RRC1 is required for epidermal phyB siganling by maintaining the circadian rhythm by suppressing the alternative splicing of core clock genes.

Purpose : The goal of this study is to identify transcriptome changes induced by rrc1 mutation under red light. We compared the transcriptome of MLB (ML1pro::phyB-GFP/phyB-9) with EMS27 grown either in darkness or continuous red light to investigate the genes that the expression and the splicing patterns are regulated by epidermal phyB and/or RRC1. The transcriptome of Col-0 and rrc1-689 was further compared to enlarge the list of putative splicing targets of RRC1. Methods : Seedlings grown under either monochromatic red light (19 μmol/m2s) or darkness for 60 hours on 1/2 MS agar plates were sampled. Total RNAs were extracted using the Spectrum Plant Total RNA Extraction Kit (Sigma-Aldrich). RNA libraries were generated and sequenced on the Illumina NextSeq 500 platform. The analysis was performed with three biological replicates. Roughly 90% of the sequencing reads were mapped to specific loci on the Arabidopsis TAIR10 reference genome using the TopHat pipeline. Mapped sequencing files were further processed with Aspli1.9 R package for Differentially Expressed Gene (DEG) analysis and Differentially Spliced Gene (DSG) analysis. Epidermal phyB-induced gene expression change was identified by comparing Dark-grown MLB with Red-grown MLB, while RRC1-induced gene expression or splicing change was identified by comparing Red-grown control line (Col-0 or MLB) with Red-grown rrc1 mutant (rrc1-689 or EMS27). All samples in triplicates.