Functional Study Of The Heat Shock Protein TMS1 In Arabidopsis

Heat stress causes heat shock response of cell,leading to accumulation of unfolded and misfolded proteins,which results in ER stress and triggers unfolded protein response(UPR).Most plant species have anthesis and seed sets in the hot summer.They can not move to avoid the environmentl stress like animals.Therefore,the plants have evolved a complex regulatory mechanism underlying the response of heat stress and ER stress.Our previous study found that the TMS1,which belongs to heat shock protein 40(Hsp40)family,was required for the heat tolerance of Arabidopsis pollen tubes and seedlings.TMS1 possesses the reductive activity similar to protein disulfide isomerase and may function in ER protein folding as a molecular chaperone.However,the regulatory mechanisms of TMS1 remain unknown.This study further analyzed the expression regulation and relationship with the heat shock protein BiP to explore the mechanism of TMS1 underlying the heat tolerance in Arabidopsis.The promoter region of TMS1 has a typical heat shock element(HSE)(aTTCgaGAAatTTCc)and an atypical HSE(cTTCtcgatttTTCtcGAAg).The result of yeast one hybrid assay showed that among the 21 heat shock transcriptin factors(Hsfs),only HsfA2 could bind to the promoter of TMS1.However,hsfa2 mutant did not affect the heat shock-induced expression level of TMS1.The hsfald hsfale double mutant of the HsfAld and HsfAle,the upstream Hsfs of HsfA2 affected heat shock-induced expression level of TMS1,implying that HsfAld and HsfAle may involve in regulation of TMS1 expression.The expression of TMS1 was induced not only by heat shock,but also by the ER stress-inducing agents:DTT and AZC,like BiP genes,indicating that TMS1 is responsive not only to heat shock,but also to ER stress.The bzip28 bzip60 double mutant of bZIP28 and bZIP60,primary UPR-responding transcription factors,could downregulate the heat shock-induced expression level of TMS1,suggesting that expression of TMSl is related to the signal pathway of bZIP28/bZIP60.In addition,bZIP28 and bZIP60 proteins could not bind to the promoter of TMS1.Thus,TMS1 is not the direct target of bZIP28 and bZIP60.Three Arabidopsis BiP proteins:BiPl,BiP2 and BiP3,share high amino acid sequence similarity.In particular,the amino acid sequence similarity between BiP1 and BiP2 is highest,up to 99%.LCI assay showed that the full length TMS1 protein and its DnaJ domain could interact with BiP1 and BiP3.GST pull-down analysis showed that the interaction of TMS1 DnaJ domain with BiPl or BiP3 was dependent on ATP;and the three conserved amino acids motif(HPD)was essential for the interaction between DnaJ and BiPs.Furthermore,the intereaction of TMS1 DnaJ domain with BiPl or BiP3 could stimulate the ATPase enzyme activity of the BiPs.Compared with wild type,12 genes were affected in the tmsl-1 seedlings grown in 22? by microarray assay,where 5 genes were downregulated and 7 genes were upregulated.Under 370C heat shock condition,6 genes were affected,where 3 genes were downregulated and 3 genes were upregulated.There were 6 genes whose expression was affected in the tmsl-1 seedlings either grown in 22? or treated by heat shock.The estimated functions of these genes are different.In conclusion,TMS1 is involved not only in heat shock response,but also in ER stress.It may function downstream of transcription factor genes bZIP28 and bZIP60,and plays important roles in heat tolerance of pollen tubes and other vegetative tissues,possibly by involvement in refolding or degradation of the unfolded and misfolded proteins as a co-chaperone of BiP.