Identification and screening of SR mutant lines for phenotypes during ABA responses
Arabidopsis 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 targets
To 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 results
During 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.
