Ectopic Expression of Multiple Chrysanthemum (Chrysanthemum × morifolium) R2R3-MYB Transcription Factor Genes Regulates Anthocyanin Accumulation in Tobacco.
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.
Project description:Chrysanthemums (Chrysanthemum morifolium Ramat.) exhibit a variety of flower colors due to their differing abilities to accumulate anthocyanins. One MYB member, CmMYB6, has been verified as a transcription regulator of chrysanthemum genes involved in anthocyanin biosynthesis; however, the co-regulators for CmMYB6 remain unclear in chrysanthemum. Here, the expression pattern of CmbHLH2, which is clustered in the IIIf bHLH subgroup, was shown to be positively correlated with the anthocyanin content of cultivars with red, pink and yellow flower colors, respectively. CmbHLH2 significantly upregulated the CmDFR promoter and triggered anthocyanin accumulation when co-expressed with CmMYB6. Yeast one-hybrid analyses indicated that CmbHLH2 was able to bind directly to the CmDFR promoter. Moreover, yeast two-hybrid assays indicated protein-protein interaction between CmbHLH2 and CmMYB6. These results suggest that CmbHLH2 is the essential partner for CmMYB6 in regulating anthocyanin biosynthesis in chrysanthemum.
Project description:Chrysanthemum is an important ornamental crop worldwide. Some white-flowered chrysanthemum cultivars produce red ray florets under natural cultivation conditions, but little is known about how this occurs. We compared the expression of anthocyanin biosynthetic and transcription factor genes between white ray florets and those that turned red based on cultivation conditions to comprehend the underlying mechanism. Significant differences in the expression of <i>CmbHLH2</i> were detected between the florets of different colors. <i>CmbHLH2</i> generated two alternatively spliced transcripts, designated <i>CmbHLH2<sup>Full</sup></i> and <i>CmbHLH2<sup>Short</sup></i> . Compared with <i>CmbHLH2<sup>Full</sup></i> , <i>CmbHLH2<sup>Short</sup></i> encoded a truncated protein with only a partial MYB-interaction region and no other domains normally present in the full-length protein. Unlike the full-length form, the splicing variant protein CmbHLH2<sup>Short</sup> localized to the cytoplasm and the nucleus and could not interact with CmMYB6. Additionally, CmbHLH2<sup>Short</sup> failed to activate anthocyanin biosynthetic genes and induce pigment accumulation in transiently transfected tobacco leaves, whereas CmbHLH2<sup>Full</sup> promoted both processes when simultaneously expressed with CmMYB6. Co-expressing CmbHLH2<sup>Full</sup> and CmMYB6 also enhanced the promoter activities of <i>CmCHS</i> and <i>CmDFR</i>. Notably, the Arabidopsis <i>tt8-1</i> mutant, which lacks red pigmentation in the leaves and seeds, could be complemented by the heterologous expression of CmbHLH2<sup>Full</sup>, which restored red pigmentation and resulted in red pigmentation in high anthocyanin and proanthocyanidin contents in the leaves and seeds, respectively, whereas expression of CmbHLH2<sup>Short</sup> did not. Together, these results indicate that CmbHLH2 and CmMYB6 interaction plays a key role in the anthocyanin pigmentation changes of ray florets in chrysanthemum. Our findings highlight alternative splicing as a potential approach to modulate anthocyanin biosynthesis in specific tissues.
Project description:'Jimba' is a white chrysanthemum cultivar, which occasionally and spontaneously produces red flower petals under natural cultivation due to cyanidin-based anthocyanin accumulation. To investigate the underlying mechanism of this process, a comparative transcriptome was analyzed between white and turning red 'Jimba'. The structural and regulatory genes of anthocyanin pathway were significantly up-regulated in turning red 'Jimba'. Among them, two alternative splicings, <i>CmbHLH2</i> and <i>CmbHLH2.1</i>, showed the most significantly up-regulated in turning red tissue. Transiently over-expressed <i>35S::CmMYB6-CmbHLH2</i> strongly induced anthocyanin accumulation in 'Jimba' flower petals, while moderate amount of anthocyanin was detected when over-expressed <i>35S::CmMYB6-CmbHLH2.1</i>. Both CmbHLH2 and CmbHLH2.1 could interact with CmMYB6 to activate <i>CmDFR</i> promoter according to Yeast two-hybrid and dual-luciferase assay. Moreover, CmMYB6-CmbHLH2 but not CmMYB6-CmbHLH2.1 could activate the <i>CmbHLH2</i> promoter to provide positive feedback loop regulation. Taken together, it suggested that both CmbHLH2 and CmbHLH2.1 involved in regulation flower color alteration in turning red 'Jimba', and CmbHLH2 played a predominant role in this process.
Project description:'Jimba', a well-known white flowered chrysanthemum cultivar, occasionally and spontaneously produces red colored petals under natural cultivation, but there is little information about the molecular regulatory mechanism underlying this process. We analysed the expression patterns of 91 MYB transcription factors in 'Jimba' and 'Turning red Jimba' and identified an R3 MYB, CmMYB#7, whose expression was significantly decreased in 'Turning red Jimba' compared with 'Jimba', and confirmed it is a passive repressor of anthocyanin biosynthesis. CmMYB#7 competed with CmMYB6, which together with CmbHLH2 is an essential component of the anthocyanin activation complex, for interaction with CmbHLH2 through the bHLH binding site in the R3 MYB domain. This reduced binding of the CmMYB6-CmbHLH2 complex and inhibited its ability to activate CmDFR and CmUFGT promoters. Moreover, using transient expression assays we demonstrated that changes in the expression of CmMYB#7 accounted for alterations in anthocyanin content. Taken together, our findings illustrate that CmMYB#7 is a negative regulator of anthocyanin biosynthesis in chrysanthemum.
Project description:<h4>Background</h4>Anthocyanins contribute to coloration and antioxidation effects in different plant tissues. MYB transcription factors have been demonstrated to be a key regulator for anthocyanin synthesis in many plants. However, little information was available about the MYB genes in the halophyte species Eutrema salsugineum.<h4>Result</h4>Here we report the identification of an important anthocyanin biosynthesis regulator EsMYB90 from Eutrema salsugineum, which is a halophyte tolerant to multiple abiotic stresses. Our phylogenetic and localization analyses supported that EsMYB90 is an R2R3 type of MYB transcription factor. Ectopic expression of EsMYB90 in tobacco and Arabidopsis enhanced pigmentation and anthocyanin accumulation in various organs. The transcriptome analysis revealed that 42 genes upregulated by EsMYB90 in 35S:EsMYB90 tobacco transgenic plants are required for anthocyanin biosynthesis. Moreover, our qRT-PCR results showed that EsMYB90 promoted expression of early (PAL, CHS, and CHI) and late (DFR, ANS, and UFGT) anthocyanin biosynthesis genes in stems, leaves, and flowers of 35S:EsMYB90 tobacco transgenic plants.<h4>Conclusions</h4>Our results indicated that EsMYB90 is a MYB transcription factor, which regulates anthocyanin biosynthesis genes to control anthocyanin biosynthesis. Our work provides a new tool to enhance anthocyanin production in various plants.
Project description:MYB transcription factors are widely involved in the development of and physiological processes in plants. Here, we isolated the chrysanthemum R2R3-MYB family transcription factor CmMYB15, a homologous gene of AtMYB15. It was demonstrated that CmMYB15 expression was induced by aphids and that CmMYB15 could bind to AC elements, which usually exist in the promoter of lignin biosynthesis genes. Overexpression of CmMYB15 in chrysanthemum enhanced the resistance of aphids. Additionally, the content of lignin and the expression of several lignin biosynthesis genes increased. In summary, the results indicate that CmMYB15 regulates lignin biosynthesis genes that enhance the resistance of chrysanthemum to aphids.
Project description:R2R3 MYB transcription factors play key functions in the regulation of secondary metabolites. In the present study, a R2R3 MYB transcriptional factor NtMYB2 was identified from Chinese narcissus (Narcissus tazetta L. var. Chinensis Roem) and functionally characterized. NtMYB2 belongs to subgroup 4 of the R2R3 MYB transcription factor family that are related to repressor MYBs involved in the regulation of anthocyanin and flavonoids. Transient expression confirmed that NtMYB2 strongly reduced the red pigmentation induced by MYB- anthocyanin activators in agro-infiltrated tobacco leaves. Ectopic expression of NtMYB2 in tobacco significantly reduced the pigmentation and altered the floral phenotypes in transgenic tobacco flowers. Gene expression analysis suggested that NtMYB2 repressed the transcript levels of structural genes involved in anthocyanin biosynthesis pathway, especially the UFGT gene. NtMYB2 gene is expressed in all examined narcissus tissues; the levels of transcription in petals and corona is higher than other tissues and the transcription level at the bud stage was highest. These results show that NtMYB2 is involved in the regulation of anthocyanin biosynthesis pathway and may act as a repressor by down regulating the transcripts of key enzyme genes in Chinese narcissus.
Project description:BACKGROUND:The primary pigments in flowers are anthocyanins, the biosynthesis of which is mainly regulated by R2R3-MYBs. Muscari armeniacum is an ornamental garden plant with deep cobalt blue flowers containing delphinidin-based anthocyanins. An anthocyanin-related R2R3-MYB MaAN2 has previously been identified in M. armeniacum flowers; here, we also characterized a novel R2R3-MYB MaMybA, to determine its function and highlight similarities and differences between MaMybA and MaAN2. RESULTS:In this study, a novel anthocyanin-related R2R3-MYB gene was isolated from M. armeniacum flowers and functionally identified. A sequence alignment showed that MaMybA contained motifs typically conserved with MaAN2 and its orthologs. However, the shared identity of the entire amino acid sequence between MaMybA and MaAN2 was 43.5%. Phylogenetic analysis showed that they were both clustered into the AN2 subgroup of the R2R3-MYB family, but not in the same branch. We also identified a IIIf bHLH protein, MabHLH1, in M. armeniacum flowers. A bimolecular fluorescence complementation assay showed that MabHLH1 interacted with MaMybA or MaAN2 in vivo; a dual luciferase assay indicated that MaMybA alone or in interaction with MabHLH1 could regulate the expression of MaDFR and AtDFR, but MaAN2 required MabHLH1 to do so. When overexpressing MaMybA in Nicotiana tabacum 'NC89', the leaves, petals, anthers, and calyx of transgenic tobacco showed intense and magenta anthocyanin pigments, whereas those of OE-MaAN2 plants had lighter pigmentation. However, the ovary wall and seed skin of OE-MaMybA tobacco were barely pigmented, while those of OE-MaAN2 tobacco were reddish-purple. Moreover, overexpressing MaMybA in tobacco obviously improved anthocyanin pigmentation, compared to the OE-MaAN2 and control plants, by largely upregulating anthocyanin biosynthetic and endogenous bHLH genes. Notably, the increased transcription of NtF3'5'H in OE-MaMybA tobacco might lead to additional accumulation of delphinidin 3-rutinoside, which was barely detected in OE-MaAN2 and control plants. We concluded that the high concentration of anthocyanin and the newly produced Dp3R caused the darker color of OE-MaMybA compared to OE-MaAN2 tobacco. CONCLUSION:The newly identified R2R3-MYB transcription factor MaMybA functions in anthocyanin biosynthesis, but has some differences from MaAN2; MaMybA could also be useful in modifying flower color in ornamental plants.
Project description:Plant R2R3-MYB transcription factor genes are widely distributed in higher plants and play important roles in the regulation of many secondary metabolites at the transcriptional level. In this study, a chrysanthemum subgroup 4 R2R3-MYB transcription factor gene, designated CmMYB1, was isolated through screening chrysanthemum EST (expressed sequence tag) libraries and using rapid application of cDNA ends (RACE) methods and functionally characterized. CmMYB1 is expressed in the root, stem, leaf and flowers, but most strongly in the stem and most weakly in the root. Its heterologous expression in Arabidopsis thaliana reduced the lignin content and altered the lignin composition. The heterologous expression also repressed the flavonoids content in A. thaliana. Together, these results suggested that CmMYB1 is a negative regulator of genes involved in the lignin pathway and flavonoid pathway, it may be a promising gene for controlling lignin and flavonoids profiles in plants.
Project description:The anthocyanin biosynthetic pathway is regulated by a transcription factor complex consisting of an R2R3 MYB, a bHLH, and a WD40. Although R2R3 MYBs belonging to the anthocyanin-activating class have been identified in many plants, and their role well elucidated, the subgroups of bHLH implicated in anthocyanin regulation seem to be more complex. It is not clear whether these potential bHLH partners are biologically interchangeable with redundant functions, or even if heterodimers are involved. In this study, AcMYB110, an R2R3 MYB isolated from kiwifruit (Actinidia sp.) showing a strong activation of the anthocyanin pathway in tobacco (Nicotiana tabacum) was used to examine the function of interacting endogenous bHLH partners. Constitutive expression of AcMYB110 in tobacco leaves revealed different roles for two bHLHs, NtAN1 and NtJAF13. A hierarchical mechanism is shown to control the regulation of transcription factors and consequently of the anthocyanin biosynthetic pathway. Here, a model is proposed for the regulation of the anthocyanin pathway in Solanaceous plants in which AN1 is directly involved in the activation of the biosynthetic genes, whereas JAF13 is involved in the regulation of AN1 transcription.