Functional Analysis Of Alkali Stress Responsive Genes TaOMT4 And TaGPAT6 From Wheat

The widely distributed salt and alkaline salts are important abiotic stress factors that greatly affect plant growth and development and severely threaten crop productivity throughout the world.Salinization and alkalization of soil are always go hand by hand.Alkaline salts(NaHCO3 and Na2CO3),which elevate soil pH,are much more destructive than neutral salts(NaCl and Na2SO4).The high pH may destroy normal soil structure;seriously affect precipitation of other ions and disrupt the ion homeostasis in plant cells;damage the structure of cell membrane,thus threatening plant's growth and photosynthesis.So far,extensive studies have been employed to uncover the signaling mechanisms and regulatory networks underlying plant tolerance to salt stress.However,few regulators have been identified to function directly in the tolerance of plants to alkali stress.Studies on the molecular mechanism of alkali resistance in wheat are few.Considering the more deteriorative effects caused by alkaline salt stress,more studies should be focused on molecular mechanisms of alkali stress responses.A serious of wheat introgression lines were generated through asymmetric somatic hybridization between bread wheat cultivar Jinan 177(JN177)and the highly tolerant related species,tall wheatgrass(Thinopyrum ponticum).Shanrong No.4(SR4)was one of the wheat introgression lines which showed strong tolerance towards alkaline salt stress.In the experiments conducted in both the field and the lab,SR4 displayed strong resistance to alkalinity which means that SR4 is an ideal plant material to explore the mechanism of alkali stress resistance in wheat.According to the transcriptome data of alkali-treated SR4,we selected two candidate alkali responsive genes for further functional analysis.One of them is TaOMT4 which encodes an O-methyltransferase,and the other is TaGPAT6 which belongs to glycerol-3-phosphate acyltransferases(GPATs).1.Functional Analysis of Wheat Alkaline Responsive Gene TaOMT4We cloned the ORF of TaOMT4 gene used cDNA of SR4 as template which has an ORF of 1072 bp and encoding a protein of 356 amino acids.TaOMT4 was up regulated in SR4 under alkaline salt stress.TaOMT4 was localized in the cytoplasm.We transferred TaOMT4 to wild type Arabidopsis(Col-0)and two TaOMT4 transgenic lines with high expression level were screened out.Overexpression of TaOMT4 does not affect plant development under control conditions.However,in the presence of NaHCO3 or with gradient pH,TaOMT4 overexpression lines showed strong alkali tolerance at the seedling stage.TaOMT4 overexpression lines also showed resistance towards H2O2 stress.DAB staining suggested that the content of H2O2 is lower in TaOMT4 overexpression lines than in Col-0.The activities of POD and CAT were also significantly improved.Thus,we speculate that TaOMT4 might enhance plant alkalinity tolerance by improving ROS scavenging enzyme activities to reduce the content of H2O2 in plants.TaOMT4 transgenic lines displayed high sensitivity to high salinity.Analysis of qRT-PCR showed that the expression level of NHX1 and NHX2 were obviously down-regulated under control conditions.Thus,we speculate that TaOMT4 might decrease plant salinity tolerance by negatively regulating ion transporters.TaOMT4 transgenic lines displayed sensitivity under MV treatment.NBT staining showed that TaOMT4 transgenic lines accumulated higher O2-.SOD activity of OE lines showed no obvious difference.All these evidence implied that higher O2-content might be involved in the sensitivity to MV.2.Functional Analysis of Wheat Alkaline Responsive Gene TaGPAT6The coding regions of TaGPAT6 gene were amplified from cDNA of SR4.The ORF had a length of 1494 bp and encoded a protein of 497 amino acids.TaGPAT6 was up-regulated in SR4 under alkaline salt stress.TaGPAT6 was localized in the cytoplasm.We transferred TaGPAT6 to wild type Arabidopsis(Col-0)and two TaGPAT6 transgenic lines with high expression level were screened out.TaGPAT6 overexpression does not affect plant development.under control conditions.However,in the presence of NaHCO3 or with gradient high pH,TaGPAT6 overexpression lines showed high sensitivity to alkali stress at the seedling stage.TaGPAT6 overexpression lines were also more sensitive towards exogenous ABA and high salt stress.Analysis of marker genes of ABA signaling pathway displayed that the expression of NCED3?RD29A and MYB2 were down-regulated in TaGPAT6 overexpression lines,while ABI4 and ABI5 which positive regulate ABA signaling pathway were up-regulated.Analysis of marker genes of ion transporters indicated that HKT1;1 was decreased significantly in overexpression lines.All these evidence suggested that TaGPAT6 might be involved in alkalinity stress through ABA signaling pathway;TaGPAT6 might down-regulated HKT1;1 through ABI4 and affected plant response towards salt stress.