The Maize Sep15-Like Involved In ER Stress In Response To Abiotic Stress

Maize(Zea mays)is a major food crop and an important industrial raw material.Due to the world population growth and the deterioration of the environment,coupled with the gradual reduction of the world’s arable land,enhancing the yield and the stress tolerance of maize have become an important and widespread topic of concern.By identifying stress-resistant genes of crops and combinating transgenic technology with traditional breeding techniques,the yield and the resistance to stress of crops could be effectively increased.Therefore,it becomes a basal work to characterize the stress-related genes of their functions and mechanisms.In this paper,ZmSep15-like gene of maize,a homolog of seleprotein15,was studied.Maize mutants,Arabidopsis and the other related materials were used to explore the function and mechanism of ZmSep15-like gene under NaCl and PEG(mannitol)by simulating the salt/drought stress in the natural environment.The main results obtained are as follows:1 Cloning and molecular characteristics of ZmSep15-likeThrough the analysis of databases information,it was found that there was a homolog of Sepl5 gene in maize.The cDNA of WT maize(W22)was used as a template,and two alternatively spliced cDNA sequences of ZmSep15-like were obtained by special primers,naming ZmSep15-like-1/2.It was shown that the Sep15 homologous sequences from animals and plants,including rice,wheat and so on,were very conserved in all species by analyzing their sequences alignment,molecular evolution,functional domains and tertiary structures.There are two conserved similar functional domains in Sep 15 sequences:a cysteine-rich domain(UGGT-binding domain)in N-terminus and a thioredoxin-like domain(TRX-like,involving in redox)in C-terminus.Subcellular localization showed that ZmSep15-like protein is mainly located in the ER.The expression patterns of ZmSep15-like gene in tissues and under salt or osmotic stress were analyzed by qRT-PCR.It was found that the expression of ZmSep15-like was not tissue-specific,but relatively higher in leaves and seeds,and also induced by salt or osmotic stress.2 The function of ZmSep15-like geneW22,hybrid(obtained by crossing between W22 and zmsepl5),and zmsep15 mutants were treated with 20%PEG and 200 mM NaCl for 48 h in three-leaf-stage seedlings.Compared to WT,the leaves of zmsepl5 seedlings appeared more strongly withered.Inspection of the relative water content(RWC%),proline,and chlorophyll and so on values recorded for the various genotypes exposed to each of the stressors showed that the zmsep15 mutants were the most strongly affected by the stress.A sample of the three-leaf-stage F2(crossed by zmsep15 and WT)seedlings was subjected to either PEG or NaCl stress,the results of which indicating that ZmSep15-like is a monogenic,dominant,and nuclear gene.By analyzing the expression of the marker genes including the bZIP family and ER stress(BiP and PDI),it was found that ZmSep15-like may also participate in the regulation of ER stress in plants.Further,the carbonyl content was increased and the peptone content was decreased significantly,the results of which indicating that the mutations of the Sep15 gene could increase ER stress in plants under stress.Meanwhile,the expression of an oxidative stress-related gene ERO1 in the ER was also significantly up-regulated,and the malondialdehyde(MDA)content was notably increased in the mutants,indicating that the oxidative stress was increased in the mutants.Further,the mutants accumulated more ROS than the WT under salt or osmotic stress,and the expression of ROS generation-related genes was significantly increased,while the activity of ROS scavenging-related enzymes was significantly reduced.These results shown that ZmSep15-like is involved in the regulation of redox balance.Exposured to either PEG or NaCl,leaf chlorosis appeared more severe and the chlorotic tissue thereafter became necrotic,and had a lower chlorophyll content in the mutants than in the WT seedlings.Exploiting the inability of compromised cells to exclude trypan blue confirmed that cell death was more widespread in the stressed mutants’ leaves than in those of WT under salt or osmotic stress treatment,and the transcription of the cell apoptosis-promoting genes was significantly increased,while the expression of cell apoptosis-inhibiting genes was decreased.In addition,the leaves of mutants exhibited an enhanced conductivity from electrolyte leakage compared with the leaves of WT.These results showed that loss of ZmSep15-like increased the sensitivity of the mutants to apoptosis under salt or osmotic stress.It was found that overexpression of ZmSepl5-like-2 in Arabidopsis could significantly enhance the resistance of Arabidopsis to mannitol or NaCl coupled with the OE lines forming larger leaves and longer roots than WT and atsep15 mutant.Further analysis found that the OE lines could reduce ER stress,ROS accumulation and apoptosis sensitivity of Arabidopsis under mannitol or NaCl treatment.These results demonstrated that ZmSep15-like was involved in above related physiological or signaling pathways.When WT and mutants seedlings were exposed for 48 h to DTT or TUN,the results showed that the two ER inhibitors could increase more ER stress and accumulate more ROS in the mutant,indicating that ER stress itself could trigger the production of ROS.3 The mechanism of ZmSepl5-like gene in response to abiotic stressThe Sep 15 homologous protein could interact with the UDP-glucose:glycoprotein glucosyltransferase(UGGT)in animals,and participate in the refolding of N-glycoprotein in the ER.There are two genes coding for UGGT homologues(ZmUGGT1/2)in Zmays.A typical UGGT protein has two functional domains:a glycosyl binding domain in the N-terminus and a glycosyltransferae domain of GT8 family in the C-terminus.The ZmUGGT1 protein bears the conserved C-terminal domain that containing the glucosyltransferase activity,however,ZmUGGT2 has a similar N-terminal domains.The Y2H,BiFC,Co-IP and genetic experiments confirmed that ZmSepl5-like-2 could interact with ZmUGGT1,and the expression of ZmSep15-like-2 could affect the activity of UGGT in maize and Arabidopsis.Y2H and BiFC assays indicated that ZmUGGT2 was able to interact with ZmUGGT1.ZmUGGT1,ZmUGGT2,and their combinations were transformed into yeast and Arabidopsis uggt mutants,respectively.It was found that the phenotypic of ZmUGGT2 alone transformed had no obvious difference compared to WT under mannitol or NaCl.However,in the presence of Sep15,the yeast and Arabidopsis phenotypes expressing ZmUGGT1 grown better under mannitol or NaCl.In addition,transgenic yeast or Arabidopsis harboring ZmUGGT1 and ZmUGGT2 grew best compared to WT and the other strains under mannitol or NaCl.These results showed that although ZmUGGT1,not ZmUGGT2,has the ability of glycosyltransferase,the ZmUGGT2 could enhance the activity of ZmUGGT1 by interacting with ZmUGGT1,so that it could better participate in the refolding of N-linked glycoprotein in the ER and relieve ER stress.