Summary: 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 short‐read RNA sequencing, single molecule long‐read sequencing and proteomic identification to unravel AS isoforms and previously unannotated proteins in response to abscisic acid (ABA) treatment. Combining the data from the two sequencing methods, approximately 83.4% of intron‐containing genes were alternatively spliced. Two 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, differentially expressed AS isoforms were more likely to be translated. ABA extensively affects the AS pattern, indicated by the increasing number of non‐conventional splicing sites. This work also identified thousands of unannotated peptides and proteins by ATI based on mass spectrometry and a virtual peptide library deduced from both strands of coding regions within the Arabidopsis genome. The results enhance our understanding of AS and alternative translation mechanisms under normal conditions, and in response to ABA treatment.

The next-generation sequencing (NGS) technology has greatly facilitated genomic and transcriptomic studies, contributing significantly in expanding the current knowledge on genome and transcriptome. However, the continually evolving variety of sequencing platforms, protocols and analytical pipelines has led the research community to focus on cross-platform evaluation and standardization. As a NGS pioneer in China, the Beijing Genomics Institute (BGI) has announced its own NGS platform designated as BGISEQ-500, since 2016. The capability of this platform in large-scale DNA sequencing and small RNA analysis has been already evaluated. However, the comparative performance of BGISEQ-500 platform in transcriptome analysis remains yet to be elucidated. The Illumina series, a leading sequencing platform in China’s sequencing market, would be a preferable reference to evaluate new platforms. To this end, we describe a cross-platform comparative study between BGISEQ-500 and Illumina HiSeq4000 for analysis of Arabidopsis thaliana WT (Col 0) transcriptome. The key parameters in RNA sequencing and transcriptomic data processing were assessed in biological replicate experiments, using aforesaid platforms. The results from the two platforms BGISEQ-500 and Illumina HiSeq4000 shared high concordance in both inter- (correlation, 0.88–0.93) and intra-platform (correlation, 0.95–0.98) comparison for gene quantification, identification of differentially expressed genes and alternative splicing events. However, the two platforms yielded highly variable interpretation results for single nucleotide polymorphism and insertion–deletion analysis. The present case study provides a comprehensive reference dataset to validate the capability of BGISEQ-500 enabling it to be established as a competitive and reliable platform in plant transcriptome analysis.

Roots are important plant organs. Lateral root (LR) initiation (LRI) and development play a central role in environmental adaptation. The mechanism of LR development has been well investigated in Arabidopsis. When we evaluated the distribution of auxin and abscisic acid (ABA) in maize, we found that the mechanism differed from that in Arabidopsis. The distribution of ABA and auxin within the primary roots (PRs) and LRs was independent of each other. Auxin localization was observed below the quiescent center of the root tips, while ABA localized at the top of the quiescent center. Furthermore, NaCl inhibited LRI by increasing ABA accumulation, which mainly regulates auxin distribution, while auxin biosynthesis was inhibited by ABA in Arabidopsis. The polar localization of ZmPIN1 in maize was disrupted by NaCl and exogenous ABA. An inhibitor of ABA biosynthesis, fluridone (FLU), and the ABA biosynthesis mutant vp14 rescued the phenotype under NaCl treatment. Together, all the evidence suggested that NaCl promoted ABA accumulation in LRs and that ABA altered the polar localization of ZmPIN1, disrupted the distribution of auxin and inhibited LRI and development.

Alternative splicing (AS) exists in eukaryotes to increase the complexity and adaptability of systems under biophysiological conditions by increasing transcriptional and protein diversity. As a classic hormone, abscisic acid (ABA) can effectively control plant growth, improve stress resistance, and promote dormancy. At the transcriptional level, ABA helps plants respond to the outside world by regulating transcription factors through signal transduction pathways to regulate gene expression. However, at the post-transcriptional level, the mechanism by which ABA can regulate plant biological processes by mediating alternative splicing is not well understood. Therefore, this paper briefly introduces the mechanism of ABA-induced alternative splicing and the role of ABA mediating AS in plant response to the environment and its own growth.

Post-transcriptional mechanisms (PTMs), including alternative splicing (AS) and alternative translation initiation (ATI), may explain the diversity of proteins involved in plant development and stress responses. Transcriptional regulation is important during the hypoxic germination of rice seeds, but the potential roles of PTMs in this process have not been characterized. We used a combination of proteomics and RNA sequencing to discover how AS and ATI contribute to plant responses to hypoxia. In total, 10 253 intron-containing genes were identified. Of these, ~1741 differentially expressed AS (DAS) events from 811 genes were identified in hypoxia-treated seeds compared with controls. Over 95% of these were not present in the list of differentially expressed genes. In particular, regulatory pathways such as the spliceosome, ribosome, endoplasmic reticulum protein processing and export, proteasome, phagosome, oxidative phosphorylation, and mRNA surveillance showed substantial AS changes under hypoxia, suggesting that AS responses are largely independent of transcriptional regulation. Considerable AS changes were identified, including the preferential usage of some non-conventional splice sites and enrichment of splicing factors in the DAS data sets. Taken together, these results not only demonstrate that AS and ATI function during hypoxic germination but they have also allowed the identification of numerous novel proteins/peptides produced via ATI.

A full-length transcriptome-based proteogenomic data set reveals the complexity of rice gene arrangement and the transcriptome’s coding ability.