In eukaryotes, mechanisms such as alternative splicing (AS) and alternative translation initiation (ATI) contribute to organismal protein diversity. Specifically, splicing factors play crucial roles in responses to environment and development cues. However, the underlying mechanisms are not well investigated in plants. Here, we report the parallel employment of RNA sequencingshort-read RNA sequencing, single molecule long-read sequencing and proteomic identification to unravel AS isoforms and novel unannotated proteins in response to abscisic acid (ABA) treatment. Combining the data of the two sequencing methods, approximately 83.4% of intron-containing genes were alternatively spliced. Two newly defined AS types, which are referred to as alternative first exon (AFE) and alternative last exon (ALE), were more abundant than intron retention (IR). However, by contrast to AS events detected under normal conditions, ABA-regulateddifferentially expressed AS isoforms were more likely to be translated. ABA extensively regulates affects the AS pattern by increasing the number of non-conventional splicing sites. This work is also the first instance in whichidentified thousands of newunannotated proteins encoded from the same mRNA through ATI were detected using a self-constructed library on the both strands of the Arabidopsis genome. The combined results thus enhance our understanding on the regulation essential function of AS and translation mechanisms under normal condition and in response to ABA treatment.