Quantitative and functional phosphoproteomics study reveals new signaling components mediating the force-induced bolting delay in arabidopsis thaliana

Abstract

Thigmorphogenesis is the response of plants to environmental mechanical stimuli (like wind, raindrops, touch etc.) which can cause significant morphogenetic changes over times. After applied periodic touching stimulus in a long term period, Arabidopsis thaliana presents a shorter inflorescence length, delayed flowering day and smaller rosette radius. Which are consistent with the previous study. In cellular level, many molecular signals and phytohormones are involved in this response. In this study, ¹⁵N-stable isotope labeling in Arabidopsis (SILIA) combined with the high-resolution mass spectrometer approach was used to study new signaling components after 40 seconds touch treatment applied (1 touch/ second). Phosphorylation changes were the mainly target for profiling because its importance. In total, 4, 895 unique phosphopeptides were identified at least twice experimentally. And 579 unique phosphopeptides were novel ones. As for the quantification result, 24 phosphopeptides driven from 34 proteins were touch regulated. Selected from these 34 touch regulated proteins, both MAP Kinase Kinase 2 (MKK2) and an previously unknown protein that being named Touch-Regulated Phosphoprotein 1 (TREPH1) were continued with the functional study. Loss-of-function mutants mkk2 and treph1-1 were insensitive to gentle touch and exhibits earlier bolting under mechanical stresses compared with wild type. Transformation of S625A mutant TREPH1 protein into the loss-of-function treph1-1 background failed to complement its abnormal thigmomorphogenesis. Furthermore, transcriptomics analysis of the loss-of-function treph1-1 and mkk2 mutants showed a reduced gene expression in touch-inducible genes. Confocal and super-resolution microscopy analysis of TREPH1 protein subcellular localization revealed that it located on the membrane of the plastid envelope and plasma membrane. All these functional data support these two proteins (especially the phosphorylation site being identified by LC-MS/ MS) are essential for Arabidopsis mechanical stimuli.

Date of Award	2019
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology