Identification And Functional Analysis Of Methylglyoxal-modified Proteins In Arabidopsis

Methylglyoxal(MG)is a highly reactive a-ketoaldehyde formed as a by-product of glycolysis,which is induced by abiotic stresses such as high salinity,drought,and heavy metals.The glyoxalase ?(GLY?)and glyoxalase ?(GLY?)protect plants from abiotic stresses.However,the role of MG in biotic stress response is still poorly unknown.In animals,MG plays important roles in many physiological processes and diseases through modifying proteins.But MG-modified proteins have not yet been reported in plants.The triphosphate tunnel metalloenzyme(TTM)superfamily can hydrolyze a range of tripolyphosphate(PPPi)substrates.Arabidopsis thaliana has three TTM genes:AtTTM1,AtTTM2,and AtTTM3.It has been reported that AtTTM2 acted as a negative regulator by suppressing SA synthesis and PR1expression in pathogen defense responses.However,the factor regulating AtTTM2 has not been reported yet.In this study,we use biochemical,molecular,and genetic approaches to explore how MG regulates biotic stress response by modifying proteins in Arabidopsis.We demonstrated that MG can be induced in pathogen infection and suppress the expression of PR1 through modifying AtTTM2,and thus reduce disease resistance of plants.The major results are summarized below:1.We verified that MG was accumulated after Pst DC3000(avrB)infection and exogenous MG treatment reduced disease resistance of wide(wild)-type Arabidopsis.qRT-PCR assay demonstated that the expression of PR1 was lower in comparison to control under MG-treated after pathogen infection,suggesting that accumulaed MG suppresses PR1 expression after pathogen infected,therefore regulates plant disease resistance.2.Monoclonal antibodies against MG-modified proteins has been successfully prepared,and we identified more than two hundreds MG-modified proteins involving in glycolysis,lipid metabolism,photosynthesis,abiotic stress,and biotic stress.These results suggest that MG may regulate growth and development,stress responses by modifying proteins in Arabidopsis.3.We choosed TTM superfamily(TTM1,TTM2,TTM3)and ADR1 family(ADR1-L1,ADR1-L2)among MG-modified proteins,for fuither study,our studies indicated that these proteins expressed and purified from E.Coli were modified by MG.4.We identified T-DNA insertion mutants of TTM1,TTM2,TTM3,ADR1-L1 and ADR1-L2,and analyzed ttm2-1 resistance to the pathogen infection.We found ttm2-1 was more resistant to the infection of Pst DC3000(avrB)than the wild type.However,when subjected to MG ttm2-1 mutant was more insensitive to pathogen infection than the wild type,indicating that MG may regulate PR1 to induce disease-susceptibility through modifying TTM2.