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.

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‘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.

Anthocyanin is the crucial pigment for the coloration of red chrysanthemum flowers, which synthesizes in the cytosol and is transported to the vacuole for stable storage. In general, glutathione S-transferases (GSTs) play a vital role in this transport. To date, there is no functional GST reported in chrysanthemums. Here, a total of 94 CmGSTs were isolated from the chrysanthemum genome, with phylogenetic analysis suggesting that 16 members of them were clustered into the Phi subgroup which was related to anthocyanin transport. Among them, the expression of CmGST1 was positively correlated with anthocyanin accumulation. Protein sequence alignment revealed that CmGST1 included anthocyanin-related GST-specific amino acid residues. Further transient overexpression experiments in tobacco leaves showed that CmGST1 could promote anthocyanin accumulation. In addition, a dual-luciferase assay demonstrated that CmGST1 could be regulated by CmMYB6, CmbHLH2 and CmMYB#7, which was reported to be related to anthocyanin biosynthesis. Taken together, we suggested that CmGST1 played a key role in anthocyanin transport and accumulation in chrysanthemums.

Loropetalum chinense var. rubrum is a common landscape plant. Because of the preference for the ‘red leaf’ trait, the ‘flower color’ trait was ignored in breeding, and the genetic diversity of L. chinense var. rubrum was replaced by a few genotypes. Therefore, the potential value of flower color traits was affected, and the genetic diversity was not fully exploited. In this study, we utilized a cultivar with mosaic-colored flowers and leaves and observed 15 phenotypic traits in its flower organs and its progeny, as well as a total of 60 different flower color materials. We analyzed the variation characteristics and diversity of flower colors, and qualitatively described and preliminarily classified flower colors using the ISCC-NBS and CIELab systems to explore the correlation with the flower color phenotype from the distribution and content of anthocyanins. The phenotypic diversity of the flower organs was enriched, and genetic differences in flower color were significant for the L. chinense var. rubrum, with most of the 15 phenotypic traits showing significant positive correlations, and the most critical phenotypes are sepal, petal and nectary color, as well as petal length. According to the CIELab color system and anthocyanin content and distribution characteristics, it is considered more accurate and convenient to classify the color phenotypes of L. chinense var. rubrum flowers into five categories. In this study, for the first time, the flower organ phenotype of L. chinense var. rubrum has been systematically analyzed, and it is explored as a reference method for breeding new cultivars of a diverse range of colors in L. chinense var. rubrum.