Many plant species can propagate asexually or be regenerated in vitro; but asexual offspring are more likely to maintain environmentally induced epigenetic marks, for instance, inheritance of the prolonged cold-induced 'vernalized state' in overwintering plants through asexual reproduction. Here we demonstrate that 'vernalized state' is reprogrammed during Arabidopsis asexual propagation through somatic embryogenesis. This overturns a long-standing idea, that the vernalized state could not be reset through asexual reproduction, and provides a strategy to erase parental effects on offspring during asexual reproduction.

Persister cells, a multidrug-tolerant bacterial subpopulation, pose challenges to disease control, with research hindered by technical limitations. In this study, single-cell Raman spectroscopy (SCRS) combined with heavy water (D2O) labeling was employed to explore the metabolic activity and compositional changes of Vibrio splendidus persisters. Raman analysis revealed distinct biochemical profiles between persisters and normal cells: persisters reduced nucleic acid-related band intensity but increased lipid and polysaccharide-related bands. Using the deuterium incorporation rate as a quantitative indicator of biosynthetic activity, we found that biosynthesis remained active under antibiotic stress during the exponential growth phase, while it declined under identical stress conditions in the stationary phase. It is revealed that persisters are not in a fully dormant state, and diverse metabolic patterns are found among persister cells. Furthermore, our screening results identified vitamin C is a potent inhibitor of persister formation in a concentration-dependent manner. It may be associated with the down-regulation of the stringent response genes relA and spoT. Collectively, this study demonstrates that SCRS combined with D2O labeling is a powerful tool for investigating the physiological characteristics of V. splendidus persisters, and provides metabolic modulation strategies for the control of persistent infection in aquaculture.

In many plants, flowering is timed by seasonal changes in the length of daylight (photoperiod). In Arabidopsis thaliana, long-day exposure results in an increasing buildup of the CONSTANS (CO) protein toward the end of daylight, which activates the expression of the major florigen gene FLOWERING LOCUS T (FT) to induce flowering. CO-mediated FT activation must be properly controlled to prevent an excessive florigen production and precocious flowering under inductive photoperiods, but the underlying mechanism remains elusive. Here, we report an auto-repression mechanism to prevent excessive FT production in inductive photoperiods. We show that the transcription factor FD is expressed in leaf veins and complexes with FT to recognize several cis-regulatory DNA motifs in FT promoter. FT-FD antagonizes CO-mediated FT activation to feedback down-regulate FT expression and thus prevent its excessive induction by long-day signals, thereby precluding precocious transition to flowering. Furthermore, we found that in the facultative short-day plant soybean, an FT homolog directly represses its own expression. Thus, the auto-repression of FT or an FT homolog is a conserved mechanism to prevent excessive production of this potent floral regulator, ensuring the floral transition at a proper time to balance vegetative growth with reproductive success and maximize plant production.

Vibrio splendidus is an important opportunistic pathogen that causes diseases in aquatic animals, and its persisters increase the difficulty of aquaculture disease control. The stringent response is a central pathway in bacteria for coping with environmental stress, and the signaling molecule (p)ppGpp, synthesized under the regulation of RelA/SpoT homologs, is closely associated with persister formation and virulence modulation. However, the regulatory mechanisms linking the stringent response to persister formation and virulence in V. splendidus remain unclear. In this study, the core gene deletion strains ΔrelA and ΔrelAΔspoT were constructed via homologous recombination. Combined with D2O single-cell Raman spectroscopy, transcriptomics, and phenotypic assays, we systematically characterized the biological effects of stringent response inactivation. The results showed that the loss of relA and spoT significantly reduced persister formation and key virulence traits while enhancing biofilm formation. Single-cell Raman spectroscopy analysis indicated that persisters remained metabolically active, accompanied by changes in different cellular components. Transcriptome analysis revealed that the absence of stringent response affected multiple pathways, including ribosomal function, energy metabolism, two-component systems, and quorum sensing. Additionally, the sigma factor RpoS may potentially exert a compensatory function in ΔrelAΔspoT strain, but this requires further validation. In conclusion, the stringent response positively regulates persister formation and virulence in V. splendidus, despite the existence of complex regulatory mechanisms. This study provides a theoretical basis for the development of anti-infective strategies targeting stringent response in aquatic pathogens.

The global decline of coral reefs underscores the urgency of understanding how corals enhance resilience in stressful environmental conditions. As metaorganisms, or holobionts, corals rely on dynamic interactions with their associated microbial communities, with bacterial restructuring proposed as a potential mechanism of holobiont adaptation. Here, we reconstructed coral symbiosis in the bleached tissues of Acropora hyacinthus by introducing beneficial bacteria and thermally domesticated Symbiodiniaceae to assess their roles in bleaching recovery. Raman spectroscopy metabolomics (RS metabolomics) enables in situ detection, providing temporal evidence of metabolic exchange within the tripartite relationship among corals, Symbiodiniaceae, and associated bacteria. This study highlights the potential of acclimation-based approaches in the development of thermotolerant Symbiodiniaceae strains. Furthermore, by manipulating this bacterial community, we identified a bacterium that enhances the thermal and light tolerances of acclimated Symbiodiniaceae, offering new insights into coral reef homeostasis strategies. Our results also indicate that the introduction of beneficial bacterial strains and thermotolerant Symbiodiniaceae, including proteins, lipids, and carbohydrates, increased nutrient levels in the coral host. This work introduces a microbial-assisted holobiont reconstitution framework that advances understanding of cross-kingdom metabolic integration and offers a mechanistic basis for engineering coral resilience under climate stress. The findings could provide insights into leveraging beneficial microbiota to mitigate thermal-induced coral bleaching, ultimately informing conservation strategies for marine ecosystems.

Microbial single-cell Raman spectroscopy (SCRS) has emerged as a powerful tool for label-free phenotyping, enabling rapid characterization of microbial diversity, metabolic states, and functional interactions within complex communities. However, high-throughput SCRS datasets often contain spectral anomalies from noise and fluorescence interference, which obscure microbial signatures and hinder accurate classification. Robust algorithms for outlier detection and microbial ramanome analysis remain underdeveloped. Here, we introduce RamEx, an R package specifically designed for high-throughput microbial ramanome analyses with robust quality control and phenotypic classification. At the core of RamEx is the Iterative Convolutional Outlier Detection (ICOD) algorithm, which dynamically detects spectral anomalies without requiring predefined thresholds. Benchmarking on both simulated and real microbial datasets—including pathogenic bacteria, probiotic strains, and yeast fermentation populations—demonstrated that ICOD achieves an F1 score of 0.97 on simulated datasets and 0.74 on real datasets, outperforming existing approaches by at least 19.8

Adult mammalian stem cells typically maintain stem cell identity through proliferative quiescence. In contrast, we demonstrate that stem cell maintenance in Arabidopsis bud precursor cells requires active cell-cycle progression. Inhibiting division silences the shoot meristem marker gene SHOOT MERISTEMLESS ( STM ) and promotes differentiation. Whereas proliferation dilutes H3K27me3 levels to counteract silencing. Meanwhile, we identified two classes of transcription factors recruiting polycomb repressive complex 2 (PRC2) to epigenetically silence STM . The balance between these forces establishes a cell cycle-coupled epigenetic “Sisyphus” mechanism that maintains pluripotency. This cell fate switch is bistable; modeling and experimental data confirm that prolonged quiescence triggers irreversible differentiation. We propose that sequence-dependent PRC2 recruitment in plants enables precise silencing of fate-determining genes, while cell proliferation sustains pluripotency by resetting epigenetic marks. ### Competing Interest Statement The authors have declared no competing interest.

Cellular metabolic state and its heterogeneity are pivotal features that determine fermentation productivity, yet label-free monitoring has generally been difficult. Employing beer fermentation by Saccharomyces pastorianusas a model, we demonstrated that temporal sampling of ramanomes, the collection of spontaneous Single-Cell Raman Spectra (SCRS) from an isogenic population, provides rich insights into the profiles and inter-conversion of both intra- and extra-cellular metabolites. Among 43 extracellular metabolic phenotypes, ramanomes successfully modeled 19 of them, including the extracellular levels of four alcohols, four esters, four amino acids, two acids, and four mono- and di-saccharide substrates, plus the alcohol-to-ester ratio. Moreover, Intra-Ramanome Correlation Analysis (IRCA) revealed potential metabolic interactions in pairs of intracellular metabolites, extracellular metabolites, and medium substrates. Specifically, carbohydrates were the most active intracellular metabolites, while proteins significantly influenced alcohol and ester synthesis on Day 1 of fermentation. Additionally, both alcohols and esters showed negative correlations with extracellular amino acids and acids. The global-IRCN average degree, reflecting metabolic network complexity, increased over time and was positively correlated with extracellular levels of key products such as n-propanol and various esters, while negatively correlated with acetic acid and certain sugars. Therefore, by enabling non-destructive, label-free, and rapid modeling of both intra- and extracellular metabolite levels, ramanomics can find wide applications in process monitoring and control.

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.

The transition from vegetative growth to reproduction in flowering plants is often timed by seasonal changes in day length (photoperiod). In the long-day (LD) plant Arabidopsis thaliana, the photoperiod pathway induces a daily rhythmic activation of the florigen gene FLOWERING LOCUS T (FT) to promote the floral transition. Under inductive LDs, FT expression is activated around dusk, but to be repressed overnight and into the early afternoon the next day. Here, we report that AtING1 and AtING2, Arabidopsis homologs of the mammalian Inhibitor of Growth (ING) proteins, read di- and tri-methylated histone-3 lysine 4 (H3K4me2/me3) on FT chromatin and further recruit Polycomb-repressive complex 2 (PRC2) to repress FT expression at night and into the early afternoon the next day, following FT activation at dusk. This prevents precocious flowering under inductive LDs. Our study reveals that the H3K4me2/me3-ING1/2-PRC2 module timely represses FT expression following the daily rhythmic FT activation, to prevent excessive FT expression and thus precisely control flowering time, in response to inductive photoperiodic signals.

Anthocyanins are plant pigments that play diverse roles in plant growth, adaptation, and stress tolerance. Anthocyanin biosynthesis is tightly regulated, but the underlying regulatory mechanisms remain unclear. Here, we identify a regulatory module composed of the DNA-binding protein VAL1 (VIVIPAROUS1/ABI3-LIKE 1) and a SIN3 (SWI-INDEPENDENT 3)-like histone deacetylase complex that dynamically regulates anthocyanin biosynthesis in Arabidopsis thaliana. Under normal growth conditions, VAL1 recruits the SNL (SIN3-Like)-HDA19 (HISTONE DEACETYLASE 19) complex (SNL-HDA19c) to the PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1) locus for histone deacetylation. Moreover, the negative regulators of jasmonic acid (JA) signaling, JASMONATE-ZIM DOMAIN (JAZ) proteins, interact with VAL1 and further stabilize the binding of VAL1 and SNL-HDA19c to PAP1 chromatin. These molecular interactions transcriptionally repress PAP1 and inhibit anthocyanin biosynthesis. Upon JA accumulation, JAZs are degraded, resulting in the release of both VAL1 and SNL-HDA19c from the PAP1 chromatin. This release leads to an immediate increase in histone acetylation, promoting transcriptional activation of PAP1 and anthocyanin production. These findings elucidate a regulatory module (VAL1-JAZ-SNL-HDA19c) that represses anthocyanin biosynthesis under normal growth conditions and further reveal how the stress hormone JA rapidly induces anthocyanin production, enabling plants to adapt to their growth conditions.

In many flowering plants, the developmental switch from vegetative growth to reproduction (flowering) is timed by seasonal changes in the length of daylight (photoperiod). Under inductive day lengths, the photoperiod pathway typically generates rhythmic expression of a transcriptional activator for florigen production. In the facultative long-day plant Arabidopsis thaliana , long-day exposure results in increasing buildup of the CONSTANS (CO) protein towards the end of daylight, and CO activates the expression of the major florigen gene FLOWERING LOCUS T ( FT ) to confer long-day induction of flowering. CO-mediated FT activation must be properly controlled to prevent an excessive florigen production and precocious flowering under inductive long days, but the underlying mechanism remains elusive. Here, we report an auto-repression mechanism to prevent excessive FT production in inductive photoperiods. We show that the transcription factor FD is expressed in leaf veins and complexes with FT to recognize several cis -regulatory DNA motifs in FT promoter. FT-FD antagonizes CO-mediated FT activation to feedback down-regulate FT expression and thus prevent its excessive induction by long-day signals, thereby precluding precocious transition to flowering. Furthermore, we found that in the facultative short-day plant soybean, an FT homolog directly represses its own expression. Thus, the auto-repression of FT or an FT homolog is a conserved mechanism to prevent excessive production of this potent floral regulator in plants, ensuring the floral transition at a proper time to balance vegetative growth with reproductive success and maximize plant production. ### Competing Interest Statement The authors have declared no competing interest. National Key R&D Program of China, , National Natural Science Foundation of China, ,

Temperature plays a pivotal role in plant growth and development, with flowering time being particularly sensitive to thermal changes. Understanding the molecular mechanisms of temperature-regulated flowering is crucial for enhancing plant adaptability and improving productivity. This review systematically summarised the molecular mechanisms underlying flowering regulation by ambient temperature fluctuations (excluding vernalisation treatments typically requiring prolonged exposure to 0°C-6°C for weeks or months) in Arabidopsis and three key short-day crops: soybean (Glycine max), rice (Oryza sativa), and maize (Zea mays). We provide a comprehensive overview of the temperature sensors involved in flowering regulation, focusing on how key molecular components, including photoreceptors, transcription factors, chromatin modifiers, miRNAs, and hormone, mediate temperature responses that regulate flowering time. Although significant insights have been gained from Arabidopsis, understanding of these mechanisms in crops remains limited, hindering advances in developing temperature-adaptive varieties. We discuss the limitations of the current study and propose future research directions, including uncovering crop-specific temperature regulation mechanisms, studying flowering responses under dynamic conditions, and exploring strategies for breeding temperature-adaptive crops. By clarifying the flowering mechanisms that respond to non-vernalisation temperatures, this review aims to guide future efforts to improve crop resilience and adaptation strategies in the face of climate change.

Polycomb protein-mediated transcriptional repression plays a crucial role in the regulation of responses to environmental stimuli in multicellular eukaryotes, but the underlying signalling events remain elusive. During Arabidopsis vernalization, prolonged cold exposure results in the formation of a Polycomb-repressed domain at the potent floral repressor FLC to confer its stable silencing upon temperature rise or epigenetic ‘memory of prolonged cold’, enabling the plants to bloom in spring. Here we report that the evolutionarily conserved casein kinase CK2 phosphorylates and thus stabilizes histone 3 lysine-27 (H3K27) methyltransferases (PRC2 subunits) to promote H3K27 trimethylation throughout the Arabidopsis genome. We found that prolonged cold induces progressive CK2 accumulation, leading to a gradual accumulation of cellular PRC2. We further show that the cold-CK2–PRC2 signalling promotes increasing PRC2 enrichment on FLC chromatin during prolonged cold exposure as well as post-cold PRC2 spreading across FLC to establish a Polycomb-repressed domain for FLC repression in warmth. Thus, this signalling cascade transduces prolonged cold exposure, but not cold spells, into epigenetic memory of prolonged cold in warmth during vernalization. CK2 phosphorylation motifs are widely present in H3K27 methyltransferases from plants and animals. Our study reveals a new layer of control of PRC2 activity in multicellular organisms. This study reports that the CK2 kinase–H3K27 methyltransferase (PRC2) signalling promotes genome-wide H3K27 trimethylation and the formation of epigenetic cold memory at a potent floral repressor in Arabidopsis in response to prolonged cold exposure.

The staple food crop winter bread wheat ( Triticum aestivum ) acquires competence to flower in late spring after experiencing prolonged cold in temperate winter seasons, through the physiological process of vernalization. Prolonged cold exposure results in transcriptional repression of the floral repressor VERNALIZATION 2 ( TaVRN2 ) and activates the expression of the potent floral promoter VERNALIZATION 1 ( TaVRN1 ). Cold-induced TaVRN1 activation and TaVRN2 repression are maintained in post-cold vegetative growth and development, leading to an epigenetic ‘memory of winter cold’, enabling spring flowering. When and how the cold memory is reset in wheat is essentially unknown. Here we report that the cold-induced TaVRN1 activation is inherited by early embryos, but reset in subsequent embryo development, whereas TaVRN2 remains silenced through seed development, but is reactivated rapidly by light during seed germination. We further found that a chromatin reader mediates embryonic resetting of TaVRN1 and that chromatin modifications play an important role in the regulation of TaVRN1 expression and thus the floral transition, in response to developmental state and environmental cues. The findings define a two-step molecular mechanism for re-establishing vernalization requirement in common wheat, ensuring that each generation must experience winter cold to acquire competence to flower in spring.

It is well known that genome organizers, like mammalian CCCTC-binding factor (CTCF) or Drosophila architectural proteins CP190 and BEAF-32, contribute to the three-dimensional (3D) organization of the genome and ensure normal gene transcription. However, bona fide genome organizers have not been identified in plants. Here, we show that EMBRYONIC FLOWER1 (EMF1) functions as a genome modulator in Arabidopsis. EMF1 interacts with the cohesin component SISTER CHROMATIN COHESION3 (SCC3), and both proteins are enriched at compartment domain (CD) boundaries. Accordingly, emf1 and scc3 show a strength decrease at the CD boundary in which these proteins colocalize. EMF1 maintains CD boundary strength, either independently or in cooperation with histone modifications. Moreover, EMF1 is required to maintain gene-resolution interactions and to block long-range aberrant chromatin loops. These data unveil a key role of EMF1 in regulating 3D chromatin structure.

Covalent modifications on DNA and histones can regulate eukaryotic gene expression and are often referred to as epigenetic modifications. These chemical reactions require various metabolites as donors or co-substrates, such as acetyl coenzyme A, S-adenosyl-l-methionine, and α-ketoglutarate. Metabolic processes that take place in the cytoplasm, nucleus, or other cellular compartments may impact epigenetic modifications in the nucleus. Here, we review recent advances on metabolic control of chromatin modifications and thus gene expression in plants, with a focus on the functions of nuclear compartmentalization of metabolic processes and enzymes in DNA and histone modifications. Furthermore, we discuss the functions of cellular metabolisms in fine-tuning gene expression to facilitate the responses or adaptation to environmental changes in plants.

The fusion of the exonuclease Trex2 with the Cas9 protein significantly enhanced the efficiency of genome editing in hexaploid common wheat, particularly for the simultaneous editing of multiple favorable alleles within a single generation, thereby facilitating genome editing-assisted breeding in polyploid crops.