Expression Of Genes On Anthocyanin Biosythesis Pathway Control Flower Coloration In Chrysanthemum And Cineraria

Chrysanthemum (Chrysanthemum×morifolium Ramat.) is one of the traditional cut flowers and pot flowers in China. Blue-flowering is the only type absent from colorful chrysanthemum varieties. Cineraria (Senecio cruentus Masson ex L'Herit; Pericallis cruentia L'Herit), which has blue varieties, is a popular potted flower in spring available at local markets in China. In this study, the difference on gene expression patterns involved in anthocyanin biosynthesis between cineraria and chrysanthemum was compared. It is helpful to explain the reasons and the molecular mechanism of the lack of blue-flowering chrysanthemum. These kinds of studies have important academic significances and practical values. There are 7 major results in this study.(1) According to phenotype of flower coloration, chrysanthemum'Regan'and cineraria'Chunchao'were divided into 4 color series. In white ray florets of cineraria, flavonoids were major pigment. There were flavonoids and anthocyanins in cineraria red, purple and blue ray florets, and in chrysanthemum pink ray florets. Carotenoids were detected in yellow ray florets of chrysanthemum.(2) Using semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR), this study isolated four internal segments and one full-length cDNA from cineraria flowers: chalcone synthase(CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H) and anthocyanidin synthase (ANS). In addition, four full-length cDNAs from chrysanthemum flowers were isolated by RT-PCR and PCR-RACE: chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H) and anthocyanidin synthase (ANS). The results of bioinformation suggested that key structual genes shared about 60%-93% homology with the accessed genes from other species in GenBank. And the putative protein contained conserved domains. CHI and F3H from chrysanthemum were considered hydrophilic proteins, F3'H and ANS may be membrane proteins.(3) The expression patterns of CHS, CHI, F3H, F3'H, DFR, flavonoid 3',5'-hydroxylase (F3'5'H) and anthocyanin 3-O-glucoside-6"-O -malonyltransferase (3MaT) in five developmental stages of cineraria with different flower colors were determined for the first time by RT-PCR. The results indicated that there is no CHS transcript in white flowers, In red flowers, F3'H mRNA was expressed at high level. But no F3'5'H transcript was found. F3'5'H was highly expressed in Stage I but no F3'H mRNA was observed in blue flowers. And the transcripts of F3'H and F3'5'H were found in purple petals. In addition, these genes were expressed at high level in early stage and underwent moderate decreases in expression. Interestingly, genes expression level increased again in Stage IV. However, in Stage V, there is little expression level could be detected in petals.(4) During seven developmental stages of chrysanthemum, most of genes were highly expressed in Stage II or Stage III and underwent moderate decreases. Little expression level could be detected in ray floret in Stage VII. CmF3'H mRNA were expressed at high level in Stage I in purple flowers. Average levels of CmF3'H expression in pink ray floret are lower than in red and purple ray florets.(5) Expression of the structral genes in different tissues showed that they were not detected in roots. DFR and ANS were floral organ-specific genes. F3'5'H transcripts was detected in ray florets and leaves. Other genes expressed both in stems and leaves.(6) After dark and high temperature treatment, chrysanthemum ray florets faded seriously. When inflorescences from Stage I were covered with aluminium foil, the gene expression of MYB and the expression of CHI, F3H, F3'H, DFR, ANS were inhibited. This results implyed that MYB was a key transcriptional factor regulating structural genes expression. However, MYB and WD40 were up-regulated after high temperature treatment, whereas the expression of most structural genes were inhibited in middle and late stages. It can be speculated that high temperature influenced the stability of anthocyanin. Furthermore, MYB and WD40 might be related to stress of chrysanthemum.(7) According to our results, it could be concluded that gene expression was the underlying causes of flower pigmentation. Besides, there are two key reasons of the lack of blue-flowering chrysanthemum:(1) F3'5'H gene deletion; (2) F3'H strongly expressed. Light and temperature were closely related to chrysanthemum ray florets pigmentation.