Involvement of Alternative Splicing in Barley Seed Germination.

Qisen Zhang,Songbo Wang,Gaofeng Zhou,Xiaoqi Zhang,Cong Tan,Chengdao Li

doi:10.1371/journal.pone.0152824

Abstract

Seed germination activates many new biological processes including DNA, membrane and mitochondrial repairs and requires active protein synthesis and sufficient energy supply. Alternative splicing (AS) regulates many cellular processes including cell differentiation and environmental adaptations. However, limited information is available on the regulation of seed germination at post-transcriptional levels. We have conducted RNA-sequencing experiments to dissect AS events in barley seed germination. We identified between 552 and 669 common AS transcripts in germinating barley embryos from four barley varieties (Hordeum vulgare L. Bass, Baudin, Harrington and Stirling). Alternative 3’ splicing (34%-45%), intron retention (32%-34%) and alternative 5’ splicing (16%-21%) were three major AS events in germinating embryos. The AS transcripts were predominantly mapped onto ribosome, RNA transport machineries, spliceosome, plant hormone signal transduction, glycolysis, sugar and carbon metabolism pathways. Transcripts of these genes were also very abundant in the early stage of seed germination. Correlation analysis of gene expression showed that AS hormone responsive transcripts could also be co-expressed with genes responsible for protein biosynthesis and sugar metabolisms. Our RNA-sequencing data revealed that AS could play important roles in barley seed germination.

Highlights

RNA sequencing (RNA-seq) generated about 26 million reads which accounted for at least 2.3 G base pairs for each barley library (S1 Table)
Genes coding for Auxin efflux carrier and Auxin responsive proteins (AUX/IAA) had many co-expressed genes, while Serine/threonine protein kinase SRK2 gene (SnRK2) had fewer co-expressed genes in protein synthesis and energy metabolism pathways (Table 3)
Since Viviparous 1 (VP1) gene played very important roles in both seed maturation and seed germination, we have closely examined the changes in its transcript level and Alternative splicing (AS) patterns

Summary

Materials and Methods

Four barley varieties of Hordeum vulgare L Bass, Baudin, Harrington and Stirling were selected for this study, since we wanted to dissect common AS events. In the data filter process, dirty reads with sequencing adapters, reads with N rates greater than 10 per cent and those with more than 50 per cent low quality (Qs < 20) were discarded. SOAPaligner/Soap (version 2.21) was chosen to align clean reads of each sample against the Morex genome (IBSC) [19] with a seed length of 32, allowing at most five mismatches in a read and maximal insert size of 1000 bp. SOAPsplice first detected the longest 5' end segment of an IUM read that was mapped to the reference, aligned the remaining segment to the reference sequences. With reads longer than 100 bp, SOAPsplice spliced the reads into multiple segments of 50 bp from the 5' end until the remaining segment was in length between 50 and 100 bp, which was spliced into two equal segments. The RNA-seq data have been submitted to NCBI GEO database (accession number GSE66024)

Results

Discussion

Conclusions