Mechanisms Underlying The Regulation Of Anthocvanin Coloration Synthesis In Kiwifruit

Kiwifruit(Actinidia spp.),a high-value fruit crop known as the 'king of fruits',is rich in nature and cultivated germplasm resources in China.Fruit color is one of the most important quality traits and plays a very important role ininfluencing the buying decisions of consumers.Due to the accumulation of different types of anthocyanin,kiwifruit can appear bright red or purplish red in skin or in flesh.The kiwifruit with red fruit coloration is favored by consumers for attractive appearance and high nutritional value.The accumulation of anthocyanin,the major pigmented group of flavonoid compounds,in fruit is mainly genetically determined,but also greatly influenced by various environmental stimuli.This study explored the metabolism of flavonoids in kiwifruit with different flesh colors,analyzed the influence of environmental factors on the fruit coloration and studied the transcriptional regulation mechanism of anthocyanin accumulation in kiwifruit.The main results were as follows:1 Comparison and analysis of flavonoids metabolism in kiwifruit with different flesh colorsThe flavonoid compounds in Actinidia chinensis and Actinidia arguta fruits investigated in present study.A total of 125 flavonoids,including 9 anthocyanins,12 catechins,17 flavanones,48 flavones(including 14 flavone C-glycosides),29 flavonols,6 isoflavones and 4 proanthocyanidins were identified in 'Hongyang' kiwifruit(red-flesh),'Jintao' kiwifruit,'Mini Amethyst' kiwiberry(purple-flesh)and 'Kuilv'kiwiberry.Thirty-nine metabolites showed significantly different contents between'Hongyang' and 'Jintao',and 38 of them showed higher content in 'Hongyang',whereas 39 metabolites showed significantly different contents between 'Mini Amethyst' and 'Kuilv',and 31 of them showed higher content in 'Mini Amethyst'.2 Transcriptome analysis of kiwifruit with different flesh colorsComparative transcriptomic analysis revealed that structural genes in the anthocyanin synthesis pathway(AcF3H,AcF3 'H,AcDFR,AcUFGT)and transcription factors(AcMYB10,AcbHLH5/42)may be involved in the pigmentation of the red-fleshed A.chinensis,whereas AaF3H,AaF3GT and AaMYB110 may play important roles in the pigmentation of the purple-fleshed A.arguta.3 Genome-wide analysis of R2R3-MYBs in the kiwifruitGenome-wide analysis identified 155 R2R3-MYBs in the 'Red 5' version of the Actinidia chinensis genome.The 155 AccR2R3-MYB genes were unevenly distributed across all 29 chromosomes except chromosome 19.A neighbor-joining phylogenetic tree was constructed by using the R2R3 conserved domain of the 155 AccR2R3-MYBs and 126 AtR2R3-MYBs and 36 AccR2R3-MYBs fall into the clade related to anthocyanin biosynthesis.Cis-acting elements analysis showed that light-,temperature-and hormone-responsive elements are enriched in the promoters of these 36 AccR2R3-MYBs.Combined with the analysis of hierarchical clustering of their expression,RT-PCR and phylogenetic analysis,Acc00493(AcMYB10)has an expression preference in the inner pericarp of fruit and has a close phylogenetic relationship with the key gene AtPAP1 regulating anthocyanin biosynthesis in Arabidopsis,indicating it may be closely related to anthocyanin accumulation in kiwifruit.4 Effects of environmental factors on the accumulation of anthocyanin in kiwifruitHormone treatment experiment showed that ABA and MeJA had no significant effect on anthocyanin accumulation in callus,they could specially have negatively regulating the expression of Acc00493;Of note,light and temperature have a significant effect on anthocyanin accumulation in callus.Strong light and low temperature can significantly promote anthocyanin accumulation associated with an upregulation of Acc00493.The experiment of fruit treatment showed that high temperature will inhibit anthocyanin accumulation in red-fleshed A.chinensis,while bagging will promote this process.Bagging all-red A.arguta fruit can accelerate the coloration process after light treatment.5 Functional and regulatory mechanism of AcMYB10(Acc00493)Anthocyanin accumulation in the inner pericarp of A.chinensis is associated with the coordinated action of AcMYB10,AcF3GT and AcLDOX during fruit development and ripening.AcMYB10 is localized in the nuclei and has transcriptional activation activity.Overexpression of AcMYB10 elevates global anthocyanin accumulation in transgenic A.chinensis.In comparison,A.chinensis fruit infiltrated with virus-induced gene silencing showed delayed red coloration,lower anthocyanin content,and lower expression of AcMYB10.The interaction between AcMYB10 and AcbHLH42 was demonstrated by yeast two-hybridization,bimolecular fluorescence complementation and dual-luciferase complementary imaging.The transient expression experiment in Nicotiana tabacum leaves and Actinidia arguta fruit indicated the interaction of AcMYB10 with AcbHLH42 might strongly activate anthocyanin biosynthesis by activating the transcription of AcLDOX and AcF3GT.