Alternative splicing (AS) of precursor mRNAs (pre-mRNAs) from multiexon genes allows organisms to increase their coding potential and regulate gene expression through multiple mechanisms. Recent transcriptome-wide analysis of AS using RNA sequencing has revealed that AS is highly pervasive in plants. Pre-mRNAs from over 60% of intron-containing genes undergo AS to produce a vast repertoire of mRNA isoforms. The functions of most splice variants are unknown. However, emerging evidence indicates that splice variants increase the functional diversity of proteins. Furthermore, AS is coupled to transcript stability and translation through nonsense-mediated decay and microRNA-mediated gene regulation. Widespread changes in AS in response to developmental cues and stresses suggest a role for regulated splicing in plant development and stress responses. Here, we review recent progress in uncovering the extent and complexity of the AS landscape in plants, its regulation, and the roles of AS in gene regulation. The prevalence of AS in plants has raised many new questions that require additional studies. New tools based on recent technological advances are allowing genome-wide analysis of RNA elements in transcripts and of chromatin modifications that regulate AS. Application of these tools in plants will provide significant new insights into AS regulation and crosstalk between AS and other layers of gene regulation.

Abiotic and biotic stresses are major limiting factors of crop yields and cause billions of dollars of losses annually around the world. It is hoped that understanding at the molecular level how plants respond to adverse conditions and adapt to a changing environment will help in developing plants that can better cope with stresses. Acquisition of stress tolerance requires orchestration of a multitude of biochemical and physiological changes, and most of these depend on changes in gene expression. Research during the last two decades has established that different stresses cause signal-specific changes in cellular Ca²⁺ level, which functions as a messenger in modulating diverse physiological processes that are important for stress adaptation. In recent years, many Ca²⁺ and Ca²⁺/calmodulin (CaM) binding transcription factors (TFs) have been identified in plants. Functional analyses of some of these TFs indicate that they play key roles in stress signaling pathways. Here, we review recent progress in this area with emphasis on the roles of Ca²⁺- and Ca²⁺/CaM-regulated transcription in stress responses. We will discuss emerging paradigms in the field, highlight the areas that need further investigation, and present some promising novel high-throughput tools to address Ca²⁺-regulated transcriptional networks.

Alternative splicing and alternative polyadenylation (APA) of pre-mRNAs greatly contribute to transcriptome diversity, coding capacity of a genome and gene regulatory mechanisms in eukaryotes. Second-generation sequencing technologies have been extensively used to analyse transcriptomes. However, a major limitation of short-read data is that it is difficult to accurately predict full-length splice isoforms. Here we sequenced the sorghum transcriptome using Pacific Biosciences single-molecule real-time long-read isoform sequencing and developed a pipeline called TAPIS (Transcriptome Analysis Pipeline for Isoform Sequencing) to identify full-length splice isoforms and APA sites. Our analysis reveals transcriptome-wide full-length isoforms at an unprecedented scale with over 11,000 novel splice isoforms. Additionally, we uncover APA of ∼11,000 expressed genes and more than 2,100 novel genes. These results greatly enhance sorghum gene annotations and aid in studying gene regulation in this important bioenergy crop. The TAPIS pipeline will serve as a useful tool to analyse Iso-Seq data from any organism.

Primary transcripts (precursor-mRNAs) with introns can undergo alternative splicing to produce multiple transcripts from a single gene by differential use of splice sites, thereby increasing the transcriptome and proteome complexity within and between cells and tissues. Alternative splicing in plants is largely an unexplored area of gene expression, as this phenomenon used to be considered rare. However, recent genome-wide computational analyses have revealed that alternative splicing in flowering plants is far more prevalent than previously thought. Interestingly, pre-mRNAs of many spliceosomal proteins, especially serine/arginine-rich (SR) proteins, are extensively alternatively spliced. Furthermore, stresses have a dramatic effect on alternative splicing of pre-mRNAs including those that encode many spliceosomal proteins. Although the mechanisms that regulate alternative splicing in plants are largely unknown, several reports strongly suggest a key role for SR proteins in spliceosome assembly and regulated splicing. Recent studies suggest that alternative splicing in plants is an important posttranscriptional regulatory mechanism in modulating gene expression and eventually plant form and function.

Summary: Precursor mRNAs with introns can undergo alternative splicing (AS) to produce structurally and functionally different proteins from the same gene. Here, we show that the pre‐mRNAs of Arabidopsis genes that encode serine/arginine‐rich (SR) proteins, a conserved family of splicing regulators in eukaryotes, are extensively alternatively spliced. Remarkably about 95 transcripts are produced from only 15 genes, thereby increasing the complexity of the SR gene family transcriptome by six‐fold. The AS of some SR genes is controlled in a developmental and tissue‐specific manner. Interestingly, among the various hormones and abiotic stresses tested, temperature stress (cold and heat) dramatically altered the AS of pre‐mRNAs of several SR genes, whereas hormones altered the splicing of only three SR genes. These results indicate that abiotic stresses regulate the AS of the pre‐mRNAs of SR genes to produce different isoforms of SR proteins that are likely to have altered function(s) in pre‐mRNA splicing. Sequence analysis of splice variants revealed that predicted proteins from a majority of these variants either lack one or more modular domains or contain truncated domains. Because of the modular nature of the various domains in SR proteins, the proteins produced from splice variants are likely to have distinct functions. Together our results indicate that Arabidopsis SR genes generate surprisingly large transcriptome complexity, which is altered by stresses and hormones.

Pre-mRNA splicing is an essential post-transcriptional process in all eukaryotes. In vitro splicing systems using nuclear or cytoplasmic extracts from mammalian cells, yeast, and Drosophila have provided a wealth of mechanistic insights into assembly and composition of the spliceosome, splicing regulatory proteins and mechanisms of pre-mRNA splicing in non-plant systems. The lack of an in vitro splicing system prepared from plant cells has been a major limitation in splicing research in plants. Here we report an in vitro splicing assay system using plant nuclear extract. Several lines of evidence indicate that nuclear extract derived from Arabidopsis seedlings can convert pre-mRNA substrate (LHCB3) into a spliced product. These include: (1) generation of an RNA product that corresponds to the size of expected mRNA, (2) a junction-mapping assay using S1 nuclease revealed that the two exons are spliced together, (3) the reaction conditions are similar to those found with non-plant extracts and (4) finally mutations in conserved donor and acceptor sites abolished the production of the spliced product. This first report on the plant in vitro splicing assay opens new avenues to investigate plant spliceosome assembly and composition, and splicing regulatory mechanisms specific to plants.