Roles Of DIN10, GHB And SKU5 On The Anthocyaninin Synthesis And Degradation In Arabidopsis Thaliana Leaves

Anthocyanins are water-soluble pigments in plants. Anthocyanins are the main determinants of flower, leaves and other plant tissue colors. The biosynthesis pathway of anthocyanins is one of the most extensively studied plant secondary metabolism. There is widespread anthocyanin degradation phenomena in flowers, fruits, leaves and other parts of the plant in their development, maturation and aging process.There are a lot of long-term researches about mechanism leading to Litchi fruit browing and synthesis of anthocyanins, but researches about degradation of anthocyanins in plant leaves are still rare. Previous studies shows that the degradation of anthocyanins in plants causes the color change of flowers, leaves and fruits and the processes may be related to Anthocyanin-beta-glucosidase, Polyphenol oxidase(PPO), Peroxidase(POD), Peroxidase(POD), Laccase. In plants, other mechanisms have been intensively studied, except for anthocyanin-?- glucosidase.The main task of this study is to see kfor the anthocyanin-beta-glucosidase or other similar enzymes in plants, and to verify their function. We used Arabidopsis(Arabidopsis thaliana) as the matierals, and studied the fuction of two glycosidase gene(DIN10, GHB)and a laccase gene(SKU5) through the observation of mutant plants, gene expression of mutant plants and function of their proteins.Main results are as follows:1. Identification and evaluation of Arabidopsis thaliana mutantsghb, DIN10 and SKU5 mutants were purchased from Tair(http://www. arabidopsis. org/). After T-DNA insertion site analysis, all three mutants are proved to be T-DNA insertion mutants. We use three pairs of primers to identify and get homozygous mutants of these three genes.2. The changes of anthocyanin content in Blue-light-induction process and low light recovery processBlue-light is continuously irradiated on WT and the mutants for 3 d, their leaves gradually change to purple, accumulation is mainly in the back of the plant leaves and stems. The content of purple of mutants are significantly higher than WT.When the blue light stops at 3 d and move to low light(16 h light / 8 h dark) recovery condition, purple pigments gradually disappear, after 5-7 d these purple pigments disappeared completely. We set seven sampling points and measured their content of anthocyanin; the results are consistent with the phenotype of blue-light- system. At the end of the blue-light treatment, anthocyanin content of ghb is more 70-80 % than the WT. Anthocyanin content of sku5 is about more 75 % than WT. And anthocyanin content of din10 is more 40-50 % than WT. From the rate of degradation of anthocyanins, the difference between these mutants and WT and is not significant.3. The changes of gene expression in Blue-light-treatment and synthesis of anthocyaninGene expression analysis showed that both WT and mutants of Arabidopsis, compared with Before Dark, anthocyanin biosynthetic genes(such as CHS, DFR, ANS and PAP1) during the blue-light- period, their expression were significantly up-regulated while reduced during the recovery period. In addition, expression of glucosidase genes(BGLU10, BGLU15), laccase gene SKU5 was a little increase and the peroxidase genes were most obvious. In mutants of Arabidopsis, GHB mutations can enhance expression of anthocyanin biosynthetic genes ANS, DFR, CHS and PAP1, but in DIN10 and SKU5 mutationswere not significantly. DIN10, GHB and SKU5 single mutations may decrease expression of other glucosidase genes(BGLU15, DIN10 and GHB). Mutation of GHB or DIN10 can increase expression of peroxidase genes(PER38, PRX, PRX33 and PRX34). While mutation of SKU5 can reduce expression of these peroxidase gene during Blue-light treatment.4. The changes of gene expression in low-light recovery treatment and degradation of anthocyaninGene expression analysis showed that in wild-type Arabidopsis, BGLU15, DIN10 and GHB these three glucosidase gene have different degrees of increase in anthocyanin degradation process, suggesting that these glycosidases may play a role in the degradation of anthocyanins, they may could influence each other: mutation of DIN10 can be very significantly improved the gene expression of BGLU15; GHB can significantly increase the gene expression of DIN10; while mutation of DIN10 can reduce the gene expression of GHB. In WT, during the recovery period of 1- 5 d, gene expression of laccase SKU5 ascends gradually and from 7 d turn to fall. And mutation of GHB can significantly increase gene expression of SKU5 at 5 d, 7 d. POD genes have been improved 10 times or even dozens of times in the degradation of anthocyanins. Deletion of glucosidase enzyme or laccase can enhance a hundred times of expression of POD genes at some point in time. Such as in din10, expressions of PRX33 and PRX34 have been increased 100 times in 1d and 7 d of recovery treatment.5. Yeast heterologous expression and substrate specificityBoth GHB and DIN10 protein can degrade ?-glycoside. Through different substrates vitality comparison, degration of 4-nitrophenyl ?-D-glucopyranoside was most suitable. In addition, the ability of DIN10 was stronger than GHB.6. Overexpression of DIN10 and GHB in ArabidopsisOverexpression vectors were constructed through Gateway technology and transform to Arabidopsis by Agrobacterium. After resistance screening test, PCR identification, quantitative PCR analysis of gene expression, 35 S: DIN10 plants get two lines, their gene expression of DIN10 were 20- 30 times higher than WT.The main conclusions of this paper are as follows:Continuous blue-light treatment can induce accumulation of anthocyanin in Arabidopsis Leaves, while during the low-light treatment, anthocyanin could be degraded. In the degradation of anthocyanin, GHB, DIN10 and SKU5 may play a role. Mutations of these genes can enhance synthesis of anthocyanin synthesis, ghb was most significantly. Single mutation of three genes can a little induce anthocyanin degradation, to some extent caused the other two genes and other anthocyanin degradation related genes upregulated. It's indicated that anthocyanin degradation system is very complex.The function of GHB, DIN10 and SKU5 in degradation process of anthocyanin need further analysis.