[Chuan-Chih Hsu] DIA-based phosphoproteomics identifies early phosphorylation events in response to EGTA and mannitol in Arabidopsis

Osmotic stress significantly hampers plant growth and crop yields, emphasizing the need for a thorough comprehension of the underlying molecular responses. Previous research has demonstrated that osmotic stress rapidly induces calcium influx and signaling, along with the activation of a specific subset of protein kinases, notably the Raf-SnRK2 kinase cascades within minutes. However, the intricate interplay between calcium signaling and the activation of RAF-SnRK2 kinase cascades remains elusive. Here in this study, we discovered that Raf-like protein (RAF) kinases undergo hyperphosphorylation in response to osmotic shocks. Intriguingly, treatment with the calcium chelator EGTA robustly activates RAF-SnRK2 cascades, mirroring the effects of osmotic treatment. This study represents the first instance where the Proteomics Core Lab utilized data-independent acquisition (DIA) technology to perform phosphoproteomics analysis, and we unveiled the global impact of EGTA on protein phosphorylation. Beyond the activation of RAFs and sucrose non-fermenting-1- related protein kinase 2s (SnRK2s), EGTA treatment also activates mitogen-activated protein kinase (MAPKs) cascades, calcium-dependent protein kinases (CDPKs), and receptor-like protein kinases, etc. Our findings illuminate the regulation of phosphorylation and cellular events by Ca2+ signaling, offering insights into the (exocellular) Ca2+ deprivation during early hyperosmolality sensing and signaling.

This project was conducted in collaboration with Dr. Pengcheng Wang’s lab at the Southern University of Science and Technology, China. The R&D scientists Chin-Wen Chen and Pei-Yi Lin from the Proteomics Core Lab are co-authors of this article. The study has been published in the journal of Molecular & Cellular Proteomics.

Sang, T.; Chen, C.-W.; Lin, Z.; Ma, Y.; Du, Y.; Lin, P.-Y.; Hadisurya, M.; Zhu, J.-K.; Lang, Z.; Tao, A. W.; Hsu, C.-C.*; Wang, P.* DIA-based phosphoproteomics identifies early phosphorylation events in response to EGTA and mannitol in Arabidopsis. Mol. Cell. Proteomics, 2024, doi.org/10.1016/j.mcpro.2024.100804.