The Regulatory Mechanism And Structural Biology Of ACS6 In Arabidopsis Thaliana

Ethylene is an important plant hormone that plays a vital role in plant growth and development as well as in responding to various biotic and abiotic stresses.The ethylene signal transduction pathway is initiated by inducing biosynthesis of ethylene.1-aminocyclopropane-l-carboxylic acid synthase(ACS)is the rate-limiting enzyme in the process of ethylene biosynthesis,which is tightly adjusted at both transcriptional and post-translational levels.ACS in Arabidopsis thaliana has a conserved catalytic domain at the N-terminus,of which type 1 ACS(ACS2 and ACS6)contain a long C-terminal flexible region including multiple phosphorylation sites.In planta studies have indicated that mitogen-activated protein kinases(MAPKs,referred as MPKs in Arabidopsis)can enhance the protein stability of ACS2 and ACS6 through phosphorylation,and may promote the gene expression of ACS2/6 to regulate ethylene synthesis induced by pathogen infection and wounding.In addition,calcium-dependent protein kinases(CDPKs,referred as CPKs in Arabidopsis)were reported to be involved in wound-induced ethylene production by modulating the gene expression of ACS2/6/8.CPKs have also been shown to phosphorylate type 1 ACS.However,the molecular mechanisms of type 1 ACS regulated by MPKs and/or CPKs remain elusive.In this study,we performed in vitro biochemical studies on the regulation of Arabidopsis MPKs and CPKs on ACS6.We first generated full-length and/or truncated constructs of MPK3/4/6,CPK3/4 and ACS6,and overexpressed in Escherichia coli system.The soluble proteins were purified by a series of chromatography methods,and the final samples of good purity and high concentration were stored at-80℃.Secondly,the phosphorylated,active MPK3/6 can phosphorylate ACS6 about three sites in vitro,and the auto-phosphorylated CPK3/4 can phosphorylate ACS6 about one site in vitro;Then,the substrate specificity of MPKs for ACS6 was verified by protein interaction assay,which showed that ACS6 can form a stable complex with MPK3/6,but little interaction,if any,with MPK4 was detected;and the C-terminal flexible region of ACS6 was indispensable for MPK6 interaction,which contains a conserved kinase interaction motif(KIM);Finally,we tried to elucidate the mode of interaction between MPK6 and ACS6 and the stability regulation mechanism of ACS6 through structural study methods.The full-length ACS6 proteins,wild-type and mutants,were subjected to crystallization trials,and some small single crystals were obtained.