Project description:The goal of this experiment was to identify the downstream targets of the GOLVEN6 peptide signaling pathway in Arabidopsis thaliana, specifically during lateral root initiation. Using an estradiol inducible GLV6 overexpression construct in wildtype and rgi1rgi5 double mutant (mutant in receptors for the GLV6 peptide) backgrounds, in combination with gravistimulation induced lateral root formation, the RGI receptor dependent transcriptional effects of GLV6 overexpression were characterized. An estradiol inducible GLV6 overexpression line in a wildtype (iGLV6) and in an rgi1rgi5 double receptor mutant background (rgi1rgi5/iGLV6) were used. 4-day old seedlings of both lines were gravistimulated (vertically grown seedlings were turned counterclockwise by 90°) to induce lateral root initiation in the resulting root bends. 8h after gravistimulation, seedlings of both lines were treated with 2µM of estradiol to induce GLV6 overexpression, or DMSO as a mock treatment. 3h and 6h after treatment, root bends were dissected and collected for RNA-sequencing. This yielded a total of 8 samples per replicate; 3h mock treated iGLV6 (IM3), 3h estradiol treated iGLV6 (IE3), 3h mock treated rgi1rgi5/iGLV6 (RM3), 3h estradiol treated rgi1rgi5/iGLV6 (RE3), 6h mock treated iGLV6 (IM6), 6h estradiol treated iGLV6 (IE6), 6h mock treated rgi1rgi5/iGLV6 (RM6), 6h estradiol treated rgi1rgi5/iGLV6 (RE6). For each sample, 4 replicates were obtained. This setup enabled the comparison of the GLV6 induced transcriptional effects between wildtype and rgi1rgi5 mutants at 2 time points after treatment, in samples that are strongly enriched for lateral root initiation events.
Project description:DELLA proteins act as hubs that relay environmental information to the multiple transcriptional circuits that control growth and development through physical interaction with transcription factors from different families. We have analyzed the presence of one DELLA protein at the Arabidopsis genome by chromatin immunoprecipitation coupled to large-scale sequencing and we find that it binds at the promoters of multiple genes. Enrichment analysis shows a strong preference for cis elements recognized by specific transcription factor families. In particular, we demonstrate that DELLA proteins are recruited by type-B ARABIDOPSIS RESPONSE REGULATORS (ARR) to the promoters of cytokinin-regulated genes, where they act as transcriptional co-activators. The biological relevance of this mechanism is underpinned by the necessity of simultaneous presence of DELLAs and ARRs to restrict root meristem growth and to promote photomorphogenesis.
Project description:DELLA proteins act as hubs that relay environmental information to the multiple transcriptional circuits that control growth and development through physical interaction with transcription factors from different families. We have analyzed the presence of one DELLA protein at the Arabidopsis genome by chromatin immunoprecipitation coupled to large-scale sequencing and we find that it binds at the promoters of multiple genes. Enrichment analysis shows a strong preference for cis elements recognized by specific transcription factor families. In particular, we demonstrate that DELLA proteins are recruited by type-B ARABIDOPSIS RESPONSE REGULATORS (ARR) to the promoters of cytokinin-regulated genes, where they act as transcriptional co-activators. The biological relevance of this mechanism is underpinned by the necessity of simultaneous presence of DELLAs and ARRs to restrict root meristem growth and to promote photomorphogenesis. Provided are 3 biological replicates analysing RGA binding sites in Arabidopsis seedlings. ChIP-seq was performed on plants expressing RGA-GFP under the native RGA promoter and on non-transgenic control plants as reference
Project description:Functionally complemented dog1-1 mutant with a YFP-DOG1 fusion protein under the control of its native promoter were used as starting material for in vivo pull-down of YFP: DOG1 and its interacting proteins from seed tissues in native conditions. DOG1 interacting proteins were identified by mass spectrometry. Pull-downs were performed in biological triplicates using dry or 24 h water imbibed seeds, directly after harvest (dormant condition) and after 7 months of dry storage (non-dormant condition).
Project description:PICKLE (PKL), a Chromodomain Helicase DNA binding domain type 3-type (CHD3) chromatin remodeler, noted for an embryonic structure called pickle root in primary root tip in pkl mutant, has been studied for decades. we obtained a comprehensive genome occupancy of PKL by Chromatin immunoprecipitation-sequencing (ChIP-seq), and found PKL co-occupy with the major repressors of seed maturation program, VIVIPAROUS1/ABI3-LIKE1/2 (VAL1/2) in genome. Furthermore, PKL physically interacts with VAL1/2 in vivo and phenotype and transcriptome data indicated that PKL and VAL1/2 function in a common pathway. Moreover, ChIP-seq and ChIP-qPCR results showed that VAL1/2 are necessary for the recruitment of PKL to co-target genes
Project description:Root exudates are composed of primary and secondary metabolites known to modulate the rhizosphere microbiota. Glucosinolates are defense compounds present in the Brassicaceae family capable of deterring pathogens, herbivores and biotic stressors in the phyllosphere. In addition, traces of glucosinolates and their hydrolyzed byproducts have been found in the soil, suggesting that these secondary metabolites could play a role in the modulation and establishment of the rhizosphere microbial community associated with this family. We used Arabidopsis thaliana mutant lines with disruptions in the indole glucosinolate pathway, liquid chromatography-tandem mass spectrometry (LC-MS/MS) and 16S rRNA amplicon sequencing to evaluate how disrupting this pathway affects the root exudate profile of Arabidopsis thaliana, and in turn, impacts the rhizosphere microbial community. Chemical analysis of the root exudates from the wild type Columbia (Col-0), a mutant plant line overexpressing the MYB transcription factor ATR1 (atr1D) which increases glucosinolate production, and the loss-of-function cyp79B2cyp79B3 double mutant line with low levels of glucosinolates confirmed that alterations to the indole glucosinolate biosynthetic pathway shifts the root exudate profile of the plant. We observed changes in the relative abundance of exuded metabolites. Moreover, 16S rRNA amplicon sequencing results provided evidence that the rhizobacterial communities associated with the plant lines used were directly impacted in diversity and community composition. This work provides further information on the involvement of secondary metabolites and their role in modulating the rhizobacterial community. Root metabolites dictate the presence of different bacterial species, including plant growth-promoting rhizobacteria. Our results suggest that alterations in the indole glucosinolate pathway cause disruptions beyond the endogenous levels of the plant, significantly changing the abundance and presence of different metabolites in the root exudates of the plants as well as the microbial rhizosphere community.