Methylglyoxal Modification Of Histone Mediates The Salt Stress Responses In Arabidopsis

Methylglyoxal (MG) as a toxic by-product of glycolysis presents ubiquitously in all cells and can be degraded by glyoxalase system. It can inhibit seed germination, root length and light-induced stomatal opening when its levels is up-elevated under stresses. For example, endogenous MG content is induced by 2-5-folds in NaCl-treated plants. However, how MG regulates salt-induced gene expression is unknown.Histone posttranslational modifications (PTMs) play important roles in many physiological processes, including cell differentiation, tissue development, cancer induction and environmental responses. Recently, many new PTMs have been identified on histone in animals with the development and application of high-sensitivity and high-resolution mass spectrometry. However, new histone PTMs and their functions have not been reported in plants yet.In this study, we explored how MG regulates the expression of salt stress response genes by modifying histone, using biochemical, genetic and molecular approaches, and demonstrated that MG functions as a new histone modifier in salt-induced gene expression.The main results are listed below:1. We verified that salt stress responsive genes were induced in MG-treated Arabidopsis seedlings by using RNA-seq and qRT-PCR assays. Whereas a T-DNA insertion mutant glyI2 with higher MG levels in comparison to WT displayed higher expression of stress responsive genes under salt stress,35S::GLYI2 35S::GLYII4 exhibited reduced expression of these genes, suggesting that MG mediates the process of salt stress-induced gene expression.2. We prepared monoclonal antibodies against MG-modified proteins and identified hundreds of MG-modified proteins, which are involved in glycolysis, tricarboxylic acid cycle, fat metabolism, photosynthesis, respiration, biotic and abiotic stress responses. These results imply that MG regulates plant growth, development and stress responses by modifying proteins. Furthermore, we found for the first time that histone H3 could be modified by MG in Arabidopsis.3. Western blot assay indicated that exogenous H3 was modified by MG. Further, methylglyoxalation of endogenous H3 was also detected by pull down assay and the modification sites of amino acid residues of H3 were identified by LC-MS/MS analysis, verifying MG as a new molecule of histone modification. In addition, ChlP-seq analysis showed that MG-modified proteins were distributed on a large number of gene loci with selective sequences. Using monoclonal antibodies against MG-modified H3R2 and H3K4, we further proved that MG can modify H3R2 and H3K4. These modifications were enhanced in gfyI2 and MG-treated wild-type plants, however 35S::GLYI235S::GLYII4 reduced the salt stress-induced modification levels of histone H3, consistent with the changes of stress responsive gene expression and ChIP-qPCR results. These results indicates that salt stress increases modification levels of histone H3 on response genes by promoting MG accumulation.4. To determine whether chromatin condensation change was caused by MG modification, we carried out MNase assays of nuclei treated with or without MG. As expected, MG-treated chromatin appeared more looser than the control. These results indicated that MG modification of histone H3 resultes in a more loose chromatin structure for change of gene expression.