The Regulation And Mechanism Of Postharvest Light On Wax Biosynthesis In Vitis Vinifera

The cuticular wax on the fruit surface plays important roles in providing fruit shine,reducing water loss,resisting pathogen infection and maintaining fruit nutrients.Light can regulate the biosynthesis and accumulation of cuticular wax,and affects the senescence and quality deterioration of postharvest fruit.However,the substance accumulation,candidate target,regulation mechanism,and interaction sites of light regulated wax biosynthesis in grape berry fruit remains unclear.In this study,high throughput sequencing of chromatin accessibility,surface plasmon resonance,structure simulation and molecular docking were applied to explore the effects of light on wax content,and fruit quality;identified key genes and regulatory factors involved in light regulated wax biosynthesis;elucidated the interaction between light responsive transcription factors and open gene sequences;and revealed the structural basis of transcription factors binding to cis-acting elements based on cell structure,metabolites,genes,and chromatin levels.This study was aimed to decipher the regulation and mechanism of postharvest light on grape wax biosynthesis and provide theoretical basis for the precise regulation of grape apparent quality and terpenoid metabolism.The main research results are as follows:（1）The crystal structure and chemical composition of surface wax of four grape varieties were clarified.The cuticular wax of 4 grape varieties（Kyoho,Muscat Hamburg,Redglobe,Zuijinxiang）appeared flaky structure,and were mainly composed of terpenoids,hydrocarbons,alcohols,fatty acids and esters.Terpenoids accounted for more than 70%of the total wax,with oleanolic acid being the most abundant terpenoid(910.68-2365.76μg mm^-2).The wax distribution detection showed that the terpenoids were mainly present in the intracuticular wax,while fatty acids were generally present in the epicuticular wax,and hydrocarbons were evenly distributed in the intracuticular wax and epicuticular wax.（2）The effects of wax on the fruit quality of grape during postharvest storage was revealed.The Zuijinxiang grape was used to establish a wax loss model.The results showed that the wax loss accelerated the fruit weight loss,softening and browning of grape during storage.The wax lossed fruit possessed higher transcription levels of key enzymes in the cell wall degradation pathway,including pectinesterase,thinner cell walls with substrate leakage,and higher relative conductivity;lower chlorophyll content,and upregulated expression level of genes related to chlorophyll degradation pathway;higher expression of genes related to wax biosynthesis and transport.（3）The effects of postharvest red light,blue light and white light on grape wax content and fruit quality were elucidated.Postharvest red light aggravated the fusion and collapse of wax crystal structure,significantly improved the fruit decay rate and accelerated the fruit stem browning.The blue and white light treated grape had higher content of wax terpenoids,such as oleanolic acid(585.53-780.01μg mm^-2 higher than the control group),maintained better wax crystal morphology,reduced the fruit decay rate and drop rate by 4.6%-4.7%,and 11.1%-12.2%,respectively.（4）The target genes and transcription factors involved in light regulated grape wax biosynthesis were identified.As chromatin accessibility increased,the gene expression levels increased.The distribution of accessible chromatin regions（ACRs）suggested that about 30%of ACRs are within 1 kb around the transcription start site.Both postharvest white light and blue light upregulated the expression of Vv TPS12 and Vv HMGR2 genes involved in the terpenoid biosynthesis pathway,and white light upregulated the chromatin accessibility level of Vv TPS12 and Vv HMGR2.Transcription factor analysis of ACRs sequence implied that b ZIP family transcription factor Vv HYH and GATA family transcription factor Vv GATA24 participated in white light regulated grape wax terpenoids biosynthesis.（5）The interaction between light responsive transcription factor Vv HYH and Vv TPS12 to regulate grape wax biosynthesis was unraveled.Transient overexpression and silence of tobacco leaves and grape fruit indicated that Vv HYH transcription factor positively regulated Vv TPS12 to affect the biosynthesis of grape wax terpenoids.Dual luciferase assay and surface plasmonic resonance experiments confirmed that the Vv HYH bound to the G-box cis-acting element in the Vv TPS12 promoter region（-2047～-1890 bp）to activate the transcription of Vv TPS12,and their equilibrium dissociation constant of 2.81 n M was determined for the first time.Molecular docking uncovered the structural basis of Arg^106A and other 23 amino acids in the interaction between Vv HYH and G-box cis-acting element.（6）The interaction between light responsive transcription factor Vv GATA24 and Vv HMGR2 to regulate grape wax biosynthesis was uncovered.Transient overexpression and silence of tobacco leaves and grape fruit indicated that Vv GATA24transcription factor positively regulated Vv HMGR2 to affect the biosynthesis of grape wax terpenoids.Dual luciferase assay and surface plasmonic resonance experiments confirmed that the Vv GATA24 bound to the GATA-box cis-acting element in the Vv HMGR2 promoter region（-1996～-1839 bp）to activate the transcription of Vv HMGR2,and their equilibrium dissociation constant of 8.59 n M was determined for the first time.Molecular docking uncovered the structural basis of Pro^218B and other 11amino acids in the interaction between Vv GATA24 and GATA-box cis-acting element.