Mechanism Of Cys-dependent Phosphorylation Of MAPK In Low Temperature Response Of Watermelon

As a fruit-based horticultural crop,watermelon(Citrullus lanatus)holds an irreplaceable position in China,and cold stress is an important environmental factor that limits its economic value.The response of plants to low temperature depends on reactive oxygen species(ROS)signal transduction,and the balance of cellular redox state plays an important role for plants in maintaining stress resistance.Thioredoxins(TRXs)are oxidoreductases encoded by multiple gene family,which contains the consensus amino acid sequence WC(G/P)PC and are widely involved in the response to stress.However,limited knowledge is available about the function of TRXs in watermelon.12 genes were identified in the watermelon genome as h-type TRXs,and selected the member ClTRX h2 that play a major role in the low temperature response.Further,the role of ClTRX h2 as well as the relationship between TRX-mediated redox balance and MAPK signaling pathway were investigated.The main research findings are as follows:1.ClTRX h2 localized at the plasma membrane is responsible for chilling tolerance in watermelon.It was found that ClTRX h2 positively regulates the chilling tolerance by VIGS,and the expression of ClTRX h2 was dynamically induced when responding to chilling stress.Subcellular localization verified that ClTRX h2 was located on the plasma membrane.The chilling physiological indexes of ClTRX h2-VIGS lines in watermelon and-overexpressed lines in tobacco were determined.It was proved that ClTRX h2 was involved in ROS scavenger under stress and positively regulated the low temperature resistance of watermelon.2.ClMPK3 activated by low temperature signal negatively regulates chilling resistance in watermelon through CBF-COR pathway.It was identified that ClMPK3 was rapidly phosphorylated after 30 min chilling treatment by LC/MS,and the chilling resistance of ClMPK3-silenced plants were higher than control,suggesting that ClMPK3 negatively regulated the response to low temperature in watermelon.Further,q RT-PCR showed that the expression levels of ClCBFs and ClCORs were significantly increased after chilling treatment,and the up-regulation ratio in ClMPK3-silenced plants were significantly higher than that in control.3.ClTRX h2 inhibits phosphorylation of ClMPK3 by interacting with ClMPKK5.The interaction between ClTRX h2 and ClMPKK5 was confirmed by yeast two-hybrid system and Firefly luciferase complementation imaging assay.It was found that the silencing of ClTRX h2 increased the phosphorylation of ClMPK3 whether control or chilling.Further more,the phosphorylation of ClMPK3 regulated by redox depends on ClTRX h2.In vitro kinase experiments showed that ClMPK3 can be phosphorylated by ClMPKK5,while the mutation in Cys-229 site inhibited this phosphorylation,proved that the Cys-229 site of ClMPKK5 was crucial for its kinase activity.In summary,this study indicates a link between thiol redox balance and MAPK cascade signaling that the redox state mediated by ClTRX h2 inhibits the phosphorylation of ClMPK3 through the interaction between ClTRX h2 and ClMPKK5,thereby activating the ClCBF-ClCOR signaling pathway under chilling stress.This study provided a new idea for the molecular mechanism of signal transduction in plants under low temperature,as well as a theoretical basis for the improvement of watermelon varieties with low temperature tolerance.