Application of SILIA-based quantitative phosphoproteomics in study of ethylene-dependent stem gravitropism in a model eukaryotic organism Arabidopsis

Abstract

Phosphorylation of proteins plays a critical role in many aspects of the cellular dynamic process such as enzyme activity, protein structural changes, and protein-protein interaction. The changes in protein phosphorylation can be detected using the combination of the high-resolution mass spectrometer and ¹⁵N-stable isotopic labeling in Arabidopsis (SILIA) quantitative proteomic approach. The SILIA quantitative phosphoproteomic approach has versatile usages in the study of growth, development and life cycle of a model plant and it preserves the in vivo post-translational modification status during the entire sample preparation procedure. In my project, a series of improvements have been made about the accuracy, repeatability, quantitation and completeness of the stable isotopic metabolic labeling method. Taking advantages of the improved method, I have exploited the phosphoproteomics changes affected by either short-term or long-term ethylene. Examination of ethylene-regulated phosphosites utilizing the group-based prediction system with a protein-protein interaction prediction capability revealed 14 kinase-substrate connections that may work in both CTR1 kinase- and PP2A phosphatase-mediated phosphor-relay pathways. Finally, a model for ethylene-controlled post-translational modifications was established. The results imply that ethylene regulates the phosphorylation of arginine/serine-rich splicing factor 41 and plasma membrane intrinsic protein 2A in a dual-and-opposing mode. Another large-scale phosphoproteomics analysis was conducted over the ethylene-dependent rapid gravitropic response. The data shows that phosphorylation of PHOT1, CESA4, CRS1 and Sec14p-like phosphatidylinositol transfer family protein is involved in the rapid gravitropic response. The follow-up physiological experiments on the transgenic plants reveal that the phot1 and phot1phot2 loss-of-function double mutant lines are deficient in rapid gravitropic response and the PHOT1 complemented double mutant line exhibits distinct rapid gravicurvature phenotypes. These results suggest that PHOT1, as a blue light receptor in mediating phototropism, also plays a significant role in ethylene-dependent gravity signaling and rapid gravicurvature response. The study presents a state-of-the-art quantitative proteomics application in discovering the active components in plant cell signaling pathways, which opening up opportunities for more efficient and holistic approach to dissect the cell signaling networks in plants.

Date of Award	2016
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology