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Genome-wide analysis of SPL7 regulated genes in Arabidopsis

ABSTRACT: To study the genes regulated by transcription factor SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (SPL7) in Arabidopsis thaliana, we conducted chromatin immunoprecipitation-based sequencing (ChIP-seq) in 35S:FALG-SPL7 transgenic plant and spl7 mutant, and genome-wide sequencing-based transcript profiling of Arabidopsis thaliana wild-type plants and of spl7 mutant under MS medium and MS supplement with 5µM CuSO4. These experiments led to the identification of genes that are direct target of SPL7 and genes differentially expressed in an SPL7-dependent or Cu-dependent manner. This study provides a framework for the identification of SPL7 regulated genes towards characterization of SPL7 in copper homeostasis.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE45213 | GEO | 2014/04/15

SECONDARY ACCESSION(S): PRJNA193223

REPOSITORIES: GEO

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Project description:To study the genes regulated by transcription factor SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (SPL7) in Arabidopsis thaliana, we conducted chromatin immunoprecipitation-based sequencing (ChIP-seq) in 35S:FALG-SPL7 transgenic plant and spl7 mutant, and genome-wide sequencing-based transcript profiling of Arabidopsis thaliana wild-type plants and of spl7 mutant under MS medium and MS supplement with 5µM CuSO4. These experiments led to the identification of genes that are direct target of SPL7 and genes differentially expressed in an SPL7-dependent or Cu-dependent manner. This study provides a framework for the identification of SPL7 regulated genes towards characterization of SPL7 in copper homeostasis. ChIP-Seq: Chromatin isolation was performed with 7-day-old whole seedlings of spl7 mutant (negative control) and 35S:FALG-SPL7 transgene (sample) grown on standard MS medium under continuous white light according to Bowler et al. (2004). The resuspended chromatin pellet was sonicated at 4°C with a Diagenode Bioruptor set at high intensity for 10 min (30 s on, 30 s off intervals). The DNA was sheared to an average size of approximately 500 bp. Chromatin was immunoprecipitated with FLAG antibody (Sigma) according to the Affymetrix Chromatin Immunoprecipitation Assay Protocol Rev.3. Reverse cross-linked DNA was purified and used for sequencing libraries construction following the manufacture’s protocol (Illumina). RNA-Seq: DNase I-treated total RNA was prepared from 7-day-old whole seedlings of wild type (Col-0) and spl7 mutant grown on standard MS medium and MS supplement with 5µM CuSO4 under continuous white light (WL). First- and second-strand cDNA were generated using SuperScript II and random hexamer primers. Double-stranded cDNA was fragmented by nebulization and used for mRNA library construction following the manufacturer’s protocol (Illumina). Tophat was used to map the sequence reads to the Arabidopsis genome and only uniquely mapped reads were used in subsequent analyses. mRNA profiles of 7-day-old whole seedlings of wild type (Col-0) or spl7 mutant grown on standard MS medium and MS supplement with 5µM CuSO4 under continuous white light (WL) were generated by deep sequencing using illumine HiSeq2000.

Project description:The transition metal copper (Cu) is an essential element for all living organisms. In plants, Cu plays key roles in electron transport chains of chloroplasts and mitochondria, as well as in cell wall metabolism, ethylene perception, molybdenum cofactor synthesis and oxidative stress protection. Because of its physiological importance, suboptimal concentrations of Cu trigger a re-organization of metabolism to economize on Cu, and pronounced Cu deficiency causes severe growth reduction and defects in male fertility. However, when present in excess, Cu can be highly toxic. Therefore, Cu uptake, utilization and cellular concentrations are strictly regulated. A number of components of the Cu-homeostatic network of Arabidopsis have already been identified. However, the mechanisms that control responses of plant gene expression to Cu-deficiency are only partly understood. In Chlamydomonas reinhardtii, the transcription factor Crr1 is required for activating and/or repressing the expression of a number of genes in response to Cu deficiency. This protein contains a plant-specific DNA-binding domain (SBP domain), ankyrin repeats and a C-terminal cysteine-rich region with similarity to a Drosophila metallothionein (MT). In Arabidopsis, there is a family of 16 proteins with SBP domains named SPL family (SBP-like) of which a subset of proteins have been implicated in flowering time control and floral development. Among all of them, AtSPL7 is the most similar to Crr1 (27 % identity), and AtSPL1 (25 % identity), AtSPL12 (24 % identity) and AtSPL14 (23 % identity) share a common protein architecture. The goal of this work was to obtain a complete account of the response of the Arabidopsis thaliana transcriptome to Cu deficiency, as well as to determine the role of AtSPL7 in the transcriptional response to Cu deficiency. For this purpose, three independent experiments were carried out including Col-0 wild-type and spl7-2 mutant plants grown in Cu-sufficient and Cu-deficient hydroponic media, respectively. Root and shoot transcriptomes were established by RNA-Seq for quantitative comparisons.

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Genome-wide analysis of SPL7 regulated genes in Arabidopsis

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