Regulatory Mechanism Of Abscisic Acid On Wound Suberization Of Kiwifruit

Wound-induced suberization is the main process of fruit wound-healing,and plays a crucial role in preventing the shriveling and deterioration of fruit caused by excessive water loss and pathogen infection.Suberin is mainly composed of SPA（suberin polyaliphatics）and SPP（suberin polyphenolics）.Genes involved in the biosynthesis of suberin have been identified in model plants of potato and Arabidopsis,however,the regulatory mechanisms of these genes are still unclear.In this study,kiwifruit（Actinidia Chinensis Planch cv.Xuxiang）was used as experimental material to explore the role of oxygen in suberin biosynthesis,and the regulation mechanism of ABA on suberin biosynthetic genes.（1）This study was conducted to investigate the role of oxygen in wound-induced suberization of half-cut kiwifruit exposed to 100%,50%,21%and 0%oxygen（balance with N₂）.By 3 days after wounding,the wound surface of kiwifruit in high oxygen（50 and 100%）appeared as a continuous layer of melanin and SPP underneath,which effectively prevent excessive water vapor loss from the fruit halves.In contrast,melanin and SPPs deposition in the wound surface in 0%oxygen was significantly reduced,with high water vapor loss.High oxygen elevated the activity of PAL,CAT,POD and PPO,resulting in the accumulation of caffeic,p-coumaric,ferulic acids and coupling with each other,which promoted the accumulation of suberin and melanin.The results suggest that oxygen is required for wound-induced melanin and suberin formation,and high oxygen is effective in promoting wound suberization of kiwifruit.（2）The regulatory mechanism of ABA on AchnFHT in wound suberization of kiwifruit was investigated.AchnFHT was found to be localized in cytosol.Transient overexpression of AchnFHT in N.benthamiana leaves induced massive production of ferulate,ω-hydroxyacids and primary alcohols,in consistent with in vitro ability of AchnFHT to catalyze acyl-transfer from feruloyl-Co A toω-hydroxypalmitic acid and1-tetradecanol.Through yeast one-hybrid assay screening,four transcription factors（TFs）,including AchnABF2,AchnMYB4,AchnMYB41 and AchnMYB107,could directly interact with AchnFHT promoter.The activation of AchnABF2,AchnMYB41and AchnMYB107,and repression of AchnMYB4 on AchnFHT promoter were detected in dual-luciferase analysis.These findings were supported by the result of transient overexpression in N.benthamiana.AchnABF2,AchnMYB41 and AchnMYB107 induced the expression of suberin biosynthetic genes（including FHT）and accumulation of suberin monomers,in contrary to AchnMYB4.Exogenous ABA induced the expressions of AchnABF2,AchnMYB41,AchnMYB107 and AchnFHT as well as suberin monomer formation,but inhibited AchnMYB4 expression.Moreover,FLD（fluridone,an inhibitor of ABA biosynthesis）was found to counter the inductive effects of ABA.Therefore,activation of suberin monomer biosynthesis by AchnFHT was coordinately controlled by both repression of AchnMYB4 and promotion of AchnABF2,AchnMYB41 and AchnMYB107 in response to ABA.（3）The regulatory mechanism of ABA on AchnCYP86A1 and AchnFAR in wound suberization of kiwifruit was investigated.AchnCYP86A1-and AchnFAR-overexpressed N.benthamiana leaves displayed that the AchnCYP86A1and AchnFAR respectively functioned as a fatty acidω-hydroxylase and fatty acyl-Co A reductase associated with biosynthesis of suberin monomer.The regulatory function of three TFs（including AchnMYC2,AchnMYB41 and AchnMYB107）on AchnCYP86A1 and AchnFAR was identified.All the three TFs could individually interact with AchnCYP86A1 and AchnFAR promoter to activate the gene transcription in yeast one-hybrid and dual-luciferase assays.The findings were further demonstrated in transient overexpressed N.benthamiana,in which all TFs notably elevated the expression of SPA biosynthetic genes including CYP86A1 and FAR,as well as the accumulation ofω-hydroxyacids,α,ω-diacids,fatty acids and primary alcohols.Moreover,exogenous ABA induced the expression of AchnMYC2,AchnMYB41 and AchnMYB107 that activated AchnCYP86A1 and AchnFAR involving in suberin monomer formation.Contrary to the inductive effects of ABA,however,FLD inhibited the three TF genes expression and suberin monomer formation.These results indicate that AchnMYC2,AchnMYB41 and AchnMYB107 positively regulate biosynthesis ofα,ω-diacids and primary alcohols by activating AchnCYP86A1 and AchnFAR promoter in response to ABA.（4）The regulatory mechanism of ABA on Achn4CL in wound suberization of kiwifruit was investigated.Achn4CL,a kiwifruit homolog of At4CL5,was isolated.Overexpression of Achn4CL in N.benthamiana leaves lead to the significantly increase in ferulate,caffeate and coumarate.Through yeast one-hybrid assay screening,three MTB TFs,including AchnMYB4,AchnMYB41 and AchnMYB107,could directly interact with Achn4CL promoter.The activation of AchnMYB41 and AchnMYB107,and repression of AchnMYB4 on Achn4CL promoter were detected in dual-luciferase analysis.The findings were further demonstrated in transient overexpressed N.benthamiana,in which AchnMYB41 and AchnMYB107 notably elevated the expression of 4CL and PAL,and the accumulation of hydroxycinnamates,ω-hydroxyacids and primary alcohols,in contrary to AchnMYB4.Exogenous ABA induced the expressions of AchnMYB41 and AchnMYB107 as well as ferulate formation,but inhibited AchnMYB4 expression.Moreover,FLD was found to counter the inductive effects of ABA.Therefore,activation of SPP monomer biosynthesis by Achn4CL was coordinately controlled by both repression of AchnMYB4 and promotion of AchnMYB41 and AchnMYB107 in response to ABA.