Study On The Mechanism Of The Regulation Of The Anthocyanin Biosynthesis In Brassica

Anthocyanins are natural pigments belonging to the flavonoid family. They are responsible for blue, purple, red colors in most flowers, fruits and seeds of higher plants. In plants, anthocyanins are believed to have a variety of functions, including defence and protection against light stress, and the pigmented anthocyanin compounds play a physiological role as attractants in plant/animal interactions. In addition, anthocyanins also have important implications in the field of human health. For example, they can reduce the incidence of cardiovascular disease, cancer, hyperlipidemias and other chronic diseases through the intake of anthocyanin-rich foods. In order to increase anthocyanin content in ornamental plants to increase its aesthetic value or to develop new anthocyanin-rich foods by biotechnology to meet the demand for our dietary consumption of anthocyanins, it is critical to have a complete understanding on the regulation of anthocyanin biosynthesis. The anthocyanin biosynthesis is mainly controlled by structural and regulatory genes. The regulatory genes encoding transcription factors control the temporal and spatial expression of the structural genes. There are numerous transcription factors responsible for regulating the anthocyanin biosynthetic pathway, such as WD40, WRKY, BZIP, MADS-box, R2R3-MYB and basic helix-loop-helix (bHLH). Among these , MYB,bHLH,WD40 have been extensively analyzed and shown to play the most important role in controlling the pathway. Anthocyanins are not always synthesized under normal growth conditions. The biosynthesis of anthocyanin is always affected by environment factors, such as light, low temperature and nutrient depletion.In this study, to investigate the mechanism of anthocyanin biosynthesis in the plant, we compared six cultivars in Brassica , the purple kale"Red Dove"and the white kale"White Dove", the red cabbage and the green cabbage, the Purple Flowering Stalk and the Pakchoi respectively. The purple kale, the red cabbage, and the Purple Flowering Stalk which are three mutations in Brassica have the strong ability to accumulate anthocyanin pigments. Meanwhile, the mechanism of abiotic stress-induced anthocyanin accumulation were also studied in these cultivars. The main results are as follows:⑴The cloning and analysis of anthocyanin related transcriptional factors in Brassica:The genes of CRY1, COP1, HY5, PAP1, PAP2, MYB114 were separately cloned from the purple kale and the white kale. They contained an open reading frame of 2034bp, 2031bp, 495bp, 750bp, 744bp and 324bp, and encoding 680, 677, 164, 249, 248, 107 amino-acid sequence respectively. Alignment of the protein sequences showed that these genes were entirely consistent with each other in two cultivars.The genes of CRY1, COP1, HY5, PAP1 were separately cloned from the red cabbage and the green cabbage. They contained an open reading frame of 2034bp, 2031bp, 495bp, and 749bp, and encoding 680, 677, 164, 250 amino-acid sequence respectively. Alignment of the protein sequences showed that CRY1 and COP1 were entirely consistent with each other in two cultivars, while there were only one different amino-acid in protein sequences of HY5 and PAP1 respectively beween two cultivars. The genes of CRY1, COP1, PAP1 were separately cloned from the Purple Flowering Stalk and the Pakchoi and the genes of PAP2 was cloned from the Purple Flowering Stalk. They contained an open reading frame of 2034bp, 2031bp, 749bp, and 744bp, and encoding 680, 677, 250, 248 amino-acid sequence respectively. Alignment of the protein sequences showed that COP1 and PAP1 were entirely consistent with each other in two cultivars, while there were only two different amino-acid in protein sequences of CRY1 beween two cultivars.⑵The purple kale (Brassica Oleracea var. acephala f. tricolor) is a mutation in kales, giving the mutant phenotype of brilliant purple color in the interior. To elucidate the molecular mechanism of the anthocyanin biosynthesis in the purple kale, we analyzed the expression of structural genes and some transcription factors associated with anthocyanin biosynthesis in the purple cultivar"Red Dove"and the white cultivar"White Dove". The result showed that nearly all the anthocyanin biosythesic genes showed higher expression levels in the purple cultivar than in the white cultivar, especially for DFR and ANS, they were barely detected in the white cultivar. Interestingly,the fact that a R2R3 MYB transcription factor named BoPAP1 was extremely up-regulated in the purple kale and induced by low temperature attracted our attention. The sequence analysis showed that BoPAP1 shared high similarity with AtPAP1. In addition, the anthocyanin accumulation in the purple kale is strongly induced by the low temperature stress. The expression of anthocyanin biosynthetic genes C4H, F3H, DFR, ANS and UFGT were all enhanced under the low temperature. These evidences strongly suggest that BoPAP1 may play an important role in activating the anthocyanin structural genes for the abundant anthocyanin accumulation in the purple kale. ⑶The color of red cabbage(Brassica oleracea var. capitata)is due to anthocyanin accumulation. To elucidate the molecular mechanism of the anthocyanin biosynthesis in the red cabbage, we analyzed the expression of structural genes and some transcription factors associated with anthocyanin biosynthesis in the seedlings of the red cabbage and the green cabbage treated by light and dark respectively. Except C4H and F3H , the expression levels of the structural genes in the red cabbage were all significantly higher than the green cabbge under not only light treatment but also dark treatment. The increased expression of the structural genes coincided with a coordinated increase in transcript levels of a MYB TFs, BoPAP2 and a bHLH TFs, BoTT8. In addition, the expressions of BoPAP2 and BoTT8 were not down-regulated by dark treatment. This may be the reasons that the red cabbage were still able to accumulate a small amount of anthocyanins under dark treatment. These results indicated that activation of these two regulatory genes up-regulate a set of anthocyanin biosynthetic genes leading to the anthocyanin accumulations in red cabbage.⑷The Purple Flowering Stalk (Brassica campestris L. ssp. chinensis L. var. purpurea Bailey) which is a mutation of Pakchoi contains high levels of anthocyanins, and the accumulations of anthocyanin in it is light-induced. To elucidate the molecular mechanism of the anthocyanin biosynthesis in the Purple Flowering Stalk, we analyzed the expression of structural genes and some transcription factors associated with anthocyanin biosynthesis in the Purple Flowering Stalk and Pakchoi. The structural genes, CHS, F3H, ANS and DFR showed higher expression levels in the stem skin than in the stem flesh of the Purple Flowering Stalk and the stem skin of the Pakchoi. This result was consistent with the phenotype that the anthocyanin was only accumulated in the skin of the Purple Flowering Stalk. Comparing the expression profiling of anthocyanin structural genes in the seedlings of the Purple Flowering Stalk and the Pakchoi treated by light and dark respectively, it showed that CHS, F3H, ANS and DFR were all extremely up-regulated by light leading to the pigmentation in the seedlings of the Purple Flowering Stalk under light treatment. Further studies showed that the increased expression of the structural genes coincided with a coordinated increase in transcript levels of a bHLH TFs, BrTT8.These results indicated that BrTT8 which could be induced by light played an important role in regulating the anthocyanin biosynthesis in the Purple Flowering Stalk.⑸The genes of MYB1 were separately cloned from the Purple Flowering Stalk and the Pakchoi. They contained an open reading frame of 1164bp, and encoding 387 amino-acid sequence. Alignment of the protein sequences showed that there were five different amino-acid in protein sequence of MYB1 beween two cultivars. To further study the fuction of the genes of BrMYB1p, a RNAi expression vector of the target gene BrMYB1p was conducted and transformed into the The Purple Flowering Stalk.There were six transgenetic plants. The color of transgenetic callus and the transgenetic plants were all turn to green. These results revealed that BrMYB1p participated in the regulation of anthocyanin biosynthesis in Purple Flowering Stalk.