Expression data of Arabidopsis seedlings in response to ARR1 or cytokinins, in the presence and absence of DELLA proteins
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ABSTRACT: DELLA proteins interact with ARR1 and modulate its activity. We have investigated the effect of DELLA proteins on transcriptional regulation by ARR1
Project description:DELLA proteins interact with ARR1 and modulate its activity. We have investigated the effect of DELLA proteins on transcriptional regulation by ARR1 A dominant hyperactive allele of ARR1 was induced with dexamethasone in seedlings preincubated with paclobutrazol (allowing DELLA acummulation) or paclobutrazol+GA3 (promoting DELLA degradation)
Project description:DELLA proteins are members of the GRAS-domain family of transcriptional regulators and modulate transcriptional activity through protein-protein interactions with at least 15 different classes of transcription factor. The plant hormone gibberellin (GA) regulates a range of growth and development responses including promoting cell elongation, by triggering the degradation of DELLA proteins. More than 70% of modern wheat (Triticum aestivum L.) varieties carry alleles encoding DELLA proteins that are resistant to GA-mediated degradation. These alleles result in constitutive growth repression in stem tissues and confer a semi-dwarf growth habit associated with improved lodging resistance. However, these alleles also have negative pleiotropic effects such as reduced nitrogen use efficiency and weak early vigour that restrict their utility in some environments. A detailed understanding of the mechanisms by which DELLA proteins regulate growth and development will be necessary to dissect these responses and develop targeted strategies to breed improved crop varieties. We identified the orthologous C2H2 Zinc-finger transcription factors INDETERMINATE DOMAIN 5 (IDD5) in wheat and SEMI-DWARF3 (SDW3) in barley (Hordeum vulgare) as positive regulators of stem and leaf expansion. Both IDD5 and SDW3 physically interact with DELLA proteins and are epistatic to these genes. We propose a mechanism by which IDD proteins integrate GA signals to modulate downstream gene transcriptional activity through a protein complex dual activation and repression activities. Changes in GA biosynthesis gene expression levels suggest that part of this response involves GA homeostasis. Plants carrying null mutations in IDD5 and SDW3 exhibit a GA-insensitive semi-dwarf growth habit comparable to the Rht-D1b ‘Green Revolution’ allele and have potential as novel dwarfing alleles in cereal breeding programmes.
Project description:The aim of this study is to identify early DELLA protein-responsive genes using a Dexamethasone (DEX)-inducible system. Two transgenic lines were used: one induces the expression of a dominant, gibberellin non-responsive DELLA protein (rga-delta17); the other is a control line that carries the same vector, but lacks the rga-delta17 transgene. By comparing the gene expression changes in the line that expresses the rga-delta17 protein in the presence or absence of DEX it is possible to identify putative targets of DELLA proteins. An empty vector transgenic line was included in this study to identify genes that might be regulated by the DEX inducible system that are not dependent on the DELLA protein. Keywords: Dexamethasone treatment, gibberellin treatment, time course, transgene effect
Project description:rs09-09_della-dark - della-regulation in darkness versus light - Identification of DELLA-dependent downstream targets in darkness - Aim was to determine downstream target of DELLA proteins involved in skotomorphogenesis. Wt, ga1-3, ga1-3_rga_gai_rgl1_rgl2_rgl3 global seeds were sterilized, sown on MS agar plates then put for stratification for 3 days at 4°C. Plates were placed in growth cabinet at 22°C for 5 days in darkness or in continous light. Keywords: gene knock out
Project description:Ovule development is a key process for plant reproduction that ensures correct seed production. Understanding the molecular mechanisms that control ovule formation will also provide new approaches to increase crop yield for breeding. Several molecular factors and plant hormones, including gibberellins, are involved in ovule initiation and development. Gibberellins control ovule development by the destabilization of DELLA proteins, whereas DELLA activity has been proved to act as a positive factor for ovule primordia emergence. But the molecular mechanism by which DELLA act remained unknown. Here we have proved that DELLA proteins control ovule initiation by the formation of a protein complex with the CUC2 transcription factor. The DELLA protein GAI requires CUC2 to promote ovule primordia formation, thus GAI would function by its direct protein-protein interaction with CUC2 in cells of the placenta that determine the boundary regions between ovules during pistil development. Analysis of GAI-CUC2 interaction and colocalization in placenta support this hypothesis. Moreover, molecular analysis of the loci at which GAI protein may act as transcriptional co-regulators in a CUC2-dependent manner identified a subset of target genes that would be regulated by the GAI-CUC2 complex and contribute to regulate ovule primordia emergence.
Project description:DELLA proteins are conserved master growth regulators that play a central role in controlling plant development in response to internal and environmental cues. DELLAs function as transcription regulators, which are recruited to target promoters by binding to transcription factors (TFs) and histone H2A via its GRAS domain. Recent studies showed that DELLA stability is regulated post-translationally via two mechanisms, phytohormone gibberellin-induced polyubiquitination for its rapid degradation, and Small Ubiquitin-like Modifier (SUMO)-conjugation to alter its accumulation. Moreover, DELLA activity is dynamically modulated by two distinct glycosylations: DELLA-TF interactions are enhanced by O-fucosylation, but inhibited by O-linked N-acetylglucosamine (O-GlcNAc) modification. However, the role of DELLA phosphorylation remains unclear. Here, we identified phosphorylation sites in REPRESSOR OF ga1-3 (RGA, an AtDELLA) purified from Arabidopsis by tandem mass spectrometry analysis, and showed that phosphorylation of the RGA LKS-peptide in the poly-S/T region enhances RGA-H2A interaction and RGA association with target promoters. Interestingly, phosphorylation does not affect RGA-TF interactions. Our study has uncovered that phosphorylation is a new regulatory mechanism of DELLA activity.
Project description:The aim of this study is to identify early DELLA protein-responsive genes using a Dexamethasone (DEX)-inducible system. Two transgenic lines were used: one induces the expression of a dominant, gibberellin non-responsive DELLA protein (rga-delta17); the other is a control line that carries the same vector, but lacks the rga-delta17 transgene. By comparing the gene expression changes in the line that expresses the rga-delta17 protein in the presence or absence of DEX it is possible to identify putative targets of DELLA proteins. An empty vector transgenic line was included in this study to identify genes that might be regulated by the DEX inducible system that are not dependent on the DELLA protein. Experiment Overall Design: Seedlings of both transgenic lines were pretreated for 16 h with 2 uM GA4 to enhance gibberellin responses. Because DELLA proteins are strong signaling repressors, this pretreatment should maximize the effect of DELLA induction. Eight-day old seedlings were treated with 2 uM GA4 or a combination of 2 uM GA4 plus 10 uM DEX to induce the rga-delta17 transgene. Three biological replicas for the transgenic line that carries the DEX-inducible rga-delta17 transgene were generated at 2h and 4h. For the empty vector line, only 2 biological replicas were generated at 4h of treatment with 2 uM GA with or without 10 uM DEX.
Project description:Arabidopsis thaliana Col-0 plants and three other genotypes (ARR1 overexpressor, arr1-1 knockout, overexpressor of ARR1-SRDX fusion protein) were grown in liquid media (1/2 MS, 1 g/L sucrose, 0.5 g/L MES, pH 5.7) in a Percival AR-66L growth chamber at 24 oC, 16:8 h day:night cycle, and 100 µE light intensity until growth stage 1.0. Plants were then treated with 5 µM 6-Benzyladenine for 0, 15, and 120 min, harvested and frozen in liquid nitrogen for RNA extraction and subsequent processing for microarray hybridization.
Project description:Purpose: The goals of this study are to identify the downstream regulated genes of ARR1. Methods: RNA was extracted from wild-type, arr1 single mutant, and AOE root explants at the nascent SAM formation stage. Library construction and RNA-Seq were performed by Biomarker Co. (www.biomarker.com.cn, Beijing, China). DESeq and the Q value were used to identify differentially transcribed genes (DTGs). Differential transcription was inferred by applying a false discovery rate threshold of 0.001 and the |log2 Ratio| ≥ 1. Functional categorization was inferred from a BLAST search of the non-redundant GenBank (http://www.ncbi.nlm.nih.gov/genbank/), KEGG Pathway (http://www.genome.jp/kegg/pathway.html), and UniProt protein databases (http://www.uniprot.org/), and was further analyzed by the Gene Ontology (http://geneontology.org/) method. Results: In the transcriptomes of arr1 vs. wild-type explants, we detected 177 up- and 260 downregulated DTGs, and in the AOE vs. wild-type transcriptomes, we detected 129 and 6 DTGs, respectively. The pathways involved in hormone signal transduction and indole alkaloid synthesis were well represented among the DTGs in arr1 vs. wild type. These DTGs also included genes involved in auxin transport and signaling, as well as IAA17, an Aux/IAA repressor gene, which was downregulated in arr1 root explants. Conclusions: Our study showed that IAA17 was downstream gene of ARR1 in root explants.