Plant de novo organogenesis depends on callus formation, yet the epigenetic mechanisms governing organ regeneration remain poorly understood. EMBRYONIC FLOWER 2 (EMF2), a core component of Polycomb Repressive Complex 2, mediates transcriptional repression through H3K27me3 and is essential for root regeneration in Arabidopsis. Here, we investigate how EMF2-mediated H3K27me3 shapes chromatin architecture and gene expression during root regeneration. We combined time-course transcriptome profiling, H3K27me3 chromatin immunoprecipitation, in situ Hi-C, and a probe hybridization-based Capture Hi-C approach to analyze chromatin organization and gene expression dynamics in wild-type and emf2 calli during root induction. Although emf2 calli lost root regeneration capacity, they retained responsiveness to root induction signals. Despite large-scale reduction of H3K27me3 across the emf2 genome, many genes remained transcriptionally inactive, coinciding with the formation of new long-range chromatin interactions and weakened intra- and peri-domain contacts. Genes with low basal transcription were preferentially derepressed following extensive H3K27me3 loss. Our results demonstrate that large-scale reduction of H3K27me3 in emf2 drives dynamic reorganization of chromatin architecture in Arabidopsis callus, providing new insights into how histone modification and three-dimensional chromatin topology coordinately regulate gene expression during plant regeneration.

Background Cannabis sativa, a dioecious species characterized by striking sexual dimorphism and exceptional sexual plasticity, presents a powerful model for exploring floral development and the evolution of dioecy. The unique male and female flowers each feature a single perianth whorl that differs significantly in structure and developmental trajectory, raising key questions about organ identity and developmental regulation that remain largely unresolved. Results Analysis of MADS-box gene expression reveals that, despite sexual dimorphism and structural reduction, both sexes conform to the canonical ABCDE model of floral organ identity, indicating unisexual flower development relies on conserved regulatory networks. We further demonstrate that male flowers undergo more extensive transcriptional reprogramming than female flowers, suggesting a more complex developmental trajectory. By integrating molecular and morphological evidence, we provide support for a petaloid interpretation of the male perianth, offering new insight into the identity of the Cannabis floral organ. Conclusions This study provides molecular and morphological evidence strongly suggesting that the male Cannabis perianth possesses petaloid characteristics, offering a new perspective on a key floral trait. Our findings demonstrate that despite structural reduction and unisexual development, Cannabis employs conserved ABCDE regulatory networks, suggesting that B-class genes link petal and stamen development. This work establishes a developmental-genetic framework for understanding the evolution of dioecy and highlights how wind pollination may drive perianth reduction while maintaining underlying genetic programs.

Plant callus cells possess a great capacity to regenerate organs or even whole plants. The mechanisms by which these cells maintain a proliferative state while retaining their pluripotent identity are poorly understood. By taking a multi-omics approach integrating epigenetic regulation (via chromatin immunoprecipitation and sequencing) with transcriptional output, we identify two complementary strategies that support callus cell pluripotency. First, callus cells prevent differentiation by promoting proliferation through activating cell cycle genes, and concurrently repress differentiation-promoting factors via H3K27me3. Second, callus cells exhibit a unique transcriptional profile enriched in diverse developmental regulators, thereby maintaining a primed pluripotent state that enables a rapid regenerative response. This strategy relies on a mechanism to silence the pluripotency network in response to regenerative stimuli, allowing a single developmental pathway to predominate. To test whether the Polycomb Repressive Complex 2 (PRC2), which mediates H3K27me3 silencing, is essential for maintaining callus identity and regenerative capacity, we analyzed the transcriptional state of Arabidopsis thaliana wild-type and PRC2 mutant emf2 calli. In emf2 mutants, many differentiation-associated transcription factors were up-regulated, and regenerative capacity was severely impaired. Our findings provide new insight into how pluripotency is regulated. We propose a novel model in which PRC2 governs callus identity and regenerative potential.

Next-generation sequencing has revolutionized epigenetics research, enabling a comprehensive analysis of DNA methylation and histone modification profiles to explore complex biological systems at unprecedented depth. Deciphering the intricate epigenetic mechanisms that regulate gene activity presents significant challenges, including the issue of analyzing heterogeneous cell populations in bulk. Bulk analysis introduces bias and can obscure crucial information by averaging readouts from distinct cells. Various approaches have been developed to address this issue, such as cell-type-specific enrichment or single-cell sequencing techniques. However, the need for transgenic lines with fluorescent markers, along with technical challenges such as efficient protoplast isolation and low yield, limits their widespread adoption and use in multi-omic studies. This review discusses the pros and cons of these approaches, providing a valuable basis for selecting the most suitable strategy to minimize heterogeneity. We will also highlight the use of cotyledon-derived callus as an ex vivo system as a simple, accessible, and robust platform for enabling high-throughput multi-omic analyses.

Plant callus cells possess a remarkable ability to regenerate organs that often differ from their respective origins or even entire new individuals. Yet, the mechanisms underlying their pluripotent state remain elusive. We propose a strategy that involves two independent mechanisms to endow callus cells with pluripotency: (1) maintaining a unique transcriptional profile, characterised by the expression of genes from diverse developmental pathways that allows rapid response to developmental cues; (2) preventing premature differentiation through H3K27 methylation-mediated silencing of key transcription factors such as WUCHEL and SPEECHLESS. This strategy relies on a mechanism to silence the pluripotency network upon regenerative stimuli, enabling a single developmental pathway to dominate. Our study reveals that the EMF2 complex, a key regulator of H3K27 tri-methylation, plays a crucial role in this process. Callus derived from the emf2 mutant, deficient in H3K27me3, exhibits severely impaired regeneration. Comparative analyses of chromatin states and transcription profiles between wild-type and emf2 calli revealed that the loss of EMF2 leads to upregulation of key transcription factors in callus, and identified the genes regulated solely by EMF2. Our findings suggest that suppressing pluripotency networks through H3K27me3 is essential for executing specific developmental programs to ensure effective regeneration.

Background: Cannabis sativa , a dioecious species characterized by striking sexual dimorphism and exceptional sexual plasticity, presents a powerful model for exploring floral development and the evolution of dioecy. The unique male and female flowers each feature a single perianth whorl that differs significantly in structure and developmental trajectory, raising key questions about organ identity and developmental regulation that remain largely unresolved. Results: We demonstrate that male flowers undergo more extensive transcriptional reprogramming than female flowers, suggesting a more complex developmental trajectory. Analysis of MADS-box gene expression reveals that, despite sexual dimorphism and structural reduction, both sexes conform to the canonical ABCDE model of floral organ identity, indicating unisexual flower development relies on conserved regulatory networks. Critically, by integrating molecular and morphological evidence, we demonstrate that the male perianth exhibits petal-like identity, addressing a longstanding uncertainty in Cannabis organ morphology. Conclusions: This study provides molecular and morphological evidence strongly suggesting that the male Cannabis perianth possesses petaloid characteristics, offering a new perspective on a key floral trait. Our findings demonstrate that despite structural reduction and unisexual development, Cannabis employs conserved ABCDE regulatory networks, suggesting that B-class genes link petal and stamen development. This work establishes a developmental-genetic framework for understanding dioecy evolution and highlights how wind pollination may drive perianth reduction while maintaining underlying genetic programs.

In plant tissue culture, callus formation serves as a crucial mechanism for regenerating entire plants, enabling the differentiation of diverse tissues. Researchers have extensively studied the influence of media composition, particularly plant growth regulators, on callus behavior. However, the impact of the physical properties of the media, a well-established factor in mammalian cell studies, has received limited attention in the context of plant tissue culture. Previous research has highlighted the significance of gelling agents in affecting callus growth and differentiation, with Agar, Phytagel, and Gelrite being the most used options. Despite their widespread use, a comprehensive comparison of their physical properties and their subsequent effects on callus behavior remains lacking. Our study provides insights into optimizing plant tissue culture media by analyzing the physical properties of gelling agents and their impact on callus induction and differentiation. We compared the phenotypes of calli grown on media composed of these different gelling agents and correlated them to the physical properties of these media. We tested water retention, examined pore size using cryo-SEM, measured the media mechanical properties, and studied diffusion characteristics. We found that the mechanical properties of the media are the only quality correlated with callus phenotype.

BACKGROUND:Cannabis sativa L. is a versatile medicinal plant known for its therapeutic properties, derived from its diverse array of secondary metabolites synthesized primarily in female flower organs. Breeding cannabis is challenging due to its dioecious nature, strict regulatory requirements, and the need for photoperiod control to trigger flowering, coupled with highly dispersible pollen that can easily contaminate nearby female flowers. This study aimed to develop a protocol for in vitro flowering in cannabis, investigate factors affecting in vitro flower production, and generate viable in vitro seeds, potentially offering a method for producing sterile cannabinoids or advancing breeding techniques. RESULTS:We show that the life cycle of cannabis can be fully completed in tissue culture; plantlets readily produce inflorescences and viable seeds in vitro. Our findings highlight the superior performance of DKW medium with 2% sucrose in a filtered vessel and emphasize the need for low light intensity during flower induction to optimize production. The improved performance in filtered vessels suggests that plants conduct photosynthesis in vitro, highlighting the need for future investigations into the effects of forced ventilation to refine this system. All tested lines readily developed inflorescences upon induction, with a 100% occurrence rate, including male flowering. We revealed the non-dehiscent trait of in vitro anthers, which is advantageous as it allows for multiple crosses to be conducted in vitro without concerns about cross-contamination. CONCLUSION:The current work developed and optimized an effective protocol for in vitro flowering and seed production in cannabis, potentially providing a platform for sterile cannabinoid production and an efficient tool for breeding programs. This system allows for the full and consistent control of plant growth conditions year-round, potentially offering the reliable production of sterile molecules suitable for pharmacological use. As a breeding strategy, this method overcomes the complex challenges of breeding cannabis, such as the need for large facilities, by enabling the production of hundreds of lines in a small facility. By offering precise control over factors such as plant growth regulators, light intensity, photoperiod, and temperature, this system also serves as a valuable tool for studying flowering aspects in cannabis.

De novo shoot apical meristem (SAM) organogenesis during regeneration in tissue culture has been investigated for several decades, but the precise mechanisms governing early-stage cell fate specification remain elusive. In contrast to SAM establishment during embryogenesis, in vitro SAM formation occurs without positional cues and is characterized by autonomous initiation of cellular patterning. Here, we report on the initial stages of SAM organogenesis and on the molecular mechanisms that orchestrate gene patterning to establish SAM homeostasis. We found that SAM organogenesis in tobacco calli starts with protuberance formation followed by the formation of an intact L1 layer covering the nascent protuberance. We also exposed a complex interdependent relationship between L1 and WUS expression and revealed that any disruption in this interplay compromises shoot formation. Silencing WUS in nascent protuberances prevented L1 formation and caused the disorganization of the outer cell layers exhibiting both anticlinal and periclinal divisions, suggesting WUS plays a critical role in the proper establishment and organization of L1 during SAM organogenesis. We further discovered that silencing TONNEAU1 prevents the exclusive occurrence of anticlinal divisions in the outermost layer of the protuberances and suppresses the acquisition of L1 cellular identity and L1 formation, ultimately impeding SAM formation and regeneration. This study provides a novel molecular framework for the characterization of a WUS/L1 interplay that mediates SAM formation during regeneration.

Chromatin is a dynamic platform within which gene expression is controlled by epigenetic modifications, notably targeting amino acid residues of histone H3. Among them is lysine 27 of H3 (H3K27), the trimethylation of which by the Polycomb Repressive Complex 2 (PRC2) is instrumental in regulating spatiotemporal patterns of key developmental genes. H3K27 is also subjected to acetylation and is found at sites of active transcription. Most information on the function of histone residues and their associated modifications in plants was obtained from studies of loss-of-function mutants for the complexes that modify them. To decrypt the genuine function of H3K27, we expressed a non-modifiable variant of H3 at residue K27 (H3.3K27A ) in Arabidopsis, and developed a multi-scale approach combining in-depth phenotypical and cytological analyses, with transcriptomics and metabolomics. We uncovered that the H3.3K27A variant causes severe developmental defects, part of them are reminiscent of PRC2 mutants, part of them are new. They include early flowering, increased callus formation and short stems with thicker xylem cell layer. This latest phenotype correlates with mis-regulation of phenylpropanoid biosynthesis. Overall, our results reveal novel roles of H3K27 in plant cell fates and metabolic pathways, and highlight an epigenetic control point for elongation and lignin composition of the stem.

Meristem maintenance, achieved through the highly conserved CLAVATA-WUSCHEL (CLV-WUS) regulatory circuit, is fundamental in balancing stem cell proliferation with cellular differentiation. Disruptions to meristem homeostasis can alter meristem size, leading to enlarged organs. Cotton (Gossypium spp.), the world's most important fiber crop, shows inherent variation in fruit size, presenting opportunities to explore the networks regulating meristem homeostasis and to impact fruit size and crop value. We identified and characterized the cotton orthologs of genes functioning in the CLV-WUS circuit. Using virus-based gene manipulation in cotton, we altered the expression of each gene to perturb meristem regulation and increase fruit size. Targeted alteration of individual components of the CLV-WUS circuit modestly fasciated flowers and fruits. Unexpectedly, controlled expression of meristem regulator SELF-PRUNING (SP) increased the impacts of altered CLV-WUS expression on flower and fruit fasciation. Meristem transcriptomics showed SP and genes of the CLV-WUS circuit are expressed independently from each other, suggesting these gene products are not acting in the same path. Virus-induced silencing of GhSP facilitated the delivery of other signals to the meristem to alter organ specification. SP has a role in cotton meristem homeostasis, and changes in GhSP expression increased access of virus-derived signals to the meristem.

We combined molecular, genomic and genetic approaches to study the molecular mechanisms underlying cell totipotency and competency to regenerate in Arabidopsis. By performing comparative analysis of mRNA-seq and chromatin landscapes between leaf differentiated cells and callus totipotent cells and between WT callus and calli derived from the emf2 mutant, exhibiting impaired regenerative capacity we revealed the following: 1 . That callus cells express numerous genes of many developmental pathways such as root, leaf, embryo, shoot, meristem and seed. This suggests a mechanism to allow rapid response to a signal by maintaining genes of all potential developmental pathways active, without the needs to release transcriptional silencing and to go through the intricate multistep process of transcription. 2 . That key transcription factors that are sufficient to derive differentiation or organogenesis are silenced and marked by the H3K27me3. 3 . That callus derived from the emf2 mutant which is impaired in setting the H3K27methylation, lost the capacity to regenerate and that 78 transcription factors from which 18 regulate flower development, where up-regulated compared with WT callus. Altogether our results suggest that competency to regenerate is achieved by keeping the chromatin of developmental genes active, and that upon a signal for cell fate switch, a mechanism to repress those genes is required to allow the one desired developmental pathway to dominate. When this mechanism is impaired the capacity to regenerate is decline.

The molecular regulation of induction and development of reproductive organs is well established in angiosperms, while it is slowly accumulating in gymnosperms. Here, we conducted comparative transcriptomic profiles at different stages of male and female cone development in Pinus halepensis. Our aim was to reveal transcription factor encoding genes involved in reproductive induction and development. For accurate developmental stage identification, histological analysis preceded the molecular analysis. The flowering induction genes FT/TFL were expressed mainly at the late developmental stages of the male cone, suggesting involvement in vegetative bud dormancy instead of flowering induction. The male cone development was associated with the expression of the C-class PhMADS2, and the B-class genes PhDAL11 and PhDAL13, while the female cone development was associated with the expression of the C-class MADS1 and DAL14 genes. This study adds valuable knowledge to the profile of transcription factors and MADS-box genes regulating cone development in gymnosperms.

Plants exhibit remarkable lineage plasticity, allowing them to regenerate organs that differ from their respective origins. Such developmental plasticity is dependent on the activity of pluripotent founder cells or stem cells residing in meristems. At the shoot apical meristem (SAM), the constant flow of cells requires continuing cell specification governed by a complex genetic network, with the WUSCHEL transcription factor and phytohormone cytokinin at its core. In this review, I discuss some intriguing recent discoveries that expose new principles and mechanisms of patterning and cell specification acting both at the SAM and prior to meristem organogenesis during shoot regeneration. I also highlight unanswered questions and future challenges in the study of SAM and meristem regeneration. Finally, I put forward a model describing stochastic events mediated by epigenetic factors to explain how the gene regulatory network might be initiated at the onset of shoot regeneration.

Sesame is an important oil-crop worldwide. Complex tradeoffs between various yield components significantly affect the outcome yield. Our aims were to characterize the effect of genotype, environment and management, and their interactions, on yield components. Wild-type line, bearing a bicarpellate-capsule and three capsules per leaf axil, and its derived mutant-line, featuring one tetracarpellate-capsule per leaf axil, were analyzed under two irrigation regimes and three sowing-stands. Dissection of flower meristems and capsules showed larger placenta size and final capsule diameter in the mutant-line. Allelic segregation of F2 population revealed that the number of carpels per capsule demonstrates monogenic inheritance, whereas the number of capsules per leaf axil is a polygenic trait. A significant effect of genotype, irrigation and stand was observed on most yield components. While wild-type had more capsules per plant, the mutant-line compensated by increased seed number per capsule and consequently accumulated the same number of seeds per plant. Under either high intra-row or inter-row density, the branches number was reduced; however, the outcome yield was compensated by number of plants per area. While some yield components showed phenotypic-plasticity (branching), other traits were genetically stable (number of capsules per leaf axil and number of carpels per capsule). Our result shed-light on tradeoffs between yield components and on their underlying mechanisms.

The WUSCHEL homeobox transcription factor is required to specify stem-cell identity at the shoot apical meristem and its ectopic expression is sufficient to induce de novo shoot meristem formation. Yet, the manner by which WUS promotes stem-cell fate is not yet fully understood. In the present research we address this question by inducing WUS function outside of its domain. We show that activation of WUS function in the root inhibits the responses to exogenous auxin and suppresses the initiation and growth of lateral roots. Using time lapse movies to follow the cell-cycle marker CYCB1;1::GFP, we also show that activation of WUS function suppresses cell division and cell elongation. In addition, activation of WUS represses the auxin-induced expression of the PLETHORA1 root identity gene and promotes shoot fate. Shoot apical meristem formation requires a high cytokinin-to-auxin ratio. Our findings provide evidence for the manner by which WUS specifies stem-cell identity: by affecting auxin responses, by reducing the cell mitotic activity and by repressing other developmental pathways. At the meristem, the stem-cells which are characterized by low division rate are surrounded by the highly proliferative meristematic cells. Our results also provide a model for WUS establishing the differential mitotic rates between two cell populations at the minute structure of the meristem.

The overall goal of this project was to elucidate the role of dissolved organic matter (DOM) in soil retention, bioavailability and plant uptake of silver and cerium oxide NPs. The environmental risks of manufactured nanoparticles (NPs) are attracting increasing attention from both industrial and scientific communities. These NPs have shown to be taken-up, translocated and bio- accumulated in plant edible parts. However, very little is known about the behavior of NPs in soil-plant system as affected by dissolved organic matter (DOM). Thus DOM effect on NPs behavior is critical to assessing the environmental fate and risks related to NP exposure. Carbon-based nanomaterials embedded with metal NPs demonstrate a great potential to serve as catalyst and disinfectors. Hence, synthesis of novel carbon-based nanocomposites and testing them in the environmentally relevant conditions (particularly in the DOM presence) is important for their implementation in water purification. Sorption of DOM on Ag-Ag₂S NPs, CeO₂ NPs and synthesized Ag-Fe₃O₄-carbon nanotubebifunctional composite has been studied. High DOM concentration (50mg/L) decreased the adsorptive and catalytic efficiencies of all synthesized NPs. Recyclable Ag-Fe₃O₄-carbon nanotube composite exhibited excellent catalytic and anti-bacterial action, providing complete reduction of common pollutants and inactivating gram-negative and gram-positive bacteria at environmentally relevant DOM concentrations (5-10 mg/L). Our composite material may be suitable for water purification ranging from natural to the industrial waste effluents. We also examined the role of maize (Zeamays L.)-derived root exudates (a form of DOM) and their components on the aggregation and dissolution of CuONPs in the rhizosphere. Root exudates (RE) significantly inhibited the aggregation of CuONPs regardless of ionic strength and electrolyte type. With RE, the critical coagulation concentration of CuONPs in NaCl shifted from 30 to 125 mM and the value in CaCl₂ shifted from 4 to 20 mM. This inhibition was correlated with molecular weight (MW) of RE fractions. Higher MW fraction (> 10 kDa) reduced the aggregation most. RE also significantly promoted the dissolution of CuONPs and lower MW fraction (< 3 kDa) RE mainly contributed to this process. Also, Cu accumulation in plant root tissues was significantly enhanced by RE. This study provides useful insights into the interactions between RE and CuONPs, which is of significance for the safe use of CuONPs-based antimicrobial products in agricultural production. Wheat root exudates (RE) had high reducing ability to convert Ag+ to nAg under light exposure. Photo-induced reduction of Ag+ to nAg in pristine RE was mainly attributed to the 0-3 kDa fraction. Quantification of the silver species change over time suggested that Cl⁻ played an important role in photoconversion of Ag+ to nAg through the formation and redox cycling of photoreactiveAgCl. Potential electron donors for the photoreduction of Ag+ were identified to be reducing sugars and organic acids of low MW. Meanwhile, the stabilization of the formed particles was controlled by both low (0-3 kDa) and high (>3 kDa) MW molecules. This work provides new information for the formation mechanism of metal nanoparticles mediated by RE, which may further our understanding of the biogeochemical cycling and toxicity of heavy metal ions in agricultural and environmental systems. Copper sulfide nanoparticles (CuSNPs) at 1:1 and 1:4 ratios of Cu and S were synthesized, and their respective antifungal efficacy was evaluated against the pathogenic activity of Gibberellafujikuroi(Bakanae disease) in rice (Oryza sativa). In a 2-d in vitro study, CuS decreased G. fujikuroiColony- Forming Units (CFU) compared to controls. In a greenhouse study, treating with CuSNPs at 50 mg/L at the seed stage significantly decreased disease incidence on rice while the commercial Cu-based pesticide Kocide 3000 had no impact on disease. Foliar-applied CuONPs and CuS (1:1) NPs decreased disease incidence by 30.0 and 32.5%, respectively, which outperformed CuS (1:4) NPs (15%) and Kocide 3000 (12.5%). CuS (1:4) NPs also modulated the shoot salicylic acid (SA) and Jasmonic acid (JA) production to enhance the plant defense mechanisms against G. fujikuroiinfection. These results are useful for improving the delivery efficiency of agrichemicals via nano-enabled strategies while minimizing their environmental impact, and advance our understanding of the defense mechanisms triggered by the NPs presence in plants.

The shoot apical meristem (SAM) of angiosperm plants is a small, highly organized structure that gives rise to all above-ground organs. The SAM is divided into three functional domains: the central zone (CZ) at the SAM tip harbors the self-renewing pluripotent stem cells and the organizing center, providing daughter cells that are continuously displaced into the interior rib zone (RZ) or the surrounding peripheral zone (PZ), from which organ primordia are initiated. Despite the constant flow of cells from the CZ into the RZ or PZ, and cell recruitment for primordium formation, a stable balance is maintained between the distinct cell populations in the SAM. Here we combined an in-depth phenotypic analysis with a comparative RNA-Seq approach to characterize meristems from selected combinations of clavata3 (clv3), jabba-1D (jba-1D) and erecta (er) mutants of Arabidopsis thaliana. We demonstrate that CLV3 restricts meristem expansion along the apical-basal axis, whereas class III HD-ZIP and ER pathways restrict meristem expansion laterally, but in distinct and possibly perpendicular orientations. Our k-means analysis reveals that clv3, jba-1D/+ and er lead to meristem enlargement by affecting different aspects of meristem function; for example, clv3 displays an increase in the stem cell population, whereas jba-1D/+ er exhibits an increase in mitotic activity and in the meristematic cell population. Our analyses demonstrate that a combined genetic and mRNA-Seq comparative approach provides a precise and sensitive method to identify cell type-specific transcriptomes in a small structure, such as the SAM.

lized by the host (19). Insertion of a TE near a gene will have little functional impact in most instances, and strongly deleterious TE insertions will be selected against. However, a subset of genes, perhaps depending on promoter strength, is susceptible to epigenetic regulation by TEs. Regulation of gene expression by means of DNA methylation could be selected for if imprinting of these genes is adaptive in the context of parental conflict or gene dosage balance in the triploid endosperm.