Chromatin remodeler SPLAYED maintains the balance of miR156 dependent developmental pathways in Arabidopsis
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ABSTRACT: In plants, micro RNAs and chromatin remodeling are both known to regulate plant development and reproduction, however evidences for molecular links between these pathways have remained sparse. The antagonistic activities of miR156 and its target mRNAs, encoding several SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors, control the juvenile-to-adult and vegetative-to-reproductive developmental phase transitions. In recent years a number of proteins were shown to act in the transcriptional control of MIR156 genes, demonstrating the complexity of the regulatory network involved in this process. Spatiotemporal control of gene expression involves interactions between transcription factors and changes in the chromatin structure. SPLAYED (SYD) is a well-characterized SWI2/SNF2 chromatin remodeling ATPase, which has been shown to be an important regulator of flowering time, flower organ identity and homeotic gene expression. In this study, we investigated the interaction between the miR156 and SYD pathways and provide evidence that SYD directly influences the transcription of several MIR156 and SPL genes in Arabidopsis. We propose that SYD maintains the balance between the miR156-dependent and miR156-independent developmental pathways. More generally, we then show that SYD may also play a very broad role in the control of gene expression by associating to most genes in Arabidopsis. We show that binding to these genes is dependent on the meristem identity factor LEAFY and the PRC2 component SWINGER. These results uncover a much more general role for SYD in regulating and balancing key developmental processes in Arabidopsis than previously assumed.
Project description:Analysis of different leaves from transgenic plants over expressing microRNA miR156. Time-dependent gene expression is essential for developmental timing control. MiR156 was characterized as a time-dependent expressed microRNA during leaf development. The level of miR156 is positive correlated to developmental time during leaf growth. We used microarray to identify genes regulated by miR156 in a time-dependent manner during leaf development. Results indicate that most time-negative genes were activated in mature leaves of miR156 over expressed plants compared with wild type. Keywords: gene expression array-based (RNA / in situ oligonucleotide) To identify the temporal regulation role of miR156 in different leaves, all leaves in the main tiller were collected separately from Md/Mh (miR156 over expressed lines) and wild type (WT) plants. The level of miR156 were checked in these samples, then three biological repeats were selected and these RNA samples of the oldest (L1) and youngest leaf (L4 or L5) were applied for microarray hybridization.
Project description:SPLAYED (SYD) is a SWItch/Sucrose Non-Fermentable (SWI/SNF)-type chromatin remodeler identified in Arabidopsis thaliana (Arabidopsis). It is believed to play both redundant and differential roles with its closest homolog BRAHMA (BRM) in diverse plant growth and development processes. To better understand how SYD functions, we profiled the genome-wide occupancy of SYD and its impact on the global transcriptome and deposition of trimethylation of histone H3 on lysine 27 (H3K27me3). To map the global occupancy of SYD, we generated a GFP-tagged transgenic line and used it for ChIP-seq experiments, by which more than 6,000 SYD target genes were identified. Through integrating SYD occupancy and transcriptome profiles, we found that SYD preferentially targets to nucleosome-free regions of expressed genes. Further analysis revealed that SYD occupancy peaks exhibit five distinct patterns, which were also shared by BRM and BAF60, a conserved SWI/SNF complex component, indicating the common target sites of these SWI/SNF chromatin remodelers and the functional relevance of such distinct patterns. To investigate the interplay between SYD and Polycomb-group (PcG) proteins, we performed a genome-wide analysis of H3K27me3 in syd-5. We observed both increases and decreases in H3K27me3 levels at a few hundred genes in syd-5 compared to wild-type (WT). Our results imply that SYD can act antagonistically or synergistically with PcG at specific genes. Together, our SYD genome-wide occupancy data and the transcriptome and H3K27me3 profiles provide a much-needed resource for dissecting SYD’s crucial roles in the regulation of plant growth and development.
Project description:The highly conserved plant microRNA, miR156, is an essential regulator for plant development. We found miR156 changed gradually during leaf development. To find how miR156 regulated genes temporally, we analyzed the transcriptome of old and young leaves from miR156-overexpressed and wild type plants. The results show that the gradual increase of miR156 regulates temporal changes of numerous gene expressions during leaf development.
Project description:Chromatin and its covalent modifications play important roles during development. However, how different types of chromatin regulators individually control gene expression in different cell types has so far not been investigated in detail. To explore their role in these processes we performed forward and reverse genetic experiments using an epigenetically silenced reporter gene in Arabidopsis. We found three chromatin regulators, each involved in different epigenetic pathways, to control reporter gene expression in distinct tissues and cell-types. Here we report on the roles of the RNA polymerase Pol V, the histone deacetylase HDA6 and the SWI/SNF class chromatin remodeler SPLAYED (SYD) in silencing expression of the reporter in different cell-types. Furthermore, we demonstrate that a highly conserved domain in HDA6 is involved in its target specificity. And we present data showing that SYD controls transcription of epigenetically silenced genes. The results presented here provide evidence supporting the notion that tissue specificity of gene expression and its fine-tuning can be influenced by the activity of different chromatin regulators.
Project description:Chromatin remodelers have been thought to be crucial in creating an accessible chromatin environment before transcription activation. However, it is still unclear how chromatin remodelers recognize and bind to the active regions. In this study, we found that chromatin remodelers SPLAYED (SYD) and BRAHMA (BRM) interact and co-occupy with the Suppressor of Ty6-like (SPT6L), a core subunit of the transcription machinery, at thousands of the transcription start sites (TSS). The association of SYD and BRM to chromatin is dramatically reduced in spt6l and can be Chromatin remodelers have been thought to be crucial in creating an accessible chromatin environment before transcription activation. However, it is still unclear how chromatin remodelers recognize and bind to the active regions. In this study, we found that chromatin remodelers SPLAYED (SYD) and BRAHMA (BRM) interact and co-occupy with Suppressor of Ty6-like (SPT6L), a core subunit of the transcription machinery, at thousands of the transcription start sites (TSS). The association of SYD and BRM to chromatin is dramatically reduced in spt6l and can be restored mainly by SPT6LΔtSH2, which binds to TSS in a RNA polymerase II (Pol II)-independent manner. Furthermore, SPT6L and SYD/BRM are involved in regulating the nucleosome and Pol II occupancy around TSS. The presence of SPT6L is sufficient to restore the association of the chromatin remodeler SYD to chromatin and maintain normal nucleosome occupancy. Our findings suggest that the two chromatin remodelers can form protein complexes with the core subunit of the transcription machinery and regulate nucleosome occupancy in the early transcription stage.
Project description:Most developmental processes associated with fruit patterning take place at the floral meristem (FM). Age-regulated microRNA156 (miR156) and gibberellins (GA) integrate to control flowering time, but it is unclear how GA and miR156 interplay during fruit patterning. Here, we used genetic, molecular, and imaging tools to demonstrate that GA and age synergistically control tomato reproductive development. We found that low levels of miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN–LIKE (SPL/SBP) transcripts or high GA responses/levels led to enlarged FMs, defects in FM determinacy, and fruits with increased locule number. Conversely, low GA responses reduced locule number and indeterminacy, and the expression of a miR156-resistant form of SlSBP15 (rSBP15) reduces FM size by decreasing cell size and cell number. Importantly, we discovered that GA levels/responses may be partially responsible for the fruit defects observed in 156OE and rSBP15 plants. GA and miR156-targeted SlSBPs regulate classical genes associated with floral determinacy, such as CRABS CLAWa (SlCRCa), and GA and SlSBPs act synergistically with tomato CLAVATA3 (SlCLV3) to control early flower and fruit development. Our findings indicate that gibberellins and age-dependent miR156-targeted SlSBPs co-operate to regulate FM activity and locule formation, thereby offering a novel mechanism of controlling fruit patterning.
Project description:Heat stress and extreme temperatures negatively affect plant development by disrupting regular cellular and biochemical functions, ultimately leading to reduced crop production. Recently, our group has shown through physiological experiments that miR156 overexpression resulted in an improved alfalfa response to heat stress. To further expand the scope of miR156 research, we employed a label-free quantification based quantitative proteomics approach to explore the effects of heat stress on protein levels in miR156OE alfalfa. Our major objective was to identify miR156-regulated gene products with differentially altered abundance under heat stress in alfalfa.
Project description:The widespread agricultural problem of pre-harvest sprouting (PHS) could potentially be overcome by improving seed dormancy. Here, we report that miR156, an important grain yield regulator, also controls seed dormancy in rice. We found that mutations in one MIR156 subfamily enhance seed dormancy and suppress PHS with negligible effects on shoot architecture and grain size, whereas mutations in another MIR156 subfamily modify shoot architecture and increase grain size but have minimal effects on seed dormancy. Mechanistically, mir156 mutations enhance seed dormancy by suppressing the gibberellin (GA) pathway through de-represssion of the miR156 target gene Ideal Plant Architecture 1 (IPA1), which directly regulates multiple genes in the GA pathway. These results provide an effective method to suppress PHS without compromising productivity, and will facilitate breeding elite crop varieties with ideal plant architectures.
Project description:Alfalfa is the most produced perennial forage crop in Canada. Drought stress is a major form of abiotic stress, affecting its productivity and annual yield. A small RNA, miR156, plays a major role in drought tolerance by downregulating downstream SPL genes, but its effects at the proteome level are unknown. In this study, the protein level perturbations of miR156 overexpression (A8) and empty vector (EV) control genotypes were compared under drought stress. Using label-free quantification, 3,000 protein groups were identified, of which 68 were upregulated in A8 and 84 were downregulated relative to EV under control conditions. Conversely, under drought stress, 610 proteins were upregulated and only 52 proteins were downregulated in A8 relative to EV. Functional analysis using PlantRegMap showed that the enriched proteins are likely involved in biological and molecular processes including antioxidant response, response to stress, signal transduction and biosynthesis of secondary metabolites. These proteins/pathways might be involved in the enhancement of drought stress tolerance mediated by miR156. Protein groups related to signaling, such as MAP kinase, calcium-dependent protein kinase, protein phosphatase 2C, and transcriptional regulators including bZIP and zinc finger proteins were found to be differentially expressed when a search was conducted against a drought stress gene database. The proteomic dataset was validated by immunoblotting of selected proteins. The results of this study provide a better understanding and insight into the role of miR156 in drought stress tolerance in alfalfa at the proteomic level.