Rapid and dynamic alternative splicing impacts the Arabidopsis cold response transcriptome

Background: Plants have adapted to tolerate and survive constantly changing environmental conditions by re-programming gene expression. The scale of the contribution of alternative splicing (AS) to stress responses has been underestimated due to limitations in RNA-seq analysis programs and poor representation of AS transcripts in plant databases. Significantly, the dynamics of the AS response have not been investigated but this is now possible with accurate transcript quantification programs and AtRTD2, a new, comprehensive transcriptome for Arabidopsis. Results: Using ultra-deep RNA-sequencing of a time-course of Arabidopsis thaliana plants exposed to cold treatment, we identified 8,949 genes with altered expression of which 2,442 showed significant differential alternative splicing (DAS) and 1,647 genes were regulated only at the level of AS (DAS-only). The high temporal resolution demonstrated the rapid induction of both transcription and AS resulting in coincident waves of differential expression (transcription) and differential alternative splicing in the first 6-9 hours of cold. The differentially expressed and DAS gene sets were largely non-overlapping, each comprising thousands of genes. The dynamic analysis of AS identified genes with rapid and sensitive AS within 3 h of transfer to the cold (early AS genes), which were enriched for splicing and transcription factors. A detailed investigation of the novel cold-response DAS-only gene, U2B2-LIKE, suggested that it regulates AS and is required for tolerance to freezing. Conclusions: Our data indicate that transcription and AS are the major regulators of transcriptome reprogramming that together govern the physiological and survival responses of plants to low temperature.