Transcription factor CmbHLH16 regulates petal anthocyanin homeostasis under different lights in Chrysanthemum

Abstract

Light is essential to plant survival and elicits a wide range of plant developmental and physiological responses under different light conditions. A low red-to-far red (R/FR) light ratio induces shade-avoidance responses, including decreased anthocyanin accumulation, whereas a high R/FR light ratio promotes anthocyanin biosynthesis. However, the detailed molecular mechanism underpinning how different R/FR light ratios regulate anthocyanin homeostasis remains elusive, especially in non-model species. Here, we demonstrate that a low R/FR light ratio induced the expression of CmMYB4, which suppressed the anthocyanin activator complex CmMYB6-CmbHLH2, leading to the reduction of anthocyanin accumulation in Chrysanthemum (Chrysanthemum morifolium) petals. Specifically, CmMYB4 recruited the corepressor CmTPL (TOPLESS) to directly bind the CmbHLH2 promoter and suppressed its transcription by impairing histone H3 acetylation. Moreover, the low R/FR light ratio inhibited the PHYTOCHROME INTERACTING FACTOR family transcription factor CmbHLH16, which can competitively bind to CmMYB4 and destabilize the CmMYB4-CmTPL protein complex. Under the high R/FR light ratio, CmbHLH16 was upregulated, which impeded the formation of the CmMYB4-CmTPL complex and released the suppression of CmbHLH2, thus promoting anthocyanin accumulation in Chrysanthemum petals. Our findings reveal a mechanism by which different R/FR light ratios fine-tune anthocyanin homeostasis in flower petals.

Figure 1

Low R/FR light ratio inhibits and high R/FR light ratio promotes anthocyanin biosynthesis by regulating multiple anthocyanin-associated structural genes. A, Phenotypes of flower color in plants treated under WL, low R/FR light ratio (WL+FR), and high R/FR light ratio (WL+R). Scale bars, 1 cm. B, Anthocyanin content in the ray flower petals. Error bars indicate the sd of three biological replicates. Significant differences are indicated with asterisks (**P < 0.01, ANOVA, Tukey’s correction). C and D, Relative expression of CmCHS, CmCHI, CmF3H, CmDFR, CmANS, CmUFGT, CmMYB6, and CmbHLH2 in the ray flower petals of plants treated under different light conditions for 6 h. Error bars indicate the sd of three biological replicates. Significant differences are indicated with asterisks (*P < 0.05, **P < 0.01, ANOVA, Tukey’s correction). CmEF1α and CmActin were used as the internal control. E and F, Protein abundance of CmMYB6 and CmbHLH2 in the ray flower petals of flowers transiently overexpressing CmMYB6 and CmbHLH2 with pSAK277-CmMYB6 (35S:CmMYB6-Flag) and pSAK277-CmbHLH2 (35S:CmbHLH2-Flag), respectively, as determined using an anti-Flag antibody. Actin in total protein extracts was used as the loading control. IB, immunoblot.

Figure 2

Low R/FR light ratio induces CmMYB4 to suppress CmbHLH2 and negatively regulates anthocyanin biosynthesis. A, ChIP-PCR of the enrichment of DNA fragments AC-II (−1702 to −1695 bp) and AC-II (−859 to −852 bp) in the CmbHLH2 promoter. The fragment (−1328 to −1193 bp) was used as a control. Error bars indicate the sd of three biological replicates. Significant differences are indicated with asterisks (**P < 0.01, ANOVA, Tukey’s correction). B, Interaction of CmMYB4 with the CmbHLH2 promoter and its mutant CmbHLH2pro(mut) in yeast cells. pGADT7-GUS was used as the negative control. SD/-T/-H/-L indicates Trp, His, and Leu synthetic dropout medium. 80 mM 3-AT was used. C, Interaction of CmMYB4 with labeled DNA probes for the AC-II element of the CmbHLH2 promoter in EMSA. His protein was used as the negative control. Increasing amounts (50× and 200×) of the unlabeled wild-type probes or the unlabeled mutant probes were added as cold competitors. “WT” indicates wild-type DNA probes and “MT” indicates mutated probes. “+” indicates presence and “−” indicates absence. D, Phenotypes of flower color in plants transiently overexpressing or suppressing genes denoted in the figure. Scale bars, 1 cm. E, Anthocyanin content in the ray flower petals. Error bars indicate the sd of three biological replicates. Samples denoted by different letters are significantly different (P < 0.01, ANOVA, Tukey’s correction).

Figure 3

CmMYB4 interacts with CmTPL to impair CmbHLH2 histone H3 acetylation. A, ChIP qPCR analysis of histone H3 acetylation level in the TSS of CmbHLH2 in the ray flower petals of transgenic flowers. pSAK277 empty vector was used as a control. Error bars indicate the sd of three biological replicates. Significant differences are indicated with asterisks (**P < 0.01, ANOVA, Tukey’s correction). B, The ratio of LUC to Renilla (REN) activity. Error bars indicate the sd of six biological replicates. Significant differences are indicated with asterisks (**P < 0.01, ANOVA, Tukey’s correction). C, Interaction between CmMYB4 or CmMYB4mEAR and CmTPL in yeast cells. SD/-T/-L indicates Trp and Leu synthetic dropout medium; SD/-T/-L/-H/-A indicates Trp, Leu, His, and Ade synthetic dropout medium. CmMYB4mEAR: mutated CmMYB4 in which the core Leu residues of the EAR motif were substituted to Ala residues. D, Interaction between CmMYB4 or CmMYB4mEAR and CmTPL in an in vitro pull-down assay. In vitro-translated GST protein was used as the negative control. “Input” indicates protein mixtures before the experiments and “Pull-down” indicates purified protein mixture. “+” indicates presence and “−” indicates absence. IB, immunoblot. Asterisks indicate nonspecific binds. E, Interaction between CmMYB4 or CmMYB4mEAR and CmTPL in BiFC assay. pSPYCE and pSPYNE empty vectors were used as the negative controls. mRFP-NLS, nuclear marker co-expressing the 35S:D53-RFP construct; YFP, images obtained in the yellow fluorescence channel; DIC, images obtained in bright light; and Merged, overlay plots. Scale bars, 20 μm.

Figure 4

CmMYB4 plays a minor role in the accumulation of anthocyanin under the high R/FR light ratio. A, Phenotypes of flower color in the transiently transformed flowers treated under different light conditions. Scale bars, 1 cm. R, red light. B, Comparisons of CmMYB4 expression levels in the ray flower petals when plants were under different light treatments. C, Anthocyanin content in the ray flower petals of transiently transformed flowers treated under different light conditions. Error bars indicate the sd of three biological replicates, significant differences are indicated with asterisks (*P < 0.05, **P < 0.01, ANOVA, Tukey’s correction) for B and C. D, Relative expression of CmbHLH2 in the ray flower petals of transiently transformed flowers treated under different light conditions. Error bars indicate the sd of three biological replicates. Significant differences are indicated with asterisks (**P < 0.01, ANOVA, Tukey’s correction). CmEF1α and CmActin were used as the internal control. E, Protein abundance of CmMYB4 in the leaves of “Nannong Fencui” cultivar cutting seedlings transiently overexpressing CmMYB4 with pSAK277-CmMYB4 (35S:CmMYB4-Flag) at different time points treated under different light conditions, as determined using an anti-Flag antibody. Actin in total protein extracts was used as the loading control. CmMYB4-Flag proteins in the leaves of wild-type cutting seedlings from a separate immunoblot were used as a negative control. IB, immunoblot. F, Quantification of CmMYB4-Flag protein levels using Image J software. The protein stability assay in E was performed three times and the protein quantities relative to the levels at initial time 0 were counted and used to calculate the sd. Samples denoted by the same letter are not significantly different (P < 0.05, ANOVA, Tukey’s correction). G, Relative expression of CmTPL in the ray flower petals of wild-type flowers in response to different light conditions for 6 h. Error bars indicate the sd of three biological replicates. CmEF1α and CmActin were used as the internal control.

Figure 5

CmbHLH16 interacts with CmMYB4 to destabilize the CmMYB4–CmTPL protein complex. A, Relative expression of CmbHLH16 in the ray flower petals of wild-type plants treated under different light conditions. Error bars indicate the sd of three biological replicates. Significant differences are indicated with asterisks (**P < 0.01, ANOVA, Tukey’s correction). CmEF1α and CmActin were used as the internal control. B, Interaction between CmMYB4 or CmTPL and CmbHLH16 in yeast cells. SD/-T/-L indicates Trp and Leu synthetic dropout medium. SD/-T/-L/-H/-A indicates Trp, Leu, His, and Ade synthetic dropout medium. C, Interaction between CmMYB4 and CmbHLH16 in an in vitro pull-down assay. Asterisks indicate nonspecific binds. D, Interaction between CmMYB4 and CmbHLH16 in a BiFC assay in N. benthamiana leaves. Scale bars, 20 μm. E, CmbHLH16 competes with CmTPL for binding to CmMYB4 in yeast cells. Met, Methionine. F, Competitive binding assays of CmTPL and CmbHLH16 to CmMYB4. The gradient indicates the increasing amount of His-CmbHLH16 added in the reaction system.

Figure 6

CmbHLH16 fine-tunes anthocyanin biosynthesis in response to different R/FR light ratios. A, The ratio of LUC to REN activity. Error bars indicate the sd of six biological replicates. Samples denoted by different letters are significantly different (P < 0.01, ANOVA, Tukey’s correction). B, Test of the interactions between CmbHLH16 and the CmbHLH2 promoter in yeast cells. pGADT7-GUS was used as a negative control. SD/-T/-H/-L indicates Trp, His, and Leu synthetic dropout medium. 3-AT concentrations: 80 mM. C, Phenotypes of flower color in the transiently transformed flowers treated under different light conditions. Scale bars, 1 cm. D, Anthocyanin content in the ray flower petals of transiently transformed flowers. Error bars indicate the sd of three biological replicates. Samples denoted by different letters are significantly different (P < 0.01, ANOVA, Tukey’s correction).

Figure 7

A model of CmbHLH16 fine-tuning anthocyanin biosynthesis in response to different R/FR light ratios in Chrysanthemum. Under the low R/FR light ratio environment, CmbHLH16 expression is suppressed and anthocyanin repressor CmMYB4 is substantially upregulated, which facilitates the formation of the anthocyanin suppression complex CmMYB4–CmTPL. This complex directly binds to and deacetylates histone H3 in the promoter of the positive anthocyanin regulator CmbHLH2, leading to the downregulation of CmbHLH2. Hence, less anthocyanin is accumulated in the petals. Under the high R/FR light ratio environment, CmbHLH16 expression is elevated, the accumulation of CmbHLH16 leads to the direct competition with CmTPL for binding CmMYB4, therefore, destabilizing the CmMYB4–CmTPL repression complex and releasing the suppression of CmbHLH2, resulting in higher anthocyanin accumulation in the ray flower petals.