The discovery of the molecular regulation and modification mechanisms of the PLT2 protein through ROS under the RGF1 peptide signaling pathway
Stem cell niche and root meristem size are maintained by signaling networks of a peptide hormone, Root Meristem Growth Factor 1 (RGF1). How RGF1 regulates root meristem development is an essential question for understanding stem cell function. Recently, we reported that RGF1 controls root development by modulating Reactive Oxygen Species (ROS) distributions along Arabidopsis root developmental zones. RGF1 increased superoxide (O2-) levels in the meristematic zone (youngest stage). In contrast, hydrogen peroxide (H2O2) distributions started to be reduced in the elongation zone (transition stage) and the differentiation zone (mature stage). The root meristem master regulator (PLT2) protein stability is modulated by these two ROS altered by RGF1. However, the mechanism of how ROS modulates the PLT2 protein stability remained obscure. In this work, we discovered the regulation and modification mechanism of the PLT2 protein stability through ROS under the RGF1 signal. 1) We showed that one of the cysteines of the PLT2 played a pivotal role in modulating their stability through ROS. Substitution of the 212th cysteine enhanced the PLT2 protein stability through ROS and generated a larger root meristem compared with the native form PLT2 protein. 2) We detected S-sulfenylation of the PLT2 protein after H2O2 treatment. H2O2 treatment reduced the native form PLT2 protein levels but not the cysteine substitution form of PLT2 suggesting that modification of PLT2 by H2O2 modulate the PLT2 stability. H2O2 oxidizes a free thiol of cysteine and generates sulfenylated cysteine. We detected more S-sulfenylation of PLT2 after the H2O2 treatment compared with the Mock treatment. 3) We found the downstream gene regulation mechanisms of the cysteine substitution form PLT2 protein. The cysteine replacement form-PLT2 line generated a larger root meristem and had broader localization of the protein. The accumulated PLT2 protein with a cysteine substitution upregulated the downstream genes related to cell proliferation and reduced the gene expression of the differentiation genes. The transcriptome analysis showed the downstream gene regulation mechanism of the cysteine substitution line.
