An R3-MYB repressor, BnCPC forms a feedback regulation with MBW complex to modulate anthocyanin biosynthesis in Brassica napus

Abstract

Background: Anthocyanins are metabolites of phenylpropanoid pathway, and involves in diverse processes of plant development and adaptation, which are regulated by the MYB-bHLH-WD40 (MBW) protein complexes. Many R2R3-MYB activators have been well characterized, but the MYB repressors in anthocyanin biosynthesis were recognized recently, which are also important in modulating phenylpropanoid metabolism in plants. The regulatory mechanism of anthocyanin biosynthesis in oil crop Brassica napus remains to be revealed.

Results: In this study, we identified an anthocyanin repressor BnCPC in B. napus. BnCPC encoded a typical R3-MYB protein containing a conserved [D/E]Lx2[R/K]x3Lx6Lx3R motif for interaction with bHLH proteins. Overexpression of BnCPC in B. napus inhibited anthocyanin accumulation, especially under anthocyanin inducible conditions. Protein-protein interaction and dual-luciferase assays confirmed that BnCPC could compete with BnPAP1 to interact with bHLHs (BnTT8 and BnEGL3), and repress the expression of anthocyanin biosynthetic genes (e.g., BnDFR) that activated by MBW complexes. Moreover, we found BnCPC inhibited the MBW complex-induced BnCPC activity.

Conclusions: Overall, this research demonstrated that BnCPC repressed anthocyanin biosynthesis by affecting the formation of MBW complex, and formed a feedback loop to regulate anthocyanin accumulation in B. napus.

Keywords: Anthocyanin; BnCPC; BnDFR; Brassica napus; MBW complex; Repressor.

Fig. 1

Characterization of BnCPC proteins. A Phylogenetic relationship of BnCPC and other known MYB repressors in plants. BnCPC homologues were highlighted with the black rhombi. Alignment of full amino acid sequences of MYB repressors was done by MUSCLE software, and phylogenetic tree was constructed using the neighbor-joining method by MEGA software 7.0. Numbers at the nodes indicated the reliability percentage of bootstrap values based on 1000 replications. B Alignment and structural domains of BnCPC and MYB repressors. Protein sequences were aligned using ClustalX. The R2 and R3 domains were indicated with grayish white and gray boxes, respectively. Three α-helices of both R2 and R3 domains were indicated in boxes. The conserved MYB–bHLH interaction motif ([D/E]Lx2[R/K]x3Lx6Lx3R) of R3 domain was underlined with a black bar. The conserved motifs in the C-terminus were highlighted with boxes and numbers. C1 domain, LIsrGIDPxT/SHRxI/L; C2 domain (the EAR repression motif), pdLNLD/ELxiG/S; C3 domain (zinc-finger motif), Cx2Cx9CxC; C4 domain, GY/FDFLGL motif; C5 domain, TLLLFR repressor motif. The following GenBank or Arabidopsis TAIR accession numbers were used, PpMYB18 (KT159234.1), VvMYBC2-L1 (JX050227), VvMYBC2-L2 (ACX50288), VvMYBC2-L3 (KM046932), FaMYB1 (AF401220), MtMYB2 (XM_003616340), PtrMYB182 (KP723392), AtMYB4 (AF062860), PhMYB27 (AHX24372), MdMYB16 (HM122617), AtMYBL2 (AEE35154), IlMYBL1 (ASR83103.1), LhR3MYB1 (BBG71951.1), LhR3MYB2 (BBG71953.1), AtCPC (AT2G46410.1), PhMYBx (AHX24371.1), MlROI1 (AGC66791.1), SlMYBATV (NP_001352307), FhMYBx (MT210095), MaMYBx (QJH86892.1)

Fig. 2

The expression pattern of A BnCPC and B–D anthocyanin biosynthetic genes (BnDFR, BnLDOX, and BnUF3GT) under anthocyanin inducible (cold and light) and non-inducible (dark) growth conditions. Expression levels were standardized to B. napus Actin-7 (NC_027775.2), and the expression levels before treatment were set at 1. Values represented the mean ± SD (n = 3)

Fig. 3

BnCPC repressed anthocyanin accumulation in B. napus. A Phenotype of J9712 and BnCPC overexpression (OE-CPC) lines under low temperature (10 °C) and normal temperature (23 °C). Scale bar represented 1 cm. B Anthocyanin content in extracts from seedlings in A. (A₅₃₀-0.25 × A₆₅₇)/gram fresh weight was considered as the relative anthocyanin content. Three biological replicates were performed, and 10 plants were pooled as one replicate. FW, fresh weight. Values represented the mean ± SD (n = 3). Different letters represented statistically significant differences (two-way ANOVA, p < 0.05). C The expression level of BnDFR, BnLDOX, and BnUF3GT in rapeseed seedlings from A. Expression levels were normalized to B. napus Actin-7 (NC_027775.2), and the expression level of J9712 under normal temperature was set at 1. Values represented the mean ± SD (n = 3)

Fig. 4

BnCPC repressed the BnDFR promoter activity by interfering with the formation of MBW complexes. A BnCPC inhibits anthocyanin accumulation mediated by MBW complexes in tobacco leaves. Tobacco leaves were infiltrated with BnPAP1 + BnTT8 ± BnCPC, BnPAP1 + BnEGL3 ± BnCPC, BnPAP1 + BnTT8 + BnTTG1 ± BnCPC, BnPAP1 + BnEGL3 + BnTTG1 ± BnCPC and cultured for 8 days. B Relative anthocyanin content in extracts from leaves in A. Independent-samples t-test was used for statistical analysis in comparison with the controls (**p ≤ 0.01). C BnCPC inhibited the MBW complex-induced BnDFR promoter activity. Values represent the mean ± SD (n = 6). Different letters represented statistically significant differences (one-way ANOVA, p < 0.05)

Fig. 5

BnCPC interacts with BnTT8 and BnEGL3. A Interaction of BnCPC with BnTT8/BnEGL3 by Y2H assay. B Interaction of BnCPC with BnTT8/BnEGL3 by BiFC assay. C Interaction of BnCPC with BnTT8/BnEGL3 by pull-down assay

Fig. 6

BnCPC competes with BnPAP1 to affect MBW formation. A BiFC analysis showed that BnCPC affected the interaction of BnPAP1 with BnTT8/BnEGL3. YFP fluorescence was observed 48 h after co-expression. Scale bar represented 100 μm. B and C qPCR analysis of BnCPC, BnTT8, BnEGL3, and BnPAP1 expression in A. Expression level was standardized to tobacco Actin (JQ256516.1). Values represented the mean ± SD (n = 3). D Competitive binding assays of BnCPC and BnPAP1 for BnTT8/BnEGL3

Fig. 7

Regulatory relationships between BnCPC and the anthocyanin activators. A–C The expression pattern of BnCPC, BnPAP1, BnTT8, and BnEGL3 under anthocyanin inducible (cold and light) and non-inducible (dark) growth conditions. Expression levels were standardized to B. napus Actin-7 (NC_027775.2), and the expression levels of BnCPC before treatment were set at 1. Values represented the mean ± SD (n = 3). D Dual-luciferase assays of BnPAP1, BnTT8, BnEGL3, BnPAP1 + BnTT8/BnEGL3, and BnCPC + BnPAP1 + BnTT8/BnEGL3 effects on the activity of BnCPC promoter. Values represent the mean ± SD (n = 6). Different letters represented statistically significant differences (one-way ANOVA, p < 0.05)

Fig. 8

Proposed working model of BnCPC regulating anthocyanin biosynthesis in rapeseed. A Under non-inducible conditions (e.g., dark or non-stress conditions), BnPAP1 was lowly expressed and BnCPC was continuously induced to repress the expression of anthocyanin biosynthetic genes (e.g., BnDFR) by inhibiting MBW formation, thereby repressed anthocyanin accumulation. B Under inducible conditions (e.g., cold and other stress conditions), BnPAP1 was induced and formed MBW complexes to activate transcription of anthocyanin biosynthetic genes (e.g., BnDFR, BnLDOX, and BnUF3GT) and promote anthocyanin accumulation. BnCPC was up-regulated under stress conditions, and competed with BnPAP1 to interact with BnTT8/BnEGL3, thus affecting the MBW complex formation. BnCPC also inhibited the MBW complex-induced BnCPC activity, and formed a feedback loop with MBWs to regulate anthocyanin biosynthesis. Arrows and blunt arrows represent positive and negative regulation, respectively