| Anthocyanin is one of the main pigments for the flower coloration, whose biosynthesis and accumulation are closely related to the plant development process and environmental conditions. However, at present the molecular mechanism of how the environmental factors regulate the anthocyanins biosynthesis has not been revealed clearly. Light is one of the most important environmental factors that affect the anthocyanin biosynthesis, thus, ornamental and commercial value of ornamental plants could be greatly improved via proper regulation of light conditions for plant growth. Therefore, revealing the molecular mechanism of anthocyanins biosynthesis regulated by light is of theoretical importance and practical value. Chrysanthemum (Chrysanthemum x morifolium Ramat.) is a worldwide famous ornamental crop with rich color variations. The simple background of pigment metabolism and the light sensitivity of pigments production make chrysanthemum an ideal model for studies of the molecular mechanism of light-induced anthocyanin biosynthesis. At present, studies of the light-induced anthocyanin biosynthesis in chrysanthemum mainly focus on phenotypic and biochemical analyses, but how the light induces the expression of key structural genes involving in the anthocyanin biosynthetic pathway is rarely reported on the molecular level. Moreover, key regulator genes that regulate the expression of structural genes in chrysanthemum have not been identified and isolated, which restricts the molecular breeding of chrysanthemum for flower colors modification by regulating light conditions. In the present study, a purple chrysanthemum cultivar’Reagan’, which is highly sensitive to light conditions, was selected as plant material. The author first contrasted the pigments composition and content in the faded samples that were induced by shading, and then inferred the molecular mechanism of light-induced anthocyanin biosynthesis in chrysanthemum after a series of bioinformatic and gene expression analyses. The main results obtained in the present study are the followings.1. The capitulum and leaves of ’Reagan’ were respectively shaded, while plants treated with light and shading on the whole plant were selected as the positive and negative controls, respectively. Analysis on the change of flower color phenotype showed that the ray florets under the three different shading treatments were all faded; in particular, fading of the ray florets under the capitulum-shading treatment was more significant than those under the leaf-shading treatment. After pigmentary analysis, we identified two cyanidin and five flavone derivatives in the ray florets; under the three shading treatments, the relative anthocyanins content showed an "increasing first and then decreasing" pattern with the capitulum development, and the decreasing significance was in consistent with the phenotypic results. The above results indicated that (1) with the capitulum development, light significantly affects the variation of the two cyanidin derivatives in the ray florets of’Reagan’; (2) both of the capitulum and leaves of this cultivar respond to light and corresponding affect the anthocyanins biosynthesis, but photoperceptivity of the capitulum was much stronger than the leaves; and (3) early stage of the capitulum was the key phase responding to light during the process of light-induced anthocyanins biosynthesis.2. Five transcriptomic libraries were constructed after shading the capitulum (the first three developmental stages) of ’Reagan’; plants treated with light were selected as the control.103,517 specific unigenes were obtained after de novo assembly of RNA sequences, of which 60,712 (58.65%) unigenes were annotated against four protein databases. After that,2,135 unigenes responding to light were identified using bioinformatic analyses. Metabolic pathway analysis showed that the anthocyanin biosynthetic pathway was the only complete metabolic pathway both modulated in response to light and related to capitulum development. Following the shading treatment, nearly all structural genes involved in the anthocyanin biosynthetic pathway were down-regulated, which was in agreement with the fading of the ray florets and the decrease of the anthocyanins content. Moreover, four key transcription factors (TFs) that might participate in the regulation of anthocyanin biosynthesis under light conditions were identified based on the construction of phylogenetic trees. Based on these results, the author inferred that the variation of the anthocyanins involved in the ray florets of ’Reagan’ is mainly owing to the light-induced expression of the 13 structural gene members and the four TFs involved in the anthocyanin biosynthetic pathway with the capitulum development.3. Based on the digital gene expression analysis, nine genes with moderate expression in the transcriptomic library were screened and used as the candidate reference genes for further study. After comprehensively analyzing the stability of gene expression with three distinct statistical algorithms, geNorm, NormFinder, and qBase plus, and further analyzing the relative expression level of the CmF3H gene in different samples for the verification of the reference genes that we selected, we found that CmF-box and CmPP2A were the most stable genes in all of the samples under different light treatments. These results provided the most suitable reference genes for the verification on the light-induced expression of the 17 candidate genes.4. After contrasting the expressive patterns of the key structural and regulator gene members that were previously screened from the transcriptomic analysis, as well as the up-stream genes involved in the light signaling pathway between the capitulum and leaves, we identified 17 candidate gene members that participate in the light-induced anthocyanin biosynthesis using the above two stable reference genes under different light treatments, including CmCHSl, CmCHS2, CmF3H, CmF3’H1, CmF3’H2, CmF3’H3, CmDFR, CmANSl, CmANS2, CmANS, Cm3GT, Cm3MTl, Cm3MT2, CmMYB5-1, CmMYB6, CmMYB7-1 and CmbHLH24. The expression of CmHY5 was positively correlated to the anthocyanins content; while the expression of five photoreceptor genes, CmPHYA, CmPHYB, CmCRYla, CmCRYlb and CmCRY2, showed weak correlation to the light-induced anthocyanin biosynthesis. The expression of CmCOP1 also presented no obvious corration to the anthocyanins content. Moreover, most of the structural and regulator genes involved in the anthocyanin biosynthetic pathway were specifically expressed in the ray florets.5. Based on the transcriptomic analysis and verification of gene expression,5,106 raw proteins were identified from the ray florets of’Reagan’using iTRAQ technique, of which 45 and 115 proteins were respectively up-and down-regulated after shading. We also found that most of the enzymes involved in the anthocyanin biosynthetic pathway were down-regulated after shading, which was consistent with the expressive change of coding genes and results of pigmentary analyses. Moreover, after shading, the expressive abundance of the light-harvesting chlorophyll proteins increased with the capitulum development. Based on the functional and pathway enrichment analyses, we inferred that GDSL lipases might play roles in the process of light-induced anthocyanin biosynthesis. These results indicated that the light-induced expression of the seven key enzymes involved in the anthocyanin biosynthetic pathway is the immediate reason for the ray florets coloration of ’Reagan’.6. Finally, the whole lengths of cDNA of four regulator genes were cloned, but only the amino acid sequence of CmMYB6 contained the conserved R39, V64, A90, and ANDV motifs for dicot anthocyanin-promoting MYBs. After promoter analysis, we found that the promoter sequence of CmMYB6 contained three photoresponsive and two bHLH-binding elements. Moreover, compared with the control plants, the flower colors of CmMYB6-transgenic tobacco plants were deepened obviously, which was consistent with the significant increase of the relative anthocyanin content and the relative expression levels of CmMYB6, NtDFR and NtANS, indicating that CmMYB6 was exactly one of the key regulator genes that promote the light-induced biosynthesis and accumulation of anthocyanins in chrysanthemum.Based on the above results, the molecular mechanism of light-induced anthocyanin biosynthesis in chrysanthemum was preliminarily inferred. After the capitulum perceiving light signal, the expression of 13 structural gene members (CmCHSl, CmCHS2, CmF3H, CmF3’H1, CmF3’H2, CrnF3’H3, CmDFR, CmANSl, CmANS2, CmANS3, Cm3GT, Cm3MTl and Cm3MT2), which encode the seven key enzymes (CmCHS, CmF3H, CmF3’H, CmDFR, CmANS, Cm3GT and Cm3MT) involved in the anthocyanin biosynthetic pathway, are activated in order via the light signaling pathway; accordingly, the biosynthesis and accumulation of seven intermediate products (chalcone, naringenin, dihydrokaempferol, dihydroquercetin, leucocyanidin, cyanidin and cyanidin-3-glucoside) and cyanidin-3-O-(6"-O-malonyl-glucoside) and cyanidin-3-O-(3",6"-O-dimalonyl-glucoside) (end products) are further improved, resulting in the coloration of the ray florets. During the light-induced expression of the 13 structural gene members, positive regulator genes CmMYB5-1, CmMYB6 and CmbHLH24 and a negative regulator gene CmMYB7-1 co-regulate the expression of structural genes after perceiving light signal. The present study lays theoretical foundations for the understanding of the molecular mechanism of light-induced anthocyanin biosynthesis in ornamental plants, and provides references for the molecular breeding of flower colors modification by regulating light conditions in chrysanthemum. |
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