The R3-Type MYB Transcription Factor BrMYBL2.1 Negatively Regulates Anthocyanin Biosynthesis in Chinese Cabbage (Brassica rapa L.) by Repressing MYB-bHLH-WD40 Complex Activity

Abstract

Chinese cabbage (Brassica rapa L.) leaves are purple in color due to anthocyanin accumulation and have nutritional and aesthetic value, as well as antioxidant properties. Here, we identified the R3 MYB transcription factor BrMYBL2.1 as a key negative regulator of anthocyanin biosynthesis. A Chinese cabbage cultivar with green leaves harbored a functional BrMYBL2.1 protein, designated BrMYBL2.1-G, with transcriptional repressor activity of anthocyanin biosynthetic genes. By contrast, BrMYBL2.1 from a Chinese cabbage cultivar with purple leaves carried a poly(A) insertion in the third exon of the gene, resulting in the insertion of multiple lysine residues in the predicted protein, designated BrMYBL2.1-P. Although both BrMYBL2.1 variants localized to the nucleus, only BrMYBL2.1-G interacted with its cognate partner BrTT8. Transient infiltration assays in tobacco leaves revealed that BrMYBL2.1-G, but not BrMYBL2.1-P, actively represses pigment accumulation by inhibiting the transcription of anthocyanin biosynthetic genes. Transient promoter activation assay in Arabidopsis protoplasts verified that BrMYBL2.1-G, but not BrMYBL2.1-P, can repress transcriptional activation of BrCHS and BrDFR, which was activated by co-expression with BrPAP1 and BrTT8. We determined that BrMYBL2.1-P may be more prone to degradation than BrMYBL2.1-G via ubiquitination. Taken together, these results demonstrate that BrMYBL2.1-G blocks the activity of the MBW complex and thus represses anthocyanin biosynthesis, whereas the variant BrMYBL2.1-P from purple Chinese cabbage cannot, thus leading to higher anthocyanin accumulation.

Keywords: BrMYBL2.1; Chinese cabbage; anthocyanin; insertional mutation; repressor; ubiquitination.

Figure 1

Phenotype and anthocyanin contents of green and purple Chinese cabbage under various cultivation conditions. (A) Phenotypes of seedlings from two Chinese cabbage cultivars were grown under low temperature or an optimal temperature, with or without 90 mM sucrose. (B) Mean anthocyanin contents of seedlings from green and purple Chinese cabbage cultivars. Results represent the mean values ± standard deviation (SD). Three independent biological replicates were performed. Different letters indicate significantly different values (p < 0.01), as determined by two-way ANOVA followed by Duncan’s multiple range tests.

Figure 2

Expression of BrMYL2.1 and other anthocyanin regulatory and biosynthetic genes under various cultivation conditions in green and purple Chinese cabbage. (A) Relative transcript levels of the anthocyanin regulatory genes. (B) Relative transcript levels of the anthocyanin biosynthetic genes. G, green Chinese cabbage; P, purple Chinese cabbage. Results represent the means ± SD from three independent biological replicates. BrEF1α was used as the reference gene. Different letters indicate significantly different values (p < 0.01), as determined by two-way ANOVA followed by Duncan’s multiple range tests.

Figure 3

Phylogenetic relationships and multiple sequence alignment among anthocyanin MYB repressors from Chinese cabbage and other plant species. (A) Phylogenetic tree of BrMYBL2.1 and other MYB repressors proteins from other plants. The phylogenetic tree was constructed using the neighbor-joining method with MEGA6 software. Numbers next to the nodes are bootstrap values from 1000 replications. The scale bar corresponds to 0.1 amino acid substitutions per site. The following GenBank or Arabidopsis TAIR accession numbers were used: Arabidopsis thaliana AtMYB4 (At4g38620), AtMYB32 (At4g34990), AtMYBL2 (At1g71030), AtCPC (At2g46410), AtTRY (At5G53200); Brassica rapa BrMYBL2.1-G (OK905440), BrMYBL2.1-P (OK905441); Fragaria × ananassa FaMYB1 (AF401220); Freesia hybrid FhMYB27 (QJW70308); Iochroma loxense IlMYBL1 (ASR83103); Lilium hybrid LhR3MYB1 (LC429593); Petunia × hybrida PhMYB27 (AHX24372), PhMYBx (AHX24371); Populus tremuloides PtMYB182 (KP723392); Solanum lycopersicum SlMYB-ATV (MF197509); Vitis vinifera VvMYBC2-L1 (JX050227). (B) Multiple sequence alignment of BrMYBL2.1s with representative MYB repressors. Absolutely conserved residues are highlighted in black and partial conservation is shown in gray. The R2 and R3 domains are indicated with light gray and dark grey boxes, respectively. The specific motif responsible for interacting with bHLH factors is indicated by the red asterisks. The conserved repression motifs in the C2 and C5 motifs are indicated by the red and blue boxes, respectively.

Figure 4

Subcellular localization of BrMYBL2.1-G, BrMYBL2.1-P, BrPAP1, and BrTT8 in Arabidopsis leaf protoplasts. (A) Schematic diagram of the constructs used in this experiment: CaMV35S, cauliflower mosaic virus 35S promoter; sGFP, soluble green fluorescent protein (GFP) gene; BrMYBL2.1-G-sGFP, BrMYBL2.1-G fused to sGFP; BrMYBL2.1-P-sGFP, BrMYBL2.1-P fused to sGFP; BrPAP1-sGFP, BrPAP1 fused to GFP; BrTT8-sGFP, BrTT8 fused to sGFP; NLS-RFP, nuclear localization signal fused to the red fluorescent protein (RFP) gene; Nos, nopaline synthase terminator. (B) In vivo localization pattern of BrMYBL2.1-G, BrMYBL2.1-P, BrPAP1, and BrTT8 in Arabidopsis protoplasts. Representative protoplasts accumulating each fusion protein are shown 16 h after transformation. GFP, GFP fluorescence; RFP, RFP fluorescence; BF, bright field and merged, merged of GFP, RFP, and bright field images. Bars, 10 μm.

Figure 5

Interaction tests between BrMYBL2.1-G, BrMYBL2.1-P, BrPAP1, and BrTT8 by yeast two-hybrid assay. AD, activation domain; BD, binding domain; SD, synthetic defined medium; 3-AT, 3-amino-1,2,4-triazole; SD − TL, SD medium lacking Trp and Leu; SD − TLH + 3AT, SD medium lacking Trp, Leu, His and containing 10 mM 3-AT.

Figure 6

Functional analysis of BrMYBL2.1-G and BrMYBL2.1-P for anthocyanin biosynthesis, as a repressor. Visual phenotype and anthocyanin contents in tobacco leaves transiently infiltrated with Agrobacterium strains harboring an empty vector or constructs BrPAP1, BrTT8, BrMYBL2.1-G, and BrMYBL2.1-P in the indicated combinations. (A) Representative photograph of a transiently infiltrated tobacco leaf 5 days after agroinfiltration. (B) Anthocyanin contents of the leaf sections shown in (A). Results are the means ± SD from three independent biological replicates. Different letters indicate significantly different values (p < 0.01), as determined by one-way ANOVA followed by Duncan’s multiple range tests.

Figure 7

Relative transcript levels of anthocyanin biosynthetic genes in tobacco leaves transiently infiltrated with BrMYBL2.1-G or BrMYBL2.1-P constructs. Relative transcript levels were determined by RT-qPCR, with NtGAPDH used as a reference gene. Results are the means ± SD from three independent biological replicates. Different letters indicate significantly different values (p < 0.01), as determined by one-way ANOVA followed by Duncan’s multiple range tests.

Figure 8

Characterization of the regulatory roles of BrMYBL2.1-G and BrMYBL2.1-P for BrCHS and BrDFR promoter activation. (A) Schematic diagrams of the effector and reporter constructs used in this assay. The effector constructs consist of the coding sequences of BrPAP1, BrTT8, BrMYBL2.1-G, and BrMYBL2.1-P driven by the 35S promoter. The reporter constructs contain the firefly luciferase reporter gene (LUC) under the control of the BrCHS and BrDFR promoters. (B) Promoter transactivation assays in Arabidopsis protoplasts transiently transfected with the constructs BrPAP1, BrTT8, BrMYBL2.1-G, and BrMYBL2.1-P in the indicated combinations. Results are the means ± SD from three independent biological replicates. Different letters indicate significantly different values (p < 0.01), as determined by one-way ANOVA followed by Duncan’s multiple range tests.

Figure 9

Degradation of BrMYBL2.1-G and BrMYBL2.1-P by the ubiquitin-proteasome system. (A) Effect of MG132 on the intensity and localization of GFP fluorescence in transiently transfected Arabidopsis protoplasts. Bars, 100 μm. (B) Immunoblot analysis of total proteins from transfected protoplasts treated with dimethyl sulfoxide (DMSO) or MG132. (C) Quantification of the GFP bands shown in B, normalized to actin abundance. Significant differences were determined by a Student’s paired t-test relative to BrMYBL2.1-G; *** p < 0.001.

Figure 10

Proposed working model of the role of BrMYBL2.1 in anthocyanin biosynthesis in green (A) and purple (B) Chinese cabbage. Arrows and blunt arrows represent positive and negative regulation, respectively.