Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Light- and Phytochrome-Dependent Regulation of Hypocotyl Elongation in Arabidopsis thaliana

ABSTRACT: To identify and characterize genes required for tissue-specific phytochrome responses during hypocotyl development in far-red-light grown bvr lines, we performed gene transcriptional profiling using bvr lines with mesophyll-specific phytochrome inactivation (cab3: :pBVR2). We identified several candidate genes whose expression is significantly altered in lines with mesophyll tissue-specific BVR expression (Cab3::pBVR2), compared to constitutive phytochrome inactivation lines, i.e. 35S-driven BVR lines (35S::pBVR3). No-0 is used as wild-type (WT) Seeds of No-0 WT, 35S::pBVR3 and CAB3::pBVR2 on MS plates were exposed to Red (R) light of 75 M-BM-5mol m-2 s-1 for 5 min and imbibing seeds were cold-stratified at 4 M-BM-0C in darkness for 3 d. Seedlings were grown under continuous far-red illumination for 7 d. Seven-day-old vegetative whole seedlings (300 M-bM-^@M-^S 500 mg) were quickly (<1 min) harvested and immediately frozen in liquid nitrogen inside the FR chamber. Seedlings were grown under continuous far-red illumination for 7 d.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Sookyung Oh

PROVIDER: E-GEOD-38989 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:FAR-RED ELONGATED HYPOCOTYL 3 (FHY3) and its homolog FAR-RED IMPAIRED RESPONSE 1 (FAR1) are two transposase-derived transcription factors initially identified as the key components in phytochrome A signaling and recently shown to function in the circadian clock. However, whether FHY3 and FAR1 are involved in other processes of plant development remains largely unknown. Here, we explored chromatin immunoprecipitation-based sequencing (ChIP-seq) analysis to identify 1745 and 1171 FHY3 direct binding target genes in darkness and far-red light conditions, respectively in the Arabidopsis thaliana genome. This analysis revealed that FHY3 preferentially binds to the gene promoters through the previously identified typical FHY3/FAR1 binding motif. Interestingly, FHY3 also binds to two novel motifs in the 178-bp repeats of the Arabidopsis centromere regions in vivo. Comparison between the ChIP-seq and microarray data indicates that FHY3 regulates the expression of 196 and 85 genes in dark and far-red respectively by directly binding to their promoters. FHY3 also co-regulates a number of common target genes with PHYTOCHROME INTERACTING FACTOR 3-LIKE 5 (PIL5) and ELONGATED HYPOCOTYL 5 (HY5). Moreover, our genome-wide identification of FHY3 direct target genes ultimately led to the discovery and validation of a new role of FHY3 in controlling chloroplast development, by directly activating the expression of ACCUMULATION AND REPLICATION OF CHLOROPLASTS5 (ARC5), a key gene regulating chloroplast constriction and division. Taken together, our data suggest that FHY3 is involved in regulating multiple facets of plant development, thus providing new insights into the functions of this type of transposase-derived transcription factors. To determine the global in vivo binding sites of FHY3, we performed ChIP-seq analysis using 35S:3FLAG-FHY3-3HA fhy3-4 transgenic lines in which the 3FLAG-FHY3-3HA fusion proteins could largely rescue the long hypocotyl phenotype of the fhy3-4 mutants The seedlings were grown in D or continuous FR light conditions for 4 days, and then chromatin fragments were prepared using the commercially monoclonal anti-FLAG antibodies. We then generated two DNA libraries, one for D and one for FR -grown samples, which were then subjected to ultra-high throughput Solexa (Illumina) sequencing

Project description:FAR-RED ELONGATED HYPOCOTYL 3 (FHY3) and its homolog FAR-RED IMPAIRED RESPONSE 1 (FAR1) are two transposase-derived transcription factors initially identified as the key components in phytochrome A signaling and recently shown to function in the circadian clock. However, whether FHY3 and FAR1 are involved in other processes of plant development remains largely unknown. Here, we explored chromatin immunoprecipitation-based sequencing (ChIP-seq) analysis to identify 1745 and 1171 FHY3 direct binding target genes in darkness and far-red light conditions, respectively in the Arabidopsis thaliana genome. This analysis revealed that FHY3 preferentially binds to the gene promoters through the previously identified typical FHY3/FAR1 binding motif. Interestingly, FHY3 also binds to two novel motifs in the 178-bp repeats of the Arabidopsis centromere regions in vivo. Comparison between the ChIP-seq and microarray data indicates that FHY3 regulates the expression of 196 and 85 genes in dark and far-red respectively by directly binding to their promoters. FHY3 also co-regulates a number of common target genes with PHYTOCHROME INTERACTING FACTOR 3-LIKE 5 (PIL5) and ELONGATED HYPOCOTYL 5 (HY5). Moreover, our genome-wide identification of FHY3 direct target genes ultimately led to the discovery and validation of a new role of FHY3 in controlling chloroplast development, by directly activating the expression of ACCUMULATION AND REPLICATION OF CHLOROPLASTS5 (ARC5), a key gene regulating chloroplast constriction and division. Taken together, our data suggest that FHY3 is involved in regulating multiple facets of plant development, thus providing new insights into the functions of this type of transposase-derived transcription factors. We used the FHY3p:FHY3-GR fhy3-4 transgenic lines expressing FHY3-glucocorticoid receptor (GR) fusion proteins under the control of the FHY3 native promoter, in which, without dexamethasone (DEX), the GR-fusion proteins localize in the cytoplasm, whereas, when treated with DEX, the fusion proteins translocate into the nucleus. The seedlings were grown in D or continuous FR light for 4 days, then treated with DEX or mock (equal volume of ethanol), and grown in the same conditions for a further 2 hours before the samples were harvested. RNA samples were extracted from these samples, and then hybridized with Affymetrix Arabidopsis ATH1 genome arrays. Four independent biological replicates for each treatment were used for the microarray analysis.

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Light- and Phytochrome-Dependent Regulation of Hypocotyl Elongation in Arabidopsis thaliana

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