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The beet Y locus encodes an anthocyanin MYB-like protein that activates the betalain red pigment pathway

Abstract

Nearly all flowering plants produce red/violet anthocyanin pigments. Caryophyllales is the only order containing families that replace anthocyanins with unrelated red and yellow betalain pigments1,2. Close biological correlation of pigmentation patterns suggested that betalains might be regulated by a conserved anthocyanin-regulating transcription factor complex consisting of a MYB, a bHLH and a WD repeat–containing protein (the MBW complex)3. Here we show that a previously uncharacterized anthocyanin MYB-like protein, Beta vulgaris MYB1 (BvMYB1), regulates the betalain pathway in beets. Silencing BvMYB1 downregulates betalain biosynthetic genes and pigmentation, and overexpressing BvMYB1 upregulates them. However, unlike anthocyanin MYBs, BvMYB1 will not interact with bHLH members of heterologous anthocyanin MBW complexes because of identified nonconserved residues. BvMYB1 resides at the historic beet pigment-patterning locus, Y, required for red-fleshed beets4. We show that Y and y express different levels of BvMYB1 transcripts. The co-option of a transcription factor regulating anthocyanin biosynthesis would be an important evolutionary event allowing betalains to largely functionally replace anthocyanins.

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Figure 1: Betalain biosynthetic pathway and analysis of BvMYB1 overexpression in beets.
Figure 2: Reverse genetic analysis of BvMYB1.
Figure 3: Analysis of BvMYB1 versus Arabidopsis anthocyanin MYBs.
Figure 4: Analysis of BvMYB1 y versus Y allele regulatory regions.

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Acknowledgements

This work was supported by US Department of Agriculture grant 2008-35301-19032 and US National Science Foundation grant MCB-1122179. We thank E. Huq, S. Sung and V. Symonds for comments on the manuscript and discussion, I. Goldman (University of Wisconsin–Madison) for the W357B beet cultivar and K. Keller for pigment analysis.

Author information

Authors and Affiliations

Authors

Contributions

G.J.H., N.A.A., R.M.S., L.E., S.C., A.H., A.G. and A.M.L. performed experiments. J.M.M. provided the BAC library, RIL mapping population and mapping expertise. G.J.H., N.A.A., R.M.S., J.M.M. and A.M.L. conceived of the project and contributed to manuscript writing and editing.

Corresponding author

Correspondence to Alan M Lloyd.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Y versus y beets.

Bull’s Blood red table beet (YY genotype) is on the left and Albina Vereduna white table beet (yy genotype) is on the right in each panel. (a) Seven-day-old seedlings. (b) Oblique hand section through 7-day-old hypocotyl. (c) Sections through same position several weeks later. (d) Fibrous roots on pot-bound plants. (e) Mature true leaves. (f) Mature beets.

Supplementary Figure 2 Alignment of BvMYB1 with two anthocyanin regulatory MYBs, AmROSEA1 from Antirrhinum majus and AtPAP1 from Arabidopsis thaliana.

Blue and red bars designate the R2 and R3 MYB domains, respectively. Black arrowheads indicate the position of introns in all three genes. A red arrowhead indicates the last amino acid in the truncated BvMYB1ΔAc. Carats indicate the amino acids involved in bHLH interaction discussed in the text. The alignment illustrates the high degree of identity throughout the MYB domains but lack of homology throughout the activation domain approximately after amino acid positions 110 to 115. The alignment was created with ClustalW2.

Supplementary Figure 3 Neighbor-joining tree of the R2R3 MYB domains of BvMYB1 and a representative selection of R2R3 MYBs across the gene family.

This tree includes all the genes from Figure 1b and at least one member from most of the subgroups previously defined7,8.

Supplementary Figure 4 Betalain gene expression is upregulated in Bull’s Blood red table beets (Y) versus Albina Vereduna white table beets (y) and C869 white sugar beets (y).

Every qRT-PCR experiment was replicated three times, and all three biological replicates are shown. Bars represent the average of five technical replicates; error bars, s.d.

Source data

Supplementary Figure 5 Betalain pigment is increased in 35S::BvMYB1 compared to 35S::GUS in Albina Vereduna.

Extracted pigment was quantified by measuring absorbance at 530 nm. Data for 35S::GUS are the average of three biological replicates with three technical replicates each. Data for 35S::BvMYB1 are from four independent biological replicates (A, B, C and D). Bars represent the average of three technical replicates each; error bars, s.d.

Source data

Supplementary Figure 6 BvMYB1 overexpression induces expression of BvDODA1 and BvCYP76AD1.

Every qRT-PCR experiment was replicated three times, and all three biological replicates (BR) are shown. Bars represent the average of five technical replicates; error bars, s.d.

Source data

Supplementary Figure 7 Virus-induced gene silencing of BvMYB1 in Bull’s Blood results in downregulation of BvDODA1 and BvCYP76AD1.

Every qRT-PCR experiment was replicated three times, and all three biological replicates are shown. Bars represent the average of five technical replicates; error bars, s.d.

Supplementary Figure 8 Dominant-negative 35S::BvMYB1ΔAc results in downregulation of BvDODA1 and BvCYP76AD1.

(a,b) qRT-PCR expression analysis of BvMYB1ΔAc (a), BvDODA1 (b) and BvCYP76AD1 (b) in 35S::BvMYB1ΔAc Bull’s Blood beet hairy roots. Every qRT-PCR experiment was replicated three times, and all three biological replicates are shown. Bars represent the average of five technical replicates; error bars, s.d.

Supplementary Figure 9 Yeast one-hybrid analysis indicates that BvMYB1 can bind to upstream regulatory regions of BvDODA1 and BvCYP76AD1.

Sequence upstream of the translation start codons for two betalain structural genes, BvDODA1 and BvCYP76AD1, and a putative phenylpropanoid gene, BvDFR, were fused upstream of the HIS3 reporter gene. The upper panels show that these upstream regions do not activate the reporter in the absence of BvMYB1 expression. The lower panels show that BvMYB1 is able to activate BvDODA1 and BvCYP76AD1 but not BvDFR.

Supplementary Figure 10 Analysis of 35S::BvMYB1-GR in Albina Vereduna hairy roots.

qRT-PCR after 4 hours of treatment for transgenic roots. Results shown as relative gene expression compared to expression in the mock (H20) sample. Bars represent the average of five technical replicates; error bars, s.d. Every experiment was replicated three times.

Source data

Supplementary Figure 11 Relative anthocyanin content of Arabidopsis thaliana expressing MYB constructs versus wild type.

Extracted pigment was quantified by measuring the absorbance at 530 nm. Each bar represents the average of five biological replicates (independent transformants); error bars, s.d.

Supplementary Figure 12 Analysis of anthocyanin gene homolog expression in Albina Vereduna beet roots expressing 35S::AtMYB114.

(a) Semiquantitative RT-PCR of AtMYB114 in roots expressing 35S::GUS versus 35S::AtMYB114. (b) qRT-PCR of BvDFR and BvLDOX in these same lines. Results are shown as relative gene expression compared to expression in the 35S::GUS sample. Every qRT-PCR experiment was replicated three times, and all three biological replicates are shown. Bars represent the average of five technical replicates; error bars, s.d.

Supplementary Figure 13 Y regulatory sequence more strongly induces GUS expression than y in both C869 beet roots and Arabidopsis.

(a) The sequences of the white (y; upper) and red (Y; lower) BvMYB1 regulatory regions used for the analysis in Figure 4 are shown. The sequences are identical for 21 bp upstream of the start codon (in red and blue). At that point, homology ends. Both sequences have approximately 70% AT content. (b,c) GUS expression in comparison of Y::GUS to y::GUS in C869 roots (b) and Arabidopsis (c). Every qRT-PCR experiment was replicated three times, and all three biological replicates are shown. Bars represent the average of five technical replicates; error bars, s.d.

Supplementary Figure 14 Representative example of antibiotic selection of transformed hairy roots.

Each plate contains kanamycin-resistant T-DNA–transformed roots on the left and no-plasmid control hairy root culture on the right. The left plate contains no antibiotic, whereas the plate on the right contains 50 mg/l kanamycin.

Supplementary Figure 15 Complete HPLC data at 530 nm, the wavelength for detecting betanin.

(a) 35S::GUS in Albina Vereduna roots. (b) 35S::BvMYB1 in Albina Vereduna roots. These HPLC data correspond to the mass spectrometry data shown in Figure 1e, f. The betanin peak is at minute 3.01.

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Hatlestad, G., Akhavan, N., Sunnadeniya, R. et al. The beet Y locus encodes an anthocyanin MYB-like protein that activates the betalain red pigment pathway. Nat Genet 47, 92–96 (2015). https://doi.org/10.1038/ng.3163

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