Cytokinin treatment of rice induces minimal effect on differential splicing compared to splicing between tissues

Abstract

Alternatively spliced genes produce multiple spliced isoforms, called transcript variants. In differential alternative splicing, the relative abundance of transcript variants differs across sample types. Differential alternative splicing is common in animal systems and influences cellular development in many processes, but its extent and significance is not as well known in plants. To investigate alternative splicing in plants, we examined rice seedling RNA-Seq data that included approximately 40 million sequence alignments per library, three libraries per sample type, and four sample types: roots and shoots, each treated with exogenous cytokinin delivered hydroponically, plus mock-treated controls. Cytokinin treatment triggered expression changes in thousands of genes, but had minimal effect on alternative splicing. However, there were many splicing differences between roots and shoots. Analysis of a similar (but less deeply sequenced) mRNA-Seq data set from Arabidopsis showed the same pattern. Quantitative fragment analysis of reverse transcriptase-PCR products made from newly prepared rice samples confirmed nine of ten differential splicing events between rice roots and shoots. In both the Arabidopsis and rice mRNA-Seq data sets, most genes annotated as alternatively spliced had small minor variant frequencies, i.e., the less frequently observed forms were supported by fewer than 20% of informative mRNA-Seq sequences. Of splicing choices where there was more abundant support for minor forms, most alternative splicing events were located within the protein-coding sequence and also maintained the annotated reading frame. A tool for visualizing protein annotations in the context of genomic sequence (ProtAnnot) together with a genome browser (Integrated Genome Browser) were used to visualize and assess effects of differential splicing on gene function. In general, differentially spliced regions coincided with conserved regions in the encoded proteins, indicating that differential alternative splicing is likely to affect protein function between root and shoot tissue in rice.