RESUMEN

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.

RESUMEN

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.

RESUMEN

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.

Asunto(s)

Medios de Cultivo , Medios de Cultivo/química , Geles , Reguladores del Crecimiento de las Plantas/farmacología , Reguladores del Crecimiento de las Plantas/metabolismo , Técnicas de Cultivo de Tejidos/métodos , Agar/química , Diferenciación Celular/efectos de los fármacos

RESUMEN

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.

Asunto(s)

Proteínas de Arabidopsis , Meristema , Meristema/metabolismo , Proteínas de Arabidopsis/metabolismo , Flores/genética , Flores/metabolismo , Frutas/genética , Frutas/metabolismo , Homeostasis , Regulación de la Expresión Génica de las Plantas , Proteínas de Homeodominio/genética

RESUMEN

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.

Asunto(s)

Arabidopsis , Histonas , Histonas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Lisina/metabolismo , Lignina/metabolismo , Metilación , Epigénesis Genética , Genes del Desarrollo

RESUMEN

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.

Asunto(s)

Proteínas de Arabidopsis , Meristema , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/genética , Redes Reguladoras de Genes , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Meristema/genética , Meristema/metabolismo , Brotes de la Planta/genética , Brotes de la Planta/metabolismo , Regeneración/genética

RESUMEN

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.

Asunto(s)

Producción de Cultivos , Interacción Gen-Ambiente , Genotipo , Sesamum/crecimiento & desarrollo , Cambio Climático , Sesamum/genética

RESUMEN

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.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Homeodominio/metabolismo , Células Madre/metabolismo , Animales , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Proteínas de Homeodominio/genética , Ácidos Indolacéticos/farmacología , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/metabolismo , Brotes de la Planta/citología , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/metabolismo , Ratas , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Células Madre/citología , Imagen de Lapso de Tiempo

RESUMEN

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.

Asunto(s)

Proteínas de Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Proteínas de Homeodominio/genética , Meristema/crecimiento & desarrollo , Proteínas Serina-Treonina Quinasas/genética , Receptores de Superficie Celular/genética , Arabidopsis/genética , Diferenciación Celular , Proliferación Celular , Meristema/citología , MicroARNs/genética , Plantas Modificadas Genéticamente , ARN Mensajero/genética , Transducción de Señal/fisiología

RESUMEN

Plants exhibit high capacity to regenerate in three alternative pathways: tissue repair, somatic embryogenesis and de novo organogenesis. For most plants, de novo organ initiation can be easily achieved in tissue culture by exposing explants to auxin and/or cytokinin, yet the competence to regenerate varies among species and within tissues from the same plant. In Arabidopsis, root explants incubated directly on cytokinin-rich shoot inducing medium (SIM-direct), are incapable of regenerating shoots, and a pre-incubation step on auxin-rich callus inducing medium (CIM) is required to acquire competency to regenerate on the SIM. However the mechanism underlying competency acquisition still remains elusive. Here we show that the chromomethylase 3 (cmt3) mutant which exhibits significant reduction in CHG methylation, shows high capacity to regenerate on SIM-direct and that regeneration occurs via direct organogenesis. In WT, WUSCHEL (WUS) promoter, an essential gene for shoot formation, is highly methylated, and its expression on SIM requires pre-incubation on CIM. However, in cmt3, WUS expression induced by SIM-direct. We propose that pre-incubation on CIM is required for the re-activation of cell division. Following the transfer of roots to SIM, the intensive cell division activity continues, and in the presence of cytokinin leads to a dilution in DNA methylation that allows certain genes required for shoot regeneration to respond to SIM, thereby advancing shoot formation.

Asunto(s)

Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Metilación de ADN/genética , Raíces de Plantas/fisiología , Brotes de la Planta/fisiología , Regeneración , Arabidopsis/efectos de los fármacos , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ciclo Celular/efectos de los fármacos , Medios de Cultivo/farmacología , Metilación de ADN/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Estudios de Asociación Genética , Mutación/genética , Organogénesis/efectos de los fármacos , Organogénesis/genética , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/ultraestructura , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/crecimiento & desarrollo , Regiones Promotoras Genéticas/genética , Regeneración/efectos de los fármacos

RESUMEN

In angiosperms, the production of flowers marks the beginning of the reproductive phase. At the emergence of flower primordia on the flanks of the inflorescence meristem, the WUSCHEL (WUS) gene, which encodes a homeodomain transcription factor starts to be expressed and establishes de novo stem cell population, founder of the floral meristem (FM). Similarly to the shoot apical meristem a precise spatial and temporal expression pattern of WUS is required and maintained through strict regulation by multiple regulatory inputs to maintain stem cell homeostasis. However, following the formation of a genetically determined fixed number of floral organs, this homeostasis is shifted towards organogenesis and the FM is terminated. In here we performed a genetic study to test how a reduction in ERECTA, CLAVATA and class III HD-ZIP pathways affects floral meristem activity and flower development. We revealed strong synergistic phenotypes of extra flower number, supernumerary whorls, total loss of determinacy and extreme enlargement of the meristem as compared to any double mutant combination indicating that the three pathways, CLV3, ER and HD-ZIPIII distinctively regulate meristem activity and that they act in parallel. Our findings yield several new insights into stem cell-driven development. We demonstrate the crucial requirement for coupling floral meristem termination with carpel formation to ensure successful reproduction in plants. We also show how regulation of meristem size and alternation in spatial structure of the meristem serve as a mechanism to determine flower organogenesis. We propose that the loss of FM determinacy due to the reduction in CLV3, ER and HD-ZIPIII activity is genetically separable from the AGAMOUS core mechanism of meristem termination.

Asunto(s)

Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Flores/crecimiento & desarrollo , Proteínas de Homeodominio/genética , Meristema/crecimiento & desarrollo , Proteínas Serina-Treonina Quinasas/genética , Receptores de Superficie Celular/genética , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Diferenciación Celular , Proliferación Celular , Proteínas Cullin/genética , Meristema/metabolismo , Factores de Transcripción/genética

RESUMEN

In plants, the shoot apical meristem (SAM) serves as a reservoir of pluripotent stem cells from which all above ground organs originate. To sustain proper growth, the SAM must maintain homeostasis between the self-renewal of pluripotent stem cells and cell recruitment for lateral organ formation. At the core of the network that regulates this homeostasis in Arabidopsis are the WUSCHEL (WUS) transcription factor specifying stem cell fate and the CLAVATA (CLV) ligand-receptor system limiting WUS expression. In this study, we identified the ERECTA (ER) pathway as a second receptor kinase signaling pathway that regulates WUS expression, and therefore shoot apical and floral meristem size, independently of the CLV pathway. We demonstrate that reduction in class III HD-ZIP and ER function together leads to a significant increase in WUS expression, resulting in extremely enlarged shoot meristems and a switch from spiral to whorled vegetative phyllotaxy. We further show that strong upregulation of WUS in the inflorescence meristem leads to ectopic expression of the AGAMOUS homeotic gene to a level that switches cell fate from floral meristem founder cell to carpel founder cell, suggesting an indirect role for ER in regulating floral meristem identity. This work illustrates the delicate balance between stem cell specification and differentiation in the meristem and shows that a shift in this balance leads to abnormal phyllotaxy and to altered reproductive cell fate.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas/fisiología , Proteínas de Homeodominio/metabolismo , Meristema/crecimiento & desarrollo , Brotes de la Planta/crecimiento & desarrollo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal/fisiología , Proteína AGAMOUS de Arabidopsis/metabolismo , Biología Computacional , Cartilla de ADN/genética , Regulación de la Expresión Génica de las Plantas/genética , Hibridación in Situ , Meristema/citología , Microscopía Electrónica de Rastreo , Mutagénesis , Brotes de la Planta/citología , Plantas Modificadas Genéticamente , Células Madre Pluripotentes/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/genética

RESUMEN

Flexible maturation rates underlie part of the diversity of leaf shape, and tomato (Solanum lycopersicum) leaves are compound due to prolonged organogenic activity of the leaf margin. The CINCINNATA-teosinte branched1, cycloidea, PCF (CIN-TCP) transcription factor lanceolate (LA) restricts this organogenic activity and promotes maturation. Here, we show that tomato APETALA1/fruitfull (AP1/FUL) MADS box genes are involved in tomato leaf development and are repressed by LA. AP1/FUL expression is correlated negatively with LA activity and positively with the organogenic activity of the leaf margin. LA binds to the promoters of the AP1/FUL genes MBP20 and TM4. Overexpression of MBP20 suppressed the simple-leaf phenotype resulting from upregulation of LA activity or from downregulation of class I knotted like homeobox (KNOXI) activity. Overexpression of a dominant-negative form of MBP20 led to leaf simplification and partly suppressed the increased leaf complexity of plants with reduced LA activity or increased KNOXI activity. Tomato plants overexpressing miR319, a negative regulator of several CIN-TCP genes including LA, flower with fewer leaves via an SFT-dependent pathway, suggesting that miR319-sensitive CIN-TCPs delay flowering in tomato. These results identify a role for AP1/FUL genes in vegetative development and show that leaf and plant maturation are regulated via partially independent mechanisms.

Asunto(s)

Proteínas de Dominio MADS/genética , Hojas de la Planta/genética , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Solanum lycopersicum/crecimiento & desarrollo , Solanum lycopersicum/metabolismo , Proteínas de Dominio MADS/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas/genética , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Factores de Transcripción/metabolismo

RESUMEN

Nucleosome remodelers of the DDM1/Lsh family are required for DNA methylation of transposable elements, but the reason for this is unknown. How DDM1 interacts with other methylation pathways, such as small-RNA-directed DNA methylation (RdDM), which is thought to mediate plant asymmetric methylation through DRM enzymes, is also unclear. Here, we show that most asymmetric methylation is facilitated by DDM1 and mediated by the methyltransferase CMT2 separately from RdDM. We find that heterochromatic sequences preferentially require DDM1 for DNA methylation and that this preference depends on linker histone H1. RdDM is instead inhibited by heterochromatin and absolutely requires the nucleosome remodeler DRD1. Together, DDM1 and RdDM mediate nearly all transposon methylation and collaborate to repress transposition and regulate the methylation and expression of genes. Our results indicate that DDM1 provides DNA methyltransferases access to H1-containing heterochromatin to allow stable silencing of transposable elements in cooperation with the RdDM pathway.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Elementos Transponibles de ADN , Proteínas de Unión al ADN/metabolismo , Heterocromatina , Histonas/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Regulación de la Expresión Génica de las Plantas , Nucleosomas/metabolismo

RESUMEN

EMBRYONIC FLOWER1 (EMF1) is a plant-specific gene crucial to Arabidopsis vegetative development. Loss of function mutants in the EMF1 gene mimic the phenotype caused by mutations in Polycomb Group protein (PcG) genes, which encode epigenetic repressors that regulate many aspects of eukaryotic development. In Arabidopsis, Polycomb Repressor Complex 2 (PRC2), made of PcG proteins, catalyzes trimethylation of lysine 27 on histone H3 (H3K27me3) and PRC1-like proteins catalyze H2AK119 ubiquitination. Despite functional similarity to PcG proteins, EMF1 lacks sequence homology with known PcG proteins; thus, its role in the PcG mechanism is unclear. To study the EMF1 functions and its mechanism of action, we performed genome-wide mapping of EMF1 binding and H3K27me3 modification sites in Arabidopsis seedlings. The EMF1 binding pattern is similar to that of H3K27me3 modification on the chromosomal and genic level. ChIPOTLe peak finding and clustering analyses both show that the highly trimethylated genes also have high enrichment levels of EMF1 binding, termed EMF1_K27 genes. EMF1 interacts with regulatory genes, which are silenced to allow vegetative growth, and with genes specifying cell fates during growth and differentiation. H3K27me3 marks not only these genes but also some genes that are involved in endosperm development and maternal effects. Transcriptome analysis, coupled with the H3K27me3 pattern, of EMF1_K27 genes in emf1 and PRC2 mutants showed that EMF1 represses gene activities via diverse mechanisms and plays a novel role in the PcG mechanism.

Asunto(s)

Proteínas de Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Epigénesis Genética , Histona Demetilasas , Proteínas Represoras/genética , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sitios de Unión , Diferenciación Celular/genética , Metilación de ADN , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Proteínas Mutantes , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Brotes de la Planta/genética , Brotes de la Planta/crecimiento & desarrollo , Complejo Represivo Polycomb 2 , Unión Proteica , Proteínas Represoras/metabolismo

RESUMEN

Natural cis-antisense siRNAs (cis-nat-siRNAs) are a recently characterized class of small regulatory RNAs that are widespread in eukaryotes. Despite their abundance, the importance of their regulatory activity is largely unknown. The only functional role for eukaryotic cis-nat-siRNAs that has been described to date is in environmental stress responses in plants. Here we demonstrate that cis-nat-siRNA-based regulation plays key roles in Arabidopsis reproductive function, as it facilitates gametophyte formation and double fertilization, a developmental process of enormous agricultural value. We show that male gametophytic kokopelli (kpl) mutants display frequent single-fertilization events, and that KPL and a inversely transcribed gene, ARIADNE14 (ARI14), which encodes a putative ubiquitin E3 ligase, generate a sperm-specific nat-siRNA pair. In the absence of KPL, ARI14 RNA levels in sperm are increased and fertilization is impaired. Furthermore, ARI14 transcripts accumulate in several siRNA biogenesis pathway mutants, and overexpression of ARI14 in sperm phenocopies the reduced seed set of the kokopelli mutants. These results extend the regulatory capacity of cis-nat-siRNAs to development by identifying a role for cis-nat-siRNAs in controlling sperm function during double fertilization.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Fertilización/genética , Regulación de la Expresión Génica de las Plantas , ARN sin Sentido/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de Arabidopsis/genética , Perfilación de la Expresión Génica , Mutación/genética , Óvulo Vegetal/crecimiento & desarrollo , Fenotipo , Polen/genética , ARN Interferente Pequeño/biosíntesis

RESUMEN

Parent-of-origin-specific (imprinted) gene expression is regulated in Arabidopsis thaliana endosperm by cytosine demethylation of the maternal genome mediated by the DNA glycosylase DEMETER, but the extent of the methylation changes is not known. Here, we show that virtually the entire endosperm genome is demethylated, coupled with extensive local non-CG hypermethylation of small interfering RNA-targeted sequences. Mutation of DEMETER partially restores endosperm CG methylation to levels found in other tissues, indicating that CG demethylation is specific to maternal sequences. Endosperm demethylation is accompanied by CHH hypermethylation of embryo transposable elements. Our findings demonstrate extensive reconfiguration of the endosperm methylation landscape that likely reinforces transposon silencing in the embryo.

Asunto(s)

Arabidopsis/embriología , Arabidopsis/genética , Metilación de ADN , Elementos Transponibles de ADN , Genoma de Planta , Impresión Genómica , Semillas/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , ADN de Plantas/genética , ADN de Plantas/metabolismo , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Genes de Plantas , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Semillas/crecimiento & desarrollo , Transactivadores/genética , Transactivadores/metabolismo , Sitio de Iniciación de la Transcripción

RESUMEN

The aerial structure of higher plants is generated dynamically throughout the life cycle through the activity of stem cells that are located at the growing shoot tip, the apical meristem. The stem cells continuously divide to renew themselves and provide cells for leaf, stem and flower formation. Stem cell maintenance is governed by intercellular communication between the apical stem cells and the underlying organizing centre. Recent advances have been made in understanding the mechanisms that induce shoot stem cell identity, and that control the position and size of the organizing centre. Elements such as chromatin remodeling factors, transcription factors and microRNAs are newly implicated in these regulatory processes. These advances provide a framework for our understanding of how signals are integrated to specify and position the stem cell niche in the shoot apical meristem.

Asunto(s)

Arabidopsis/citología , Arabidopsis/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Brotes de la Planta/citología , Brotes de la Planta/crecimiento & desarrollo , Células Madre/fisiología , Arabidopsis/genética , Cromatina/genética , Cromatina/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Brotes de la Planta/genética

RESUMEN

Plant development is characterized by precise control of gene regulation, leading to the correct spatial and temporal tissue patterning. We have characterized the Arabidopsis jabba-1D (jba-1D) mutant, which displays multiple enlarged shoot meristems, radialized leaves, reduced gynoecia and vascular defects. The jba-1D meristem phenotypes require WUSCHEL (WUS) activity, and correlate with a dramatic increase in WUS expression levels. We demonstrate that the jba-1D phenotypes are caused by over-expression of miR166g, and require the activity of the RNase III helicase DCL1. miR166g over-expression in jba-1D plants affects the transcripts of several class III homeodomain-leucine zipper (AtHD-ZIP) family target genes. The expression of PHABULOSA (PHB), PHAVOLUTA (PHV) and CORONA (CNA) is significantly reduced in a jba-1D background, while REVOLUTA (REV) expression is elevated and ATHB8 is unchanged. In addition, we show that miR166 has a dynamic expression pattern in wild-type and jba-1D embryos. Our analysis demonstrates an indirect role for miRNAs in controlling meristem formation via regulation of WUS expression, and reveals complex regulation of the class III AtHD-ZIP gene family.

Asunto(s)

Arabidopsis , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Meristema/fisiología , MicroARNs/metabolismo , Morfogénesis , Secuencia de Aminoácidos , Arabidopsis/anatomía & histología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Hibridación in Situ , MicroARNs/genética , Datos de Secuencia Molecular , Fenotipo , Alineación de Secuencia

RESUMEN

Two classes of small RNAs, microRNAs and short-interfering RNA (siRNAs), have been extensively studied in plants and animals. In Arabidopsis, the capacity to uncover previously uncharacterized small RNAs by means of conventional strategies seems to be reaching its limits. To discover new plant small RNAs, we developed a protocol to mine an Arabidopsis nonannotated, noncoding EST database. Using this approach, we identified an endogenous small RNA, trans-acting short-interfering RNA-auxin response factor (tasiR-ARF), that shares a 21- and 22-nt region of sequence similarity with members of the ARF gene family. tasiR-ARF has characteristics of both short-interfering RNA and microRNA, recently defined as tasiRNA. Accumulation of trans-acting siRNA depends on DICER-LIKE1 and RNA-DEPENDENT RNA POLYMERASE6 but not RNA-DEPENDENT RNA POLYMERASE2. We demonstrate that tasiR-ARF targets three ARF genes, ARF2, ARF3/ETT, and ARF4, and that both the tasiR-ARF precursor and its target genes are evolutionarily conserved. The identification of tasiRNA-ARF as a low-abundance, previously uncharacterized small RNA species proves our method to be a useful tool to uncover additional small regulatory RNAs.

Asunto(s)

Proteínas de Arabidopsis/genética , Arabidopsis/genética , Biología Computacional/métodos , Proteínas de Unión al ADN/genética , Bases de Datos Genéticas , Familia de Multigenes/genética , Proteínas Nucleares/genética , ARN Interferente Pequeño/genética , Proteínas de Arabidopsis/metabolismo , Secuencia de Bases , Proteínas de Ciclo Celular/metabolismo , Cartilla de ADN , Datos de Secuencia Molecular , Técnicas de Amplificación de Ácido Nucleico , ARN Polimerasa Dependiente del ARN/metabolismo , Complejo Silenciador Inducido por ARN/genética , Complejo Silenciador Inducido por ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleasa III/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN