Funtional Analysis Of CC-type MeGRXs And MeDREB1D Genes From Cassava In Drought Tolerance

Cassava(Manihot esculenta Crantz)is an important root crop in the tropical and subtropical areas where drought and poor soil fertility are the main constraints to crop productivity,owing to its ability to survive and produce under these adverse environments.Thus far,there is limited information about gene function related to the drought resistance mechanism in cassava.In our study,MeGRXs and DREB1 D were significantly and positively induced by drought stress.In order to understand the function of drought tolerance for MeGRXs and MeDREB1 D in cassava,the MeGRXs in cassava genome were analyzed,and we transformed MeGRXs and DREB1 D via the genetic transformation system to identify their function under drought stress.The main results are as follows:1 A total of 38 MeGRX genes were selected from cassava genome by sequence alignment with arabidopsis thaliana sequences.According to the results of phylogenetic tree analysis,38 MeGRXs could be divided into four major classes,including the CC-type,the CPYC,the CGFS and the GRL.The number of the CC-type GRXs which are exclusive of terrestrial plants is 17,being the maximum in the number of the four major classes.The results of gene expression profile showed that 14 members of 17 CC-type MeGRXs were induced by drought stress,6 members(MeGRX232,MeGRX360,MeGRX785,MeGRX058,MeGRX892 and MeGRX496)of them were significantly and positively induced by drought stress in the leaves of SC124 which is a cassava variety with stronger drought tolerance.2 To evaluate the function of MeGRX785 and MeGRX058 under drought stress,they were transformed into cassava plant.Over-expressing MeGRX785 in cassava showed sensitive to drought,while RNAi on MeGRX785 in cassava displayed stronger drought tolerance.Interesting,the performances of over-expressing MeGRX058 and RNAi on MeGRX058 in cassava plants were opposite to MeGRX785.Meanwhile,we transformed MeGRX785 and MeGRX058 into Arabidopsis to evaluate their function under drought stress.Same as the results in cassava,over-expressing MeGRX785 in Arabidopsis displayed sensitive to drought,while over-expressing MeGRX058 in Arabidopsis performed drought tolerance.This consistency indicated the feasibility of verifying cassava genes function in transgenic Arabidopsis.Based on the above results,we transformed Me GRX232 and MeGRX360 into Arabidopsis to test their drought tolerance.The results showed over-expressing MeGRX232 Arabidopsis displayed sensitive to drought,while over-expressing MeGRX360 Arabidopsis presented drought tolerance,which corresponded to the results of MeGRX785 and MeGRX058.It indicated that different MeGRX genes performed different function in drought tolerance regulation of cassava.Analysis of expression profile of related genes in transgenic Arabidopsis showed that ERF and GA2OX6 related to ethylene regulation were up-regulated in over-expressing MeGRX232 and MeGRX785 Arabidopsis,which revealed that MeGRX might regulated the drought tolerance of cassava through ethylene signal pathway.3 To anlyze the difference of drought tolerance between MeGRX785 and MeGRX058,the sequences of their amino acid were aligned.The result showed difference in ALWL motif of C terminus might contribute to the different drought tolerance between them.Yeast auto-activation experiment was conducted on MeGRX785 and MeGRX058.Auto-activation can be found in MeGRX785,but no auto-activation effect in MeGRX058.Deletion and mutation were used to analyze the effect of ALWL motif.The auto-activation effect of MeGRX785 was lost by deleting ALWL motif or mutating the first alanine(A)into glycine(G).Conversely,MeGRX058 acquired the auto-activation effect by mutating the first glycine(G)into alanine(A).These results suggested the potential importance of ALWL motif on the function of MeGRX785,and the difference between A and G is the main reason for the different function between MeGRX785 and MeGRX058.Over-expressing MeGRX058 with ALWL motif mutated(058mC)in Arabidopsis displayed sensitive to drought,which suggested the difference on A of ALWL motif is the main reason for the different drought tolerance between MeGRX785 and MeGRX058.Amino acid mutation and genetic transformation system of yeast were using to test the effect of CDMC,L**LL and P****G motifs in MeGRX785.MeGRX785 lost auto-activation effect by deleting the last two L of L**LL motif or mutating the last two L into NN.The same result was found by mutating P****G motif into L****G and P****D or mutating CDMC motif into ADMA.In addition,over-expressing MeGRX785 with L**LL and P****G motif mutation(785mLL and 785mG)in Arabidopsis lost the sensitive to drought stress.These findings indicated the function of MeGRX785 not only depended on ALWL motif but also depended on other motifs.To test the difference of drought tolerance between MeGRX232 and MeGRX360,we aligned the protein sequences of them.The result showed there was no difference in function domains.However,the subcellular localization was different between MeGRX232 and MeGRX360.The protein of MeGRX232 was located in nucleus and cytoplasm,but MeGRX232 was only in nucleus.Whereas,if we want to know whether the difference of subcellular localization was the reason explaining the different drought tolerance of MeGRX232 and MeGRX360,there will be more works to do.4 MeDREB1 D was significantly and positively induced by drought stress and two allelic genes of it were identified,MeDREB1D(R-2)and MeDREB1D(Y-3)with one amino acid difference in NLS domain.Subcellular localization showed MeDREB1D(R-2)-GFP fusion protein was exclusively located in the nucleus,while MeDREB1D(Y-3)-GFP fusion protein not only emerged green fluorescent signal in nucleus but also outside of the nucleus.Function analysis showed that over-expressing MeDREB1D(R-2)and MeDREB1D(Y-3)in Arabidopsis displayed stronger tolerance to drought and cold stress than WT.However,Under normal growth conditions,MeDREB1D(R-2)over-expression plants performed dwarf phenotype and the time to flowering was also significantly delayed,while there were no obvious phenotypic differences between the wild-type(WT)and MeDREB1D(Y-3)over-expression plants.RNA-Seq analysis was conducted in the transgenic and wild-type plants.The results showed several POD genes were attractively up-regulated in transgenic plants,which may play a vital role in drought and cold tolerance.Pathway analysis of the DEGs showed “Photosynthesis”and“Photosynthesis-antenna proteins” pathways were common but significantly diferent in MeDREB1D(Y-3)and MeDREB1D(R-2)over-expression plants.In MeDREB1D(R-2)over-expression plants,28 DEGs were significantly enriched in “Photosynthesis”and “Photosynthesis-antenna proteins” pathways.However,only 5 DEGs were enriched in “Photosynthesis-antenna proteins” and “Photosynthesis” pathways in MeDREB1D(Y-3)over-expression plants.These differences are possibly related to the growth differences between MeDREB1D(Y-3)and MeDREB1D(R-2)over-expression plants.In conclution,MeGRXs and MeDREB1 D genes play an important role in molecular mechanism of drought tolerance regulation for cassava.Nevertheless,different MeGRX genes perform different function in drought tolerance regulation.These results will be good references for other cassava genes researching and also provide a basis for drought tolerance studying and variety breeding in cassava.