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DkMyb2 wound-induced transcription factor of persimmon (Diospyros kaki Thunb.), contributes to proanthocyanidin regulation

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Abstract

Proanthocyanidins (PAs) are secondary metabolites that contribute to the protection of a plant against biotic and abiotic stresses. Persimmon (Diospyros kaki) accumulates abundant PAs in each plant organ, and some potential Myb-like transcription factors (Myb-TFs) involved in the production of PAs have been isolated. In this study, we aimed to molecularly characterize one of them, DkMyb2, which was placed in a subclade including a PA regulator of Arabidopsis (Arabidopsis thaliana), TRANSPARENT TESTA2 (TT2), and was co-induced with PA pathway genes after wound stress. Ectopic DkMyb2 overexpression caused significant up-regulation of PA pathway genes in transgenic persimmon calluses and significant accumulation of PA, and increased mean degree of polymerization of PAs in transgenic kiwifruit calluses. Analysis of the DNA-binding ability of DkMyb2 by electrophoretic mobility shift assays showed that DkMyb2 directly binds to the AC-rich cis-motifs known as AC elements in the promoters of the two PA pathway genes in persimmon, DkANR, and DkLAR. Furthermore, a transient reporter assay using a dual-luciferase system demonstrated direct transcriptional activation of DkANR and DkLAR by DkMyb2. We also discuss subfunctionalization of two PA regulators in persimmon, DkMyb2 and DkMyb4, as well as PA regulators in other plant species from the viewpoint of their ability to bind to cis-motifs and their functions in transcriptional activation. Our results provide insight into the multiple regulatory mechanisms that control PA metabolism by Myb-TFs in persimmon.

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Abbreviations

ANR:

Anthocyanidin reductase

ANS:

Anthocyanidin synthase

CHS:

Chalcone synthase

DFR:

Dihydroflavonol 4-reductase

F3′H:

Flavanone 3′-hydroxylase

F3′5′H:

Flavanone 3′5′-hydroxylase

LAR:

Leucoanthocyanidin reductase

PAL:

Phenylalanine ammonia-lyase

PA:

Proanthocyanidin

TF:

Transcription factor

References

  • Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell 15:63–78

    Article  CAS  PubMed  Google Scholar 

  • Abrahams S, Tanner GJ, Larkin PJ, Ashton AR (2002) Identification and biochemical characterization of mutants in the proanthocyanidin pathway in Arabidopsis. Plant Physiol 130:561–576

    Article  CAS  PubMed  Google Scholar 

  • Akagi T, Ikegami A, Suzuki Y, Yoshida J, Yamada M, Sato A, Yonemori K (2009a) Expression balances of structural genes in shikimate and flavonoid biosynthesis cause a difference in proanthocyanidin accumulation in persimmon (Diospyros kaki Thunb.) fruit. Planta 230:899–915

    Article  CAS  PubMed  Google Scholar 

  • Akagi T, Ikegami A, Tsujimoto T, Kobayashi S, Sato A, Kono A, Yonemori K (2009b) DkMyb4 is a Myb transcription factor involved in proanthocyanidin biosynthesis in persimmon fruit. Plant Physiol 151:2028–2045

    Article  CAS  PubMed  Google Scholar 

  • Akagi T, Suzuki Y, Ikegami A, Kamitakahara H, Takano T, Nakatsubo F, Yonemori K (2010) Condensed tannin composition analysis in persimmon (Diospyros kaki Thunb.) fruit by acid catalysis in the presence of excess phloroglucinol. J Jpn Soc Hort Sci 79:275–281

    Article  CAS  Google Scholar 

  • Aron PM, Kennedy JA (2008) Flavan-3-ols: nature, occurrence and biological activity. Mol Nutr Food Res 52:79–104

    Article  CAS  PubMed  Google Scholar 

  • Bagchi D, Bagchi M, Stohs SJ, Das DK, Ray SD, Kuszynski CA, Joshi SS, Pruess HG (2000) Free radicals and grape seed proanthocyanidin extract: importance in human health and disease prevention. Toxicology 148:187–197

    Article  CAS  PubMed  Google Scholar 

  • Ban Y, Honda C, Hatsuyama Y, Igarashi M, Bessho H, Moriguchi T (2007) Isolation and functional analysis of a MYB transcription factor gene that is a key regulator for the development of red coloration in apple skin. Plant Cell Physiol 48:958–970

    Article  CAS  PubMed  Google Scholar 

  • Baudry A, Heim MA, Dubreucq B, Caboche M, Weisshaar B, Lepiniec L (2004) TT2, TT8, and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana. Plant J 39:366–380

    Article  CAS  PubMed  Google Scholar 

  • Baxter IR, Young JC, Armstrong G, Foster N, Bogenschutz N, Cordova T, Peer WA, Hazen SP, Murphy AS, Harper JF (2005) A plasma membrane H + -ATPase is required for the formation of proanthocyanidins in the seed coat endothelium of Arabidopsis thaliana. Proc Natl Acad Sci USA 102:2649–2654

    Article  CAS  PubMed  Google Scholar 

  • Bogs J, Jaffé FW, Takos AM, Walker AR, Robinson SP (2007) The grapevine transcription factor VvMYBPA1 regulates proanthocyanidin synthesis during fruit development. Plant Physiol 143:1347–1361

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Debeaujon I, Peeters AJ, Leon-Kloosterziel KM, Koornneef M (2001) The TRANSPARENT TESTA12 gene of Arabidopsis encodes a multidrug secondary transporter-like protein required for flavonoid sequestration in vacuoles of the seed coat endothelium. Plant Cell 13:853–871

    Article  CAS  PubMed  Google Scholar 

  • Debeaujon I, Nesi N, Perez P, Devic M, Grandjean O, Caboche M, Lepiniec L (2003) Proanthocyanidin-accumulating cells in Arabidopsis testa: regulation of differentiation and roll in seed development. Plant Cell 15:2514–2531

    Article  CAS  PubMed  Google Scholar 

  • Deluc L, Bogs J, Walker AR, Ferrier T, Decendit A, Merillon JM, Robinson SP, Barrieu F (2008) The transcription factor VvMYB5b contributes to the regulation of anthocyanin and proanthocyanidin biosynthesis in developing grape berries. Plant Physiol 147:2041–2053

    Article  CAS  PubMed  Google Scholar 

  • Dixon RA, Paiva NL (1995) Stress-induced phenylpropanoid metabolism. Plant Cell 7:1085–1097

    Article  CAS  PubMed  Google Scholar 

  • Dixon RA, Xie D-Y, Sharma SB (2005) Proanthocyanidins—a final frontier in flavonoid research? New Phytol 165:9–28

    Article  CAS  PubMed  Google Scholar 

  • Downey MO, Harvey JS, Robinson SP (2003) Analysis of tannins in seeds and skins of Shiraz grapes throughout berry development. Aust J Grape Wine Res 9:15–27

    Article  CAS  Google Scholar 

  • Espley R, Hellens R, Putterill J, Stevenson D, Kutty-Amma S, Allan A (2007) Red coloration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10. Plant J 49:414–427

    Article  CAS  PubMed  Google Scholar 

  • Harborne JB, Grayer RJ (1993) Flavonoids and insects. In: Harborne JB (ed) The flavonoids: advances in research since 1986. Chapman & Hall, London, pp 589–618

    Google Scholar 

  • Hartmann U, Sagasser M, Mehrtens F, Stracke R, Weisshaar B (2005) Differential combinatorial interactions of cis-acting element recognized by R2R3-MYB, BZIP, and BHLH factors control light-responsive and tissue-specific activation of phenylpropanoid biosynthesis genes. Plant Mol Biol 57:155–171

    Article  CAS  PubMed  Google Scholar 

  • Haslam E (1977) Symmetry and promiscuity in procyanidin biochemistry. Phytochemistry 16:1625–1640

    Article  CAS  Google Scholar 

  • Hatton D, Sablowski R, Yung MH, Smith C, Schuch W, Bevan M (1995) 2 Classes of cis sequences contribute to tissue-specific expression of a pal2 promoter in transgenic tobacco. Plant J 7:859–876

    Article  CAS  PubMed  Google Scholar 

  • Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res 27:297–300

    Article  CAS  PubMed  Google Scholar 

  • Jiang C, Gu X, Peterson T (2004) Identification of conserved gene structures and carboxy-terminal motifs in the Myb gene family of Arabidopsis and Oryza sativa L. ssp. indica. Genome Biol 5:R46

    Article  PubMed  Google Scholar 

  • Kennedy JA, Jones GP (2001) Analysis of proanthocyanidin cleavage products following acid-catalysis in the presence of excess phloroglucinol. J Agric Food Chem 49:1740–1746

    Article  CAS  PubMed  Google Scholar 

  • Kitamura S, Shikazono N, Tanaka A (2004) TRANSPARENT TESTA 19 is involved in the accumulation of both anthocyanins and proanthocyanidins in Arabidopsis. Plant J 37:104–114

    Article  CAS  PubMed  Google Scholar 

  • Koshita Y, Kobayashi S, Ishimaru M, Funamoto Y, Shiraishi M, Azuma A, Yakushikji H, Nakayama M (2008) An anthocyanin regulator from grapes, VlmybA1–2, produces reddish-purple plants. J Jpn Soc Hort Sci 77:33–37

    Article  CAS  Google Scholar 

  • Lees GL (1992) Condensed tannins in some forage legumes: their role in the prevention of ruminant pasture bloat. Basic Life Sci 59:915–934

    CAS  PubMed  Google Scholar 

  • Lepiniec L, Debeaujon I, Routaboul JM, Baudry A, Pourcel L, Nesi N, Caboche M (2006) Genetics and biochemistry of seed flavonids. Annu Rev Plant Biol 57:405–430

    Article  CAS  PubMed  Google Scholar 

  • Leyva A, Liang X, Pintor-Toro JA, Dixon RA, Lamb CJ (1992) Cis-element combinations determine phenylalanine ammonia-lyase gene tissue-specific expression patterns. Plant Cell 4:263–271

    Article  CAS  PubMed  Google Scholar 

  • Li YG, Tanner G, Larkin P (1996) The DMACA-HCl protocol and the threshold proanthocyanidin content for bloat safety in forage legumes. J Sci Food Agric 70:89–101

    Article  CAS  Google Scholar 

  • Matsuo T, Ito S (1978) The chemical structure of kaki-tannin from immature fruit of the persimmon (Diospyros kaki L.). Agric Biol Chem 42:1637–1643

    CAS  Google Scholar 

  • Matsuta N, Iketani H, Hayashi T (1993) Transformation in grape and Kiwifruit. In: Hayashi T, Omura M, Scott NS (eds) Techniques on gene diagnosis and breeding in fruit trees. Fruit Tree Research Station, Tsukuba, pp 184–192

    Google Scholar 

  • Matus JT, Aquea F, Arce-Jhonson P (2008) Analysis of the grape MYB R2R3 subfamily reveals expanded wine quality-related clades and conserved gene structure organization across Vitis and Arabidopsis genomes. BMC Plant Biol 8:83

    Article  PubMed  Google Scholar 

  • McMahon LR, McAllister TA, Berg BP, Majak W, Acharya SN, Popp JD, Coulman BE, Wang Y, Cheng KJ (2000) A review of the effects of forage condensed tannins on ruminal fermentation and bloat in grazing cattle. Can J Plant Sci 80:469–485

    CAS  Google Scholar 

  • Mellway RD, Tran LT, Prouse MB, Campbell MM, Constabel CP (2009) The wound-, pathogen-, and UV-B -responsive MYB134 gene encodes an R2R3 MYB transcription factor that regulates proanthocyanidin synthesis in poplar. Plant Physiol 150:924–941

    Article  CAS  PubMed  Google Scholar 

  • Miranda M, Ralph SG, Mellway R, White R, Heath MC, Bohlmann J, Constabel CP (2007) The transcriptional response of hybrid poplar (Populus trichocarpa × P. deltoides) to infection by Melampsora medusae leaf rust involves induction of flavonoid pathway genes leading to the accumulation of proanthocyanidins. Mol Plant Microbe Interact 20:816–831

    Article  CAS  PubMed  Google Scholar 

  • Nakagawa T, Kurose T, Hino T, Tanaka K, Kawamukai M, Niwa Y, Toyooka K (2007) Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. J Biosci Bioeng 104:34–41

    Article  CAS  PubMed  Google Scholar 

  • Naoumkina MA, He XZ, Dixon RA (2008) Elicitor-induced transcription factors for metabolic reprogramming of secondary metabolism in Medicago truncatula. BMC Plant Biol 8:132

    Article  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Pang Y, Peel GJ, Wright E, Wang Z, Dixon RA (2007) Early steps in proanthocyanidin biosynthesis in the model legume Medicago truncatula. Plant Physiol 145:601–615

    Article  CAS  PubMed  Google Scholar 

  • Pang Y, Peel GJ, Sharma SB, Tang Y, Dixon RA (2008) A transcript profiling approach reveals an epicatechin-specific glucosyltransferase expressed in the seed coat of Medicago truncatula. Proc Natl Acad Sci USA 105:14210–14215

    Article  CAS  PubMed  Google Scholar 

  • Paolocci F, Robbins MP, Madeo L, Arcioni S, Martens S, Damiani F (2007) Ectopic expression of Basic Helix-Loop-Helix gene transactivates parallel pathways of proanthocyanidin biosynthesis. Structure, expression analysis, and genetic control of leucoanthocyanidin 4-reductase and anthocyanidin reductase gene in Lotus corniculatus. Plant Physiol 143:504–516

    Article  CAS  PubMed  Google Scholar 

  • Patzlaff A, McInnis S, Courtenay A, Surman C, Newman LJ, Smith C, Bevan MW, Mansfield S, Whetten RW, Sederoff RR, Campbell MM (2003) Characterisation of a pine MYB that regulates lignification. Plant J 36:743–754

    Article  CAS  PubMed  Google Scholar 

  • Peters DJ, Constabel CP (2002) Molecular analysis of herbivore-induced condensed tannin synthesis: cloning and expression of dihydroflavonol reductase from trembling aspen (Populus tremuloides). Plant J 32:701–712

    Article  CAS  PubMed  Google Scholar 

  • Ramsay NA, Walker AR, Mooney M, Gray JC (2003) Two basic-helix-loop-helix genes (MYC-146 and GL3) from Arabidopsis can activate anthocyanin biosynthesis in a white-flowered Matthiola incana mutant. Plant Mol Biol 52:679–688

    Article  CAS  PubMed  Google Scholar 

  • Rogers LA, Campbell MM (2004) The genetic control of lignin deposition during plant growth and development. New Phytol 164:17–30

    Article  CAS  Google Scholar 

  • Ronquist F, Huelsenbeck JP (2003) MrBayes 3: bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574

    Article  CAS  PubMed  Google Scholar 

  • Sainz MB, Grotewold E, Chandler VL (1997) Evidence for direct activation of an anthocyanin promoter by the maize C1 protein and comparison of DNA binding by related Myb domain proteins. Plant Cell 9:611–625

    Article  CAS  PubMed  Google Scholar 

  • Sharma SB, Dixon RA (2005) Metabolic engineering of proanthocyanidins by ectopic expression of transcription factors in Arabidopsis thaliana. Plant J 44:62–75

    Article  CAS  PubMed  Google Scholar 

  • Shirley BW, Hanley S, Goodman HM (1992) Effects of ionizing radiation on a plant genome: analysis of two Arabidopsis transparent testa mutations. Plant Cell 4:333–347

    Article  CAS  PubMed  Google Scholar 

  • Solano R, Nieto C, Avila J, Canas L, Diaz I, Paz-Ares J (1995) Dual DNA binding specificity of a petal epidermis-specific MYB transcription factor (MYB.Ph3) from Petunia hybrida. EMBO J 14:1773–1784

    CAS  PubMed  Google Scholar 

  • Taira S, Matsumoto N, Ono M (1998) Accumulation of soluble and insoluble tannins during fruit development in nonastringent and astringent persimmon. J Jpn Soc Hort Sci 67:572–576

    Article  CAS  Google Scholar 

  • Takos AM, Jaffe 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

    Article  CAS  PubMed  Google Scholar 

  • Tanner GJ, Francki KT, Abrahams S, Watson JM, Larkin PJ, Ashton AR (2003) Proanthocyanidin biosynthesis in plants. purification of legume leucoanthocyanidin reductase and molecular cloning of its cDNA. J Biol Chem 278:31647–31656

    Article  CAS  PubMed  Google Scholar 

  • Tao R, Dandekar AM, Sandra LU, Vail PV, Tebbets JS (1997) Engineering genetic resistance against insects in Japanese persimmon using the cryIA(c) gene of Bacillus thuringiensis. J Am Soc Hort Sci 122:764–771

    CAS  Google Scholar 

  • Terrier T, 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

    Article  CAS  PubMed  Google Scholar 

  • Urao T, Yamaguchi-Shinozaki K, Urao S, Shinozaki K (1993) An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence. Plant Cell 5:1529–1539

    Article  CAS  PubMed  Google Scholar 

  • Wan CY, Wilkins TA (1994) A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.). Anal Biochem 223:7–12

    Article  CAS  PubMed  Google Scholar 

  • Wei YL, Li JN, Lu J, Tang ZL, Pu DC, Chai YR (2007) Molecular cloning of Brassica napus TRANSPARENT TESTA 2 gene family encoding potential MYB regulatory proteins of proanthocyanidin biosynthesis. Mol Biol Rep 34:105–120

    Article  CAS  PubMed  Google Scholar 

  • Weisshaar B, Jenkins GI (1998) Phenylpropanoid biosynthesis and its regulation. Curr Opin Plant Biol 1:251–257

    Article  CAS  PubMed  Google Scholar 

  • Winkel-Shirley B (2001) Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol 126:485–493

    Article  CAS  PubMed  Google Scholar 

  • Xie D-Y, Dixon RA (2005) Proanthocyanidin biosynthesis—still more questions than answers? Phytochemistry 66:2127–2144

    Article  CAS  PubMed  Google Scholar 

  • Xie D-Y, Sharma SB, Paiva NL, Ferreira D, Dixon RA (2003) Role of anthocyanidin reductase, encoded by BANYULS in plant flavonid biosynthesis. Science 299:396–399

    Article  CAS  PubMed  Google Scholar 

  • Yoshida K, Iwasaka R, Kaneko T, Sato S, Tabata S, Sakuta M (2008) Functional differentiation of Lotus japonicus TT2s, R2R3-MYB transcription factors comprising a multigene family. Plant Cell Physiol 49:157–169

    Article  CAS  PubMed  Google Scholar 

  • Zhao J, Dixon RA (2009) MATE transporters facilitate vacuolar uptake of epicatechin 3′-O-glucoside for proanthocyanidin biosynthesis in Medicago truncatula and Arabidopsis. Plant Cell 21:2323–2340

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Laboratory of Pomology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan

    Takashi Akagi & Keizo Yonemori

  2. Laboratory of Pomology, Department of Bioproduction Sciences, Ishikawa Prefectural University, Nonoichi, Ishikawa, 921-8836, Japan

    Ayako Ikegami

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  1. Takashi Akagi
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  2. Ayako Ikegami
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Correspondence to Keizo Yonemori.

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Akagi, T., Ikegami, A. & Yonemori, K. DkMyb2 wound-induced transcription factor of persimmon (Diospyros kaki Thunb.), contributes to proanthocyanidin regulation. Planta 232, 1045–1059 (2010). https://doi.org/10.1007/s00425-010-1241-7

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