An expression-based functional screening highlights the importance of splicing and growth-regulation processes for osmotic stress tolerance

Drought and soil salinity limit available land for agriculture and reduce crop yields by imposing osmotic stress on plants. The identification of genes involved in plant tolerance mechanisms is a key goal to develop crops better capable to handle these stresses. Previously, several genes whose expression is differentially regulated in Solanum tuberosum culture cells adapted to increasing concentrations of polyethylene glycol (PEG) were identified. Here, the functional role of fifty of these genes was verified by studying their orthologues in Arabidopsis thaliana. Homozygous knockout lines for each gene were subjected to a large-scale phenotype screening in order to identify genes involved in adaptation to osmotic stress. Using this strategy, we have identified two genes whose function in stress response was so far unexplored: a splicing factor, which we named IAG1 (INSENSITIVE TO ABA IN GERMINATION1) and the putative negative regulator of TOR pathway, which we named XSA1 (EXTRA SENSITIVE TO ABA1). The Arabidopsis IAG1 is induced upon long-term exposure to abscisic acid (ABA) and PEG and is mainly expressed in trichomes and stomata, organs controlling transpiration. iag1-1 knockout mutants were highly insensitive to ABA treatments, germinating and developing fully expanded cotyledons in presence of high concentrations of the hormone, while IAG1 over-expressing plants showed a significant hypersensitivity to ABA. IAG1 protein interaction with SUA, another important splicing factor, suggests an involvement of IAG1 in pre-mRNA splicing of ABA effectors such as ABI3, a major regulator of ABA-related seed maturation. Similarly, XSA1 possibly affects several pathways in the ABA-mediated response to osmotic stress. XSA1 is constitutively expressed in vascular tissues and is up-regulated by long-term exposure to NaCl and ABA. The abolished expression of XSA1 leads to ABA hypersensitive phenotypes at germination and seedling stage as well as severe reduction in root development, indicating alteration in ABA biosynthesis and/or perception. Taken together, our results reveal promising mechanisms of plant acclimation to stress. IAG1 and XSA1 are active participants in the response to drought and salinity and represent interesting targets for future use in crop species.