| Anthocyanins,as important subclass of flavonoids, are the main water-soluble pigments which are widely distributed among higher plants. The red, blue and purple colors found in plant tissues including flowers, leaves, fruits and roots are always attributed to the accumulation of these kind of vacuole pigments. Apart from the well-known physiological function of serving as pollinator and seed disperser attractant, anthocyanins also play essential roles in protecting plants against the damages from UV radiation, coldness, drought stress and microbial agents. In addition, most plants synthesize anthocyanins as sunscreen which can absorb UV light and serve as free radical scavengers to cancel out the damaging consequence of irradiation. Growing evidences indicate that regular intake of anthocyanins can reduce the risk of suffering from artherosclerosis and related diseases by inhibiting the low-density lipid oxidation. Besides, anthocyanins can also provide protection against cancer and other chronic illnesses. The health-promoting effects of anthocyanins are usually believed to be closely linked with the high antioxidant activities and the capacity of eliminating reactive oxygen species. Recent articles show that this kind of second metabolites is able to modulate signaling pathways in mammalian cell for the explanation of some beneficial biological effects. As an important subclass of flavonoids, anthocyanins not only play important roles in the physiological plant processes of coloration and adaption to various environmental conditions, but also act as a health-promoting supplement for human diet. Transcriptional regulation of anthocyanin biosynthetic genes by associated transcription factors appears to be the major mechanism in regulating anthocyanin biosynthesis in plant. The pathway gwnwa of anthocyanin biosynthesis is regulated by several different families of regulatory genes including MYB transcriptional factors, b HLH transcriptional factors,WD40-like proteins,b ZIP and MADS box transcriptional factors. Besides, the biosynthesis of anthocyanin is also regulated by enviromental factors including light, droughtness and temperature.Chinese cabbage and eggplant are important dietary vegetables. They are cultivated and consumed extensively in the world. Compared with the corlorless varieties, purple Chinese cabbage and purple eggplant are more popular due to the beautiful color and the rich amount of health-promoting ingredients. In this paper, we focus on the molecular mechanism underlying the anthocyanin biosynthesis in purple Chinese cabbage. Then we analyze the molecular mechanism of the anthocyanin accumulation induced by high-light in vegative and reproductive tissues of transgenic tomato in which heterougenous gene Br TT8 expressed. In addition, the molecular mechanism of ligh-indued anthocyanin biosynthesis in the purple fruit skin of eggplant and the freezing tolerance of transgenic eggplant were studied. Main results of this study are exhibitied with four parts as follows:Part 1:fifteen kinds of anthocyanins were separated and identified from a purple bok choy cultivar(Zi He) by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. To investigate the molecular mechanisms underlying anthocyanin accumulation in bok choy, the expression profiles of anthocyanin biosynthetic and regulatory genes were analyzed in seedlings and leaves of the purple cultivar and the green cultivar(Suzhouqing). Compared with the other tissues, Br TT8 and most of anthocyanin biosynthetic genes were significantly up regulated in the leaves and light-grown seedlings of Zihe. The results that heterologous expression of Br TT8 promotes the transcription of partial anthocyanin biosynthetic genes in regeneration shoots of tomato indicate that Br TT8 plays an important role in the regulation of anthocyanin biosynthesis.Part 2:The transgenic tomato generated in the former study, which displays significantly enhanced, light-induced vegetative and reproductive pigmentation, was used to investigate how high light regulates anthocyanin accumulation. Transgenic tomato plants displayed intense anthocyanin pigmentation throughout the leaves, stems, flowers and fruits when grown under high-light conditions, yet remain colorless when grown under low-light conditions. The coloured phenotypes in high-light treated transgenic plants matched well with an accumulation of anthocyanins, as well as the activation of the anthocyanin biosynthetic genes required for pigment production. In addition, high-light also induced a modest amount of anthocyanin to accumulate in wild-type tomato plants, as well as the activation of anthocyanin biosynthetic genes. In the high-light treated plants of transgenic and wild-type tomato, where the b HLH factor Sl TT8 and MYB factor Sl AN2 and Sl MYBL2 are all significantly up-regulated, expression of the b HLH(Br TT8) and other components of the anthocyanin regulatory system remained stable, these results suggest that the high-light-induced anthocyanin accumulation in trasgenic tomato plants is regulated coordinately by endogenous and heterogeneous transcription factors. However, studies on the expression levels of anthocyanin biosynthetic and regulatory genes in high-light-treated plants under different time rule out the role Sl MYBL2 might participate in the activation of structural gene during the early stage of anthocyanin biosynthesis. In addition, the results of yeast two hybrid indicate that Sl AN2 can interact with Br TT8 with specific domain. Furthermore, the co-expression of Sl AN2 and Br TT8 under 35 S promoter induced abuandant anthocyanin accumulation in tranegenic tomato plants. These result presented here show that high-light-induced Sl AN2 and heterogeneous Br TT8 are account for the large amount of anthocyanin accumulation in transgenic tomato plant.Part 3:two kinds of anthocyanin were separated and identified from purple cultivar(Zi Chang) by HPLC-ESI-MS/MS. To investigate the molecular mechanisms of anthocyanin accumulation in eggplant, the transcripts of anthocyanin biosynthetic and regulatory genes were analyzed in fruit skin and flesh of the purple cultivar and white cultivar(Bai Xue). Compared with the other tissues, Sm MYB1 and all anthocyanin biosynthetic genes except Sm PAL were dramatically up-regulated in the fruit skin of purple cultivar. Over-expression of Sm MYB1 activated abundant anthocyanin accumulation in the regenerating shoots of eggplant. These results prove that transcriptional activation of Sm MYB1 accounts for constitutive up-regulation of most anthocyanin biosynthetic genes and the onset of anthocyanin biosynthesis in the purple cultivar.In addition, the production of white-peeled fruit by dark treatment of the purple eggplant showed that the biosynthesis of anthocyanins in the purple eggplant is strictly dependent on light. q PCR analysis showed that the expression of structural genes in the peel of the purple eggplant is regulated strictly by light, while the expression of associated regulatory gene was not affected by darkness. These results suggested that light probaly regulate the functional activity of anthocyanin biosynthesis related transcription factors at the level of protein to adjust the accumulation of anthocyanin in fruit skin.Part 4: Previously, we reported that Sm MYB1 gene encoding a R2R3 MYB transcription factor participated in anthocyanin biosynthesis regulation in the peel of eggplant. Here, we introduced Sm MYB1 into wild eggplant(Solanum aethiopicum group Gilo) via Agrobacterium-mediated transformation. Genetically engineered plants exhibited high concentrations of anthocyanin accumulation in leaves, petals, stamens, and peels under normal growth conditions, especially in fleshed fruit. Furthermore, highly methylated anthocyanins, malvidin 3-(p-coumaroyl)rhamnoside(glucoside)-5-glucoside and malvidin 3-(feruloyl) rhamnoside(glucoside)-5-glucoside, were separated and identified from the purple flesh by HPLC-ESI-MS/MS. q PCR analysis revealed that most of anthocyanin structural genes were dramatically up-regulated in the tissues of transgenic lines, compared with non transformed plants. In addition, the transgenic eggplant seedlings presented greater tolerance to freezing stress and better recovery under re-warming conditions. These results provide a good foundation for the breeding of new eggplant cultivars with more excellent agronomic traits in the future. |
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