The Study On Carotenoid Mechanism In Grape Berries And The Function Of Its Key Gene VvPSY1/2

Grapes(Vitis vinifera L.) are world-wide fruit trees with a wide range of cultivation,rich in table and processed varieties.The skin color is an important appearance quality that affects the commerciality of fruit.Depending on the type of coloration,grapes can be mainly divided into red and white(yellow-green)varieties,of which red is mainly affected by anthocyanins,and yellow-green is mainly affected by chlorophyll and carotenoids.Carotenoids belong to the C40 isoprenoid compound.In plants,carotenoids not only participate in the capture and absorption of light and energy transfer,but also act as a photoprotective agent to prevent photo-oxidative damage to the photosynthetic apparatus caused by strong light.Carotenoids can also be used as precursors for the biosynthesis of many physiologically active substances such as phytohormone abscisic acid and strigolactone,defense compounds and volatile aromas,and participate in the adaptive response of plants to the environment and the formation of unique flavor quality of plants.In view of the important role of carotenoids in animal and plant life activities,and the significance of carotenoid metabolism in coordinating other metabolic balances in plants,this project uses grape as a test material to carry out the study of carotenoids metabolism and its regulatory factors.The main research methods and results are as follows:1.In order to clarify the difference of main pigment components and the changes of carotenoid components in different coloring types of grape berries,Sixteen different coloring types of grape varieties such as 'Champion','Moldova','Guixiangyi','Cabernet Gernischet','Jingya','Summer Blacky',' Wase Takasumi ','Fenghou','Muscat Humburg','Benizuiho','Manicure Finger','Xinnongle','Benni fuji','Shine Muscat','Manai' and 'Golden Finger' were chosen for this study.Lutein,?-carotene and ?-cryptoxanthin were the main carotenoids types in grape skin.At various stages of fruit development,the content of lutein and ?-carotene was relatively high,while that of ?-cryptoxanthin was relatively low.With the development of grape berries,the content of carotenoids in grape varieties showed a significant downward trend.At the maturity stage,the highest content of lutein was found in the berry of ' Champion ',the highest content of cryptoxanthin was found in 'Moldova',and the highest content of ?-carotene found in 'Cabernet Gernischet'.At the maturity stage,there were differences in the types and contents of anthocyanins in different grape varieties.Malvidin-type anthocyanins were the most abundant components of the blue-black,purple-black,and purple-red grape varieties.On the whole,with the development of grape berries,anthocyanins are cumulative pigments,while chlorophyll and carotenoids are degradable pigments.The content of pigments in grape skins of different coloring types is quite different,There is no significant correlation between anthocyanins,chlorophyll and carotenoids contents.2.In order to clarify the effects of fruit bagging and abscisic acid(ABA)on carotenoids metabolism during fruit development,the yellow-green variety 'Shine Muscat'and the red variety 'Jumeigui' were used as materials to study the application effect of fruit bags in different color and ABA treatment in different concentration.(1)Under the control,white bags and red bags,the berries of 'Shine Muscat' was yellowish,while the fruit treated by black bags and green bags was more green.The berries of the 'Jumeigui' treated under the control,white bags,red bags,green bags and black bags became purple black,purple red,purple,light purple and light red,respectively.For 'Shine Muscat',the ratio of chlorophyll content to carotenoids content in the skins under green bags was the highest.From post-verasion stage to maturity,the expression levels of VvGGPPS,VvPSY1,VvLCYB,VvVDE,VvPORa under bagging treatments were higher than those of the control treatment.Compared with other bagging treatments,the expression levels of various genes under the treatment of black bags and green bags were relatively low.(2)The ABA treatment had no significant effect on grape berry size,but affected the coloration of the fruit.For 'Shine Muscat',the grape berries became yellowish after ABA treatment and became greenish when treated with the ABA inhibitor.For 'Jumeigui',the grape berries were more reddish after ABA treatment,while the ABA inhibitor treated berries were lighter red.ABA treatment accelerated the degradation of carotenoids in the berries of the two cultivars,and the effect of high concentration treatment was more obvious.The treatment of ABA inhibitors slowed the degradation of carotenoids.In the 'Shine Muscat' skin,the expression levels of VvDXS1,VvDXR,VvGGPPS,VvPSY1,VvPSY2,VvPDS1,VvLCYB,VvZEP1,VvHO3,VvFe2,VvPORa were higher under the control,low concentration ABA and the ABA inhibitor treatment in the pre-verasion stage,while the expression of VvCCD1,VvCLH1,and VvCLH2 were higher under the medium and high concentrations of ABA treatment.3.The storage and preservation technology directly affects the commercial value of grape berries.This study compared the effects of chitosan,low temperature and light-filling treatment on the quality of grape berries and carotenoids metabolism during storage.The results indicated that the content of TSS,TA,chlorophyll and carotenoids,and the fruit hardness in 'Shine Muscat' and 'Jumeigui' berries decreased during storage,while the fruit decay rate increased.Chitosan and low temperature treatment all delayed the changes of fruit quality traits.Compared with other treatments,the low temperature treatment and the concentration of 1%,the degree of polymerization of 150,000(Chi1-15)chitosan treatment was the best.During normal temperature storage,the supplemental light of different light qualities affects the appearance color of grape berries,The supplemental light promoted the degradation of chlorophyll and slowed down the degradation of carotenoids.Under the red light treatment,the content ratio of chlorophyll to carotenoids was the smallest,and the berries was yellowish.Under dark treatment(control),the ratio was the largest and the fruit was the greenest.During the storage of 'Shine Muscat',the expression levels of carotenoids and chlorophyll biosynthesizing genes,such as VvDXR,VvGGPPS,VvPSY1,VvPD1,VvVDE,VvZEPI,VvPORa,in Chil-15 treated skins were higher than those in control,while the expression levels of VvCCD1 VvCLHl and VvCLH2 were lower.Except for some developmental stages,the expression levels of carotenoids and chlorophyll metabolism genes under white and red light treatments were higher than those in the control,while the expression level of VvCCD1 was lower.4.The VvPSY1 and VvPSY2 genes in the grape were cloned and transformed into tomato and tobacco by Agrobacterium for functional study.The phenotype of VvPSY1,VvPSY2 overexpressing tomato was significantly different from that of the wild.The plant height of VvPSY1 and VvPSY2 overexpressing tomato was significantly lower than that of wild type plants.Additionally,the leaves,fruit stalks and cobs of transgenic plants showed yellowing.Compared with wild type,the fruit of VvPSY1 transgenic tomato was light yellow in young fruit stage,and dark red in maturity,with high level of lycopene and?-carotene content,but the fruit of the VvPSY2 overexpressing plant was orange in the mature stage and the lycopene content was low.RT-qPCR results showed that the expression levels of PSY1,PSY2 and GGPPS in VvPSY1 transgenic lines were significantly increased,while the expression of these genes in VvPSY2 transgenic lines were lower.Co-inhibition may occur in VvPSY2 transgenic fruits.In transgenic tobacco,the content of carotenoids in leaves of VvPSY1 and VvPSY2 overexpressing tobacco increased.Taken together,VvPSY1 and VvPSY2 in grapes have the function of participating in carotenoids biosynthesis.5.Phytochrome-interacting factors(PIFs),as the basic helix-loop-helix(bHLH)transcription factors,are the primary signaling partners for phytochromes(PHY)that play a key role in PHY-mediated light signal transduction.In order to clarify the status of PIFs in grapevines,we identified members of the grape PIFs family and conducted phylogenetic and expression analysis.We identified PIF1,PIF3,PIF4,and PIF7 in PIFs families of the grapevine(Vitis vinifera L.),which were distributed on four different chromosomes with similar gene structures.The photoreceptor-related elements in the VvPIFs promoter region were the most abundant.The relative expression levels of the VvPIFs showed diverse patterns in the various organs at different developmental stages,of which PIF4 was most highly expressed.Prior to maturation,the expression of PIF4 and PIF7 in the skin of the different cultivars increased,while the expression of all PIFs in the flesh decreased.The transcription level of PIFs in grape leaves was sensitive to changes in lighting and shading.Shading treatment was beneficial for enhancing the transcription level of VvPIFs,but the effect on VvPIF3 and VvPIF4 was time-controlled.The results of yeast one-hybrid system and transgenic tobacco showed that VvPIFl can bind to the promoters of VvPSY1 and VvPSY2,and inhibit the formation of chlorophyll and carotenoids in transgenic tobacco.Taken together,the main carotenoid components in grape berries are lutein,?-carotene and ?-cryptoxanthin.Both VvPSY1 and VvPSY2 in grapes were involved in the biosynthesis of carotenoids.VvPIF1 plays a role in the negative regulation of chlorophyll and carotenoids biosynthesis and could bind to the promoters of VvPSY1 and VvPSY2.Bagging could delay the degradation of carotenoids,and ABA treatment could accelerate the degradation of carotenoids.The content of carotenoids in berries during storage under low temperature treatment was relatively stable.The content of carotenoids in berries under red light treatment was the highest during short-term storage.