Functional Analysis Of Alkaline Stress Response Genes TaCCD1 And TaStpk-B In Wheat Introgression Line SR4

The alkaline stress of plants resulted from Na2CO3 and NaHCO3 in the soil.Compared with the effect of salinity stress,the effect of alkaline stress may be more destructive.Alkaline stress not only involves ion injury,osmotic stress,but also includes high pH stress.So far,the mechanisms of plants tolerance to alkaline stress have not been exhaustively studied,with the fact that more attention has been paid to study salinity resistance mechanisms.It's urgent to exploit how the plants cope with alkaline stress,especially for the crops.An saline-alkaline tolerant wheat line SR4 was selected from the derivatives of an asymmetric somatic hybridization between the salinity sensitive line,bread wheat cultivar Jinan 177(JN177)and the highly tolerant related species,tall wheatgrass(Thinopyrum ponticum).Experinments in both the field and the lab both demonstrated that SR4 displaying a superior tolerance of alkalinity stress than JN177.We carried out the transcriptome sequencing of alkali stressed SR4 and screened many alkaline-responsive genes.Based on the transcriptome sequencing data,we selected two genes for further study.One is Ca2+-binding protein gene TaCCD1,the other is serine/threonine kinase gene TaStpk-B.1.Functional identification of a wheat Ca2+-binding protein TaCCD1The ORF of TaCCDl was cloned from SR4 cDNA which include about 600 bp.TaCCD1 encoding a Ca2+-binding protein with an EF-hand motif which displayed different expression level under various abiotic stress treatment.TaCCD1 was localized in the nucleus and cytoplasm membrane.Through the Agrobacterium-mediated transformation method,TaCCD1 overexpression(OE)lines were generated from the wild type(WT)Arabidopsis ecotype Col-0.We selected two homozygous transgenic lines for further study.After treatment with alkaline pH,no difference was observed in seed germination rate between the TaCCD1 OE lines and the WT.However,the TaCCD1 OE lines presented higher cotyledon greening degree than the WT.In the seedling stage,after treatment with alkaline pH,the primary root length of the OE lines was longer than the WT.They all demonstrated that TaCCDl conferred alkaline tolerance in Arabidopsis.When treated with ABA,the TaCCDl OE lines displayed longer main root than the WT in the seedling stage,which indicated the TaCCD1 OE lines were resistant to ABA.When treated with NaCl,the TaCCD1 OE lines displayed longer main root than the WT in the seedling stage,which indicated the TaCCD1 OE lines were resistant to salinity stress.When treated with H2O2,the TaCCD1 OE lines displayed much lateral root than the WT,which indicated that TaCCD1 OE lines were resistant to oxidative stress.The ROS content of OE lines was lower than WT,and the ROS scavenage enzyme activity of the OE lines was much higher than the WT which enable the OE lines have less ROS.For further study,we analyzed the expression level of some marker genes of ABA pathway and ion transport in the TaCCD1 OE lines and WT.The MYB2,RAB18 and RD29B which were linked to ABA-dependent pathway were upregulated,while the ABI5 which belongs to the positive factor of the ABA signaling pathway.The SOS2 and HKT were upregualted in OE lines.At the same time,we analysed the relative expression level of chlorophyl synthesis and degredation genes.It showed that the HEMF2,HEME2,HEMB2,HEME1,CRD1,GSA1,PORA,PORB were downregualted.And.the chlorophyll content of the OE lines were higher than the WT.Combining the above results,we can draw a conclusion that TaCCDl may confer the Arabidopsis alkaline,salinity and the oxidation tolerance by regualting the accumulation of ROS and transporting of Na+,which are relevant with the regulation of the ABA siganling pathway.Meanwhile,TaCCD1 may confer the OE lines alkaline stress tolerance by regulating the chlorophyll synthesis and degredation genes.2.Functional characterization of a wheat serine/threonine kinase TaStpk-BWe cloned the ORF of TaStpk-B,with a length of about 1200 bp from SR4 cDNA.TaStpk-B encods a serine/threonine protein kinase which displayed diverse expression pattern under different abiotic treatment.TaStpk-B was localized in cytoplasm membrane.The Agrobacterium-mediated transformation method was applied to generate the TaStpk-B OE(overexpression)lines in WT(wild type)Arabidopsis ecotype Col-0.We eventually selected two homozygous lines for further study.After treatment with alkaline pH,the OE lines' main root was longer than the WT.The experinment demonstrated that TaStpk-B conferred alkaline stress tolerance in Arabisopsis.Under NaCl and mannitol condition,the TaStpk-B OE lines displayed shorter main root than the WT,elucidating the TaStpk-B OE lines was sensitive to salt and osmatic stress.The OE lines had less ROS than the WT.The OE lines had higher ROS scavenage enzyme activity than the WT which enable the OE lines have less ROS.The expression of genes of ion transport and SOS pathway showed no difference with the WT.The proline content of OE lines was lower than the wild type in NaC1 contion.However,the expression of ABA2 involved in ABA synthesis were upregulated in OE lines.The ABI3,ABI4 which belong to ABA-dependent pathway were downregulated,while the RD29A which was involved in ABA indepedent pathways were downregulated.With the results above,TaStpk-B confers the plant alkaline tolerance stress by scavenging ROS,which are correlated with ABA signaling pathway.It may confer the salt and osmatic sensitivity by negatively regualting the RD29A gene.Besides that,TaStpk-B made the proline content low under NaCl condition,thus conferring the OE lines salt sensitivity.