Periodic Reporting for period 1 - AS-ABA (Alternative splicing and SR proteins in ABA-mediated plant stress responses) Reporting period: 2017-09-01 to 2019-08-31 Summary of the context and overall objectives of the project Abiotic stresses such as drought, high salinity, and extreme temperatures represent the most pervasive causes of loss of crop productivity worldwide. Therefore, a major goal in plant science is to understand how plants respond and withstand environmental stresses successfully. An adequate response to osmotic stress in plants involves endogenous accumulation of the hormone abscisic acid (ABA), which induces the activation of downstream effectors such as transcription factors and ion channels, resulting in the implementation of adaptive responses to withstand reduced water availability. While important research efforts have efficiently identified the transcriptional and posttranslational changes associated with plant responses to ABA, the posttranscriptional regulation of this hormonal pathway remains poorly explored.This project aims at understanding the role of alternative splicing, a process by which the same gene can generate multiple transcripts, during plant stress responses. This question is being addressed through the study of established important upstream modulators of this process, serine/arginine-rich (SR) proteins. SR proteins constitute a highly conserved family of RNA-binding proteins and are established important regulators of splicing. They affect splice sites in a concentration and phosphorylation-dependent manner and are conserved in all genomes known to undergo alternative splicing, being therefore widely recognized as major modulators of this process. Thus, the central hypothesis of the AS-ABA proposal is that posttranscriptional networks, SR protein nodes in particular, act as central coordinators of plant abiotic stress responses, namely by targeting ABA pathway components.Overview of the action: In view of the evidence linking AS to plant environmental responses and the recent findings by the Duque group implicating Arabidopsis SR proteins in stress responses regulated the ABA phytohormone, the overall objectives of this project are to uncover novel SR protein players in ABA-mediated plant stress tolerance and identify the splicing targets underlying their mode of action.Conclusions of the action:In this project, we identified the SR34a splicing factor as an important negative regulator of ABA-mediated stress responses during early seedling development. Indeed, using reverse genetic approaches, we showed that SR34a might regulate stress responses by decreasing sensitivity to ABA during seed germination and the post-germination transition from heterotrophic to autotrophic growth. In an attempt to address the molecular mode of action of SR34a, we first showed that this nuclear protein can bind RNA in vivo. Current ongoing work is being done in order to identify RNA targets and elucidate the exact molecular mechanisms by which SR34a controls those ABA-mediated stress responses.This project could have important socio-economic implications. Indeed, ABA-related stresses such as drought and high-salinity are severe environmental stresses that constitute the primary causes of crop losses worldwide and reduce average yields of most major crops by more than 50%. Thus, understanding how plants withstand environmental stresses successfully is a major objective in plant science. The control of plant stress responses by alternative splicing modulation is a relatively recent field of research holding much promise in providing new potential solutions for agricultural losses. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Identification and screening of SR mutant lines for phenotypes during ABA responsesArabidopsis mutant lines for nine SR genes were ordered from available Arabidopsis mutant collections, and were screened for differential phenotypes in response to ABA during early stages of development (developmental stages particularly sensitive to the phytohormone). We could observe a clear ABA-hypersensitive phenotypes for a mutant in SR34a and SCL28 during seed germination and early seedling development. Indeed, when plated on ABA-supplemented medium or on medium that mimics osmotic stress, the sr34a-1 showed strong hypersensitivity during germination (Fig 1 A-C). In addition to germination, the sr34a-1 mutant showed severe hypersensitivity to ABA during post-germination growth (Figure 1 D-E), which was correlated with a higher level of expression of several ABA-marker genes (Figure 1 F-H), and this phenotype was rescued in mutant seedlings recapitulating the wild-type expression of SR34a. Together, these data indicate that SR34a is involved in decreasing sensitivity to ABA during seed germination and early seedling development.Identification of SR34a RNA targetsTo uncover the direct targets of SR34a, I am using the individual nucleotide resolution Crosslinking ImmunoPrecipitation (iCLIP) technique, which relies on the immunoprecipitation of UV-induced covalently bonded SR34a-RNA complexes, and provides additional detailed information on the site of interaction. This is currently being done at the Bielefeld University (Germany, secondment) in collaboration with Dr. Staiger and Dr. Köster who have successfully established this protocol in plant systems. Preliminary results from this experiment indicate that SR34a, which is mainly accumulated in the plant cell nucleus, can bind RNA in vivo. We will now identify the nature of the SR34a RNA targets by sequencing the RNAs associated with SR34a, and unravel key information on the molecular mode of action of this splicing factor during ABA-mediated stress responses.Dissemination of the resultsDuring the implementation of this project, I actively participated in the dissemination of my research results. My first step was to communicate my research plans and results for discussion during weekly lab meetings including all members of the host laboratory. At the host institute, I presented my data in the Annual Postdoc Retreat organized by the IGC Postdoc Committee, of which I was a member between 2017 and 2018. This was for me a good opportunity to highlight the importance of plant research to my co-workers from other research fields, and to exchange innovative ideas with them. I also attended and presented posters at the 8th National RNA Meeting (Porto, 2019) and the Society of Experimental Botany (SEB) Meeting (Seville, 2019). This fellowship also allowed me to fund my participation in the International Conference on Arabidopsis Research (ICAR) that will take place in Seattle in July 2020 and in which I will apply for an oral presentation. Finally, the work that was done during this fellowship is expected to be published in the near future in high-ranked journals. In addition, we wrote a review on how plants use alternative splicing to control ABA-mediated stress responses which was published in Trends in Plant Science in 2018. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) This report is my final fellowship report. The results gathered from this project, as well as the potential economic impacts have been presented in the previous sections.