Salt And Cadium Tolerance Mechanism Analysis Of ThVHAc1 From Tamarix Hispida

VHAc(vacuolar H+-ATPase c subunit)is a vacuolar H+-ATPase(V-ATPase)subunit,mainly functioned in the formation of proton channel,responsible for proton transport and essential for V1-V0 assemble.VHAc are 16 kDa proteins,and among many species show high similarity and homology.Previous study showed VHAc gene mainly response to salt,heavy metal,drought stresses and so on.In the current study,the V-ATPase activity,the relationship between the activity and its subunits of Tamarix hispida exposed to salt and heavy metals were determined,the results showed that the 15 subunits of T.hispida V-ATPase were all effected by6,12 d stresses,and under both NaCl and CdCl2 for a short time(less than 3 days),the expression of ThVHAcl was induced to 227.34,36.74-fold respectively.Western blotting showed ThVHAcl protein was highly induced by salt and Cd stresses,indicating that ThVHAcl may be important for salt and cadmium resistence.To further understand the salt resistence of ThVHAcl,ThVHAc1,was overexpressed in poplar,under salt stress the transgenic poplar showed higher biomass accumulation than WT.The Na+,H+,K+ transport in the root tip meristematic zones of WT and transgenic lines under NaCl treated for 0,1,10 d by noninvasively using scanning ion-selective electrode technique(SIET)(the SIET system BIO-001 A;Younger USA Sci.& Tech),V-ATPase activity and other salt resistence indicators were tested.The results showed that ThVHAcl regulated the Na+,H+,K+ ratio and transport,improved V-ATPase,SOD,POD,GST activities and decreased MDA content,ROS accumulation,cell damage compared to WT,indicating ThVHAcl improved salt tolerance of transgenic poplar by regulating V-ATPase to supply energy for ions transport and increasing protecting activities to scavenge ROS accumulation.ThVHAc1 was also overexpressed in Arabidopsis to analyze Cd tolerance.ThVHAcl transgenic Arabidopsis showed higher seeds germination(average 2.54 fold to WT and ck),biomass and chlorophyll content,protecting enzymes activity and lower MDA content,H2O2,relative electron leakage and ROS accumulation than WT under CdCl2 treatment.DAB,NBT,Evans blue and PI stainings were also showed the ThVHAcl lines generated lower ROS content and less cell damage than control lines.The results showed that overexpression of ThVHAcl effectively improved Arabidopsis Cd tolerance.To understand heterogenous ThVHAcl whether play a role in V-ATPase activity and its subunits,the expression of 28 subunits of Arabidopsis V-ATPase under NaCl and CdCl2 stresses in transgenic Arabidopsis lines were analyzed by real time RT-PCR.The results showed that under both stresses,all the 28 subunits were effected by overexpression of ThVHAcl,and the V-ATPase activity was also increased,indicating both of the V-ATPaserelated activities and the subunits were all regulated by overexpression of ThVHAcl.Meanwhile,the 5 c subunits of Arabidopsis V-ATPase were all showed low expression level just reverse to ThVHAcl,indicating overexpression of heterogenous VHAc may complementary to the in-house VHAc gene.Overexpression of ThVHAcl also induced some VATPase(AHA,ACA)and abiotic resistence(LOX1,CSD,APX,RBOHC)related genes.What’s more,ThVHAcl was transient overexpressed in Thispida,and the overexpression lines showed highest V-ATPase activity and improved resistence indicators under Cd stress compared to RNAi::ThVHAc1 and T-ck.The V-ATPase subunits of T.hispida were also effected by overexpression of ThVHAc1 under salt and Cd stresses,but their expression profiles were different to transgenic Arabidopsis,indicating species expression specificity.Some other related genes(CAX,CSB,ADP,GLH,NADPH)were also induced by ThVHAcl overexpression.These results further confirmed the result in transgenic lines.Further,a 1164 bp promoter of ThVHAcl was amplified by genomic walking method,and was inserted into pCAMBIA1301 generated p1301-ThVHAcl,which was further transformed into Arabidopsis to analyze the spatial and temporal expression of ThVHAcl.The T4 generation was used in the analysis.The results showed that ThVHac1 was expressed in different growth and development stage and different tissues excepting the fresh pod and immature seed,indicating the ThVHac1 promoter posses GUS expression activity and spatial and temporal expression profiles.Analysis of the 1164 bp ThVHAcl promoter by PLACE(http://www.dna.affrc.go.jp/PLACE/signalscan.html)showed the promoter including many ciselements,such as DOFCOREZM,MYCCONSENSUSAT,WBOX,WRKY710 S,MYBCORE,which were related to abiotic stresses regulation.Yeast one-hybird assay screened the special recognition cis-elements DOFCOREZM,MYBCORE and WRKY in ThVHAc1 promoter by upstream regulators ThDof2,ThMYB3 and ThWRKY7.The expression profiles of ThDof2,ThMYB3 and ThWRKY7 under NaCl,CdCl2,PEG,ABA stresses were analyzed by real time RT-PCR and the results showed that these three upstream regulators showed similar expression patterns with ThVHAc1 under the same stress,indicating these upstream regulators controlled or functioned together with ThVHAcl in salt and Cd tolerance regulation.