Differential splicing fine‐tunes guard cell gene expression and is required for drought tolerance in <i>Arabidopsis thaliana</i>

The increasing global prevalence of droughts due to climate change poses a major threat to food security, necessitating the development of drought-tolerant crops. To respond appropriately to environmental perturbations, plants undergo widespread transcriptional reprogramming at both the gene expression and splicing levels. Differential splicing dynamically alters the ratio of splice isoforms, generating transcriptomic diversity that can be translated into novel protein isoforms or fine-tune gene expression levels through post-transcriptional regulatory pathways. In this study, we examine the role of differential splicing in Arabidopsis thaliana guard cells during progressive drought using nuclear RNA sequencing. Using this data set and RT-PCR on different RNA pools, we demonstrated that many alternative transcripts are degraded by nonsense-mediated mRNA decay or are retained in the cell's nucleus, processes largely regulating gene expression rather than generating proteomic diversity. We investigated differences between differentially expressed and differentially spliced genes and revealed a potential role for gene pleiotropy in determining the preferred regulatory pathway. We then showed that differential splicing of a novel drought-responsive gene is required for optimal drought tolerance. This analysis highlights both the utility of differential splicing analyses in identifying important stress-responsive genes and the diverse cellular fates of physiologically relevant differential splicing events.