Functional Study Of Salt-alkaline Stress Responsive Gene TaGPAT6 From Wheat

Soil saline-alkalinization has become a major problem of agriculture and it is also one of the main abiotic stress factors restricting crop production.Improving the salt-alkali tolerance ability of crops is the most effective way to utilize saline-alkaline lands.Identifying genes for salt-alkali tolerance and exploiting their mechanisms will provide a theoretical basis and practical value for the cultivation and breeding of salt-alkali tolerant crops.Shanrong No.4(SR4)is a new salt-alkali tolerant wheat variety selected from the introgression lines derived from asymmetric somatic hybridization.Previously,the salt-alkali responsive gene for glycerol-3-phosphate acyltransferase(TaGPAT6)was cloned from SR4.Overexpression of TaGPAT6 in Arabidopsis lead to sensitive to saline-alkali stress,but the mechanism is still unclear and worthy of further discussion.In this work,the function and mechanism of TaGPAT6 involved in abiotic stress responses was further studied.The main results are as follows:1.Expression and enzyme activity analysis of TaGPAT6The TaGPAT6 open reading frame is 1494 bp in length and encodes a protein with 497 amino acids which localized to the endoplasmic reticulum and Golgi apparatus.By comparing with the Chinese Spring genome sequences,it is speculated that this gene is located on the 3B chromosome of wheat,and there are homologous genes with extremely high similarity but low expression level on the 3A and 3D chromosomes.TaGPAT6 was found to highly expressed in flower buds,which may imply that TaGPAT6 is associated with wheat fertility.TaGPAT6 encodes a glycerol-3-phosphate acyltransferase with both an acyltransferase active domain and a phosphatase domain,and it has all of the key amino acids at the active sites of these two functional domains.Detection for the lipids of Arabidopsis thaliana overexpression lines indicated that the content of sn-2 MAG was significantly increased.This result indicates that TaGPAT6 has both acyltransferase activity and phosphatase activity.2.TaGPAT6 is involved in the response of wheat to saline-alkaline stressThe transgenic wheat lines were constructed by shoot apical meristem transformation method,and the transgenes were identified by PCR.the two transgenic wheat lines with higher expression level were selected to exploiting their tolerant ability to saline-alkaline stress.The results showed that there was no significant difference between the transgenic and nontransgenic wheats under the control conditions,but under salt stress and alkali stress,the wheat overexpression lines showed sensitive to stress.This suggests that TaGPAT6 is involved in the response of wheat to saline-alkaline stress.3.Overexpression of TaGPAT6 reduces Arabidopsis resistance to abiotic stressWe identified the phenotype of TaGPAT6 heterologous overexpression lines,gpat4,gpat6 mutants and their complements with TaGPAT6 under abiotic stress.The results showed that overexpression of TaGPAT6 significantly reduced the resistance of plants to saline-alkaline and ABA stress.However,the mutations of gpat4 and gpat6 in Arabidopsis showed certain resistance to saline-alkaline and ABA stress;Arabidopsis gpat4 and gpat6 mutations relieved the sensitivity of Arabidopsis to saline-alkaline and ABA stress caused by the overexpression of TaGPAT6.4.TaGPAT6 may respond to saline-alkaline stress by influencing PAGPAT can acylate G3P to form LPA,and then LPA was modified to PA under the action of lysophosphatidic acid acyltransferase.We detected the expression of genes related to phosphatidic acid synthesis and degradation,and found that the expression levels of genes related to PA synthesis and degradation have a certain change.The PA content of Arabidopsis overexpression lines was found to be lower than wild type plants.We also found that the sensitive phenotype of Arabidopsis overexpression lines under salt,alkali and ABA stress can be recovered through exogenously applying PA.Therefore,we hypothesized that TaGPAT6 may participate in plant response to stress by influencing the synthesis and degradation of PA.5.TaGPAT6 may respond to saline-alkaline stress by affecting ABA signalingThe Arabidopsis overexpression lines of TaGPAT6 showed sensitive to exogenous applying of ABA,and the Arabidopsis thaliana mutant showed a little resistance.We detected the expression of the marker genes related to ABA synthesis and signal transduction,and results showed that the expression levels of many marker genes have changed significantly.Recent studies have shown that PA,as a signaling molecule,is also involved in mediating the transduction of ABA signaling,so we speculate that TaGPAT6 may respond to saline-alkaline stress by affecting ABA signaling.6.Overexpression of TaGPAT6 leads to accumulation of ROS in plantsArabidopsis overexpression lines are sensitive to MV,and results for NBT and DAB staining showed that the content of H2O2 and O2-was significantly increased in the overexpression lines.The expression of ROS synthesis related genes,like AtRBOHC,AtRBOHD and AtRBOHF was found to be increased in the overexpression lines.In addition,the expression level and activity of ROS scavenging enzymes including CAT,POD and APX was suppressed in overexpression lines,although that of SOD was increased.Physiological assay results showed that the overexpression lines have decreased proline content and increased MDA content.These results suggest that TaGPAT6 may contribute to the accumulation of ROS in plants by reducing ROS scavenge,resulting to be sensitive to saline-alkaline stress.