Molecular Mechanism Of McWRKY71 Regulating The Biosynthesis Of Anthocyanin And Proanthocyanidin In Malus Crabapple In Response To Ozone Stress

Ozone（O₃）pollution is widespread in many parts of the world and caused a series of oxidative damage to plants,which seriously threatened the growth and development of plants.Anthocyanins and proanthocyanidins are widely found in plants as secondary metabolites.They play an antioxidant role in plants and can remove excess reactive oxygen species in plants to protect plants.The WRKY family is a family of transcription factors unique to plants.It plays an important role in plant growth and regulation of abiotic stress response.However,the molecular mechanism of how WRKY genes regulate anthocyanin and procyanidin synthesis under O₃stress is still unclear.In this study,transcriptome sequencing was conducted with Malus‘Hongjiu’crabapple as the experimental material,and it was found that the transcription factor McWRKY71 was induced by O₃.Subsequently,function verification,interaction gene screening and downstream target gene identification were carried out,and results as follow:1.Under O₃stress,the leaves of Malus hongju showed obvious reddishan-brown lesions.NBT staining showed that ROS was accumulated,and with the extension of mitigation time,the activity of antioxidant enzymes first increased and then decreased,while the content of MDA was always increased.The contents of anthocyanins and proanthocyanidins were significantly increased and reached the peak at 12h after stress.q RT-PCR was used to detect the expression levels of anthocyanin synthesis structural genes,and it was found that the expression levels of McCHI,McANS,McF3H and McCANR were also significantly up-regulated and reached the peak at 12h after stress,and then decreased.2.The apple callus overexpressed with McWRKY71 and the callus transgenic with GFP carrier were treated with O₃.On the 20th day after stress,the transgenic apple callus had higher antioxidant enzyme activity,and its MDA content was lower than that of the control.Overexpression of McWRKY71 also promoted the accumulation of anthocyanins and proanthocyanidins in apple callus,and significantly increased the expression levels of structural genes Md ANS,Md CHI,Md ANR and Md UFGT.3.Yeast two-hybrid and Pull-down analysis showed that McWRKY71 could interact with McMYB12 to form heterodimer.4.The combination of McWRKY71 with W-box element of Pro McANR was verified by yeast single hybridization and EMSA experiments to promote the accumulation of proanthocyanidins in apple callus.This study demonstrated that McWRKY71,as a positive regulator of anthocyanins and procyanidins,is involved in the regulation of anthocyanin and procyanidin accumulation to improve the resistance of Malus to O₃stress,providing a new discovery for WRKY transcription factors in the regulation of anthocyanin and procyanidin accumulation in Malus to O₃stress.