Abiotic stresses are known to dramatically affect plant growth by influencing the capacity of cells to expand properly, which substantially limits crop production worldwide. Therefore, a detailed understanding of how cell expansion regulators are modulated in response to environmental cues is crucial in the development of efficient strategies to improve crop production, particularly given the threatening environmental fluctuations imposed by global climate change. Alternative splicing (AS) is a key posttranscriptional mechanism that generates multiple mRNAs from the same gene, thus regulating gene expression and expanding proteomic diversity, which is emerging as crucial in allowing plants to adapt their development to stressful environments. This project aimed at investigating the impact of this understudied regulatory layer on genes modulating cell expansion during plant growth under stress conditions.
SR (serine/arginine-rich) proteins belong to a highly conserved family of splicing factors that plays a crucial role in regulating AS. Research in the host laboratory had contributed to determine that both the expression and function of plant SR proteins are stress regulated at multiple levels, with previous work showing that loss-of-function mutations in plant SR protein genes affect the capacity of seedlings to develop under abiotic stress. Our analyses of publicly available RNA-seq data have also revealed that AS events in cell expansion regulators are differentially regulated by environmental cues that impact plant growth. Together, these results encourage us to investigate a role for SR protein-mediated AS of cell expansion regulator genes in plant growth under abiotic stress.
We proposed to focus on three major tasks. In Task 1, we implemented a computational pipeline to detect and quantify all types of AS events in Arabidopsis. This pipeline was then applied on available RNA-seq data to generate an AS database, thus allowing the identification of AS events in genes involved in cell growth during plant development and in response to environmental stresses. The implementation of these tools was done in collaboration with the group of Manuel Irimia from the Centre for Genomic Regulation (CRG) in Barcelona, who has pioneered this method in several animal species, achieving accurate quantification and high validation rates. In parallel, Task 2 focused on generating mutant and transgenic Arabidopsis lines with altered SR protein levels to analyze their growth response to abiotic stresses. For plant lines with altered growth, we are investigating the SR regulation of specific AS events by means of RNA-seq and RT-qPCR. Finally, in Task 3, and based on the results from the two previous tasks, reverse genetics were planned to be used to functionally validate the predicted role of individual splice variants in plant growth adaptation to environmental stress.
