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Isolation and characterization of a novel anthocyanin-promoting MYBA gene family in Citrus

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MYB transcription factors play an important role in the activation and regulation of anthocyanin biosynthesis. Many such transcription factor genes from various plant species were well characterized at the molecular level. However, none were reported in Citrus. Here, we demonstrated the isolation, molecular characterization, and functional evaluation of a large number of Citrus genes belonging to a previously unidentified MYBA gene family. All these genes were recovered from up to nine genotypes in the genus Citrus by PCR amplification using primers corresponding to VvMybA1 of grapevine (Vitis vinifera). Among the 56 genes analyzed, 35 genes contained full-length coding sequences and showed a 98.3% to 100% amino acid sequence homology with VvMybA1 (referred to as MybA1 genes), whereas 21 genes contained a 209-bp deletion in the exon-3 region similar to a related mutant gene VvMybA3 of grapevine. Citrus MybA1genes are composed of three exons ranging in sizes from 115, 130, to 508 bp and two introns of 87 and 119 bp, respectively, and encode a polypeptide of 250 residues. Unique single-residue substitution mutations allowed the categorization of these genes into three groups. Transient transgene expression analysis using grapevine somatic embryos revealed that genes from group a failed to induce anthocyanin production. Many members from groups b and c were fully functional and capable of activating anthocyanin biosynthesis in non-anthocyanic explants. Reversion mutagenesis experiments indicate that an R93K substitution unique to group a genes is the single loss-of-function mutation. This study confirmed, for the first time, the existence of highly homologous regulatory genes involved in the anthocyanin pathway among taxonomically distant species.

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  1. Acinas SG, Sarma-Rupavtarm R, Klepac-Ceraj V, Polz MF (2005) PCR-induced sequence artifacts and bias: insights from comparison of two 16S rRNA clone libraries constructed from the same sample. App Eng Microbiol 71:8966–8969

  2. Allan AC, Hellens RP, Laing WA (2008) MYB transcription factors that colour our fruit. Trends Plant Sci 13:99–102

  3. Azuma A, Kobayashi S, Mitani N, Shiraishi M, Yamada M, Ueno T, Kono A, Yakushiji H, Koshita Y (2008) Genomic and genetic analysis of Myb-related genes that regulate anthocyanin biosynthesis in grape berry skin. Theor Appl Genet 117:1009–1019

  4. Batie SS, Ervin DE (2001) Transgenic crops and the environment: missing markets and public roles. Environ Dev Econ 6:435–457

  5. Boss PK, Davies C, Robinson SP (1996) Expression of anthocyanin biosynthesis pathway genes in red and white grapes. Plant Mol Biol 32:565–569

  6. Chandler BV (1958) Anthocyanin of blood oranges. Nature 182:933

  7. Cultrone A, Cotroneo PS, Recupero GR (2010) Cloning and molecular characterization of R2R3-MYB and bHLH_MYC transcription factors from Citrus sinensis. Tree Genetics & Genomes 6:101–112

  8. Cutanda-Perez MC, Ageorges A, Gomez C, Vialet S, Terrier N, Romieu C, Torregrosa L (2009) Ectopic expression of VlmybA1 in grapevine activates a narrow set of genes involved in anthocyanin synthesis and transport. Plant Mol Biol 69:633–648

  9. Dombrecht B, Xue GP, Sprague SJ, Kirkegaard JA, Ross JJ, Reid JB, Fitt GP, Sewelam N, Schenk PM, Manners JM, Kazan K (2007) MYC2 differentially modulates diverse jasmonate-dependant functions in Arabidopsis. Plant Cell 19:2225–2245

  10. Dubos C, Stracke R, Grotewold E, Weisshaar B, Martin C, Lepiniec L (2010) MYB transcription factors in Arabidopsis. Trends Plant Sci 15:573–581

  11. Espley RV, Brendolise C, Chagné D, Kutty-Amma S, Green S, Volz R, Putterill 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

  12. Fournier-Level A, Lacombe T, Le Cunff L, Boursiquot JM, This P (2010) Evolution of the VvMYbA gene family, the major determinant of berry colour in cultivated grapevine (Vitis vinifera L.). Heredity 104:351–362

  13. Gallivan JP, Dougherty DA (1999) Cation- interactions in structural biology. PNAS 96:9459–9464

  14. Gould K (2004) Nature’s Swiss army knife: the diverse protective roles of anthocyanins in leaves. J Biomed Biotech 5:314–320

  15. Grotewold (2006) The genetics and biochemistry of floral pigments. Annu Rev Plant Biol 57:761–780

  16. Hatier JB, Gould K (2009) Anthocyanin function in vegetative organs. In: Gould K, Davies K, Winefield C (eds) Anthocyanins: biosynthesis, functions, and applications. Springer, New York, pp 1–20

  17. Jin H, Martin C (1999) Multifunctionality and diversity within the plant MYB-gene family. Plant Mol Biol 41:577–585

  18. Kobayashi S, Goto-Yamamoto N, Hirochika H (2004) Retrotransposon-induced mutations in grape skin color. Science 304:982

  19. Kortstee AJ, Khan SA, Helderman C, Trindade LM, Wu Y, Visser RGF, Allan A, Schouten HJ, Jacobsen E (2011) Anthocyanin production as a potential visual selection marker during plant transformation. Transgenic Res 20:1253–1264

  20. Kui LW, 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

  21. Li Z, Jayasankar S, Gray DJ (2001) Expression of a bifunctional green fluorescent protein (GFP) fusion marker under the control of three constitutive promoters and enhanced derivatives in transgenic grape (Vitis vinifera). Plant Sci 160:877–887

  22. Li Z, Jayasankar S, Gray DJ (2004) Bi-directional duplex promoters with duplicated enhancers significantly increase transgene expression in grape and tobacco. Transgenic Res 13:143–154

  23. Li ZT, Dhekney SA, Dutt M, Gray DJ (2008) An improved protocol for agrobacterium-mediated transformation of grapevine (Vitis vinifera L.). Plant Cell Tissue Organ Cult 93:311–321

  24. Li ZT, Dhekney SA, Gray DJ (2011a) Use of the VvMybA1 gene for non-destructive quantification of promoter activity via color histogram analysis in grapevine (Vitis vinifera) and tobacco. Transgenic Res 20:1087–1097

  25. Li ZT, Dhekney SA, Gray DJ (2011b) PR-1 gene family of grapevine: a uniquely duplicated PR-1 gene from a Vitis interspecific hybrid confers high level resistance to bacterial disease in transgenic tobacco. Plant Cell Rep 30:1–11

  26. Lo Piero AR, Consoli A, Puglisi I, Orestabo G, Reforgiato Recupero G, Petrone G (2005) Anthocyaninless cultivars of sweet orange lack to express the UDP-glucose flavonoid 3-O- Glucosyl transferase. J Plant Biochem Biotech 14:1–6

  27. Martin C, Paz-Ares J (1997) MYB transcription factors in plants. Trends Genet 13:67–73

  28. Matus TJ, Aquea F, Arce-Johnson 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

  29. 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

  30. Ogata K, Hijo H, Aimoto S, Nakai T, Nakamuta H, Sarai A, Ishii S, Nishimuta Y (1992) Solution structure of a DNA-binding unit of Myb: a helix-turn-helix-related motif with conserved tryptophans forming a hydrophobic core. PNAS 89:6428–6432

  31. Pires N, Dolan L (2010) Origin and diversification of basic-helix-loop-helix proteins in plants. Mol Biol Evol 27:862–874

  32. Rommens CM, Haring MA, Swords K, Davies HV, Belknap WR (2007) The intragenic approach as a new extension to traditional plant breeding. Trends Plant Sci 12:397–403

  33. Schwinn K, Venail J, Shang Y, Mackay S, Alm V, Butelli E, Oyama R, Bailey P, Davies K, Martin C (2006) A small family of MYBregulatory genes controls floral pigmentation intensity and patterning in the genus Antirrhinum. Plant Cell 18:831–851

  34. Stracke R, Werber M, Weisshaar B (2001) Th7e R2R3-MYB gene family in Arabidopsis thaliana. Curr Opin Plant Biol 4:447–456

  35. Talon M, Gmitter FG Jr (2008) Citrus genomics. Int J Plant Genom 2008:528361

  36. 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

  37. 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

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

  39. Winkel-Shirley B (2006) The biosynthesis of flavonoids. In: Grotewold E (ed) The science of flavonoids. Springer Science & Business Media, New York, pp 71–95

  40. Yadav V, Mallappa C, Gangappa SN, Bhatia S, Chattopadhyay S (2005) A basic helix-loop-helix transcription factor in Arabidopsis, MYC2, acts as a repressor of blue light-mediated photomorphogenic growth. Plant Cell 17:1953–1966

  41. Zarba AS, Pulvirenti G (2006) The consumption of Sicilian red oranges: implications for firms involved in commercialization. J Bus Chem 3:22–41

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This research was supported in part by the Florida Citrus Production Research Advisory Council, the Florida Agricultural Experiment Station and the Florida Department of Agriculture, and Consumer Services’ Viticulture Trust Fund. The authors gratefully acknowledge Drs. Qibin Yu and Xiuli Shen of CREC and Mr. Matthew Creech and Matthew McKinley, Ms. Angela Adamitis, Natalia Cimino, and Marlene Saldivar of MREC for their excellent technical support.

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Correspondence to Dennis J. Gray.

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Communicated by A. Dandekar

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Li, Z.T., Gmitter, F.G., Grosser, J.W. et al. Isolation and characterization of a novel anthocyanin-promoting MYBA gene family in Citrus . Tree Genetics & Genomes 8, 675–685 (2012). https://doi.org/10.1007/s11295-011-0455-6

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  • Anthocyanin biosynthesis
  • MYB transcription factor
  • Genetic diversity