Functional Charaterization And Underlying Mechanisms Of The Transcription Factor MdbHLH108 Conferred Drought Tolerance In Apple

Apple(Malus × domestica Borkh.)is one of the most cultivated fruit crops in China.However,apple tree frequently suffer from large-area and different-degree drought,which affect the quality and yield of apple,which has become an important problem to be solved in apple production and cultivation.The basic helix-loop-helix(b HLH)transcription factor is plays important roles in plant growth and development,and regulating abiotic stress tolerance.At present,the studies on b HLH transcription factors are mainly focused on model plants,while the physiological and regulatory functions of b HLH transcription factors in most nonmodel plants,especially perennial fruit trees,are still unclear.In our previous study,apple b HLH gene family was identified and analyzed at the whole genome,and Mdb HLH108,a member of apple b HLH gene family,was isolated and significantly up-regulated by drought In this study,Mdb HLH108 gene was transformed into Arabidopsis thaliana and apple for identification of drought resistance,and the regulatory mechanism was also analyzed.The main results are as follows:1.The CDS of Mdb HLH108 was 783 bp,which encoded a b HLH protein of 261 amino acids.The promoter sequence of Mdb HLH108 contained a number of cis-acting elements related to stress response,including ABRE and MYC and other cis-acting elements in response to abscisic acid.The expression of Mdb HLH108 was induced by drought and ABA.Mdb HLH108 protein does not have significant self-activating activity in yeast cells,and Mdb HLH108 protein was localized in the nucleus.2.In order to identify the drought resistance function of Mdb HLH108 gene,Arabidopsis thaliana Atb HLH38 mutant,overexpressed of Mdb HLH108 and wild type were used to drought and ABA treatments.The results showed that the malondialdehyde content and relative electrical conductivity of the mutant were lower than those of the wild type under drought stress,and stomatal density also decreased.Compared with the mutants,alondialdehyde content and relative conductivity were increased in the overexpressed Arabidopsis mutant.The results showed that the mutant lines tm1 and tm2 had stronger drought resistance,while the overexpression mutant had lower drought resistance,suggesting that Mdb HLH108 negatively regulated drought resistance of Arabidopsis thaliana.3.In order to further identify the function of Mdb HLH108 gene in drought resistance gene in apple,we obtained positive ‘Qrin’ apple callus by overexpressing Mdb HLH108 and carried out drought(200 m M Mannitol)and ABA(200 μM ABA)treatments.The results showed that the phenotype of Mdb HLH108 overexpressed callus showed high sensitivity to drought and ABA.Compared with the wild type,the fresh weight of overexpressedMdb HLH108 ‘Qrin’ callus decreased,while the malondialdehyde content and relative electrical conductivity increased.The content of hydrogen peroxide and superoxide anion in wild-type calli was lower than that in transgenic lines under drought stress,but higher under ABA treatment.These results indicated that the overexpression of Mdb HLH108 decreased the drought resistance and increased the ABA sensitivity of apples,suggesting that Mdb HLH108 negatively regulated the drought resistance of apple.4.Functional verification of gene indicated that Mdb HLH108 negatively regulated of drought resistance in both Arabidopsis thaliana and apple callus,and it is highly likely that Mdb HLH108 regulates drought tolerance by mediating ABA signaling.Based on the above results and guesses,we quantitatively analyzed three important genes in ABA signaling pathway,Md NCED3,Md CYP707A1 and Md CYP707A2,in Arabidopsis thaliana and apple,and verified their interactions by yeast single hybridization and double luciferase tests.The results showed that Mdb HLH108 interacted with the promoter of Md NCED3,and Mdb HLH108 could effectively activate the promoter of Md NCED3 to regulate the downstream gene Md NCED3,and the transcription level of Md NCED3 in apple overexpressed callus was also significantly increased.The promoter cis-acting elements of Md NCED3 were analyzed,and the acting elements that might interact with Mdb HLH108 were selected.Based on the above results,we speculated that Mdb HLH108 affected ABA level by inducing the expression of Md NCED3,a gene related to ABA synthesis,and then affected its drought resistance.