Response Mechanism Of Amur Grape VaMYB4a In Response To Cold Stress

Grapes(Vitis spp.)are one of the world’s commercially significant fruit crops.The frequent occurrence of harmful cold temperatures in winter and late spring frost have severely affected the healthy and sustainable growth of grapevines and the wine industry,resulting in substantial economic losses.Therefore,it is essential to perform a comprehensive analysis of the biological functions and regulatory mechanisms in the low-temperature responses of grapes,as well as to screen the candidate gene resources of important agronomic traits in grapes,and to establish a theoretical foundation for the molecular breeding of cold-resistant grape varieties.Based on prior research and our team’s findings,the transcription factor VaMYB4a has been identified as a possible interacting protein with the low temperature positive regulator VaCIPK18.Using yeast two-hybrid(Y2H)and ChIP-seq assay,the downstream essential components of VaMYB4a’s low temperature signaling network were identified.These findings give a reference for elucidating the mechanism of this transcription factor implicated in low-temperature response and developing novel cold-tolerant grapevine cultivars.The primary outcomes are as follows:1.There is a protein-protein interaction between VaMYB4a and VaCIPK 18 in Vitis amurensis.In vivo Y2H and BiFC(bimolecular fluorescence complementation)and in vitro CoIP(coimmunoprecipitation)tests were used to investigate the relationships between VaMYB4a and VaCIPK18.VaMYB4a interacts with VaCIPK18 in yeast and planta,as determined by Y2H,BiFC,and Co-IP assays.VaMYB4a is a possible substrate for phosphorylation by VaCIPK18.2.VaMYB4a positively regulates plant response to cold stress.Low temperature stress enhanced VaMYB4a expression,homodimer formation,and posttranslational modification.The overexpression of VaMYB4a provided tolerance to low-temperature stress to yeast and plant.After treatment at-5℃,the survival rate of VaMYB4a over-expression transgenic Arabidopsis was significantly higher than that of the wild type(WT).Under cold stress,VaMYB4a-overexpressing transgenic Arabidopsis showed higher cell integrity,soluble sugar content and antioxidant enzymes(SOD and POD)than WT.VaMYB4a-overexpressing transgenic plants were hypersensitive to ABA and induced stomatal closure.VaMYB4a controlled plant response to cold stress through CBF-dependent route and CBF-independent pathway.Concurrently,VaMYB4a controlled plant responded to cold stress by changing the expression of critical genes in the ABA signaling system.3.VaMYB4a might interact with the downstream interacting-proteins VaCOL2,VaCOL4 and VaCOL5.A yeast homogenized cDNA library was constructed with the leaves of ’Zuoshan-1’ under cold stress.VaMYB4a113-252 was used as ’bait’,and 18 candidate interacting-proteins were screened from the library.We discovered that VaCOL2,VaCOL4,and VaCOL5 interact with the full-length and C-terminal region of VaMYB4a.4.VaMYB4a interacts with the downstream target gene VaPIF3Using genome-wide ChIP-sequencing methods,the putative target genes of VaMYB4a have been identified.In addition,Y2H and BiFC assays revealed that VaMYB4a might interact with VaPIF3 at the protein level.The yeast one hybrid experiment revealed that VaMYB4a could bind to the ’CTTC’ motif(481～529 bp,1856～2021 bp,and 2459～2550 bp)on the VaPIF3 promoter.5.The VaMYB4a-VaPIF3-VaCBF4 molecular module is involved in responding to low temperature stress.VaPIF3 interacts with VaCBF4 at the genetic and protein level,as shown by Y1H and Y2H assays.In Arabidopsis and yeast,VaMYB4a interacts with VaCBF4;hence,a network of VaMYB4a-VaPIF3VaCBF4 molecular module implicated in response to low temperature stress was developed.