Functional Characterization Of Apple MdbHLH122 In Response To Drought Stress

Apple is one of the world’s four fruits,and our country is the largest producer and consumer of apples.The apple industry has played a great role in Chinese farmer’s gains,ecological environment optimization and agricultural structure adjustment.The north-west Loess Plateau apple production area is the largest apple growing area in China,and drought deficiency is one of the main environmental factors limiting the development of the apple industry in this area.The bHLH family is the second largest family of transcription factors in plants and is widely involved in abiotic stress response and resistance regulation in plants.Currently,there are fewer functional studies on the regulation of drought tolerance by bHLH transcription factors in apple,and the related regulatory mechanisms are also unclear.Researching drought resistant related bHLH family genes in apple and identifying their functions in drought stress response in detail through transgenic technology may provide potential gene resources and theoretical basis for stress resistance molecular breeding and variety improvement in apple.In this study,we identified a bHLH family gene,MdbHLH122,from apple in response to drought and characterized its gene structure,expression pattern,protein conserved functional domains,subcellular localization,and transcriptional activation activity.A variety of transgenic Arabidopsis thaliana,apple calli,and apple plants were obtained by genetic transformation,and these transgenic materials were used to provide a detailed characterization of the regulatory functions of MdbHLH122 in drought tolerance.The main results are as follows:1.The apple MdbHLH122 gene ORF is 690 bp in length and encodes a protein with229 amino acids.Sequence analysis indicates that the MdbHLH122 protein has a conserved bHLH domain,and subcellular localization analysis indicates that the MdbHLH122 protein localizes to the nucleus.The MdbHLH122 promoter sequence was analyzed and found to contain several cis-acting elements related to light response,hormone and stress response to adversity.Promoter activity analysis showed that MdbHLH122 significantly responded to ABA,mannitol and PEG treatment.In addition,the transcriptional activation identification results in yeast indicated that the MdbHLH122 protein has strong transcriptional activation activity,consistent with its transcription factor properties.2.Transgenic Arabidopsis heterologously expressing MdbHLH122 was obtained by genetic transformation and functionally characterized under drought treatment.First,we analyzed seed germination under mannitol treatment and found that heterologous overexpression of MdbHLH122 reduced the sensitivity of Arabidopsis seeds to osmotic stress and significantly increased seed germination rates under the treatment.Secondly,250 m M mannitol treatment on Arabidopsis seedlings revealed that the growth of MdbHLH122 transgenic Arabidopsis seedlings under the treatment was better than that of the wild type,and both the primary root length and fresh weight were significantly greater than those of the wild type.Further,adult plants of various lines of Arabidopsis were drought treated,and the results showed that heterologous expression of MdbHLH122 significantly enhanced drought tolerance in Arabidopsis,showing better growth status,higher accumulation of chlorophyll and proline,and lower ion leakage and ROS levels.The above results suggest that MdbHLH122 positively regulates plant drought tolerance.3.To identify the function of MdbHLH122 in apple,transgenic calli overexpressing MdbHLH122 were first obtained.Both the growth state and fresh weight of MdbHLH122 transgenic calli under PEG simulated drought treatment were higher than those of the wild type,indicating that it positively regulated the resistance of calli to osmotic stress.Transgenic Malus hupehensis lines overexpressing or interfering with MdbHLH122 expression in the root system were obtained by Agrobacterium rhizogenes mediated genetic transformation and were drought stress treated,respectively.The growth status of the overexpression lines after treatment was better than that of the control,and both antioxidant enzyme activities and proline contents were significantly higher than that of the control,whereas the interference expression lines showed the opposite trend,which indicated that overexpression of MdbHLH122 in the root system enhanced drought tolerance in apple plants.The above results indicated that MdbHLH122 played a positive regulatory role in drought stress response in apple.