Abstract
A complex of R2R3-MYB and bHLH transcription factors, stabilized by WD40 repeat proteins, regulates gene transcription for plant cell pigmentation and epidermal cell morphology. It is the MYB component of this complex which specifies promoter target activation. The Arabidopsis MYB TT2 regulates proanthocyanidin (PA) biosynthesis by activating the expression of ANR (anthocyanidin reductase), the gene product of which catalyzes the first committed step of this pathway. Conversely the closely related MYB PAP4 (AtMYB114) regulates the anthocyanin pathway and specifically activates UFGT (UDP-glucose:flavonoid-3-O-glucosyltransferase), encoding the first enzyme of the anthocyanin pathway. Both at the level of structural and regulatory genes, evolution of PA biosynthesis proceeded anthocyanin biosynthesis and we have identified key residues in these MYB transcription factors for the evolution of target promoter specificity. Using chimeric and point mutated variants of TT2 and PAP4 we found that exchange of a single amino acid, Gly/Arg39 in the R2 domain combined with an exchange of a four amino acid motif in the R3 domain, could swap the pathway selection of TT2 and PAP4, thereby converting in planta specificity of the PA towards the anthocyanin pathway and vice versa. The general importance of these amino acids for target specificity was also shown for the grapevine transcription factors VvMYBPA2 and VvMYBA2 which regulate PAs and anthocyanins, respectively. These results provide an insight into the evolution of the different flavonoid regulators from a common ancestral gene.
Similar content being viewed by others
References
Albert S, Delseny M, Devic M (1997) BANYULS, a novel negative regulator of flavonoid biosynthesis in the Arabidopsis seed coat. Plant J 11:289–299
Bogs J, Jaffe FW, Takos AM, Walker AR, Robinson SP (2007) The grapevine transcription factor VvMYBPA1 regulates proanthocyanidin synthesis during fruit development. Plant Physiol 143:1347–1361
Borevitz JO, Xia Y, Blount J, Dixon RA, Lamb C (2000) Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. Plant Cell 12:2383–2394
Bradshaw HD, Schemske DW (2003) Allele substitution at a flower colour locus produces a pollinator shift in monkeyflowers. Nature 426:176–178
Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD (2003) Multiple sequence alignment with the Clustal series of programs. Nucl Acids Res 31:3497–3500
Czemmel S, Stracke R, Weisshaar B, Cordon N, Harris NN, Walker AR, Robinson SP, Bogs J (2009) The grapevine R2R3-MYB transcription factor VvMYBF1 regulates flavonol synthesis in developing grape berries. Plant Physiol 151:1513–1530
Dare AP, Schaffer RJ, Lin-Wang K, Allan AC, Hellens RP (2008) Identification of a cis-regulatory element by transient analysis of co-ordinately regulated genes. Plant Methods 4:17
Debeaujon I, Nesi N, Perez P, Devic M, Grandjean O, Caboche C, Lepiniec L (2003) Proanthocyanidin-accumulating cells in Arabidopsis testa: regulation of differentiation and role in seed development. Plant Cell 15:2514–2531
Espley RV, Hellens RP, Putterill J, Stevenson DE, Kutty-Amma S, Allan AC (2007) Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10. Plant J 49:414–427
Espley RV, Brendolise C, Chagné D, Kutty-Amma S, Green S, Volz R, Putteril J, Schouten HJ, Gardiner SE, Hellens RP, Allan AC (2009) Multiple repeats of a promoter segment causes transcription factor autoregulation in red apples. Plant Cell 21:168–183
Foresti O, daSilva LLP, Denecke J (2006) Overexpression of the Arabidopsis syntaxin PEP12/SYP21 inhibits transport from the prevacuolar compartment to the lytic vacuole in vivo. Plant Cell 18:2275–2293
Frampton J, Gibson TJ, Ness SA, Doderlein G, Graf T (1991) Proposed structure for the DNA-binding domain of the MYB oncoprotein based on model building and mutational analysis. Protein Eng 4:891–901
Gabrielsen O, Sentenac A, Fromageot P (1991) Specific DNA binding by c-MYB: evidence for a double helix-turn-helix-related motif. Science 253:1140–1143
Gleave AP (1992) A versatile binary vector system with a T-DNA organizational- structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol Biol 20:1203–1207
Gonzalez A, Zhao M, Leavitt JM, Lloyd AM (2008) Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant J 53:814–827
Grotewold E, Drummond BJ, Bowen B, Peterson T (1994) The myb-homologous P gene controls phlobaphene pigmentation in maize floral organs by directly activating a flavonoid biosynthetic gene subset. Cell 76:543–553
Hichri I, Deluc L, Barrieu F, Bogs J, Mahjoub A, Regad F, Gallois B, Granier T, Trossat-Magnin C, Gomes E, Lauvergeat V (2011) A single amino acid change within the R2 domain of the VvMYB5b transcription factor modulates affinity for protein partners and target promoters selectivity. BMC Plant Biol 11:117
Horstmann V, Huether CM, Jost W, Reski R, Decker EL (2004) Quantitative promoter analysis in Physcomitrella patens: a set of plant vectors activating gene expression within three orders of magnitude. BMC Biotechnol 4:13
Hsu F-C, Wirtz M, Heppel SC, Bogs J, Krämer U, Khan MS, Bub A, Hell R, Rausch T (2011) Generation of Se-fortified broccoli as functional food: impact of Se fertilization on S metabolism. Plant, Cell Environ 34:192–207
Jia L, Clegg MT, Jiang T (2003) Excess non-synonymous substitutions suggest that positive selection episodes occurred during the evolution of DNA-binding domains in the Arabidopsis R2R3-MYB gene family. Plant Mol Biol 52:627–642
Kobayashi S, Goto-Yamamoto N, Hirochika H (2004) Retrotransposon-induced mutations in grape skin color. Science 304:982
Koes RE, Quattrocchio F, Mol JNM (1994) The flavonoid biosynthetic pathway in plants: function and evolution. BioEssays 16:123–132
Lin-Wang K, Bolitho K, Grafton K, Kortstee A, Karunairetnam S, McGhie TK, Espley RV, Hellens RP, Allan AC (2010) An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in rosaceae. BMC Plant Biol 10:50
Mehrtens F, Kranz H, Bednarek P, Weisshaar B (2005) The Arabidopsis transcription factor MYB12 is a flavonol-specific regulator of phenylpropanoid biosynthesis. Plant Physiol 138:1083–1096
Nesi N, Debeaujon I, Jond C, Pelletier G, Caboche M, Lepiniec L (2000) The TT8 gene encodes a basic helix-loop-helix domain protein required for expression of DFR and BAN genes in Arabidopsis siliques. Plant Cell 12:1863–1878
Nesi N, Jond C, Debeaujon I, Caboche M, Lepiniec L (2001) The Arabidopsis TT2 gene encodes an R2R3 MYB domain protein that acts as a key determinant for proanthocyanidin accumulation in developing seed. Plant Cell 13:2099–2114
Ogata K, Morikawa S, Nakamura H, Sekikawa A, Inoue T, Kanai H, Sarai A, Ishii S, Nishimura Y (1994) Solution structure of a specific DNA complex of the MYB DNA-binding domain with cooperative recognition helices. Cell 79:639–648
Ogata K, Kanei-Ishii C, Sasaki M, Hatanaka H, Nagadoi A, Enari M, Nakamura H, Nishimura Y, Ishii S, Akinori S (1996) The cavity in the hydrophobic core of MYB DNA-binding domain is reserved for DNA recognition and trans-activation. Nat Struct Biol 3:178–187
Paz-Ares J, Ghosal D, Wienand U, Peterson PA, Saedler H (1987) The regulatory c1 locus of Zea mays encodes a protein with homology to MYB proto-oncogene products and with structural similarities to transcriptional activators. EMBO J 6:3553–3558
Quattrocchio F, Wing JF, van der Woude K, Mol JN, Koes R (1998) Analysis of bHLH and MYB domain proteins: species-specific regulatory differences are caused by divergent evolution of target anthocyanin genes. Plant J 13:475–488
Saikumar P, Murali R, Reddy EP (1990) Role of tryptophan repeats and flanking amino acids in MYB-DNA interactions. Proc Natl Acad Sci USA 87:8452–8456
Solano R, Fuertes A, Sánchez-Pulido L, Valencia A, Paz-Ares J (1997) A single residue substitution causes a switch from the dual DNA binding specificity of plant transcription factor MYB.Ph3 to the animal c-MYB specificity. J Biol Chem 272:2889–2895
Stafford HA (1991) Flavonoid evolution: an enzymic approach. Plant Physiol 96:680–685
Stracke R, Werber M, Weisshaar B (2001) The R2R3-MYB gene family in Arabidopsis thaliana. Curr Opin Plant Biol 4:447–456
Takos AM, Jaffé FW, Jacob SR, Bogs J, Robinson SP, Walker AR (2006) Light-induced expression of a MYB gene regulates anthocyanin biosynthesis in red apples. Plant Physiol 142:1216–1232
Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599
Tanikawa J, Yasukawa T, Enari M, Ogata K, Nishimura Y, Ishii S, Sarai A (1993) Recognition of specific DNA sequences by the c-MYB protooncogene product: role of three repeat units in the DNA-binding domain. Proc Natl Acad Sci USA 90:9320–9324
Terrier N, Torregrosa L, Ageorges A, Vialet S, Verries C, Cheynier V, Romieu C (2009) Ectopic expression of VvMYBPA2 promotes proanthocyanidin biosynthesis in grapevine and suggests additional targets in the pathway. Plant Physiol 149:1028–1041
Thévenin J, Dubos C, Xu W, Gourrierec JL, Kelemen Z, Charlot F, Nogué F, Lepiniec L, Dubreucq B (2012) A new system for fast and quantitative analysis of heterologous gene expression in plants. New Phytol 193:504–512
Tominaga R, Iwata M, Okada K, Wad T (2007) Functional analysis of the epidermal-specific MYB genes CAPRICE and WEREWOLF in Arabidopsis. Plant Cell 19:2264–2277
Tominaga-Wada R, Nukumizu Y, Wada T (2012) Amino acid substitution converts WEREWOLF function from an activator to a repressor of Arabidopsis non-hair cell development. Plant Sci 183:37–42
Walker AR, Davison PA, Bolognesi-Winfield AC, James CM, Srinivasan N, Blundell TL, Esch JJ, Marks MD, Gray JC (1999) The TRANSPARENT TESTA GLABRA1 locus, which regulates trichome differentiation and anthocyanin biosynthesis in Arabidopsis, encodes a WD40 repeat protein. Plant Cell 11:1337–1350
Walker AR, Lee E, Bogs J, McDavid DAJ, Thomas MR, Robinson SP (2007) White grapes arose through the mutation of two similar and adjacent regulatory genes. Plant J 49:772–785
Williams CE, Grotewold E (1997) Differences between plant and animal MYB domains are fundamental for DNA binding activity, and chimeric MYB domains have novel DNA binding specificities. J Biol Chem 272:563–571
Winkel-Shirley B (2001) Flavonoid biosynthesis: a colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol 126:485–493
Xie D-Y, Sharma SB, Paiva NL, Ferreira D, Dixon RA (2003) Role of anthocyanidin reductase, encoded by BANYULS in plant flavonoid biosynthesis. Science 299:396–399
Zhang F, Gonzalez A, Zhao M, Payne CT, Lloyd A (2003) A network of redundant bHLH proteins functions in all TTG1-dependent pathways of Arabidopsis. Development 130:4859–4869
Zimmermann IM, Heim MA, Weisshaar B, Uhrig JF (2004) Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B-like bHLH proteins. Plant J 40:22–34
Acknowledgments
We thank Lauren Hooper, Karin Sefton, Monika Kupke and Michael Bartoschek for excellent technical assistance. Arabidopsis seeds were supplied by the ABRC, Ohio. This work was supported by a Grant (AH01015: Key Genes for Horticultural Markets) from Horticulture Australia Ltd, CSIRO Plant Industry and the Landesgraduiertenkolleg Baden-Württemberg, Germany.
Author information
Authors and Affiliations
Corresponding author
Additional information
Simon C. Heppel and Felix W. Jaffé contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Heppel, S.C., Jaffé, F.W., Takos, A.M. et al. Identification of key amino acids for the evolution of promoter target specificity of anthocyanin and proanthocyanidin regulating MYB factors. Plant Mol Biol 82, 457–471 (2013). https://doi.org/10.1007/s11103-013-0074-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11103-013-0074-8