Study On The Function Of Apple MdbHLH134 In Response To Drought Stress

The drought is one of the primary environmental agents affecting the production of crops,with nearly half of the world’s regions suffering from varying degrees of drought stress year-round.China is the biggest apple producing and consuming nation,with the world’s largest cultivated area and output.The northwest loess plateau apple production area is the biggest apple production area in China,with high altitude,temperature difference and other environment conditions to foster high quality apple.Yet,the low rainfall and the uneven distribution in the yearly cycle in the region make the drought stress a critical environmental factor that limits the yield and quality enhancement of apples in this producing area.Thus,cultivating new apple varieties and rootstocks with excellent drought resistance is important for the development of China’s apple industry.bHLH transcription factors are the second largest transcription factor family in plants and are widely involved in the process of plant growth and development and response to adversity.At present,there are few studies on the functions of bHLH transcription factors regulating drought tolerance in apple,and the related regulatory mechanisms are not clear.In this study,a bHLH family gene MdbHLH134 was identified from apple in response to drought stress.MdbHLH134 transgenic Arabidopsis thaliana,transgenic apple healing and root transgenic apple seedlings were obtained by Agrobacterium-mediated genetic transformation.Functional analysis of the transgenic materials under drought treatment revealed that MdbHLH134 was regulating drought tolerance in apple and might be related to its regulatory role on leaf and root development.The results of the study can provide a theoretical basis and genetic resources for the breeding of apple for drought tolerance.The main results are as follows:1.The cloning and characterization of the MdbHLH134 gene.the full length of the ORF of the MdbHLH134 gene is 1098 bp,encoding 366 aminos.The genome sequence contains 2exons and 1 intron,and the gene structure is similar to the homologous gene AtbHLH30 in Arabidopsis.Conservative domain analysis and three-dimensional structural prediction of proteins revealed that MdbHLH134 contains a typical bHLH domain.The subcellular localization analysis showed that MdbHLH134 protein was targeted in the cell nucleus.The gene expression analysis showed that MdbHLH134 expression was the highest in the roots and its expression was significantly induced by the drought,salinity,cold and other non biotic stress.The yeast two hybrid experiment showed that MdbHLH134 can interact with itself to form homologous dimers,which conforms to the characteristics of bHLH transcription factors.In addition,it also interacts with the drought resistant key protein Md OST1 in apples.2.Identification of drought resistance in MdbHLH134 transgenic Arabidopsis.Several transgenic Arabidopsis lines expressing MdbHLH134 heterologously were obtained by infection,and functional analysis was carried out under drought treatment.Transgenic Arabidopsis plants under drought showed better growth condition,higher leaf water content,and lower relative ion leakage and MDA content compared with wild type,suggesting that MdbHLH134 is a positive regulatory factor in plant response to drought stress.In addition,the accumulation of reactive oxygen species H₂O₂ and O₂^-in the leaves of MdbHLH134 transgenic plants under drought is lower,while the activity of antioxidant enzymes POD and SOD is higher,indicating that MdbHLH134 can promote the clearance of excess ROS under drought by enhancing antioxidant enzyme activity,thereby improving plant drought resistance.3.MdbHLH134 gene responds to auxin（IAA）to regulate leaf and root development.Observing the leaf morphology of Arabidopsis,it was found that the MdbHLH134 transgenic Arabidopsis exhibits a leaf curling phenotype,similar to the Arabidopsis functional acquired mutant abs5-1D.Comparing the water loss rates of leaves of various plant lines under in vitro conditions,it was found that the water loss rates of transgenic Arabidopsis leaves were significantly lower than those of wild-type plants,indicating that MdbHLH134 can enhance plant drought resistance by reducing leaf water loss.In addition,external application of IAA can eliminate the leaf roll phenotype of transgenic Arabidopsis,indicating that the impact of MdbHLH134 on leaf development is related to IAA.Comparing the root growth of Arabidopsis under IAA treatment,it was found that transgenic Arabidopsis had significantly more lateral roots than wild-type plants,indicating that MdbHLH134 may promote plant water absorption by affecting root development.4.MdbHLH134 transgenic heal and apple plants were identified for drought resistance.Invasion was obtained to overexpress MdbHLH134 transgenic apple healing tissue.Under drought treatment simulated by mannitol,the growth of transgenic healing wound was better than wild type and the fresh weight was significantly greater than that of wild type,suggesting that MdbHLH134 was regulating the resistance of the healing wound to osmotic stress.Transgenic apple（Malus hupehensis）plants overexpressing or interfering with MdbHLH134expression in the root system were obtained by using Agrobacterium tumefaciens infection.Long term cultivation of plants under moderate drought conditions revealed that overexpression of MdbHLH134 significantly promoted root development,while interference with MdbHLH134 expression significantly inhibited root development.In addition,overexpression of MdbHLH134 in the root system significantly promoted the aboveground biomass accumulation of plants,indicating that MdbHLH134 may enhance water absorption by promoting root development,thereby improving plant drought resistance and water use efficiency.