Research On The Chromoplast Differentiation And The Related Genes Expression Pattern Analysis Among Different Flesh Color Watermelon Accessions

Watermelon?Citrullus lanatus?is a popular horticultural crop with important economic and nutritional value.Watermelon flesh contains a large amount of antioxidant carotenoids,which can promote human health and also give watermelon different flesh colors.The flesh color is the key factor to determine the quality of watermelon.Carotenoids are synthesized and stored in the chromoplast,so the formation of chromoplast seemed as an important role in the carotenoids accumulation.Some important secondary metabolites and hormones are also stored in the chromoplasts.Plastid lipid-associated protein?PAPs?genes involved in the formation of the lipoprotein structure,and is the main protein of the fibrous structure formed by chromoplast.Phytofluene synthase?PSY?is a speed-limiting enzyme in the synthesis of carotenoids,and the expression of PSY plays an important role in the synthesis of carotenoids.Therefore,it is of great theoretical significance and application value to study the differentiation characteristics of the chromoplast and the related genes expression pattern analysis among different flesh color watermelon accessions.In this study,five different watermelon accessions:white-fleshed“PI 186490”,pale yellow-fleshed“Cream of Saskatchewan”,short for“COS”,canary yellow-fleshed“PI 635597”,orange-fleshed“PI192938”and red-fleshed“LSW-177”,were selected for this experiment.Samples of fruit flesh were collected 10 DAP?days after pollination?,with the intervals of 8 days until 42 DAP,for a total of five stages.Chromoplast differentiation during fruit ripening for different cultivars were invested by optical and transmission electron microscopy.The composition of carotenoids in flesh at the mature stage were analyzed by LC-MS/MS among various flesh color watermelon accessions.In addition,the expression patterns of Cl PSYs?Cl PSY1,Cl PSY2,Cl PSY3?and Cl PAPs?Cla006670,Cla010946,Cla008831,Cla014416,Cla021506,Cla003468,Cla003198?were evaluated by real-time quantitative PCR.Finally,the coding region and promoter sequence were cloned from different materials for sequence alignment and cis-acting element analysis.The main research contents and results are as follows:?1?The total carotenoid content in the mature stage is relevant to the content of phytoene.The red-fleshed“LSW-177”is mainly based on the accumulation of lycopene,violaxanthin may be the main factor causing the color difference between pale yellow-fleshed“COS”and canary yellow-fleshed“PI 635597”.Orange-fleshed“PI 192938”mainly accumulates?-carotene and violaxanthin.?2?In red-fleshed“LSW-177”,crystalloid chromoplast was formed,while in white-fleshed“PI186490”,pale yellow-fleshed“COS”,canary yellow-fleshed“PI 635597”and orange-fleshed“PI192938”,the chromoplast appeared as globular structures.?3?The expression of Cl PSY1 conforms to the accumulation trend of phytoene and total carotenoids,while the expression of Cl PSY2 and Cl PSY3 were irregular during the whole cycle of fruit development,and not significantly different across the different varieties.The expression trend of Cla014416 was almost identical to that of Cl PSY1,and the expression of Cla006670,Cla010946,Cla008831,Cla021506,Cla003468 and Cla003198 were much lower than that of Cla014416,and there was no obvious rules to follow.?4?Compared with the red-fleshed“97103”,there were two non-synonymous mutations of Cl PSY1 in orange-fleshed“PI 192938,”causing a change from asparagine to aspartic acid at 133^thamino acid,and from lysine to glutamate at 148^th amino acid,and the amino acid sequence alignment of Cla014416 showed non-synonymous mutations in white-fleshed“PI 186490”(glycine to arginine at 113^th amino acid),pale yellow-fleshed“COS”(antagonine to isoleucine at242^nd amino acid),and orange-fleshed“PI 192938”(proline to leucine at 138^th amino acid).?5?The difference in the starting codon and number of MYB?pigment response elements?in Cl PSY1 promoter sequence may be the main reason for the significant differences in gene expression levels.