Loss of the R2R3 MYB Transcription Factor RsMYB1 Shapes Anthocyanin Biosynthesis and Accumulation in Raphanus sativus

Abstract

The red or purple color of radish (Raphanus sativus L.) taproots is due to anthocyanins, which have nutritional and aesthetic value, as well as antioxidant properties. Moreover, the varied patterns and levels of anthocyanin accumulation in radish roots make them an interesting system for studying the transcriptional regulation of anthocyanin biosynthesis. The R2R3 MYB transcription factor RsMYB1 is a key positive regulator of anthocyanin biosynthesis in radish. Here, we isolated an allele of RsMYB1, named RsMYB1^Short, in radish cultivars with white taproots. The RsMYB1^Short allele carried a 4 bp insertion in the first exon causing a frame-shift mutation of RsMYB1, generating a truncated protein with only a partial R2 domain at the N-terminus. Unlike RsMYB1^Full, RsMYB1^Short was localized to the nucleus and the cytoplasm and failed to interact with their cognate partner RsTT8. Transient expression of genomic or cDNA sequences for RsMYB1^Short in radish cotyledons failed to induce anthocyanin accumulation, but that for RsMYB1^Full activated it. Additionally, RsMYB1^Short showed the lost ability to induce pigment accumulation and to enhance the transcript level of anthocyanin biosynthetic genes, while RsMYB1^Full promoted both processes when co-expressed with RsTT8 in tobacco leaves. As the result of the transient assay, co-expressing RsTT8 and RsMYB1^Full, but not RsMYB1^Short, also enhanced the promoter activity of RsCHS and RsDFR. We designed a molecular marker for RsMYB1 genotyping, and revealed that the RsMYB1^Short allele is common in white radish cultivars, underscoring the importance of variation at the RsMYB1 locus in anthocyanin biosynthesis in the radish taproot. Together, these results indicate that the nonsense mutation of RsMYB1 generated the truncated protein, RsMYB1^Short, that had the loss of ability to regulate anthocyanin biosynthesis. Our findings highlight that the frame shift mutation of RsMYB1 plays a key role in anthocyanin biosynthesis in the radish taproot.

Keywords: MBW complex; RsMYB1; anthocyanin; frameshift mutation; radish.

Figure 1

Phenotypes and anthocyanin contents of the six radish cultivars used in this study. (A) Representative photographs of white- and red-taproot radishes at the mature stage, with whole taproots on the left and taproots sectioned to show flesh color on the right of each image. (B) Anthocyanin levels in white and red taproot radishes. Results are mean values ± SD from three independent biological replicates. Different letters above the bars indicate significantly different values (p < 0.0001, one-way ANOVA followed by Duncan’s multiple range test).

Figure 2

Relative transcript levels of anthocyanin biosynthetic and regulatory genes in the six radish cultivars. (A) Anthocyanin biosynthetic regulators. (B) Anthocyanin biosynthetic genes. RsRPII was used as a reference gene for evaluating the target gene expression. Results are mean values ± SD from three independent biological replicates. Different letters above the bars indicate significantly different values (p < 0.05, two-way ANOVA followed by Duncan’s multiple range test).

Figure 3

Genomic structure of RsMYB1 alleles and phylogenetic tree of RsMYB1 proteins from red and white radish cultivars and R2R3 MYB proteins from other plants. (A) Schematic diagram of the genomic structure of RsMYB1. The predicted protein encoded by each mature mRNA is indicated below with their functional domains. White boxes, exons; black lines, introns. The 4 bp insertion site in RsMYB1^Short is indicated by a triangle. Premature termination codon (PTC) is indicated by a circle. (B) Multiple protein sequence alignment of the R2 and R3 domains across R2R3 MYB proteins from other plants. The conserved residues of the DNEI and ANDV motifs are shown in the red and blue boxes, respectively. Inverted blue triangles indicate the conserved residues forming the inner hydrophobic core of the R2 and R3 domains.

Figure 4

Subcellular localization of RsMYB1^Short, RsMYB1^Full, and RsTT8 in Arabidopsis leaf protoplasts. (A) Schematic diagrams of the constructs: CaMV35S, cauliflower mosaic virus 35S promoter; sGFP, soluble green fluorescent protein (GFP) gene; RsMYB1^Short-sGFP, RsMYB1^Short fused to sGFP; RsMYB1^Full-sGFP, RsMYB1^Full fused to sGFP; RsTT8-sGFP, RsTT8 fused to sGFP; NLS-RFP, nuclear localization signal fused to the red fluorescent protein (RFP) gene; Nos, nopaline synthase terminator. (B) RsMYB1^Short, RsMYB1^Full, and RsTT8 localize to the nucleus in Arabidopsis protoplasts. Data are representative of protoplasts accumulating each fusion protein 16 h after transfection. Scale bars = 10 μm.

Figure 5

Physical interactions among RsMYB1^Full, RsMYB1^Short, and RsTT8. (A) Schematic diagrams of the constructs used in the Y2H assay. The amino acid positions of the fragments are shown. (B) Protein–protein interactions among RsMYB1^Full, RsMYB1^Short, and RsTT8, as revealed by Y2H analysis. SD/−LT, synthetic defined medium lacking Leu and Trp; SD/−LTH+3AT, SD medium lacking Leu, Trp, and His but containing 10 mM 3-amino-1,2,4-triazole (AT). AD and BD indicate the GAL4 activation domain and binding domain, respectively.

Figure 6

Functional analysis of RsMYB1^Short and RsMYB1^Full for anthocyanin biosynthesis. (A) Transient Agrobacterium-mediated infiltration assay of constructs expressing RsMYB1 in radish cotyledons. (B) Anthocyanin contents in transiently infiltrated radish cotyledons shown in A. Results are mean values ± SD from three independent biological replicates. ** p < 0.01, as determined by Student’s paired t-test relative to the empty vector.

Figure 7

Visible phenotypes and anthocyanin contents of tobacco leaves transiently expressing RsMYB1 constructs. (A) Tobacco leaves transiently infiltrated with constructs carrying the genomic sequence from RsMYB1^Short or RsMYB1^Full alone or together with RsTT8. (B) Tobacco leaves transiently infiltrated with constructs carrying the cDNAs from RsMYB1^Short or RsMYB1^Full alone or together with RsTT8. (C) Anthocyanin contents of the leaf sectors shown in A and B. Results are mean values ± SD from three independent biological replicates. * p < 0.05; and *** p < 0.001, as determined by Student’s paired t-test relative to the empty vector.

Figure 7

Visible phenotypes and anthocyanin contents of tobacco leaves transiently expressing RsMYB1 constructs. (A) Tobacco leaves transiently infiltrated with constructs carrying the genomic sequence from RsMYB1^Short or RsMYB1^Full alone or together with RsTT8. (B) Tobacco leaves transiently infiltrated with constructs carrying the cDNAs from RsMYB1^Short or RsMYB1^Full alone or together with RsTT8. (C) Anthocyanin contents of the leaf sectors shown in A and B. Results are mean values ± SD from three independent biological replicates. * p < 0.05; and *** p < 0.001, as determined by Student’s paired t-test relative to the empty vector.

Figure 8

Relative transcript levels of anthocyanin biosynthetic genes in tobacco leaves transiently expressing RsMYB1 constructs. NtGAPDH was used as a reference gene. Results are mean values ± SD from three independent biological replicates. * p < 0.05; *** p < 0.001, as determined by Student’s paired t-test relative to the empty vector.

Figure 9

Transcriptional activation assay of the RsCHS and RsDFR promoters by RsMYB1^Full, RsMYB1^Short, and RsTT8. (A) Schematic diagrams of effector and reporter constructs used in the transcriptional activation assay. The effector construct harbors the coding sequences of RsMYB1^Full, RsMYB1^Short, and RsTT8, respectively, driven by the 35S promoter and NOS terminator. The reporter constructs carry the RsCHS and RsDFR promoters driving GUS expression. (B) Regulatory consequences of the transient expression of RsMYB1^Short, RsMYB1^Full, and RsTT8 on RsCHS and RsDFR promoter activity. Results are mean values ± SD from three independent biological replicates. *** p < 0.001, as determined by Student’s paired t-test relative to the control.

Figure 10

Sequence variation at RsMYB1 predicts taproot color across 20 radish cultivars. (A) Representative photographs of radish taproots showing the different colors of the indicated cultivars. (B) Gel electrophoresis of amplified fragments of the RsMYB1 gene. (C) Genotyping results of the 20 radish cultivars with the CAPS marker for RsMYB1. FC and SC are PCR amplicons from plasmids harboring RsMYB1^Short or RsMYB1^Full, respectively, as positive controls. M: 100 bp DNA size marker.