Abstract
The generation of chrysanthemum (Chrysanthemum × morifolium) flower color is mainly attributed to the accumulation of anthocyanins. In the anthocyanin biosynthetic pathway in chrysanthemum, although all of the structural genes have been cloned, the regulatory function of R2R3-MYB transcription factor (TF) genes, which play a crucial role in determining anthocyanin accumulation in many ornamental crops, still remains unclear. In our previous study, four light-induced R2R3-MYB TF genes in chrysanthemum were identified using transcriptomic sequencing. In the present study, we further investigated the regulatory functions of these genes via phylogenetic and alignment analyses of amino acid sequences, which were subsequently verified by phenotypic, pigmental, and structural gene expression analyses in transgenic tobacco lines. As revealed by phylogenetic and alignment analyses, CmMYB4 and CmMYB5 were phenylpropanoid and flavonoid repressor R2R3-MYB genes, respectively, while CmMYB6 was an activator of anthocyanin biosynthesis, and CmMYB7 was involved in regulating flavonol biosynthesis. Compared with wild-type plants, the relative anthocyanin contents in the 35S:CmMYB4 and 35S:CmMYB5 tobacco lines significantly decreased (p < 0.05), while for 35S:CmMYB6 and 35S:CmMYB7, the opposite result was obtained. Both in the 35S:CmMYB4 and 35S:CmMYB5 lines, the relative expression of several anthocyanin biosynthetic genes in tobacco was significantly downregulated (p < 0.05); on the contrary, several genes were upregulated in the 35S:CmMYB6 and 35S:CmMYB7 lines. These results indicate that CmMYB4 and CmMYB5 negatively regulate anthocyanin biosynthesis in chrysanthemum, while CmMYB6 and CmMYB7 play a positive role, which will aid in understanding the complex mechanism regulating floral pigmentation in chrysanthemum and the functional divergence of the R2R3-MYB gene family in higher plants.
Highlights
Anthocyanin biosynthesis is primarily controlled by a complex consisting of R2R3-MYB, basic helix–loop–helix, and WD40 proteins that acts as the master regulator to coordinate the transcription of anthocyanin biosynthetic genes
CmMYB4 was closely related to general phenylpropanoid repressor MYBs, such as AmMYB308 (Antirrhinum majus), AtMYB4, AtMYB32, and AtMYB6, while CmMYB5 was similar to flavonoid MYB repressors, e.g., FaMYB1, VvMYBC2-L1 (Vitis vinifera), and PtMYB182 (Populus tremula)
Lines, the relative anthocyanin content in tobacco corolla significantly increased, accompanied by the deepening of flower color. These results indicated the repressive function of CmMYB4 and CmMYB5 and the activating role of CmMYB6 and CmMYB7 for anthocyanin accumulation in the chrysanthemum
Summary
Ornamental plants with novel colors and coloration patterns are of great commercial value in the floricultural industry, as they are among the most important aesthetic characters for consumers [1].Pigmentation in flowers is mainly attributed to anthocyanins, a type of flavonoid; the major pigments imparting a great variety of colors from light yellow, red, dark red, magenta, purple, to blue in ornamental plants have been investigated extensively [2,3,4].Based on their DNA binding domains, MYB transcription factors (TFs) are generally classified into four main types, namely, R2, R2R3, R3, and R4 types, of which R2R3-MYBs are closelyGenes 2019, 10, 777; doi:10.3390/genes10100777 www.mdpi.com/journal/genesGenes 2019, 10, 777 related to anthocyanin biosynthesis [5]. Pigmentation in flowers is mainly attributed to anthocyanins, a type of flavonoid; the major pigments imparting a great variety of colors from light yellow, red, dark red, magenta, purple, to blue in ornamental plants have been investigated extensively [2,3,4]. Based on their DNA binding domains, MYB transcription factors (TFs) are generally classified into four main types, namely, R2, R2R3, R3, and R4 types, of which R2R3-MYBs are closely. Zhang et al 2019 identified three novel R2R3-MYB members of anthocyanin regulators in the genome of the purple flowering Petunia inflate, which were expressed with the development of various tissues; transcript levels of many anthocyanin biosynthetic genes, such as CHSaI (chalcone synthase), CHSj, F3H (flavanone 3-hydroxylase), F30 50 H (flavonoid-30 ,50 -hydroxylase), DFR (dihydroflavonol 4-reductase), and ANS (anthocyanin synthase), were increased in the overexpression lines of P. hybrida line M1 compared to untransformed controls [15]
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