Purple Foliage Coloration In Tea (Camellia Sinensis L.) Arises From Activation Of An R2R3-MYB Transcription Factor CsAN1

Anthocyanins are the largest group of water-soluble pigments in the plant kingdom and belong to the family of compounds known as flavonoids, are display a wide range of biological functions such as attracting pollinators, seed dispersers and protecting plants against various biotic and abiotic stresses. As the natural antioxidants, anthocyanins also can protect the body from free radicals and other harmful substances, increase the elasticity of blood vessels, prevent disease of heart head blood-vessel, protect the liver. In addition, as a harmless pigment, anthocyanins has broad application prospects in the edible pigment development. Although there are large numbers of researches on the mechanisms underlying controlling anthocyanin production in flowers, fruits, and model plants, only few have focused on tea plants. Purple foliage ‘Zijuan' tea was a somatic mutant selected from Yunnan Daye cultivar(Camellia sinensis var. assamica(Mast.) Kitamura), which anthocyanin content was reported to be almost three times of that found in the other Chinese purple tea cultivars. In order to get more insight into the high levels of anthocyanin accumulation in ‘Zijuan', a comparative transcriptome strategy was used, with the aim of revealing the underlying molecular mechanisms. Main results are shown as follows:1. Altered MBW complex expression changes anthocyanin accumulation in N.benthamiana.Pigmentation was evident at infiltration points for CsAN1/Cs GL3/Cs TTG1. However, no coloring was observed at infiltration sites after transformation with empty vector. The q RT-PCR result revealed that LBGs transcript accumulation were slightly increased transformed transfected alone CsAN1, compared with the empty vector.2. CsAN1 is a transcription activator and can interaction with the promoters Cs LDOX1 and Cs LDOX2.The transactivation assay indicated that CsAN1 has transactivation activity. Y1 H assays results showed that CsAN1 associated with MYB-binding site of Cs LDOX1 and Cs LDOX2,supporting the hypothesis that CsAN1 was the transcription factor regulating the expression of Cs LDOX1 and Cs LDOX2.3. CsAN1 is a menber of the MBW complex.Y2H and Bi FC assay showed that CsAN1 interacts with Cs GL3 and Cs EGL3 respectively,but not with Cs TTG1 protein. Similarly, Cs TTG1 also interacts with Cs GL3 and Cs EGL3.These results implied that CsAN1 and Cs TTG1 proteins physically interact with the b HLH to formation a MYB/b HLH/WD-repeat complex.4. Cs LDOX2 promoter activity was regulated by MBW protein complexesDual luciferase assay results showed that CsAN1 increase Cs LDOX2 promoter activity.When transformed simultaneously with CsAN1, Cs TTG1 and Cs GL3, the highest activity was observed, this emphasizing the fact that these three proteins act together to activate their target promoters.5. The hypomethylation in the CsAN1 promoter resulted in its higher expression in red coloration leavesThe bisulfite sequencing(BSP)-PCR results indicated that the promoter of CsAN1 methylation level was significant decreased in the high anthocyanin cultivars than in that of low anthocyanin cultivars. After treated the tea plants with 5-aza-2' deoxycytidine(5-aza-d C), an inhibitor of DNA methylation, the expression of CsAN1 increased, and lead to activation anthocyanin LBGs expression. These results indicate that the DNA methylation in the 5' upstream sequence of CsAN1 plays an essential role in the regulation of CsAN1 expression.6. Temperature and light alter the expression of MBW protein complexs and anthocyanin biosynthesis genesLonger light and low temperatures promoted red coloration in tea leaves from the ‘Zijuan'.CsAN1, Cs GL3, Cs EGL3 and all essential anthocyanin biosynthetic genes transcripts were increased. Thus, Longer light and low temperatures enhanced anthocyanin biosynthesis and accumulation in leaves.