The detection of differentially expressed gene and atlas construction of pre-mRNA alternative splicing during seed germination of Arabidopsis thaliana

Abstract

Seed germination is the initial phase of life cycle for plants and plays a pivotal role in regeneration of plant population, continuation of plant species and crop yield. However, seed germination is an extremely complex biological process and its regulatory mechanisms remain unclear. It is well known that gene expression and pre-mRNA alternative splicing are vital to the central dogma. In Arabidopsis genome, ~60% multi-exon genes are alternatively spliced. Alternative splicing is involved in the response to environmental cues, including abiotic and biotic stresses, in the regulation of crucial developmental processes such as flowering, and in circadian timekeeping. The present paper studies the effect of gene expression and alternative splicing on seed germination in Arabidopsis . We constructed RNA-seq datasets from dry seeds, germinating seeds 1–3, Young seeds and Seeds in Arabidopsis and carried out quality control for the raw reads by using FastQC and Trimmomatic. StringTie estimated transcript and gene expression from a mapping to the Arabidopsis genome obtained from Hisat2. We next created the gene expression matrix for Arabidopsis , which includes 37336 genes along the rows and 12 samples along the columns, and employed DESeq2 to systematically identify differentially expressed genes between different stages during seed germination. To elucidate the biological functions of differentially expressed genes, we performed a functional enrichment analysis of these genes using clusterProfiler tool. The results showed that many genes involved in metabolic processes, such as glycolysis and DNA synthesis, were activated during the seed germination. It ensures the reactivation of metabolism and basic cellular activities of seeds. Some genes involved in environmental responses, such as oxidative stress, toxic substance, heat, and water, were inhibited. This is essential for seeds to be successfully activated from a dormant state. Furthermore, rMATS was utilized to detect the pre-mRNA alternative splicing events and differential alternative splicing events under different conditions. We constructed the atlas of pre-mRNA alternative splicing at different stages of seed germination and analyzed the distribution characteristics of alternative splicing patterns across every stage. Besides, we detected the differential alternative splicing events between different germination stages and analyzed the biological functions of differential alternative splicing events during seed germination using clusterProfiler. A total of 18 genes, in which differential retained intron events were identified between dry seeds and germinating seeds 3, were enriched on splicing process. By visualization of alternative splicing events in AT4G25500 , which was done by using the rmats2sashimiplot tool, the inclusion levels and functions of differential alternative splicing between different stages of seed germination were clearly illuminated. Overall, we investigated the dynamic changes of gene expressed levels and alternative splicing events during seed germination of Arabidopsis based on bioinformatics methods and dissected omics features and regulated mechanisms during seed germination from the perspective of gene expression and alternative splicing of pre-mRNA. This study is expected to be helpful for elucidating the regulated mechanisms of seed germination and to provide a guideline for relevant experimental works.