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Activation of anthocyanin biosynthesis by expression of the radish R2R3-MYB transcription factor gene RsMYB1

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Abstract

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RsMYB1, a MYB TF of red radish origin, was characterized as a positive regulator to transcriptionally activate the anthocyanin biosynthetic machinery by itself in Arabidopsis and tobacco plants.

Abstract

Anthocyanins, providing the bright red-orange to blue-violet colors, are flavonoid-derived pigments with strong antioxidant activity that have benefits for human health. We isolated RsMYB1, which encodes an R2R3-MYB transcription factor (TF), from red radish plants (Raphanus sativus L.) that accumulate high levels of anthocyanins. RsMYB1 shows higher expression in red radish than in common white radish, in both leaves and roots, at different growth stages. Consistent with RsMYB1 function as an anthocyanin-promoting TF, red radishes showed higher expression of all six anthocyanin biosynthetic and two anthocyanin regulatory genes. Transient expression of RsMYB1 in tobacco showed that RsMYB1 is a positive regulator of anthocyanin production with better efficiency than the basic helix-loop-helix (bHLH) TF gene B-Peru. Also, the synergistic effect of RsMYB1 with B-Peru was larger than the effect of the MYB TF gene mPAP1D  with B-peru.  Arabidopsis plants stably expressing RsMYB1 produced red pigmentation throughout the plant, accompanied by up-regulation of the six structural and two regulatory genes for anthocyanin production. This broad transcriptional activation of anthocyanin biosynthetic machinery in Arabidopsis included up-regulation of TRANSPARENT TESTA8, which encodes a bHLH TF. These results suggest that overexpression of RsMYB1 promotes anthocyanin production by triggering the expression of endogenous bHLH genes as potential binding partners for RsMYB1. In addition, RsMYB1-overexpressing Arabidopsis plants had a higher antioxidant capacity than did non-transgenic control plants. Taken together, RsMYB1 is an actively positive regulator for anthocyanins biosynthesis in radish plants and it might be one of the best targets for anthocyanin production by single gene manipulation being applicable in diverse plant species.

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Abbreviations

4CL:

4-coumarate-CoA ligase

bHLH:

basic helix-loop-helix

ANS:

anthocyanidin synthase

CHI:

chalcone isomerase

CHS:

chalcone synthase

DFR:

dihydroflavonol 4-reductase

F3H:

flavanone 3-hydroxylase

F3′H:

flavonoid 3′-hydroxylase

PAL:

phenylalanine ammonia lyase

qRT-PCR:

quantitative real time polymerase chain reaction

TF:

transcription factor

TT2:

TRANSPARENT TESTA2

TT8:

TRANSPARENT TESTA8

TTG1:

TRANSPARENT TESTA GLABRA1

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Acknowledgments

This work was supported by a fund from the National Academy of Agricultural Science (PJ01002701) and a grant from the Next-Generation BioGreen 21 Program (PJ01109402), Rural Development Administration, Republic of Korea.

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Authors and Affiliations

  1. National Academy of Agricultural Science, Rural Development Administration, Jeonju, 54874, Republic of Korea

    Sun-Hyung Lim, Ji-Hye Song, Da-Hye Kim, Jong-Yeol Lee & Young-Mi Kim

  2. Division of Life Sciences and Bio-Resource and Environmental Center, Incheon National University, Incheon, 22012, Republic of Korea

    Jae Kwang Kim

  3. Department of Genetic Engineering and Graduate School of Biotechnology, Kyung Hee University, Yongin, 17104, Republic of Korea

    Sun-Hwa Ha

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  1. Sun-Hyung Lim
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  2. Ji-Hye Song
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Correspondence to Sun-Hyung Lim or Sun-Hwa Ha.

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Communicated by Y.-I. Park.

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299_2015_1909_MOESM1_ESM.tif

Supplementary Figure legends, Figure 1. The genomic structure of radish RsMYB1 and multiple alignment of the amino acid sequences among R2R3-MYBs. (A) Schematic representation of the structure of RsMYB1. The black boxes represent exons and solid lines represent introns. (B) Multiple sequence alignment of the R2R3-MYB transcription factors related to anthocyanin production that were used in the neighbor-joining phylogenetic tree (Fig. 1). Arrows indicate specific residues that contribute to a motif implicated in the interaction with the bHLH co-factor in Arabidopsis. The red box and black box represent the conserved ANDV motif and the C-terminal-conserved KPRPR[S/T]F motif of anthocyanin-promoting MYBs, respectively. Numbers on the right denote the number of amino acid residues. Residues identical for all the sequences in a given position are in white text on a black background and residues identical for at least over 80 % in a given position are presented in white text on a dark grey background and residues identical ranged from 60 to 80 % in given position are presented in white text on a light gray background. The following sequences were included in the analysis: AmROSEA1 (ABB83826) and AmVENOSA (ABB83828) in Antirrhinum majus, AtPAP1 (NP_176057) and AtPAP2 (NP_176813) in Arabidopsis, BoMYB2 (ADP76650) in Brassica oleracea, GhMYB10 (CAD87010) in Gerbera hybrida, LeANT1 (AAQ55181) in Lycopersicon esculentum, MdMYB10 (ABB84753) in Malus  ×  domestica, OsC1 (CAA75509) in Oryza sativa, PhAN2 (AAF66727) in Petunia  ×  hybrida, RsMYB1 (KR706195) in Raphanus sativus and ZmPl (AAA19821) and ZmC1 (P10290) in Zea mays (TIFF 1926 kb)

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Lim, SH., Song, JH., Kim, DH. et al. Activation of anthocyanin biosynthesis by expression of the radish R2R3-MYB transcription factor gene RsMYB1 . Plant Cell Rep 35, 641–653 (2016). https://doi.org/10.1007/s00299-015-1909-3

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  • DOI: https://doi.org/10.1007/s00299-015-1909-3

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