Project description:Deep sequencing provided evidence that a novel subset of small RNAs were derived from the chloroplast genome of Chinese cabbage (Brassica rapa) and Arabidopsis (Ler). The chloroplast small RNAs (csRNAs) include those derived from mRNA, rRNA, tRNA and intergenic RNA. The rRNA-derived csRNA were preferentially located at the 3â-ends of the rRNAs, while the tRNA-derived csRNAs were mainly located at 5â-termini of the tRNAs. After heat treatment, the abundance of csRNAs decreased in chinese cabbage seedlings, except those of 24 nt in length. The novel heat-responsive csRNAs and their locations in the chloroplast were verified by Northern blotting. The regulation of some csRNAs to the putative target genes were identified by real-time PCR. Our results indicated that high temperature regulated the production of some csRNAs, which may have potential roles in transcriptional or post-transcriptional regulation, and affected putative target genes expression in chloroplast.
Project description:We performed chloroplast ChIP-seq (cpChIP-seq) to identify the possible DNA-binding sites of mTERF5 in Arabidopsis thaliana. To this end, we generated transgenic Arabidopsis plants expressing mTERF5 carrying an HA tag under the control of the CaMV 35S promoter. Then, We used the polyclonal antibody (abcam, ab9110, lot GR304617-8 ) against HA tag which conjugated to ChIP-Grade protein A/G agarose (Thermo scientific, 26161, lot QJ223903) to perform cpChIP assay. The obtained chromatin immunoprecipitated DNA of chloroplasts were used to build DNA libaries for high-throughput sequencing. Finally, we showed that three potenssial DNA regions across the chloroplast genome compared to the control group were enriched by mTERF5.
Project description:Because the minimal chloroplast genome carries very limited genetic information, plants rely on signals sent from the chloroplasts to the nucleus for proper chloroplast development as well as for recovery from photoinhibition and response to photo-oxidative stress. In this study, we report the discovery of several factors involved in the reduced PQ pool-driven chloroplast-to-nucleus signaling process. High-throughput RNA-Seq expression profiling of tanorexia-1 (tnr-1) mutants in comparison to wild-type. From these experiments, we found out that the HSF and HAC1 transcription factors have broad effects on HL-driven nuclear gene expression. The DEAD-box RNA helicase 38, CRY1 and a previously uncharacterized G-patch domain-containing protein are also involved in the signaling.
Project description:The project aims to use photocycle mutants to understand the importance of phot2 autophosphorylation for signaling leading to chloroplast accumulation and avoidance. The second part is focused on the identification of new interacting proteins for phot2.
Characterization of proteins important for signaling leading to chloroplast avoidance. PHOT2-GFP wild type and PHOT2-V392L-GFP muteins were immunoprecipitated from leaves of transgenic Arabidopsis plants, either dark-adapted or irradiated with low or high blue light. Proteins identified by Mass Spectrometry as interacting with wild-type PHOT2-GFP and PHOT2-V392L-GFP were compared to identify proteins putatively involved in chloroplast avoidance. Phosphorylation profiles of PHOT2-GFP wild type and PHOT2-V392L-GFP were analyzed to distinguish between phosphorylation sites characteristic for chloroplast accumulation and avoidance.
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.
Project description:To explore the role and target of chloroplast proteases under heat stress, thylakoid membranes were isolated from wild-type and mutant chloroplast thylakoid membrane-localized proteases after heat stress and subjected to comparative quantification by LC-MS/MS analysis using the spectral counting method.
Project description:Deep sequencing provided evidence that a novel subset of small RNAs were derived from the chloroplast genome of Chinese cabbage (Brassica rapa) and Arabidopsis (Ler). The chloroplast small RNAs (csRNAs) include those derived from mRNA, rRNA, tRNA and intergenic RNA. The rRNA-derived csRNA were preferentially located at the 3M-CM-"M-BM-^@M-BM-^Y-ends of the rRNAs, while the tRNA-derived csRNAs were mainly located at 5M-CM-"M-BM-^@M-BM-^Y-termini of the tRNAs. After heat treatment, the abundance of csRNAs decreased in chinese cabbage seedlings, except those of 24 nt in length. The novel heat-responsive csRNAs and their locations in the chloroplast were verified by Northern blotting. The regulation of some csRNAs to the putative target genes were identified by real-time PCR. Our results indicated that high temperature regulated the production of some csRNAs, which may have potential roles in transcriptional or post-transcriptional regulation, and affected putative target genes expression in chloroplast. Examination of two replicates of heat treated (HT) and control (MT) Chinese cabbage sample respectively, and one Arabidopsis (Ler) RNA sample.
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.