleoproteins, transient RNA-based systems, and nanocarrier-mediated delivery platforms, highlighting their potential to generate non-integrative edits while reducing prolonged nuclease exposure. In addition, we discuss regeneration reprogramming strategies based on developmental regulators and morphogenic modules, including BBM-WUS, GRF-GIF, de novo meristem induction, and somatic embryogenesis, as enabling technologies for overcoming cultivar-dependent regeneration barriers. Importantly, this review proposes an integrated AI-to-field framework that connects target discovery, editability prediction, DNA-free editing, regeneration reprogramming, phenotypic validation, and breeding deployment into a unified soybean improvement pipeline. We further highlight emerging opportunities in multi-omics-guided target discovery, genotype-aware prediction models, regeneration-aware editing strategies, and closed-loop machine-learning systems that continuously improve editing decisions through experimental feedback. Collectively, these convergent innovations provide a practical foundation for accelerating the development of climate-resilient, nutritionally enhanced, and industry-ready soybean cultivars. Full article

Soil salinization represents one of the most severe abiotic constraints on global agricultural productivity, threatening crop yields and food security across increasingly large areas of cultivated land. Among the molecular mechanisms underlying plant physiological adaptation to salinity, histone methylation has emerged as a central epigenetic regulatory layer governing salt-responsive transcriptional reprogramming through the coordinated and opposing actions of histone methyltransferases, demethylases, and reader proteins at specific chromatin loci. Recent advances reveal how dynamic changes in activating marks, principally H3K4me3 and H3K36me3, and repressive marks, H3K9me2 and H3K27me3, orchestrate the activation of stress-responsive gene networks and the silencing of growth-incompatible programs under salt stress. How these modifications establish and sustain stress memory across somatic and transgenerational timescales is discussed. Recent technological advances, including single-cell epigenomics, CUT&RUN, CUT&Tag, and spatial transcriptomics, are assessed as future research priorities. The application of CRISPR/dCas9-based epigenome editing and epigenetic breeding strategies for improving crop salt tolerance is further explored. Full article

Drought stress is the most pervasive abiotic constraint on global crop productivity, with projected intensification under climate change threatening the yields of staple crops including wheat, rice, maize, and legumes. Conventional breeding approaches have delivered limited gains against drought tolerance, constrained by the polygenic and multifactorial nature of stress adaptation, the complexity of genotype-by-environment interactions, and the inadequacy of field-based phenotyping under variable stress conditions. Omics technologies, including genomics, transcriptomics, proteomics, metabolomics, epigenomics, and phenomics, have substantially advanced the molecular dissection of drought tolerance by enabling high-resolution characterization of stress-responsive genes, regulatory networks, adaptive proteins, and metabolic reprogramming pathways. Specific traits targeted include root system architecture and depth, osmotic adjustment capacity through proline and glycine betaine accumulation, antioxidant defense mechanisms, ABA-mediated stomatal regulation, LEA protein accumulation, epigenetic stress memory, and yield stability under water deficit. This review systematically examines omics-based strategies for drought stress mitigation across major crops, highlighting individual omics contributions, multi-omics integration frameworks, computational tools including machine learning and AI-driven predictive modelling, and translational breeding applications. Case studies in wheat, rice, maize, and legumes illustrate how omics-driven approaches accelerate precision breeding for drought resilience through marker-assisted selection, genomic selection, and CRISPR-based gene editing. Challenges including data integration complexity, high implementation costs, limited cross-species transferability, and the need for field-scale validation of microbiome-based strategies are critically addressed. Future perspectives encompassing single-cell and spatial omics, AI-driven predictive breeding, digital agriculture integration, and international data governance frameworks are discussed. By aligning with climate-smart agriculture principles, multi-omics approaches provide a robust and transformative foundation for developing drought-resilient crop cultivars suitable for water-limited production systems worldwide. Full article

Epigenetic regulation plays a significant role in controlling plant secondary metabolism and offers a promising strategy to enhance medicinal alkaloid production without altering the genomic sequence. This review highlights major plant epigenetic mechanisms, including DNA methylation, histone modifications, chromatin remodeling, and non-coding RNAs, and their influence on alkaloid biosynthetic pathways. Particular emphasis is placed on chemical epigenetic modulators, such as DNA methyltransferase and histone deacetylase inhibitors, which can activate silent metabolic genes in a rapid, reversible, and non-GMO manner. Evidence from medicinal plants demonstrates tissue-, dose-, and time-dependent enhancement of alkaloid production. The review further discusses emerging approaches including CRISPR-based epigenome editing, multi-omics integration, and advanced bioinformatics for systems-level understanding of epigenetic regulation. Despite challenges, such as limited species-specific datasets, and concerns regarding long-term stability, epigenetic modulation represents a promising tool for sustainable alkaloid production with important pharmaceutical and industrial applications. Full article

Cucurbit crops—including cucumber (Cucumis sativus), watermelon (Citrullus lanatus), and melon (Cucumis melo)—are of major economic and nutritional importance worldwide. Yet their productivity and quality are severely compromised by foliar fungal diseases, particularly powdery mildew (PM), downy mildew (DM), and target leaf spot (TLS). While PM and DM have been extensively studied, TLS has emerged as an increasingly prevalent and damaging disease in key production regions, yet it remains comparatively understudied—especially with respect to its molecular basis and comparative pathobiology relative to PM and DM. Current reliance on chemical fungicides is hampered by escalating pathogen resistance and concerns over residual toxicity, whereas conventional breeding approaches face inherent limitations in pyramiding durable, broad-spectrum resistance against multiple pathogens. In this context, cucumber has emerged as a pivotal model species for dissecting foliar disease resistance mechanisms in cucurbits, supported by a high-quality reference genome, extensive resequencing datasets, diverse germplasm collections, and an efficient Agrobacterium-mediated transformation system. Despite these advantages, existing reviews predominantly address PM or DM resistance in isolation; comprehensive syntheses integrating TLS resistance advances—and critically, cross-disease comparisons of genetic architecture, transcriptional reprogramming, and defense signaling—are notably scarce. Furthermore, the translational pipeline—from gene discovery and functional validation to deployment in marker-assisted or genome-edited breeding—lacks systematic evaluation. Here, we provide a focused, cucumber-centered review that (i) synthesizes recent progress in mapping QTLs and GWAS loci, and characterizing key resistance-associated gene families (such as NLRs, RLKs, PR genes) conferring resistance to PM, DM, and TLS; (ii) integrates transcriptomic, epigenomic, and proteomic evidence to delineate conserved versus pathogen-specific host responses; (iii) highlights breakthroughs and unresolved questions in TLS resistance research, including the roles of novel susceptibility factors and non-canonical immune regulators; and (iv) critically assesses bottlenecks in translating resistance genes into practical breeding outcomes—such as linkage drag, functional redundancy, and genotype-by-environment interactions—and proposes empirically grounded strategies for accelerating molecular design of multi-disease-resistant cultivars. Collectively, this review aims to bridge fundamental insights with applied breeding goals, offering a conceptual and strategic framework for integrated management of foliar fungal diseases and the development of durable, broad-spectrum resistance in cucurbits. Full article