Gene Expression And Regulation Of Ultraviolet A-specific Induction Of Anthocyanin Biosynthesis In Brassica Rapa 'Tsuda'

Anthocyanin biosynthesis in higher plants is regulated by light. Photoreceptors receive lightand are involved in light signal transduction to stimulate the metabolism of anthocyaninaccumulation. In Arabidopsis anthocyanin biosynthesis is induced by red, UV-A, blue, and UV-B light through phytochromes, cryptochromes and UV-B receptor, respectively. In Brassicarapa 'Tsuda', however, only UV-A induced anthocyanin biosynthesis. In this study, 'Tsuda'plants at the stage of 4 days or 3 months old were used as materials. The anthocyaninbiosynthesis character as induced by light, the analysis of gene expression profile, the cloningand expression of regulated gene, the cloning and analysis of promoter of CHS wereinvestigated. The main results were obtained as follows.1. UV-A specific induction of anthocyanin biosynthesis in 'Tsuda' plants.Anthocyanin biosynthesis was induced by sunlight in 'Tsuda' plants. When lights withspecific wave length were irradiated such as UV-B, UV-A, blue, red, far-red, blue plus red, UV-A plus red, and UV-A plus far-red lights, only UV-A induced anthocyanin biosynthesis. UV-Ainduced anthocyanin biosynthesis in both of hypcotyls of seedlings and swollen hypocotyls of'Tsuda' plants.2. UV-A specific induction of gene expression profile of anthocyanin biosynthesis andprotein phosphorylation in 'Tsuda' plants.Microarray analysis showed that gene expression profile induced by UV-A had obviousdifference from those by blue and UV-B. Among a set of many genes induced by UV-A, theexpression of CHS, which was a key gene of anthocyanin biosynthesis, showed up-regulation.Genes for anthocyanin biosynthesis, including chalcone synthase, flavanone 3-hydroxylase,glutathione S-transferase, cytochrome P450, bHLH transcription factor, zinc finger-like protein,and Myb-like DNA binding protein were screened out by UV-A induction.Both cytoplasmic and membrane proteins were phosphorylated in response to UV-Aexposure. The phosphorylation of these cytoplasmic and membrane proteins became detectablewithin 5 minutes after UV-A exposure. The phosphorylation extent increased gradually until30-45 min after exposure, and then declined to an almost undetectable level by 120 min. Itsuggested that the phosphorylation of proteins in response to UV-A exposure might be involvedin light signal transduction.3. The expression character of putative CRY1, COP1, HY5 induced by UV-A in 'Tsuda'plants.Homolog cDNAs of CRY1, COP1, and HY5 in 'Tsuda' were isolated and the amino acidresidues deduced from each gene showed over 90ï¼…sequence identity to that of Arabidopsis CRY1, COP1, and HY5, respectively. Photolyase and FAD-binding domains in CRY1, Ringand WD-40 domains in COP1, and bZIP domain in HY5 were separately found in putativeCRY1, COP1, and HY5 of 'Tsuda' plants. The transient expression of CRY1, COP1, and HY5in protoplast showed that CRY1 and COP1 were distributed in the whole protoplast and HY5was in the area of nucleus. In the seedlings of 'Tsuda'plants, the level of CRY1, COP1, HY5expression was higher than that in mature plants, but they were not induced by light. In matureplants, in contrast, the expression of COP1, HY5 was induced by UV-A. It suggested that itmight be related to the expression of anthocyanin biosynthesis genes. Although CRY1 was notshown to be a photoreceptor of UV-A signal transduction in anthocyanin biosynthesis of B.rapa 'Tsuda', the pseudo-UV-A photoreceptor and CRY1 photoreceptor were considered toshare potentially the same regulators, ie., COP 1 and HY5.4. The expression of CHS was induced specifically by UV-A and LRE was localized in its5'-untranslation regionThe sequence of CHS 5' UTR in 'Tsuda' plants had high identity to that of the promoter ofCHS in Arabidopsis. It contained CAAT box, TATA box and light-induced motif such as ACE,ATCT-motif, Boxâ…¡, G-Box, GATA-motif, GT1-motif, MRE, SP1 and TCT-motif. Theexpression character of CHS induced by light with different wavelengths showed that only UV-A induced the expression of CHS in 'Tsuda' plants. It may be possible that a UV-A signaltransduction way specifically induces the expression of CHS in B. rapa 'Tsuda'.