SPLICING FACTOR FOR PHYTOCHROME SIGNALING promotes photomorphogenesis by regulating alternative splicing in Arabidopsis

Purpose: The goals of this study are to compare the transcriptome profiling and alternative splicing (AS) profiling between Col-0 wild type and SFPS knockout mutant (sfps-2) through RNA-seq to determine the molecular mechanisms of how splicing factor SFPS regulates photomorphogenesis in Arabidopsis. Results: Using an optimized data analysis workflow, we mapped about 100 million sequence reads per sample to the Arabidopsis genome (TAIR10) and identified 1495 differentially expressed genes between Col-0 and mutant dark samples; 1361 differentially expressed genes between Col-0 and mutant red light treated samples; 4291 differentially expressed genes between Col-0 dark and red light treated samples; and 4479 differentially expressed genes between mutant dark and red light treated samples. Except for gene expression, we also discovered 788 differentially spliced bins between Col-0 and mutant dark samples; 827 differentially spliced bins between Col-0 and mutant red light treated samples; 610 differentially spliced bins between Col-0 dark and red light treated samples; and 405 differentially spliced bins between mutant dark and red light treated samples. Altered splicing of 9 genes was confirmed with qRT-PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Conclusions: Our study represents the first detailed analysis of SFPS mutant transcriptomes, with biologic replicates, generated by RNA-seq technology. Our results show that SFPS regulates photomorphogenesis in Arabidopisis through regulating the splicing activity of light signaling genes, which helps us. Overall design: Methods: Total RNA (rRNA was depleted) is extracted from 4-day-old Col-0 wild-type (WT) and SFPS knockout mutant (sfps-2) dark grown seedlings with or without 3hrs continuous red light irradiation (7µmolm-2s-1). The RNA-seq libraries are generated according to the commercial handbook from the Illumina kit (TruSeq Stranded Total RNA with Ribo-Zero Plant). The transcriptome and alternative splicing profiles are generated from Col_D, Col_Rc, mut_D, and mut_Rc samples, in triplicate. The sequence reads were analyzed to find the differentially expressed genes or differentially spliced bins by the following packages: TopHat, Cufflinks, and ASpli. qRT–PCR validation was performed using TaqMan and SYBR Green assays.