Functional Analysis Of SEC In Response To Abiotic Stresses In Arabidopsis

O-linked ?-N-acetylglucosamine(O-GlcNAc)modification is widespread on Ser/Thr residues of nuclear-cytoplasmic protein in animals and plants.In animal,O-GlcNAc modifieation is regulated by two highly conservative enzymes,O-GlcNAc transferase(OGT)and O-GlcNAcase(OGA).One is to O-GlcNAcylate protein,OGT Transferase,another is to make the protein to get rid of O-GlcNAc modified enzyme OGA.OGT is highly conservative from nematode to mammal and plants and plays functional roles in response to various stresses,such as UV radiation,ethanol,NaCl andReactive oxygen species(ROS).There are two OGT homologs in Arabidopsis thaliana,SPY(SPINDLY)and SEC(SECRET AGENT).SPY shares the similarity of 35-39%with SEC,which is at the equal level of SPY and OGT.Nevertheless,SEC shares 63%similarity with OGT in c-terminal conservative catalytic activity domain.Unlike SPY,SEC has been proved to have catalytic activity.The full length ORF of SEC gene is 2934 bp,encoding 977 amino acids.As a homolog of OGT,SEC also has two function domains.One is TPR structure at the amino-terminal which is involved in the interaction between proteins.The other domain locates at the carboxy-terminal and is responsible for substrate catalysis.Bioinformatics prediction showed that there are multiple stress responsive cis-elements,including three ABREs,one MBS,one TC-rich repeats and one W-box.Analysis of SEC expression patterns at transeriptional level indicated that SEC is induced by drought and NaCl,but not responsive to ABA and cold stress.So we speculated that SEC plays a role in abiotic stress response.To further investigate the biological function of SEC,two mutants,sec-3 and sec-4 were obtained from the SALK T-DNA collection,and homozygous mutants were identified for further analysis.Compared with Col-0,the mutant sec-4 shows more tolerance to drought and salt stresses by analyzing the germination and survival rate.Furthermore,overexpression of SEC rescued the phenotype of sec-4 under drought and salt stress treatment.Based on biological software prediction,ERD5 is a potential candidate of interaction protein of SEC.The interaction between SEC and ERD5 was confirmed by yeast two-hybrid and Co-IP assays.ERD5,the proline dehydrogenase,participates in proline degradation process.We found that both SEC and ERD5 located in nucleus,cytoplasm and cytomembrane,which provides space possibility for protein interaction.Using WGA pull down and O-GlcNAc specific antibody CTD 110.6 to enrich O-GlcNAc modified proteins and identify particular O-GlcNAc sites,We confirmed two O-GlcNAc modified sites of ERD5 in proline dehydrogenase domain.The T-DNA insertion mutant of ERD5 i5 also responsive to abiotic stress.erd5 enhanced tolerance of Arabidopsis to abiotic stresses.We suggested that s SEC plays a role in abiotic stress by interacting with ERD5 to regulate proline synthesis.We analyzed the proline content in plants treated with Alloxan,a glycosyl transferase inhibitor.Our data showed that proline content was increased after Alloxan treatment.To further analyze the molecular mechanism of SEC responding to abiotic stress,the expression level of stress related marker genes,KIN1,COR15,RD29A,RD29B and P5CS were detected in Col-0 and sec-3 under drought and salt stresses condition.The expression level of these genes in sec-3 were significantly higher than that of wild type.Interestingly,RD29A was signifieantly up-regulated under both drought and salt stresses.Further analysis showed that the histone modification(H3K4me3,H3K27me3 and H3Ac)of RD29A were changed obviously in sec-4 comparing with Col-0 by ChIP under drought and salt stresses.We also identified that the binding abundance of SEC on different regions of 7RD29A was varied after drought and salt stresses.In conclusion,we speculated that SEC is involved in abiotic stress response mainly by two pathways.Firstly,SEC O-GlcNAcylates ERD5 to affect proline synthesis.Secondly,SEC regulates RD29A expression by influencing the histone modifications at RD29A chromotin regions under stress condition.