Molecular Cloning And Functional Identification Of ABA Degradation Gene MdCYP707A1 In Apple

The plant hormone abscisic acid(ABA)is essential for plant growth and development,and it can regulate various physiological processes of plants.On the one hand,as a negative regulator in plant growth and development,ABA inhibits plant growth,accelerates leaf senescence and death,promotes the fall of flowers and fruit,inhibits seed germination,regulate root development.On the other hand,ABA can increase plant resistance to biotic and abiotic stress and protect plants from the external environment.In higher plants,the ABA signaling pathway mediated by the PYR/PYL/RCAR complex is involved in the regulation of stress.The ABA receptor PYR/PYL/RCA senses ABA signaling,activates downstream gene expression and improves the ability of plant resistance to adverse environment.The content of ABA in plants is determined by the combination of ABA synthesis and degradation rate.The enzymes and related regulatory factors in the ABA synthesis pathway have been studied more clearly,while the enzymes in ABA catabolism are less studied.Studies have shown that in higher plants,oxidative inactivation of ABA is the main way of ABA degradation.In this process,the methyl hydroxylation pathway of 8? position of ABA dominates.CYP707 A family members encode 8?-hydroxylase,which is a key enzyme in ABA catabolism and belongs to cytochrome P450 monooxygenase,whose activity is induced by ABA and regulated by various environmental stresses.In this study,from the perspective of seed dormancy and stress response,four MdCYP707 A genes in apples were compared according to the AtCYP707 A family gene in Arabidopsis.Phylogenetic tree analysis revealed that the phylogenetic relationship between MdCYP707A1(MDP0000859733)and AtCYP707A1 was the most similarity.MdCYP707A1 responded most obviously to ABA and drought.Therefore,MdCYP707A1 gene was selected for functional analysis.We obtained MdCYP707A1 transgenic apple callus and Arabidopsis.Functional analysis of its sensitivity to ABA and resistance to stress was performed.Based on our results,we draw the following conclusions:1.Relationship analysis of MdCYP707A1The MdCYP707A1 coding region is 1425 bp and encodes 474 amino acids.Phylogenetic analysis revealed that MdCYP707A1 is most closely related to AtCYP707A1.Protein conserved domain analysis revealed that MdCYP707A1 contains a cytochrome P450 monooxygenase domain,and the software predicted that the conserved amino acid sequence of this domain is FGXGXHXCPG,indicating that this gene may be involved in the coding of 8'-hydroxylase.The cis-acting element analysis of the promoter revealed that the promoter of MdCYP707A1gene contained ABA,stress and other response elements.These indicat that the gene may be involved in the regulation of ABA and stress.2.MdCYP707A1 regulates seed dormancy and promotes seed germinationReal-time quantitative PCR was used to detect the expression of MdCYP707A1 in different tissues and during seed maturation in apple.It was found that the expression of MdCYP707A1 was the highest in seeds and reached the peak in the seeds of 60 days after flowering,indicating that MdCYP707A1 may participate the regulation of seed maturation.Expression analysis of water swelling and stratification of apple seeds showed that all four genes of MdCYP707 A family were involved in the regulation of seed release dormancy.Overexpression of MdCYP707A1 in Arabidopsis thaliana,under ABA treatment conditions,the germination rate of overexpressing plants was significantly higher than that of wild type.These indicated that MdCYP707A1 may promote seed germination.3.MdCYP707A1 reduced sensitivity to ABAWild type(Col-0)and MdCYP707A1 overexpressing Arabidopsis thaliana(OX-2,OX-4,OX-6)were treated on MS+ABA medium,the root length,fresh weight and chlorophyll content of MdCYP707A1 overexpressing line were significantly higher than wild type.These indicated that MdCYP707A1 reduced sensitivity to ABA.4.MdCYP707A1 reduced resistance to abiotic stressThe wild type and transgenic 'Wanglin' apple callus were treated with PEG6000,mannitol and NaCl,the physiological indexes such as fresh weight,electrical conductivity and MDA content were detected.It was found that the transgenic apple callus decreased the resistance to PEG6000,mannitol and NaCl.Likewise,wild type(Col-0)and MdCYP707A1 overexpressing in Arabidopsis thaliana(OX-2,OX-4,OX-6)were treated with NaCl and PEG6000,the root length,fresh weight and chlorophyll content of transgenic Arabidopsis thaliana were significantly lower than wild type.The above results indicated that MdCYP707A1 reduces resistance to abiotic stress.