Project description:Purpose: Using RNA-seq and differential expression analysis, we examined the NAE-type and organ-specific genetic pathways involved in transducing ear;y signals into downstream physiological changes involved in Arabidopsis seedling growth. Methods: Intact seedlings and dissected cotyledon and root mRNA profiles of 3-day-old Arabidopsis seedlings treated with DMSO, 40 µM NAE 18:2 or 80 µM NAE 18:3 were generated by deep sequencing, in triplicate, using the Illumina Next-Seq 500 system. The sequence reads that passed quality filters were analyzed using STAR followed by DESeq2. qRT–PCR validation was performed using SYBR Green assays Results: Using RNA-seq and differential expression analysis, we identified early (1 h) transcriptional changes induced by the exogenous treatment of NAE 18:2 and NAE 18:3 in cotyledons, roots and intact seedlings. These two treatments led to a significant enrichment in ABA-response and chitin-response genes in organs where the treatments led to changes in development. In Arabidopsis seedlings, NAE 18:2 treatment led to the repression of genes involved in cell wall biogenesis and organization in roots and seedlings. In addition, cotyledons, roots, and seedlings treated with NAE 18:3 also showed a decrease in transcripts that encode proteins involved in growth processes. NAE 18:3 also led to changes in the abundance of transcripts involved in the modulation of chlorophyll biosynthesis and catabolism in cotyledons. Overall, NAE 18:2 and NAE 18:3 treatment led to lipid-type and organ-specific gene expression changes that include overlapping and non-overlapping gene sets. These data will provide future, rich opportunities to examine the genetic pathways involved in transducing early signals into downstream physiological changes in seedling growth. Conclusions: A detailed transcriptional analyses provided insight into the early organ-specific molecular responses to NAE 18:2 and NAE 18:3. Using this experimental and computational approach, we gained an unbiased view of the NAE 18:2- and NAE 18:3-modulated transcriptome changes that are activated and lead to downstream changes in seedling development. NAE 18:2 and NAE 18:3 induced the expression of several genes also induced by ABA or chitin treatment. The overlap with ABA-response genes corroborates previously work; however, this is the first time chitin-response genes have been identified as part of the NAE-modulation of seedling growth. Overall, the bioinformatic analyses presented here supports the hypothesis that NAE 18:2 and NAE 18:3 elicit organ-specific and signal- specific molecular changes that precede developmental changes in Arabidopsis seedlings. Further, these data provide novel insights into the genetic programs that are modulated by NAE 18:2 and NAE 18:3 at the organ-specific level in Arabidopsis seedlings, and will facilitate future studies of the corresponding signaling networks.

Project description:The first described feedback loop of the Arabidopsis circadian clock is based on reciprocal regulation between TOC1 and CCA1/LHY. CCA1 and LHY are MYB transcription factors that bind directly to the TOC1 promoter to negatively regulate its expression. Conversely, the activity of TOC1 has remained less well characterized. Genetic data supports that TOC1 is necessary for the reactivation of CCA1/LHY, but there is little description of its biochemical function. Here we show that TOC1 occupies specific genomic regions in the CCA1 and LHY promoters. Purified TOC1 binds directly to DNA through its CCT domain, which is similar to known DNA binding domains. Chemical induction and transient overexpression of TOC1 in Arabidopsis seedlings cause repression of CCA1/LHY expression demonstrating that TOC1 can repress direct targets, and mutation or deletion of the CCT domain prevents this repression showing that DNA binding is necessary for TOC1 action. Furthermore, we use the Gal4/UAS system in Arabidopsis to show that TOC1 acts as a general transcriptional repressor, and that repression activity is in the Pseudoreceiver (PR) domain of the protein. To identify the genes regulated by TOC1 on a genomic scale, we couple TOC1 chemical induction with microarray analysis and identify new potential TOC1 targets and output pathways. Together these results define the biochemical action of the core clock protein TOC1 and refine our perspective on how plant clocks function. Keywords: Expression profiling by array wild type (Col-0) and ALC::TOC1 were sown on Murashige-Skoog with 0.8% agar, stratified for 48 hours and grown in12:12 light:dark (LD) for 12 days and either left in LD or transferred to constant light (LL) and then grown for one more day before the start of the experiment. Tissue was submerged in Murashige-Skoog media supplemented with 2.5% ethanol or no ethanol (mock) and with 20mM MG132 for 3 hours and harvested at ZT1. Three replicates each of the seedlings were collected and frozen in liquid nitrogen.

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Repression of bZIP11 expression in Seedlings

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