Characterization of Upstream Regulators Controlling Light-Mediated Alternative Splicing During Photomorphogenesis

Abstract

Alternative splicing (AS) is increasingly recognized to play a role in plant development, including photomorphogenesis. Photomorphogenesis is accompanied by massive reprogramming of gene expression via changes in total transcript levels, transcription start sites, translational control, and AS. Previously, it has been shown that external sugar supply and the inhibition of kinase signaling cause similar AS changes in dark grown Arabidopsis thaliana seedlings as observed upon illumination. These findings suggested that light-mediated AS involves metabolic and kinase signaling. To further dissect the upstream regulatory mechanisms, we first analyzed the role of the two major energy sensor kinases ‘SNF1-related kinase1’ (SnRK1) and ‘target of rapamycin’ (TOR) during skoto- and photomorphogenesis. Using inducible artificial microRNAs, we found similar AS changes in etiolated seedlings upon SnRK1 and TOR knockdown, as observed in response to light or sugar treatment. Furthermore, phenotypical analyses revealed that both kinase mutants display shortened hypocotyls in darkness, but also exhibit delayed cotyledon opening during de-etiolation in light. These findings demonstrate that both SnRK1 and TOR are indispensable for proper skoto- and photomorphogenic growth. Given the role of SnRK1 and TOR in light-responsive AS regulation, we proposed that an altered kinase signaling might trigger phosphorylation-dependent changes in the activity of splicing regulators and hence, the AS response. Using phosphoproteomics, we identified splicing regulators from the RS subfamily of serine/arginine-rich (SR) proteins that are specifically phosphorylated upon sugar and light treatment. The RS subfamily is specific to plants and comprises four members in Arabidopsis: RS31, RS31a, RS40 and RS41. Knocking out all four RS genes resulted in almost complete male sterility, suggesting that RS proteins are essential for reproductive processes. In addition, we found that RS proteins are crucial for skoto- and photomorphogenic growth, as they control hypocotyl elongation and cotyledon opening. We could demonstrate that RS-induced cotyledon opening involves brassinosteroid signaling and further found that rs mutants display an altered red-light sensitivity. Splicing pattern studies revealed that RS proteins contribute to the regulation of light-mediated AS events in darkness. Furthermore, we showed that RS proteins localize to the nucleus and undergo a re-localization from the nucleoplasm into biomolecular condensates in response to light and sugar. Together, our results provide novel insights into mechanisms and biological functions of SnRK1, TOR and RS proteins in light-dependent plant development.

Die Dissertation ist gesperrt bis zum 04. Oktober 2024 !