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. 2023 Nov 10;5(3):100744. doi: 10.1016/j.xplc.2023.100744

RHA2b-mediated MYB30 degradation facilitates MYB75-regulated, sucrose-induced anthocyanin biosynthesis in Arabidopsis seedlings

Huapeng Zhou 1,4,∗, Jiaxian He 2,4, Yiyi Zhang 1, Hongyun Zhao 3, Xia Sun 1, Xi Chen 1, Xinrui Liu 1, Yuan Zheng 3,∗∗∗, Honghui Lin 1,∗∗
PMCID: PMC10943538  PMID: 37946410

Abstract

Anthocyanins play diverse roles in plant physiology and stress adaptation. In Arabidopsis, the MYB–bHLH–WD40 (MBW) complex has a crucial role in the regulation of anthocyanin synthesis. Here, we report that the R2R3-MYB transcription factor MYB30 and the ubiquitin E3 ligase RHA2b participate in anthocyanin biosynthesis through regulation of the MBW complex. MYB30 was found to negatively regulate sucrose-induced anthocyanin biosynthesis in Arabidopsis seedlings. Expression of multiple genes involved in flavonoid or anthocyanin biosynthesis was affected in the myb30 mutant, and MYB30 directly repressed the expression of MYB75, which encodes a core component of the MBW complex, by binding to its promoter. Moreover, MYB30 physically interacted with MYB75 to inhibit its activity by repressing MBW complex assembly. In addition, sucrose treatment significantly promoted MYB30 degradation via the action of RHA2b. The ubiquitination and degradation of MYB30 were significantly attenuated in the rha2b mutant under high-sucrose treatment, and further analysis showed that MYB75 directly promoted RHA2b expression in response to high sucrose. Our work thus reveals an anthocyanin biosynthetic regulatory module, RHA2b–MYB30, that controls the function of the MBW complex via MYB75. The repression of MYB75 by MYB30 is released by MYB75-induced RHA2b expression, thus ensuring the self-activation of MYB75 when anthocyanin synthesis is needed.

Key words: anthocyanin biosynthesis, high sucrose, MBW complex, MYB30, MYB75, RHA2b

This study reports that the R2R3-MYB transcription factor MYB30 and the ubiquitin E3 ligase RHA2b participate in sucrose-induced anthocyanin biosynthesis via MYB75 in Arabidopsis seedlings. Repression of MYB75 by MYB30 is released by MYB75-induced RHA2b expression and subsequent MYB30 degradation mediated by RHA2b under high-sucrose conditions, thereby enhancing anthocyanin biosynthesis.

Introduction

Anthocyanins are widely distributed in plants, giving them a variety of colors (Loreti et al., 2008; Tanaka and Ohmiya, 2008; Das et al., 2012). Importantly, anthocyanins also function as defense molecules for plants under environmental stresses (Rolland et al., 2006; Solfanelli et al., 2006). In terms of evolution, accumulation of anthocyanins in flowers and fruits improves plant reproductive efficiency by attracting pollinators and seed-dispersing animals (Rabino and Mancinelli 1986; Sicilia et al., 2020). Of interest to humans is the finding that anthocyanins may promote health by acting as effective free radical scavengers or antioxidants (Winkel-Shirley, 2000; Zhang et al., 2014; Huang et al., 2019). Anthocyanin biosynthesis in plants is stimulated by multiple factors, including intracellular signals such as sucrose, phytohormones, and specific developmental stimuli, as well as exogenous environmental stressors such as water deficit, high light, and nutrient depletion (Shi and Xie, 2014; Li et al., 2016; Imtiaz et al., 2018; Sun et al., 2021). In challenging environments, anthocyanins can effectively neutralize free radicals via hydroxyl groups in their ring structures, making them helpful for plant survival (Solfanelli et al., 2006; Saigo et al., 2020).

Anthocyanin biosynthetic pathways in model plants have been well elucidated (Zhang et al., 2003; Ramsay and Glover, 2005; Gonzalez et al., 2008; Shi and Xie, 2011; Zhou et al., 2012; Liu et al., 2014). Phenylalanine and tyrosine are the precursors of anthocyanins; three sequential reaction steps are responsible for the transformation of phenylalanine or tyrosine into anthocyanins and are catalyzed by a variety of enzymes (Shi and Xie, 2014; Zhang et al., 2014). Anthocyanin biosynthesis–related enzymes are mainly encoded by a variety of structural genes, which can be divided into early biosynthetic genes (EBGs), including CHS, CHI, F3H, F3′H, and F3′5′H, and late biosynthetic genes (LBGs), including DFR, ANS, UF3GT, and other related genes (Shan et al., 2009; Shi and Xie, 2014; Gu et al., 2018; Sun et al., 2021). The spatial and temporal expression of EBGs and LBGs is determined by a combination of regulatory components and/or their interactions (Zhang et al., 2017; Sun et al., 2021). Previous studies have shown that expression of EBGs in Arabidopsis thaliana (Arabidopsis) is regulated by specific R2R3-MYB transcription factors (TFs) such as MYB11, MYB12, and MYB111 (Stracke et al., 2010). By contrast, expression of LBGs is regulated by a functional protein complex composed of R2R3-MYB TFs, MYC-like basic helix–loop–helix (bHLH) TFs, and WD40 repeat proteins (the MYB–bHLH–WD40 [MBW] complex) (Pattanaik et al., 2007; Shi and Xie, 2011; Zhou et al., 2012; Liu et al., 2014; Jain and Pandey, 2018; Sun et al., 2021). MYB75/PAP1, MYB90/PAP2, MYB113, and MYB114 have been characterized as MYB TFs in the Arabidopsis MBW complex (Yao et al., 2017; Zheng et al., 2020), and bHLH TFs for MBW assembly include TT8, GL3, EGL3, and MYC1 (Hichri et al., 2010; Wen et al., 2018). Only one WD40 protein, TTG1, is involved in the flavonoid metabolic pathway in Arabidopsis. Functionally, the transcriptional specificity of the MBW complex toward target genes is determined by the MYB and bHLH components rather than the WD40 protein (Jain and Pandey, 2018); nonetheless, the WD40 protein is indispensable for a functional MBW complex, although its expression shows no tissue specificity (An et al., 2012). Transcriptional and posttranslational modulations of the MBW complex have been implicated in fine-tuning plant anthocyanin biosynthesis in ever-changing environments (Shi and Xie, 2014; Li et al., 2016; Colanero et al., 2020; Saigo et al., 2020; Sun et al., 2021).

Sugars are involved in signaling networks related to anthocyanin biosynthesis (Teng et al., 2005; Solfanelli et al., 2006; Smeekens et al., 2010; Ruan, 2012; Sun et al., 2021). In Arabidopsis, sucrose is the most effective sugar inducer of anthocyanin biosynthesis (Solfanelli et al., 2006). When Arabidopsis seedlings are treated with sucrose, transcription of structural genes related to anthocyanin biosynthesis is induced at high levels, resulting in increased anthocyanin biosynthesis (Teng et al., 2005; Solfanelli et al., 2006; Smeekens et al., 2010). Sucrose-induced anthocyanin production may depend on sucrose transport mediated by sucrose transporter 1, but the exact mechanisms remain unclear (Sauer and Stolz, 1994; Sivitz et al., 2008). In Arabidopsis, sucrose-specific induction of anthocyanin biosynthesis has been reported to require MYB75/PAP1 (Teng et al., 2005). Sucrose can repress the gibberellin–mediated degradation of DELLA proteins, thereby activating MYB75/PAP1 expression and increasing anthocyanin biosynthesis (Li et al., 2014). By contrast, sucrose-mediated induction of the YDA–EIN3/EIL1 complex negatively regulates anthocyanin biosynthesis by directly repressing expression of TT8 (Meng et al., 2018). Therefore, the underlying mechanisms of sucrose-induced anthocyanin biosynthesis need to be fully clarified.

In this study, we report that the Arabidopsis R2R3-MYB TF MYB30 negatively regulates sucrose-induced anthocyanin biosynthesis in seedlings. MYB30 modulates gene expression throughout the Arabidopsis genome in response to sucrose treatment. It negatively regulates MYB75 transcription by directly binding to the MYB75 promoter. Further analysis showed that MYB30 interacts with MYB75 to repress assembly of the MBW complex that is critical for expression of anthocyanin LBGs. High sucrose levels reduce MYB30 stability via the RING-type ubiquitin E3 ligase RHA2b, and transcription of RHA2b is directly activated by MYB75 under high-sucrose conditions. Overall, our work demonstrates that the RHA2b–MYB30 module functions as a switch that regulates MYB75 function under high-sucrose conditions and that direct upregulation of RHA2b by MYB75 permits the rapid amplification of sucrose signals for anthocyanin biosynthesis in plants.

Results

MYB30 negatively regulates sucrose-induced anthocyanin accumulation

Previous studies have revealed that sucrose treatment induces high levels of anthocyanin biosynthesis in Arabidopsis (Teng et al., 2005; Solfanelli et al., 2006). To better understand the mechanisms underlying this process, we first identified an appropriate sucrose treatment concentration. Our phenotypic data showed that wild-type (WT, Col-0) seedlings grown in half-strength Murashige and Skoog medium (1/2 MS) with a high level of sucrose (4% or 6% sucrose) produced more anthocyanins than those grown in 1/2 MS with 2% sucrose (Control) (Supplemental Figures 1A and 1B). Because exogenously supplied sugars might cause osmotic stress in plants, we investigated whether 6% sucrose application resulted in severe osmotic stress by assessing the transcription of osmotic stress–responsive genes in Col-0 plants. We indeed observed induction of NCED3, RD29A, and RD26 in Col-0 plants in response to 6% sucrose treatment, but this induction was much less marked than that observed in plants treated with 0.3 M mannitol, which was used to cause typical osmotic stress (Supplemental Figure 2A). This suggested that 6% sucrose treatment did not lead to severe osmotic stress in plants. By contrast, our phenotypic data showed that 0.18 M mannitol, which results in comparable osmolarity in media such as 6% sucrose or even 0.3 M mannitol, indeed repressed plant growth, but neither 0.18 M nor 0.3 M induced the amount of anthocyanin accumulation induced by 6% sucrose (Supplemental Figure 2B). Together, these results suggest that the anthocyanin biosynthesis induced by 6% sucrose was attributable to sugar effects rather than osmotic stress.

Next, we screened mutants involved in this process and determined that the R2R3-MYB TF MYB30 plays an important role in sucrose-induced anthocyanin biosynthesis. The phenotypic data showed that myb30-2 seedlings accumulated more anthocyanins than Col-0 seedlings when both were grown in 1/2 MS containing 4% or 6% sucrose, and the 6% sucrose treatment led to a greater difference in anthocyanin accumulation between myb30-2 and Col-0 seedlings (Supplemental Figures 1A and 1B). To evaluate the role of MYB30 in high sucrose–induced anthocyanin accumulation in greater detail, we performed phenotypic assays using MYB30-related mutants and transgenic plants (Supplemental Figure 3A) (Zheng et al., 2012; Liao et al., 2017). The results showed that two independent lines of MYB30 loss-of-function mutants (myb30-1 and myb30-2) accumulated significantly higher levels of anthocyanins, whereas plants overexpressing MYB30 (MYB30-OE, lines #1 and #2) accumulated lower levels of anthocyanins than Col-0 under 6% sucrose conditions (Figures 1A and 1B). Under high-sucrose treatment, anthocyanin levels of two independent complementation lines (myb30-C, lines #1 and #2) were comparable to those of Col-0, implying that the anthocyanin-rich phenotype of the myb30 mutant is due to loss of MYB30 function (Figures 1A and 1B). We also examined the transcript levels of selected anthocyanin biosynthetic structural genes, including DFR, LDOX, and UF3GT, in these tested plants. The RT–qPCR data consistently showed that expression of the selected genes was much higher in myb30 mutants but lower in MYB30-overexpressing plants than in Col-0 plants under high-sucrose treatment (Figures 1C–1E). Recently, the sugar status of the cell was shown to be a decisive factor for activation of anthocyanin biosynthesis under high-light conditions (Zirngibl et al., 2023), indicating that MYB30 might also participate in the regulation of high light–induced anthocyanin biosynthesis. As predicted, we found that myb30 plants accumulated higher levels of anthocyanins than Col-0 plants when exposed to intense light (Supplemental Figures 4A and 4B). These results together demonstrated that MYB30 negatively regulates sucrose-induced anthocyanin accumulation in plants.

Figure 1.

Figure 1

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MYB30 negatively regulates sucrose-induced anthocyanin accumulation in plants.

(A) Phenotypic assays. Mature seeds of Col-0, myb30, myb30-C (#1 and #2), and MYB30-OE (#1 and #2) were germinated and grown on 1/2 MS containing 2% sucrose (Control) or 1/2 MS supplemented with 6% sucrose. Photographs were taken and anthocyanin accumulation was measured after one week of growth. Scale bar, 2 mm.

(B) Anthocyanin content in extracts of seedlings in (A). Approximately 30 seedlings of each sample were pooled for subsequent measurement. Three independent experiments were performed, and similar results were obtained. Error bars represent ± SD (n = 3). Different lowercase letters indicate significant differences based on ANOVA (P < 0.05). FW, fresh weight.

(C–E) RT–qPCR analysis of the expression of selected anthocyanin biosynthetic genes using the seedlings in (A). Expression of DFR (C), LDOX (D), and UF3GT (E) in 7-day-old seedlings, left untreated or treated with 6% sucrose, was measured by RT–qPCR. ACTIN2 was used as the internal control. Error bars represent ± SD (n = 3). Different lowercase letters indicate significant differences based on ANOVA (P < 0.05).

MYB30 regulates multiple genes during the sucrose response

For genome-wide analysis of the regulatory role of MYB30 in sucrose-induced anthocyanin biosynthesis, we performed RNA sequencing to obtain transcriptomic profiles of Col-0 and myb30-2 grown in 1/2 MS containing 2% sucrose (control) or 6% sucrose (Suc). In total, 989 differentially expressed genes (DEGs) were identified in Col-0 Suc compared with Col-0, whereas 3258 DEGs were identified in myb30-2 Suc compared with myb30-2 (Figure 2A). In addition, we identified 857 DEGs in myb30-2 compared with Col-0 under control conditions and 2007 DEGs in Col-0 Suc compared with myb30-2 Suc (Figure 2A). Importantly, 280 DEGs that were identified in both the Col-0 Suc/Col-0 and myb30-2/Col-0 comparisons showed the same pattern of change (Figure 2A). Biological processes involved in anthocyanin and flavonoid biosynthesis were consistently and substantially overrepresented in these 280 genes (Figure 2B). As shown in the heatmap, selected genes involved in anthocyanin biosynthesis (MYB75/PAP1, DFR, MYB90/PAP2, and UF3GT) and flavonoid biosynthesis (MYB111, UGT78D1, and UGT75C1) were induced at high levels in myb30-2 plants but not in Col-0 plants under high-sucrose conditions (Figure 2C). Interestingly, several genes related to jasmonate (JA) signaling (AOC1, TAT3, JOX1, and MYC2) (Li et al., 2020) also showed changes in expression due to loss of MYB30 function (Figure 2C). Subsequent RT–qPCR assays confirmed that the expression of MYB75, PAP2, GSTF12, 5MAT, MYB111, and UGT78D1 was upregulated to a greater extent in myb30-2 than in Col-0 (Figure 2D). Taken together, the results of our transcriptomic investigation confirmed a possible regulatory role of MYB30 in sucrose-induced anthocyanin biosynthesis.

Figure 2.

Figure 2

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MYB30 fine-tunes sucrose-induced anthocyanin biosynthesis by regulating a battery of genes.

(A) Venn diagram of DEGs identified in comparisons of Col-0 Suc vs. Col-0, myb30-2 Suc vs. myb30-2, myb30-2 vs. Col-0, and Col-0 Suc vs. myb30-2 Suc. Suc, 6% sucrose.

(B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the DEGs identified in (A).

(C) Expression heatmap of genes related to anthocyanin and flavonoid biosynthesis based on RNA sequencing data. The blue-to-red scale represents the log10 (fold change) values of fragments per kilobase of transcript per million mapped reads.

(D) RT–qPCR assays for the expression of representative genes related to anthocyanin biosynthesis in 7-day-old Col-0 and myb30-2 plants treated with 1/2 MS containing 2% sucrose (Control) or 6% sucrose (Sucrose) for 3 h. ACTIN2 was used as the internal control. Data were normalized by the gene expression level in Col-0 under control conditions. Error bars represent ± SD (n = 3). Different lowercase letters indicate significant differences based on ANOVA (P < 0.05).

(E) Transactivation assay. The MYB75pro::LUC construct was transiently expressed with the 35Spro::Flag-MYB30 or 35Spro::Flag plasmid in myb30-2 protoplasts, and the luciferase activity was determined. Error bars represent ± SD (n = 3). Statistical significance was determined by Student’s t-test. ∗P < 0.05.

(F) ChIP–qPCR analysis. Ten-day-old seedlings of myb30-2 and MYB30pro::MYB30-Flag/myb30-2 were used for chromatin isolation. MYB30 protein was purified with anti-Flag agarose, and immunoprecipitated DNA and input DNA were analyzed by RT–qPCR to determine MYB75 enrichment. Error bars represent ± SD (n = 3). Different lowercase letters indicate significant differences based on ANOVA (P < 0.05).

(G) EMSA for the determination of MYB30 interaction with the MYB75 promoter. Recombinant GST-MYB30N and double-stranded oligonucleotide probes containing MBS sequences from the MYB75 promoter were used for probe binding reactions. Experimental details are provided in the methods.

MYB75 is a direct target of MYB30

Consistent with the findings of a previous study in which MYB75 expression was induced by sucrose to promote anthocyanin biosynthesis (Teng et al., 2005), our data showed that sucrose treatment enhanced MYB75 transcription in both Col-0 and myb30-2 plants (Figures 2C and 2D). Transcript levels of MYB75 were much higher in myb30-2 than in Col-0 under sucrose treatment, implying that MYB30 is a negative regulator of MYB75 transcription (Figures 2C and 2D). To evaluate whether MYB75 is a direct target of MYB30, we performed transactivation assays in Arabidopsis leaf protoplasts. The promoter of MYB75 was fused to the luciferase (LUC) reporter gene, and the resulting MYB75pro::LUC construct (Figure 2E, top) was transiently expressed with the 35Spro::MYB30-Flag or 35Spro::Flag plasmid in myb30-2 leaf protoplasts. Relative luciferase activity measurements demonstrated that MYB30 significantly repressed MYB75pro::LUC expression (Figure 2E, bottom). Sequence predictions revealed the presence of two MYB30-binding motifs (MBSs) (Li et al., 2009; Liao et al., 2017; Yan et al., 2020) in the MYB75 promoter (MBS1, −1608[AACAAAC]−1602; MBS2, −270[GTTTGTT]−264) (Figure 2F, top, and Supplemental Figure 5A). Subsequent electrophoretic mobility shift assays (EMSAs) using the N-terminal DNA-binding domain of MYB30 (MYB30N) (Liao et al., 2017) (Supplemental Figure 5C) confirmed that the recombinant GST-MYB30N protein bound to both MBS1 and MBS2 in the MYB75 promoter region (Figure 2G). Next, we performed a chromatin immunoprecipitation (ChIP) assay to detect the DNA-binding activity of MYB30 at specific regions of the MYB75 promoter in planta using 35Spro::MYB30-Flag/myb30-2 transgenic plants (Zheng et al., 2012). The MYB30 protein and DNA fragments were cross-linked, and the MYB30-Flag proteins were then immunoprecipitated with anti-Flag antibody–conjugated agarose. The ChIP–qPCR results revealed that MYB30 enriched the F1 and F2 fragments of the MYB75 promoter containing the MBS motif but not the NB fragment with no MBS motif (Figure 2F, bottom). These results together demonstrated that MYB75 is a direct target of MYB30 and that MYB30 represses MYB75 transcription under high-sucrose conditions.

MYB30 interacts with MYB75

The ternary MBW complex plays an essential part in anthocyanin biosynthesis by increasing the transcription of anthocyanin biosynthesis genes (Zhu et al., 2009; Shi and Xie, 2014). We therefore asked whether there was a functional link between MYB30 and the MBW complex. First, we performed yeast two-hybrid (Y2H) assays for possible interactions between MYB30 and components of the MBW complex. The results showed that MYB30 interacted specifically with MYB75 but not with TT8 or EGL3 (Figure 3A and Supplemental Figure 6A). We next performed a co-immunoprecipitation (Co-IP) assay to study the interaction between MYB30 and MYB75 in planta. Flag-HA-MYB30 and MYB75-GFP or GFP alone were transiently expressed in Arabidopsis leaf protoplasts, and Flag-HA-MYB30 proteins were then immunoprecipitated using anti-Flag antibody–conjugated agarose. MYB30 and its interacting protein(s) were detected by immunoblot analysis using anti-Flag and anti-GFP antibodies, respectively. The data showed that MYB75-GFP, but not GFP alone, was coimmunoprecipitated by MYB30, implying that MYB30 interacts with MYB75 in planta (Figure 3B). To more accurately determine the region of MYB30 that interacts with MYB75, we divided MYB30 into two sections (Supplemental Figure 5C). The data showed that the N terminus of MYB30 (MYB30N, containing the MYB-DNA binding domain) interacted with MYB75 in bimolecular fluorescence complementation (BiFC) and pull-down assays (Figures 3C and 3D and Supplemental Figure 6B). By contrast, no physical interaction between MYB30 and MYB90/PAP2 was observed in our Co-IP assays (Supplemental Figure 6C). Taken together, these results confirmed that MYB30 interacts with MYB75.

Figure 3.

Figure 3

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MYB30 represses formation of the MBW protein complex through direct interaction with MYB75.

(A) Y2H assay for the interaction between MYB30 and MYB75. Indicated plasmid combinations were transformed into yeast strain AH109, and the yeast transformants were plated on minimal SC-Trp/Leu medium (DDO) or SC-Trp/Leu/His/Ade medium (QDO) for further growth at 28°C. The interaction between MYB75 and TT8 served as the positive control.

(B) Co-IP analysis of the interaction between MYB30 and MYB75. Flag-HA-MYB30 and MYB75-GFP or GFP alone were co-expressed in Arabidopsis leaf protoplasts. Flag-HA-MYB30 proteins were precipitated with anti-Flag antibody–conjugated agarose, and the products were detected with anti-GFP and anti-Flag antibodies. IP, immunoprecipitation.

(C) BiFC assay in tobacco leaves for the interaction between MYB30 and MYB75. MYB30 truncations (MYB30N and MYB30C) and MYB75 were fused with the N and C termini of yellow fluorescent protein (YFP), respectively. The resulting constructs were transiently expressed in tobacco leaves for 3 days. The YFP signal was visualized by confocal microscopy. The interaction between MYB75 and TT8 served as the positive control. Scale bar, 100 μm.

(D)In vitro pull-down assay to examine the MYB30 and MYB75 interaction. MBP-tagged MYB75 was incubated with GST-tagged MYB30N or GST alone. MBP-MYB75 was isolated using amylose resin, and MBP-MYB75 and its interacting proteins were detected by immunoblotting with anti-MBP and anti-GST antibodies, respectively.

(E) Transactivation assay. The DFRpro::LUC construct (upper panel) was transiently expressed with MYB75, TT8, or both in Arabidopsis leaf protoplasts with or without MYB30, and the luciferase activity was determined. Error bars represent ± SD (n = 3). Different lowercase letters indicate significant differences based on ANOVA (P < 0.05).

(F) BiFC assays of the inhibitory effect of MYB30 on the interaction between MYB75 and TT8 or EGL3. MYB75 and TT8 or EGL3 were fused with the YFP C terminus and YFP N terminus, respectively, and transiently expressed in tobacco leaves with or without Flag-HA-tagged MYB30 for 3 days. The 35Spro::Flag-HA vector was used as the negative control. Scale bar, 100 μm.

(G) Quantification of YFP fluorescence intensity in (F). Error bars represent ± SD (n = 3). Different lowercase letters indicate significant differences based on ANOVA (P < 0.05).

(H) Quantification of the transient expression level of proteins for BiFC as well as MYB30 in (F) using RT–qPCR analysis. Error bars represent ± SD (n = 3). Different lowercase letters indicate significant differences based on ANOVA (P < 0.05). ns, not significant.

(I and J)in vitro pull-down assays of the inhibitory effect of MYB30 on the interaction between MYB75 and TT8 or EGL3. Recombinant His-MYB75 and TT8-MBP or EGL3-MBP were subjected to pull-down assays with GST or GST-tagged MYB30N. Immunoblotting with anti-MBP, anti-GST, and anti-His antibodies was used to detect TT8/EGL3-MBP, GST-MYB30N, and His-MYB75, respectively.

MYB30 represses formation of the MBW complex

The physical interaction between MYB30 and MYB75 prompted us to examine their functional connection in greater detail. First, we investigated the possible effect of MYB30 on MYB75 transcriptional activity. The promoter sequence of DFR, a target of MYB75 in anthocyanin biosynthesis, was cloned and inserted into the pGreenII-0800-LUC vector for a transactivation assay (Figure 3E, top). The results showed that MYB75 effectively enhanced LUC expression driven by the DFR promoter (DFRpro), whereas MYB30 repressed MYB75-induced DFRpro::LUC expression (Figure 3E, bottom), consistent with the negative role of MYB30 in MYB75 expression (Figures 2C and 2D). When MYB75 was expressed together with TT8, another MBW complex component, we observed much higher induction of DFRpro::LUC expression than that induced by MYB75 alone; however, relative luciferase activity was significantly attenuated when MYB30 was co-expressed with MYB75 and TT8 in the transactivation assay (Figure 3E).

MYB75 contributes to formation of the MBW protein complex with other components, such as TT8 and EGL3, for the activation of LBGs involved in anthocyanin biosynthesis (Li et al., 2014; Shi and Xie, 2014). Because MYB30 attenuates the activation of DFRpro::LUC by MYB75 (alone or together with TT8) and MYB30 does not interact with TT8 (Figure 3E and Supplemental Figure 6A), we speculated that MYB30 might compete with TT8 for binding to MYB75, thus inhibiting MBW complex formation. To test this hypothesis, we performed BiFC assays by transiently expressing MYB75-YFPC and TT8-YFPN or EGL3-YFPN in tobacco leaves with or without Flag-HA-MYB30. Microscopy observations showed that co-expression of Flag-HA-MYB30 with MYB75-YFPC/TT8-YFPN or MYB75-YFPC/EGL3-YFPN produced a much weaker YFP fluorescence signal in infiltrated tobacco leaves than did the MYB75-YFPC/TT8-YFPN or MYB75-YFPC/EGL3-YFPN combinations, suggesting that MYB30 might play a negative role in MBW complex formation (Figures 3F and 3G). It should be noted that variations in the fluorescence signal were not caused by differential expression of proteins for BiFC (Figure 3H). We also performed in vitro competitive pull-down assays to further test this hypothesis. Consistent with previous findings, our data confirmed that MYB75 interacted with TT8 and EGL3 in pull-down assays, and the interaction between His-MYB75 and TT8-MBP or EGL3-MBP was gradually attenuated by increasing amounts of GST-MYB30N protein (Figures 3I and 3J). These results demonstrated that MYB30 is a negative regulator of MBW complex assembly.

MYB30 represses sucrose-induced anthocyanin accumulation through MYB75

To examine the genetic relationship between MYB30 and MYB75 in regulation of sucrose-induced anthocyanin biosynthesis, we performed phenotypic assays using Col-0, myb30-2, myb75-c (Li et al., 2016), and myb30-2 myb75-c plants (Supplemental Figure 3B). Consistent with the positive role of MYB75 in anthocyanin biosynthesis (Li et al., 2016; Zheng et al., 2020), myb75-c plants with MYB75 defects accumulated fewer anthocyanins than Col-0 plants under high-sucrose treatment (Figures 4A and 4B). Moreover, the higher levels of anthocyanins in myb30-2 under high-sucrose conditions were nearly abolished by the additional myb75-c mutation, and anthocyanin accumulation in myb30-2 myb75-c seedlings was comparable to that in myb75-c plants (Figures 4A and 4B). We also assessed the transcription of anthocyanin biosynthetic genes in these tested plants and found that their sucrose-induced expression in the myb30-2 mutant was significantly reduced by the MYB75 mutation in myb30-2 myb75-c plants (Figures 4C–4E). Moreover, myb75-c and myb30-2 myb75-c plants exhibited similar trends in the transcript levels of anthocyanin biosynthetic genes in response to high sucrose (Figures 4C–4E). Collectively, these results demonstrated that MYB30 negatively regulates sucrose-induced anthocyanin accumulation through MYB75.

Figure 4.

Figure 4

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MYB30 represses sucrose-induced anthocyanin accumulation through MYB75.

(A) Phenotypic assays. Mature seeds of Col-0, myb30-2, myb75-c, and myb30-2 myb75-c were germinated and grown on 1/2 MS containing 2% sucrose (Control) or 1/2 MS supplemented with 6% sucrose. Photographs were taken and anthocyanin accumulation was measured after 1 week of growth. Scale bar, 2 mm.

(B) Anthocyanin content in extracts of seedlings in (A). Approximately 30 seedlings of each sample were pooled for subsequent measurement. Three independent experiments were performed, and similar results were obtained. Error bars represent ± SD (n = 3). Different lowercase letters indicate significant differences based on ANOVA (P < 0.05).

(C–E) RT–qPCR analysis of the expression of selected anthocyanin biosynthetic genes in the seedlings in (A). Expression of DFR (C), LDOX (D), and UF3GT (E) in 7-day-old seedlings, left untreated or treated with 6% sucrose, was measured by RT–qPCR. ACTIN2 was used as the internal control. Error bars represent ± SD (n = 3). Different lowercase letters indicate significant differences based on ANOVA (P < 0.05).

RHA2b mediates the degradation of MYB30 under high-sucrose conditions

We next investigated the possible regulation of MYB30 under high-sucrose conditions. High-sucrose treatment had no obvious effect on the transcription of MYB30 (Supplemental Figure 3C), but MYB30 protein content gradually decreased with long-term high-sucrose treatment (24 h and 36 h), indicating that high sucrose may induce degradation of MYB30 (Figure 5A). When the protein synthesis inhibitor cycloheximide (CHX) was applied, clear sucrose-induced MYB30 degradation was observed, even at 6 h post treatment, whereas additional supplementation with MG132, an inhibitor of the 26S proteasome, largely attenuated the MYB30 degradation induced by high sucrose (Figure 5B). By contrast, our immunoblotting data showed that MYB30 remained stable under control conditions (Supplemental Figures 7A and 7B). Together, these results suggested that MYB30 undergoes proteasomal degradation in response to high-sucrose treatment.

Figure 5.

Figure 5

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RHA2b mediates sucrose-induced MYB30 degradation.

(A) Determination of MYB30 abundance under high-sucrose conditions. Ten-day-old seedlings of 35Spro::Myc-MYB30/Col-0 and 35Spro::Myc-MYB30/rha2b-1 were treated with 6% sucrose for the indicated times. The content of MYB30 protein was detected with anti-Myc antibody. Actin was used as the loading control.

(B) Immunoblotting assays for MYB30 abundance. Ten-day-old 35Spro::Flag-MYB30/Col-0 seedlings were left untreated or treated with 6% sucrose for the indicated times, together with 300 μM CHX, with or without 50 μM MG132. Immunoblotting with anti-Flag antibody was performed to determine MYB30 abundance. Actin was used as the loading control.

(C) RT–qPCR assays for the expression of RHA2b in 7-day-old Col-0, myb75-c, and myb30-2 seedlings treated with 1/2 MS containing 2% sucrose (Control) or 6% sucrose (Sucrose) for 3 h. Error bars represent ± SD (n = 3). Different lowercase letters indicate significant differences based on ANOVA (P < 0.05).

(D) Ubiquitination status of MYB30. Ten-day-old seedlings of 35Spro::Myc-MYB30/Col-0 and 35Spro::Myc-MYB30/rha2b-1 were left untreated or treated with 6% sucrose for 6 h. Then, Myc-MYB30 proteins were immunoprecipitated with anti-Myc antibody–conjugated agarose. Immunoblotting assays using anti-Myc and anti-UBQ antibodies were performed to detect MYB30 ubiquitination. IP, immunoprecipitation.

(E) Split-Luc analysis. MYB30 was fused with the Luc N terminus, and RHA2b was fused with the Luc C terminus. The fusion proteins were transiently expressed in tobacco leaves. The infiltrated leaves were left untreated or treated with 6% sucrose for 30 min. Error bars represent ± SD (n = 3). Different lowercase letters indicate significant differences based on ANOVA (P < 0.05).

(F) Transactivation assay. The RHA2bpro::LUC construct was transiently expressed with the 35Spro::Flag-MYB75 or 35Spro::Flag plasmid in rha2b-1 protoplasts, and the luciferase activity was determined. Error bars represent ± SD (n = 3). Statistical significance was determined by Student’s t-test. ∗P < 0.05. ns, not significant.

(G) ChIP–qPCR analysis. Ten-day-old seedlings of Col-0 and 35Spro::Flag-MYB75/Col-0 were used for chromatin isolation. MYB75 protein was purified with anti-Flag agarose, and immunoprecipitated DNA and input DNA were analyzed by RT–qPCR to determine RHA2b enrichment. Error bars represent ± SD (n = 3). Different lowercase letters indicate significant differences based on ANOVA (P < 0.05).

(H) EMSA for the association of MYB75 with the RHA2b promoter. Recombinant His-MYB75 and double-stranded oligonucleotide probes containing MYB-binding sequences (MYB1–MYB3) from the RHA2b promoter were used for probe binding reactions. Experimental details are provided in the methods.

The RING-type ubiquitin E3 ligase RHA2b has been shown to regulate MYB30 stability during seed germination (Zheng et al., 2018). Importantly, its expression was found to be induced by 6% sucrose treatment. RT–qPCR results revealed that transcript levels of RHA2b in Col-0 were ∼1.7-fold higher under high-sucrose treatment compared with normal growth conditions (Figure 5C). Consistent with this finding, when RHA2bpro::Flag-RHA2b and 35Spro::GFP were transiently expressed in tobacco leaves subjected to control (2% sucrose) or 6% sucrose treatment, we observed clear accumulation of Flag-RHA2b protein, but not GFP, under high-sucrose conditions (Supplemental Figure 7C). We investigated whether RHA2b was involved in regulating MYB30 stability under high-sucrose conditions using 35Spro::Myc-MYB30/Col-0 and 35Spro::Myc-MYB30/rha2b-1 plants (Zheng et al., 2018). Immunoblotting results revealed that MYB30 accumulated to a higher level in rha2b-1 mutant plants than in Col-0 plants under 6% sucrose treatment, demonstrating that RHA2b participates in sucrose-induced MYB30 degradation (Figure 5A). We next asked whether RHA2b mediated MYB30 ubiquitination for further degradation under high-sucrose conditions. Ten-day-old 35Spro::Myc-MYB30/Col-0 and 35Spro::Myc-MYB30/rha2b-1 plants were left untreated or treated with 6% sucrose for 6 h. Myc-MYB30 proteins were immunoprecipitated with anti-Myc antibody–conjugated agarose, and the ubiquitinated form of MYB30 was detected using an anti-ubiquitin antibody. Immunoblotting assays clearly revealed that MYB30 underwent ubiquitination in response to high-sucrose exposure when RHA2b was functional (Figure 5D). By contrast, presence of defective RHA2b significantly attenuated the level of MYB30 ubiquitination (Figure 5D). A split-luciferase complementation (split-Luc) analysis consistently demonstrated that high sucrose enhanced the interaction between MYB30 and RHA2b (Figure 5E), indicating that sucrose-induced MYB30 ubiquitination and degradation are mediated by RHA2b. Taken together, these results revealed that RHA2b is a functional mediator of sucrose-induced MYB30 degradation.

Further analysis showed that induction of RHA2b expression by high-sucrose treatment was nearly abolished in the myb75-c mutant (Figure 5C), suggesting that MYB75 might be involved in the transcriptional regulation of RHA2b. We next determined whether MYB75 directly regulated the expression of RHA2b by transiently expressing the RHA2bpro::LUC construct (Figure 5F, top) with the 35Spro::Flag-MYB75 or 35Spro::Flag plasmid in rha2b-1 leaf protoplasts for a transactivation assay. The results showed that the presence of MYB75 dramatically enhanced LUC activity (Figure 5F, bottom). A sequence analysis revealed the presence of three MYB-binding sites (MYBs; MYB1, −1108[CAGTTG]−1103; MYB2, −1871[AACAAAC]−1865; MYB3, −1914[CAGTTG]−1909) in the RHA2b promoter (Figure 5G, top, and Supplemental Figure 5B). We therefore performed a ChIP assay to determine whether MYB75 directly bound to the promoter of RHA2b. ChIP–qPCR results revealed that the F3 and F5 fragments containing MYB motifs were significantly enriched by MYB75 (Figure 5G, bottom). EMSA was also performed to determine whether MYB75 bound to MYBs in the RHA2b promoter, and the results showed that recombinant His-MYB75 specifically bound to MYB1–MYB3 in the RHA2b promoter region (Figure 5H). Together, these results suggested that the MBW complex directly upregulates RHA2b expression via the action of MYB75 under high-sucrose treatment.

The RHA2b–MYB30 module fine-tunes sucrose-induced anthocyanin biosynthesis

Finally, we investigated the regulatory role of RHA2b in sucrose-induced anthocyanin biosynthesis and the genetic relationship between MYB30 and RHA2b using Col-0, myb30-2, rha2b-1, myb30-2 rha2b-1, and 35Spro::MYB30-Flag/rha2b-1 plants (Zheng et al., 2018). The phenotypic data showed that rha2b-1 plants accumulated lower levels of anthocyanins than Col-0 plants under high-sucrose conditions (Figures 6A and 6B and Supplemental Figures 1A and 1B), consistent with the higher abundance of MYB30 in rha2b-1 plants exposed to high sucrose (Figure 5A). Importantly, the myb30-2 rha2b-1 double mutant and the myb30-2 single mutant accumulated comparable levels of anthocyanins, implying that RHA2b regulates anthocyanin biosynthesis via the action of MYB30 (Figures 6A and 6B). We also examined the transcripts levels of anthocyanin-specific biosynthetic genes in these tested plants. Expression of DFR and UF3GT was much lower in rha2b-1 than in Col-0, whereas myb30-2 and myb30-2 rha2b-1 exhibited comparable transcript levels of these genes, which were much higher than those in Col-0 (Figures 6C–6E). Importantly, we found that expression of MYB75 was much lower in rha2b-1 plants but much higher in myb30-2 and myb30-2 rha2b-1 plants than in Col-0 plants (Figure 6F), supporting the conclusion that the RHA2b–MYB30 module regulates MYB75 at the transcriptional level. Taken together, these results demonstrated that RHA2b participates in sucrose-induced anthocyanin accumulation through the action of MYB30.

Figure 6.

Figure 6

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MYB30 functions downstream of RHA2b in sucrose-induced anthocyanin biosynthesis.

(A) Phenotypic assays. Mature seeds of Col-0, myb30-2, rha2b-1, myb30-2 rha2b-1, and MYB30-Flag/rha2b-1 were germinated and grown on 1/2 MS containing 2% sucrose (Control) or 1/2 MS supplemented with 6% sucrose. Photographs were taken and anthocyanin accumulation was measured after 1 week of growth. Scale bar, 2 mm.

(B) Anthocyanin content in extracts from seedlings in (A). Approximately 30 seedlings of each sample were pooled for subsequent measurement. Three independent experiments were performed, and similar results were obtained. Error bars represent ± SD (n = 3). Different lowercase letters indicate significant differences based on ANOVA (P < 0.05).

(C–F) RT–qPCR analysis of the expression of selected anthocyanin biosynthetic genes and MYB75 in the seedlings in (A). Expression of DFR (C), LDOX (D), UF3GT (E), and MYB75 (F) in 7-day-old seedlings, left untreated or treated with 6% sucrose, were measured by RT–qPCR. ACTIN2 was used as the internal control. Error bars represent ± SD (n = 3). Different lowercase letters indicate significant differences based on ANOVA (P < 0.05).

Discussion

Because anthocyanins are beneficial for both plants and humans (Shi and Xie, 2014; Zhang et al., 2014; Sun et al., 2021), multiple studies have investigated the network of anthocyanin biosynthesis, with the aim of developing anthocyanin-rich crops through genetic modification (GM) approaches (Zhang et al., 2014; Sun et al., 2021). Transcriptional regulation of the MBW complex is essential for fine-tuning of anthocyanin biosynthesis in ever-changing environments, and several transcriptional regulators of the MBW complex have been characterized (Sun et al., 2021). For instance, GLABRA2 (GL2) represses expression of MYB75, MYB90, and TT8; therefore, transgenic Arabidopsis plants overexpressing GL2 accumulate fewer anthocyanins, whereas the GL2 loss-of-function mutant gl2 accumulates more anthocyanins (Chen and Wang, 2019). Likewise, the three lateral organ boundary domain proteins LBD37, LBD38, and LBD39 also inhibit transcription of MYB75 and MYB90 in Arabidopsis, reducing the synthesis of anthocyanins when they are overexpressed (Petridis et al., 2016; Sun et al., 2021). In this study, we found that MYB30 negatively regulates sucrose-induced anthocyanin biosynthesis by directly repressing MYB75 transcription. MYB30 can specifically bind MBSs in the MYB75 promoter region, inhibiting MYB75 transcription and thus leading to reduced expression of anthocyanin biosynthetic genes.

Previous studies have shown that MYB30 can serve as a repressor through interactions with other TFs such as ABI5, a key activator of abscisic acid (ABA)-responsive genes (Nie et al., 2022). In this study, we found that MYB30 can form a complex with MYB75, but it does not physically interact with other MBW components such as TT8 and EGL3, suggesting that the function of MYB30 in sucrose-induced anthocyanin biosynthesis relies on MYB75. We found that MYB30 competes with TT8 and EGL3 to interact with MYB75, therefore inhibiting the assembly of the MBW complex and repressing the expression of LBGs under high-sucrose conditions. Many regulators, including the R3-MYB protein MYBL2, the miR156-targeted squamosa promotor binding protein-like 9 (SPL9), and the conserved regulator of Pi response SPX4, have been shown to regulate anthocyanin biosynthesis by affecting formation of the MBW complex (Dubos et al., 2008; Matsui et al., 2008; Gou et al., 2011; He et al., 2021). For instance, SPL9 interacts with MYB75 to destabilize formation of the MBW complex, and it negatively regulates anthocyanin accumulation by directly repressing the expression of DRF. Our results demonstrated that MYB30 transcriptionally regulates MYB75 and inhibits assembly of the MBW complex through physical interaction with MYB75, suggesting another essential role for MYB30 in MYB75-mediated anthocyanin biosynthesis.

Compared with other MYB TFs, MYB75 plays a predominant role in the regulation of anthocyanin biosynthesis in seedlings, but there may still be functional redundancy among MYB proteins. Our data revealed a difference in MYB90 expression between Col-0 and the myb30 mutant, implying that MYB30 might also participate in anthocyanin synthesis by regulating MYB90. Sequence analysis showed that two MBSs (−1032[TTTGGTT]−1038 and −1060[TTTGGTT]−1066) were present upstream of the start codon in the MYB90 promoter (Supplemental Figure 8A) (Liao et al., 2017; Yan et al., 2020), suggesting the possible direct transcriptional regulation of MYB90 by MYB30. At the protein level, however, we found no evidence of physical interaction between MYB30 and MYB90. Analysis of the MYB75 and MYB90 protein sequences showed that their N-terminal MYB-DNA binding domains share ∼94% (106/113) similarity, whereas their C-terminal regions share only ∼66% (90/136) similarity (Supplemental Figure 8B). We speculate that differences in the C terminus, which is the regulatory region of the MYB protein, may lead to differences in the binding proteins of MYB90 and MYB75.

Our results showed that MYB75 binds directly to the promoter of the ubiquitin E3 ligase gene RHA2b to activate its expression. Accumulation of RHA2b is responsible for the induction of MYB30 degradation under high-sucrose conditions. In Col-0 plants, MYB30 ubiquitination is induced at high levels by high sucrose, leading to MYB30 degradation, whereas sucrose-induced MYB30 ubiquitination and degradation are attenuated in the rha2b-1 mutant. Consistent with these results, anthocyanin accumulation was lower in rha2-1 plants than in Col-0 plants under high-sucrose conditions. Furthermore, expression of MYB75, DFR, and UF3GT, which is repressed by MYB30, was also reduced in the rha2-1 mutant. Our results reveal a role for MYB75 in regulating anthocyanin biosynthesis via self-activation under high-sucrose conditions.

Unexpectedly, there was no significant difference in LDOX expression between the rha2-1 mutant and Col-0 (Figure 7D). Our immunoblotting data clearly showed that MYB30 is still degraded in the rha2b-1 mutant background (Figure 6), suggesting that additional E3 ligases may be involved in the sugar-dependent regulation of MYB30 abundance. It has been reported that the stability of MYB30 is also controlled by the RING-type ubiquitin E3 ligase MIEL1 during the hypersensitive response and the ABA-mediated inhibition of seed germination (Marino et al., 2013; Nie et al., 2022). Recently, another RING-type ubiquitin E3 ligase, COP1, has been reported to integrate light signaling and post-submergence stress through the ubiquitination and degradation of MYB30 (Xie et al., 2023). COP1/SPA has been reported to control anthocyanin accumulation by regulating the stability of MYB75 and MYB90 (Maier et al., 2013). Further investigations are needed to determine whether additional E3 ligases, such as MIEL1 and COP1, participate in sucrose-induced anthocyanin accumulation by mediating the ubiquitination and degradation of MYB30.

Figure 7.

Figure 7

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A working model for the RHA2b–MYB30 module in sucrose-induced anthocyanin biosynthesis of Arabidopsis seedlings.

Under normal conditions, MYB30 represses the function of MYB75 by binding to the MYB75 promoter and interacting with MYB75 to inhibit formation of the MBW complex, thus restricting the expression of genes for anthocyanin biosynthesis. Under high-sucrose conditions, MYB75 transcriptionally activates RHA2b, which promotes degradation of MYB30 and thus releases MYB75 to enable precise regulation of anthocyanin synthesis.

Our transcriptomic analysis revealed that expression of multiple genes involved in anthocyanin biosynthesis was altered in the myb30-2 mutant, including flavonoid biosynthetic genes such as UGT78D1 and UGT75C1, consistent with the finding that MYB TFs have critical roles in flavonoid biosynthesis. We also found that several non-MBW complex targets were regulated by MYB30 under high-sucrose conditions, some of which were found to participate in JA signaling (Figure 2C). JA is a critical hormone that induces anthocyanin biosynthesis (Shan et al., 2009; Qi et al., 2011). Recent studies have shown that accumulation of anthocyanins under moderate salt stress is partially dependent on JA signaling, which induces degradation of the JA repressor JAZ proteins (Li et al., 2020). In addition, the EAR domain-containing protein ECAP not only interacts with MYB75 to repress its activity but also promotes the interaction of JAZ6/8 and TPR2, thus repressing MBW complex formation to suppress JA-mediated anthocyanin accumulation (Li et al., 2022). The fact that MYB30 promotes expression of the JA catabolic gene JOX1 but represses the JA biosynthesis gene AOC1 (Figure 2C) suggests that MYB30 might negatively regulate JA signaling in plants. COI1-mediated JA signaling has previously been shown to stimulate anthocyanin biosynthesis in the presence of high sucrose (Loreti et al., 2008). The same phenomenon could occur in the myb30 mutant, which accumulates anthocyanins when grown under high-sucrose conditions. MYB30 connects light and JA signaling pathways via MYC2 stabilization and activation (Xie et al., 2023). Given the close association of MYB30 and JA signaling and the important role of JA signaling in anthocyanin accumulation, we cannot exclude the possibility that high sucrose stimulates anthocyanin biosynthesis through MYB30-mediated JA signaling.

In addition, MYB30 may act as a conditional regulator of plant anthocyanin biosynthesis. The MYB30 loss-of-function mutant fails to accumulate high levels of anthocyanins under normal growth conditions because stimuli for anthocyanin biosynthesis, such as high sucrose, are absent, although higher AOC1 expression (and possibly high JA levels) is observed in myb30. MYB30 and other negative regulators may transcriptionally repress MYB75 function under low-sucrose conditions to ensure low anthocyanin accumulation. In the presence of high sucrose, RHA2b-mediated MYB30 degradation releases MYB75, leading to upregulation of anthocyanin biosynthetic genes and accumulation of anthocyanins. More data are needed to confirm these processes in the future.

In summary, our work reveals a signaling network involved in the regulation of sucrose-induced anthocyanin biosynthesis in Arabidopsis seedlings. Under normal conditions, MYB30 directly represses MYB75 transcription and inhibits formation of the MBW complex via direct interaction with MYB75, thus repressing the expression of anthocyanin biosynthetic genes. Under high-sucrose conditions, MYB75 transcriptionally activates RHA2b expression, which promotes degradation of MYB30. In this way, the function of MYB75 is further released for precise regulation of anthocyanin synthesis in plants (Figure 7). Importantly, transcriptomic analysis revealed that MYB30 negatively regulates the expression of anthocyanin biosynthetic genes throughout the Arabidopsis genome. MYB30 therefore functions as a versatile regulator of sucrose-induced anthocyanin biosynthesis in Arabidopsis seedlings, making it a promising target for development of anthocyanin-rich food sources using GM methods.

Methods

Mutants and transgenic plants

The Arabidopsis Columbia (Col-0) ecotype was used as the WT, and all mutants in this study were in the Col-0 background. Most MYB30-related mutants and transgenic plants were kindly provided by Dr. Yan Guo of China Agricultural University. The myb75-c mutant was generated using the CRISPR–Cas9 system in Jin-Long Qiu’s laboratory (Li et al., 2016), and rha2b-1 (SALK_014943) was obtained from the NASC/ABRC. We generated homozygous myb30-2 myb75-c and myb30-2 rha2b-1 mutants for further investigations. Primers used for mutant validation and transgenic plant construction are listed in Supplemental Table 1.

Plant growth, phenotypic analysis, and anthocyanin measurement

Sterilized seeds were sown on 1/2 MS containing 2% sucrose (Control), and plates were kept at 4°C for 2 days. Seeds were germinated and grown under constant illumination at 22°C. For phenotypic analysis of plant growth under high-sucrose conditions, Col-0, mutant, and related transgenic plants were germinated and grown on 1/2 MS with 6% sucrose. Photographs were taken 7 days after germination, and then anthocyanin accumulation and expression of anthocyanin-specific biosynthetic genes (DFR, LDOX, and UF3GT) were analyzed. High light–induced anthocyanin accumulation was investigated as described previously (Li et al., 2016). Col-0 and myb30 mutant seeds were germinated and grown on 1/2 MS with 2% sucrose under 40 μmol m−2 s−1 white light (Control) or 180 μmol m−2 s−1 white light (high light). Photographs were taken 7 days after germination, and anthocyanin measurement was performed as described previously. In brief, Arabidopsis seedlings were incubated in extraction buffer (methanol containing 1% HCl) overnight at 4°C in the dark. Next, the samples were centrifuged, and the supernatants were collected for absorbance quantification at 530 and 657 nm. Relative anthocyanin content was calculated as A530 − 0.25 × A657 per gram fresh weight.

Y2H assay

To examine the interaction between MYB30 and MYB75 in yeast, the coding sequences of MYB30 and TT8/EGL3 were cloned into the pGADT7 vector, and the coding sequence of MYB75 was cloned into the pGBKT7 vector. Plasmids of various AD/BD combinations were transformed into yeast strain AH109 (MATα type) according to the standard manual. The yeast transformants were then plated on minimal SC/Trp-Leu medium or SC/Trp-Leu-His-Ade medium and grown for 3 days at 28°C. Primer sequences used for plasmid construction are listed in Supplemental Table 1.

BiFC assay

BiFC assays were performed to examine the interaction between MYB30 and MYB75 and to determine the inhibitory effect of MYB30 on formation of the MBW complex in planta. The coding sequence of MYB75 was cloned into the pSPYCE(M) vector, and the coding sequences of MYB30N (1–159 aa) and MYB30C (160–323 aa) were cloned into the pSPYNE(R)173 vector. We also cloned the coding sequences of TT8/EGL3 into the pSPYNE(R)173 vector. The resulting plasmids and the pSPYNE(R)173 and pSPYCE(M) empty vectors were transiently expressed in tobacco leaves for 3 days in various pSPYCE(M)/pSPYNE(R)173 combinations. The YFP fluorescence signal was detected using a Leica SP5 confocal microscope. Primer sequences used for plasmid construction are listed in Supplemental Table 1.

In vitro pull-down assays

In vitro pull-down assays were performed as described previously. In brief, the coding sequence of MYB75 was cloned into the pMAL-C2X or pET28a vector for purification of maltose-binding protein (MBP)-tagged or 6×His-tagged fusion proteins. The coding sequences of TT8 and EGL3 were cloned into the pMAL-C2X vector, and MYB30N was recombinantly fused with a glutathione S-transferase (GST) tag. For in vitro pull-down assays, the indicated proteins were incubated in the pull-down buffer, and the selected protein was isolated using specific antibody–conjugated agarose or amylose resin (New England Biolabs). Its interacting proteins were detected by immunoblot analysis using anti-MBP, anti-GST, or anti-His antibodies. Primer sequences used for plasmid construction are listed in Supplemental Table 1.

Co-IP assay

To confirm the interaction between MYB30 and MYB75 or MYB90 in planta, the coding sequence of MYB75 or MYB90 was cloned into the pCAMBIA1300-GFP vector, and the coding sequence of MYB30 was cloned into the pCAMBIA1307-Flag-HA vector. The resulting plasmids were purified with a plasmid extraction kit for subsequent transient expression. Arabidopsis leaf protoplast preparation, transformation, and immunoprecipitation were performed as described previously (Zhou et al., 2014). The protein complex was probed by immunoblot analysis using anti-GFP and anti-Flag antibodies. Primer sequences used for plasmid construction are listed in Supplemental Table 1.

Determination of sucrose-induced MYB30 degradation

To determine sucrose-induced MYB30 degradation, 10-day-old seedlings of 35Spro::Myc-MYB30/Col-0 and 35Spro::Myc-MYB30/rha2b-1 treated for the indicated times were used for protein extraction. The protein content of MYB30 was detected with anti-Myc antibody, and actin content was also detected with anti-actin antibody as a loading control. For drug treatment, 10-day-old seedlings of 35Spro::Flag-MYB30/Col-0 seedlings were left untreated or treated with 6% sucrose for the indicated times along with 100 μM CHX or 100 μM CHX + 50 μM MG132. MYB30 abundance was then determined by immunoblot assays with anti-Flag antibody. Rubisco served as the loading control.

In vivo ubiquitination assay

Ten-day-old seedlings of 35Spro::Myc-MYB30/Col-0 and 35Spro::Myc-MYB30/rha2b-1 treated with 6% sucrose for 0 h or 6 h were used for protein extraction. Following centrifugation at 12 000 × g for 15 min at 4°C, the supernatant was incubated with anti-Myc agarose (Sigma) for 2 h to purify MYB30 protein. The immunoprecipitated Myc-MYB30 protein, which was detected with anti-Myc antibody, was used for determination of protein ubiquitination with anti-ubiquitin antibody (Santa Cruz Biotechnology).

Split-Luc complementary assay

Coding sequences of MYB30 and RHA2b were fused with the coding sequences of the luciferase N terminus and C terminus, respectively. For the split-Luc assay, nLuc-MYB30 was co-expressed with cLuc-RHA2b in Nicotiana benthamiana via Agrobacterium tumefaciens and incubated for 2 d. MG132 was injected into leaf tissues 12 h before observation. For sucrose treatment, leaves were divided into small discs and then incubated in water (control) or 6% sucrose with 100 μM D-luciferin, and luminescence intensities were detected after 30 min.

Transactivation assays in Arabidopsis leaf protoplasts

The promoter of MYB75, RHA2b, or DFR was cloned into the pGreenII-0800-LUC vector, and the resulting constructs were used as reporters. The indicated TFs were then co-expressed with the reporters in Arabidopsis leaf protoplasts. After overnight incubation at 23°C, luciferase activities were determined using the Promega dual-luciferase reporter assay system and a GloMax 20-20 luminometer (Promega). Relative luciferase activity was defined as firefly luciferase activity divided by Renilla luciferase activity. Protoplast preparation and transformation were performed as described previously (Yan et al., 2020). The primers used for plasmid construction are listed in Supplemental Table 1.

ChIP–qPCR analysis

Transgenic plants overexpressing MYB30 or MYB75 were used for ChIP analysis. In brief, chromatin was isolated from the indicated plants (myb30-2 and 35Spro::MYB30-Flag/myb30-2 for MYB30 IP; Col-0 and 35Spro::Flag-MYB75/Col-0 for MYB75 IP). Monoclonal anti-Flag antibody (Sigma) was used for protein immunoprecipitation. DNA fragments in input and immunoprecipitated samples (output) were quantified by qRT–PCR. The ACTIN2 promoter was used as the negative control (Liao et al., 2017). At least three independent experiments were performed. The primer sequences are listed in Supplemental Table 2.

EMSA

The 1–477-bp coding sequence of MYB30 and the full-length coding sequence of MYB75 were amplified and cloned into the pET28a vector. The resultant GST-MYB30N and His-MYB75 recombinant proteins were purified. For EMSA, MBS fragments from the promoters of MYB75 and RHA2b were obtained by annealing with biotin-labeled or unlabeled primers. Unlabeled fragments of the same sequences were used as competitors. Each 10-μL reaction mixture contained 2 μL 5× EMSA/Gel-Shift Binding Buffer (Beyotime), 1 μg purified protein, 1 μL biotin-labeled probe, and ultrapure water. The reactions were incubated at 22°C for 30 min, then fractionated on a 6% native polyacrylamide gel in 0.5× TBE buffer (45 mM Tris, 45 mM boric acid, 1 mM EDTA). The EMSA was performed using a Chemiluminescent EMSA Kit (Beyotime) following the manufacturer’s protocol. The primer sequences are listed in Supplemental Table 2.

Transcriptomic analysis

Ten-day-old Col-0 and myb30-2 plants, left untreated or treated with 6% sucrose, were pooled for total RNA isolation and transcriptome sequencing. RNA concentration and purity were determined using a NanoDrop 2000 instrument (Thermo Fisher Scientific). RNA integrity was assessed using the RNA Nano 6000 Assay Kit for the Agilent Bioanalyzer 2100 system (Agilent Technologies). RNA libraries were constructed and deep sequenced on the Illumina NovaSeq 6000 platform (BioMarker Technologies, Inc.) according to standard procedures. The clean reads from each sample were mapped to the TAIR10 reference genome using HISAT2 with default parameters (Xiao et al., 2021). DEGs were identified using edgeR (Yan et al., 2020) with the criteria fold-change ≥2 and false discovery rate <0.01. Gene Ontology enrichment analysis of DEGs was performed as described previously (Tian et al., 2021). The RNA sequencing data are provided as Supplemental File 1.

RT–qPCR and statistical analysis

Total DNA and RNA extraction, cDNA synthesis, and RT–qPCR were performed as described previously. RT–qPCR analysis was performed using SYBR Green PCR Master Mix as described in the accompanying manual. Three independent experiments were performed with similar results, and statistical significance was determined using Student’s t-test or ANOVA. A difference at P < 0.05 was considered significant. ACTIN2 was used as the internal control. The primer sequences are listed in Supplemental Table 2.

Accession numbers

Sequences of the genes described in this article can be found at the Arabidopsis Genome Initiative under the following accession numbers: MYB30 (AT3G28910), MYB75 (At1g56650), TT8 (At4g09820), EGL3 (At1g63650), RHA2b (AT2G01150), DFR (At5g42800), LDOX (At4g22880), UF3GT (At5g54060), JOX1 (AT3G11180), UGT75C1 (AT4G14090), MYB114 (AT1G66380), NCED3 (AT3G14440), RD29A (AT5G52310), RD26 (AT4G27410), GDH2 (AT5G07440), GAD (AT5G17330), ASN2 (AT5G65010), GSR2 (AT1G66200), and ACTIN2 (AT3G18780).

Data and code availability

All relevant data can be found within the manuscript and its supporting materials. The raw sequencing data are available at the China National Genomics Data Center (https://ngdc.cncb.ac.cn) under BioProject accession number PRJCA021040.

Funding

We sincerely thank Dr. Yan Guo from China Agricultural University for kindly providing seeds of MYB30-related mutants and transgenic plants. This work was supported by the National Natural Science Foundation of China (grants 32170295 and 31870241 to H.Z.), the China Postdoctoral Science Foundation (grant 2023M730776 to J.H.), and the Institutional Research Fund of Sichuan University (grant 2020SCUNL212 to H.L.).

Author contributions

H.P.Z., J.H., and Y.Z. designed the project; J.H., Y.Y.Z., H.Y.Z., and X.S. performed the experiments with the assistance of X.C. and X.L.; H.P.Z., J.H., and Y.Z. analyzed the data and wrote the manuscript. All the authors contributed to discussions. H.P.Z. and H.L. supervised the project.

Acknowledgments

No conflict of interest is declared.

Published: November 10, 2023

Footnotes

Published by the Plant Communications Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and CEMPS, CAS.

Supplemental information is available at Plant Communications Online.

Contributor Information

Huapeng Zhou, Email: zhouhuapeng@scu.edu.cn.

Yuan Zheng, Email: zhengyuan051@163.com.

Honghui Lin, Email: hhlin@scu.edu.cn.

Supplemental information

Document S1. Supplemental Figures 1–8 and Supplemental Tables 1 and 2
mmc1.pdf (1.1MB, pdf)
Supplemental File 1. RNA-seq data
mmc2.pdf (601.2KB, pdf)
Supplemental File 2. Raw data supporting IB and EMSA
mmc3.xls (1.7MB, xls)
Document S2. Article plus supplemental information
mmc4.pdf (4.4MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Supplemental Figures 1–8 and Supplemental Tables 1 and 2
mmc1.pdf (1.1MB, pdf)
Supplemental File 1. RNA-seq data
mmc2.pdf (601.2KB, pdf)
Supplemental File 2. Raw data supporting IB and EMSA
mmc3.xls (1.7MB, xls)
Document S2. Article plus supplemental information
mmc4.pdf (4.4MB, pdf)

Data Availability Statement

All relevant data can be found within the manuscript and its supporting materials. The raw sequencing data are available at the China National Genomics Data Center (https://ngdc.cncb.ac.cn) under BioProject accession number PRJCA021040.

Articles from Plant Communications are provided here courtesy of Elsevier

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