Proteogenomic Analysis Reveals Abscisic Acid Response Through Alternative Splicing And Alternative Translation In Arabidopsis Seedlings

In eukaryotes,mechanisms such as alternative splicing(AS)and translation(ATI)contribute to the protein diversity of organisms.Specifically,splicing factors play crucial roles during stress treatments,in hormone responses and during plant development.However,the underlying mechanisms are not well investigated in plants.Here,we employed a parallel approach using RNA sequencing and proteomic identification by constructing specific libraries to unravel AS isoforms and novel proteins in response to abscisic acid(ABA)treatment.Our results suggest that approximately 78% of intron-containing genes were alternatively spliced.Amongst total141,419 idenfied AS events,two newly defined AS types,which are referred to as alternative first exon(TSS)and alternative last exon(TTS),were more abundant than intron retention(IR),exon skipping(SKIP),and multi-exon skipping(MSKIP)in Arabidopsis.Genes located in the ABA pathways were altered at the transcript level in ABA-treated samples compared with a control.Interestingly,approximately 40% of the AS isoforms were regulated independently from their DE values,indicating that a new layer of AS regulation may play an important role in the ABA response.Categories,such as the tricarboxylic acid(TCA)cycle,glycolysis,photosynthesis,etc,enriched or depleted during the treatment may represent long-term AS regulation in response to ABA.Conventional splicing sites(5'-GT-AG-3')accounted for 96% of AS sites using all AS events identified from previous findings.However,the percentage of GT/AG decreased to 89% when subject to ABA-treatment,whereas the percentage of other non-canonical AS sites increased by 1.3 to 11.1-fold,suggesting that the alternation of ABA-regulated AS events may result from the manipulation of the splicing sites.In comparison to AS events detected under normal condition,ABA-regulated AS isoforms were more likely to be translated into proteins.Subsequent bioinformatic and structural analysis indicates that the alternation of splicin g factor AS isoforms was crucial in response to ABA treatment.ABA extensivelyregulates the AS pattern in Arabidopsis by increasing the number of non-conventional splicing sites.This work is also the first instance in which thousands of newproteins encoded from the same m RNA through ATI or 3' to 5' translation were detected using a self-constructed library on the sense and antisense strand of the Arabidopsis genome.All of these results expand our knowledge with respect to understanding the regulation of AS and translation mechanisms under normal condition and in response to ABA treatment.In this study,a proteogenomic approach has been used for the first time to simultaneously analyse both transcriptome and proteome datasets in planta.As ABA is a popular plant hormone that can participate in variable stress responses,we treated seedlings with ABA to study its molecular function in Arabidopsis.Valuable information has been obtained from bioinformatic analysis using customized databases followed by experimental validation.The entire workflow is applicable for other plant species.