Study On The Mechanism Of Cold Resistance Via β-amylase Regulating Sugar Metabolism In Grapes

Northwest China has vastly regional area,abundant sunlight,grate temperature variation between day and night,and less the pests and diseases,which are conducive to product high quality grape.However,in this area,the extremely freezing and dryness environmental condition result in grape plants need to be buried with soil to avoid cold harmful in winter and unearthed growth in spring.The planting pattern leads to increase planting costs and damage to ecological environment,which seriously restricts the development of the grape industry.Therefore,exploring the cold-resistant mechanism of grapes and cultivating cold-resistant varieties has become one of the primary tasks of grape planting in Northwest China.However,for long time,there has been little report on the mechanism of sugar involved in regulating grape cold tolerance.Therefore,in order to elucidate the metabolism mechanism of sugar in response to cold,this work selected a Vitis amurensis(‘Zuoshan I’,marked as LR)with strong cold tolerance and ‘Merlot’(marked as S)with weak cold tolerance subjected to cold acclimation under natural conditions.There are five different air temperature were programmed,including growth phase(28 ± 1 °C,labled A),early cold resistance phase(5 ± 1 °C,labled B),middle cold resistance phase(0 ± 1 °C,labled C),late cold resistance phase(-5 ± 1 °C,labled D),and deep dormancy(-10 ± 1 °C,labled E),followed determinated content of soluble sugar and hormone,and related enzymatic activities in branches phloem,and performed transcriptomics sequencing.The transcriptome data were analyzed to obtain candidate cold resistance genes and identifiy function in tomato,Arabidopsis and grape calli,and to screen interacted proteins of candidate gene,and the final results is the following:(1)The contents of glucose,fructose,sucrose and ABA were gradually increased in the phloem of LR and S with decreasing temperature under cold acclimation.The activities of amylase,sucrose synthase(Su Sy),sucrose phosphate synthase(SPS)and acid invertase(AI)showed a trend of enhancing first and then reducing,while the content of IAA gradually decreasing.The content peaks of starch appeared in the B phase in LR and S,which was57.63 mg/g FW and 48.80 mg/g FW,respectively.The activity peaks of Su Sy,SPS and AI also appeared in the B phase.The activity peak of total amylase and BAM appered in C phase.In addition,the above physiological indicators in LR were higher than S during the whole cold acclimation.(2)Transcriptome sequencing of the phloem of LR and S during the cold acclimation phase showed that the number of upregulated differentially expressed genes(DEGs)in LR was higher than downregulated DEGs in S only under the B and E phases.In addition,a total of 11,343 DEGs were identified from LR and S,including 4,913 upregualted and 6,431 downregulated DEGs.In ABA biosynthesis and signal transduction pathway,CRTISO,AOG,PYL4-1,PYL4-2,P2C08,SAPK2,TRAB1 and DPBF3 genes were upregulated in LR.In starch and sucrose metabolism pathways,HXK1,PGMP,GLGL1,SUS6-1,VCINV,CWINV1,BAM1,BAM3,and TPS6 were higher expressing in LR than that of S.(3)A total of 6 BAM gene members were identified in the grape genome data and clustered into 3 subfamilies.The protein sequence showed that the these BAM members contained the glucoside hydrolase domain and a catalytic residue Glu186.q RT-PCR results showed that Va BAM1 was gradually upregulated in LR during cold acclimation.In addition,compared with untransformed plants,the Va BAM1-overexpressing tomato and Arabidopsis plants had higher the activity of antioxidant enzyme,amylase,and higher soluble sugar content after low temperature stress,and the starch content was significantly reduced,the number of autophagosomes also increased significantly in leaves of transgenic tomato plants.(4)Va BAM1-overexpressing grape calli(Va BAM1-OEs)grew new callus around Va BAM1-OEs,and the activity of amylase was higher than that of untransformed and edited Va BAM1 callus under low temperature stress.The content of soluble sugar was increased,while the starch significantly decreased,and the tolerance of overexpressing calli was enhanced.In addition,Vv CBF3,Vv CBF4,Vv CBF6,Vv COR27 and Vv COR41 were all upregulated in Va BAM1-overexpressing grape calli after low temperature stress,but downregulated in edited calli.(5)A protein Va SR1,interacting with Va BAM1,was identified by yeast two-hybrid and bimolecular fluorescence complementation experiments.Subcellular localization showed that Va SR1 was mainly expressed in the nucleus.Compared with Va BAM1-OEs,Va SR1-OEs did not grow and the starch content increased,while the amylase activity and soluble sugar content decreased after low temperature stress,the expression of BAM1 was inhibited,and calli tolerance reducing.In addition,compared with untransformed plants,Va SR1-overexpressing Arabidopsis showed increased starch content in leaves after low temperature stress,while amylase activity and soluble sugar content decreased,and plant tolerance was weakened.