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Functional characterization of the Gentiana lutea zeaxanthin epoxidase (GlZEP) promoter in transgenic tomato plants

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The accumulation of carotenoids in plants depends critically on the spatiotemporal expression profiles of the genes encoding enzymes in the carotenogenic pathway. We cloned and characterized the Gentiana lutea zeaxanthin epoxidase (GlZEP) promoter to determine its role in the regulation of carotenogenesis, because the native gene is expressed at high levels in petals, which contain abundant chromoplasts. We transformed tomato (Solanum lycopersicum cv. Micro-Tom) plants with the gusA gene encoding the reporter enzyme β-glucuronidase (GUS) under the control of the GlZEP promoter, and investigated the reporter expression profile at the mRNA and protein levels. We detected high levels of gusA expression and GUS activity in chromoplast-containing flowers and fruits, but minimal levels in immature fruits containing green chloroplasts, in sepals, leaves, stems and roots. GlZEP-gusA expression was strictly associated with fruit development and chromoplast differentiation, suggesting an evolutionarily-conserved link between ZEP and the differentiation of organelles that store carotenoid pigments. The impact of our results on current models for the regulation of carotenogenesis in plants is discussed.

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  1. Agarwal S, Jha S, Sanyal I, Amla DV (2009) Effect of point mutations in translation initiation context on the expression of recombinant human α1-proteinase inhibitor in transgenic tomato plants. Plant Cell Rep 28:1791–1798

  2. Ahrazem O, Rubio-Moraga A, López RC, Gómez-Gómez L (2010) The expression of a chromoplast-specific lycopene beta cyclase gene is involved in the high production of saffron’s apocarotenoid precursors. J Exp Bot 61:105–119

  3. Alquezar B, Zacarías L, Rodrigo MJ (2009) Molecular and functional characterization of a novel chromoplast-specific lycopene beta-cyclase from Citrus and its relation to lycopene accumulation. J Exp Bot 60:1783–1797

  4. Ampomah-Dwamena C, McGhie T, Wibisono R, Montefiori M, Hellens RP, Allan AC (2009) The kiwifruit lycopene beta-cyclase plays a significant role in carotenoid accumulation in fruit. J Exp Bot 60:3765–3779

  5. Audran C, Borel C, Frey A, Sotta B, Meyer C, Simonneau T, Marion-Poll A (1998) Expression studies of the zeaxanthin epoxidase gene in Nicotiana plumbaginifolia. Plant Physiol 118:1021–1028

  6. Bai C, Twyman RM, Farre G, Sanahuja G, Christou P, Capell T, Zhu C (2011) A golden era–pro-vitamin A enhancement in diverse crops. In Vitro Cell Dev Biol-Plant 47:205–221

  7. Bartley GE, Scolnik PA (1993) cDNA cloning, expression during development, and genome mapping of PSY2, a second tomato gene encoding phytoene synthase. J Biol Chem 268:25718–25721

  8. Bartley GE, Scolnik PA (1995) Plant carotenoids: pigments for photoprotection, visual attraction, and human health. Plant Cell 7:1027–1038

  9. Blas AL, Ming R, Liu Z, Veatch OJ, Paull RE, Moore PH, Yu Q (2010) Cloning of the papaya chromoplast-specific lycopene beta-cyclase, CpCYC-b, controlling fruit flesh color reveals conserved microsynteny and a recombination hot spot. Plant Physiol 152:2013–2022

  10. Bradford MM (1976) Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

  11. Carvalho RF, Campos ML, Pino LE, Crestana SL, Zsögön A, Lima JE, Benedito VA, Peres LE (2011) Convergence of developmental mutants into a single tomato model system: ‘Micro-Tom’ as an effective toolkit for plant development research. Plant Methods 7:18

  12. Corona V, Aracri B, Kosturkova G, Bartley GE, Pitto L, Giorgetti L, Scolnik PA, Giuliano G (1996) Regulation of a carotenoid biosynthesis gene promoter during plant development. Plant J 9:505–512

  13. Creelman RA, Zeevart JA (1984) Incorporation of oxygen into abscisic acid and phaseic acid from molecular oxygen. Plant Physiol 75:166–169

  14. Dalal M, Chinnusamy V, Bansal KC (2010) Isolation and functional characterization of lycopene β-cyclase (CYC-B) promoter from Solanum habrochaites. BMC Plant Biol 10:61

  15. Demmig-Adams B, Adams WW (2002) Antioxidants in photosynthesis and human nutrition. Science 298:2149–2153

  16. Devitt LC, Fanning K, Dietzgen RG, Holton TA (2010) Isolation and functional characterization of a lycopene beta-cyclase gene that controls fruit colour of papaya (Carica papaya L.). J Exp Bot 61:33–39

  17. Egea I, Barsan C, Bian W, Purgatto E, Latché A, Chervin C, Bouzayen M, Pech JC (2010) Chromoplast differentiation: current status and perspectives. Plant Cell Physiol 51:1601–1611

  18. Facella P, Lopez L, Carbone F, Galbraith DW, Giuliano G, Perrotta G (2008) Diurnal and circadian rhythms in the tomato transcriptome and their modulation by cryptochrome photoreceptors. PLoS One 3:e2798

  19. Farre G, Sanahuja G, Naqvi S, Bai C, Capell T, Zhu C, Christou P (2010) Travel advice on the road to carotenoids in plants. Plant Sci 179:28–48

  20. Forth D, Pyke KA (2006) The suffulta mutation in tomato reveals a novel method of plastid replication during fruit ripening. J Exp Bot 57:1971–1979

  21. Frank HA, Cogdell RJ (1996) Carotenoids in photosynthesis. Photochem Photobiol 63:257–264

  22. Galpaz N, Ronen G, Khalfa Z, Zamir D, Hirschberg J (2006) A chromoplast-specific carotenoid biosynthesis pathway is revealed by cloning of the tomato white-flower locus. Plant Cell 18:1947–1960

  23. Giuliano G, Tavazza R, Diretto G, Beyer P, Taylor MA (2008) Metabolic engineering of carotenoid biosynthesis in plants. Trends Biotechnol 26:139–145

  24. Gomez-Roldan V, Fermas S, Brewer PB, Puech-Pagès V, Dun EA, Pillot JP, Letisse F, Matusova R, Danoun S, Portais JC, Bouwmeester H, Bécard G, Beveridge CA, Rameau C, Rochange SF (2008) Strigolactone inhibition of shoot branching. Nature 455:189–194

  25. Goodwin TW (1980) The biochemistry of the carotenoids, vol. 1, 2nd edn. Chapman and Hall, London

  26. Haroldsen VM, Chi-Ham CL, Kulkarni S, Lorence A, Bennett AB (2011) Constitutively expressed DHAR and MDHAR influence fruit, but not foliar ascorbate levels in tomato. Plant Physiol Biochem 49:1244–1249

  27. He Y, Zhu C, Wang D, Kong D, Sun J (2002) Cloning of plastid division gene GlFtsZ from Gentiana lutea and its expression during petal development. Prog Nat Sci 12:592–597

  28. Jefferson AR, Kavanagh TA, Bevan MW (1987) GUS fusion: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

  29. Kloer DP, Schulz GE (2006) Structural and biological aspects of carotenoid cleavage. Cell Mol Life Sci 63:2291–2303

  30. Kosugi S, Ohashi Y, Nakajima K, Arai Y (1990) An improved assay for β-glucuronidase in transformed cells: methanol almost completely suppresses a putative endogenous β-glucuronidase activity. Plant Sci 70:133–140

  31. Lescot M, Dehais P, Moreau Y, De Moor B, Rouze P, Rombauts S (2002) PlantCARE: a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res Database Issue 30:325–327

  32. Li F, Vallabhaneni R, Yu J, Rocheford T, Wurtzel ET (2008) The maize phytoene synthase gene family: overlapping roles for carotenogenesis in endosperm, photomorphogenesis, and thermal stress tolerance. Plant Physiol 147:1334–1346

  33. Li Q, Farre G, Naqvi S, Breitenbach J, Sanahuja G, Bai C, Sandmann G, Capell T, Christou P, Zhu C (2010) Cloning and functional characterization of the maize carotenoid isomerase and β-carotene hydroxylase genes and their regulation during endosperm maturation. Transgen Res 19:1053–1068

  34. Liu F, Vantoai T, Moy LP, Bock G, Linford LD, Quackenbush J (2005) Global transcription profiling reveals comprehensive insights into hypoxic response in Arabidopsis. Plant Physiol 137:1115–1129

  35. Marin E, Nussaume L, Quesada A, Gonneau M, Sotta B, Hugueney P, Frey A, Marion-Poll A (1996) Molecular identification of zeaxanthin epoxidase of Nicotiana plumbaginifolia, a gene involved in abscisic acid biosynthesis and corresponding to the ABA locus of Arabidopsis thaliana. EMBO J 15:2331–2342

  36. Mattanovich D, Rüker F, Machado AC, Laimer M, Regner F, Steinkellner H, Himmler G, Katinger H (1989) Efficient transformation of Agrobacterium spp. by electroporation. Nucleic Acids Res 17:6747

  37. Meissner R, Jacobson Y, Melaned S, Levyatuv S, Shalev G, Ashri A, Elkind Y, Levy A (1997) A new model system for tomato genetics. Plant J 12:1465–1472

  38. Mendes AF, Chen C, Gmitter FG Jr, Moore GA, Costa MG (2011) Expression and phylogenetic analysis of two new lycopene β-cyclases from Citrus paradisi. Physiol Plant 141:1–10

  39. Nilsson L, Müller R, Nielsen TH (2010) Dissecting the plant transcriptome and the regulatory responses to phosphate deprivation. Physiol Plant 139:129–143

  40. Orzaez D, Mirabel S, Wieland WH, Granell A (2006) Agroinjection of tomato fruits. A tool for rapid functional analysis of transgenes directly in fruit. Plant Physiol 140:3–11

  41. Peremarti A, Twyman RM, Gómez-Galera S, Naqvi S, Farré G, Sabalza M, Miralpeix B, Dashevskaya S, Yuan D, Ramessar K, Christou P, Zhu C, Bassie L, Capell T (2010) Promoter diversity in multigene transformation. Plant Mol Biol 73:363–378

  42. Pfitzner AJ (1998) Transformation of tomato. Methods Mol Biol 81:359–363

  43. Ronen G, Carmel-Goren L, Zamir D, Hirschberg J (2000) An alternative pathway to β-carotene formation in plant chromoplasts discovered by map-based cloning of Beta and old-gold color mutations in tomato. Proc Natl Acad Sci USA 97:11102–11107

  44. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Plainview

  45. Scott JW, Harbaugh BK (1989) Micro-Tom—a miniature dwarf tomato. Florida Agric Exp Station Circ 370:1–6

  46. Seo M, Koshiba T (2002) Complex regulation of ABA biosynthesis in plants. Trends Plant Sci 7:41–48

  47. Simkin A, Zhu C, Kuntz M, Sandmann G (2003) Light-dark regulation of carotenoid biosynthesis in pepper (Capsicum annuum) leaves. J Plant Physiol 160:439–443

  48. Thompson AJ, Jackson AC, Parker RA, Morpeth DR, Burbidge A, Taylor IB (2000) Abscisic acid biosynthesis in tomato: regulation of zeaxanthin epoxidase and 9-cis-epoxycarotenoid dioxygenase mRNAs by light/dark cycles, water stress and abscisic acid. Plant Mol Biol 42:833–845

  49. Umehara M, Hanada A, Yoshida S, Akiyama K, Arite T, Takeda-Kamiya N, Magome H, Kamiya Y, Shirasu K, Yoneyama K, Kyozuka J, Yamaguchi S (2008) Inhibition of shoot branching by new terpenoid plant hormones. Nature 455:195–200

  50. Von Lintig J, Vogt K (2004) Vitamin A formation in animals: molecular identification and functional characterization of carotene cleaving enzymes. J Nutr 134:251S–2516S

  51. Von Lintig J, Welsch R, Bonk M, Giuliano G, Batschauer A, Kleinig H (1997) Light dependent regulation of carotenoid biosynthesis occurs at the level of phytoene synthase expression and is mediated by phytochrome in Sinapis alba and Arabidopsis thaliana seedlings. Plant J 12:625–634

  52. Welsch R, Medina J, Giuliano G, Beyer P, von Lintig J (2003) Structural and functional characterization of the phytoene synthase promoter from Arabidopsis. Planta 216:523–534

  53. Welsch R, Maass D, Voegel T, DellaPenna D, Beyer P (2007) Transcription factor RAP2.2 and its interacting partner SINAT2: stable elements in the carotenogenesis of Arabidopsis leaves. Plant Physiol 145:1073–1085

  54. Welsch R, Wust F, Bar C, Al-Babili S, Beyer P (2008) A third phytoene synthase is devoted to abiotic stress-induced abscisic acid formation in rice and defines functional diversification of phytoene synthase genes. Plant Physiol 147:367–380

  55. Zhu C, Yamamura S, Koiwa H, Nishihara M, Sandmann G (2002) cDNA cloning and expression of carotenogenic genes during flower development in Gentiana lutea. Plant Mol Biol 48:277–285

  56. Zhu C, Yamamura S, Nishihara M, Koiwa H, Sandmann G (2003) cDNAs for the synthesis of cyclic carotenoids in petals of Gentiana lutea and their regulation during flower development. Biochim Biophys Acta 1625:305–308

  57. Zhu C, Bai C, Sanahuja G, Yuan D, Farre G, Naqvi S, Shi L, Capell T, Christou P (2010) The regulation of carotenoid pigmentation in flowers. Arch Biochem Biophys 504:132–141

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Our work was supported by grants from the Spanish Ministry of Science and Innovation (MICINN) (BFU2007-61413); European Research Council Advanced Grant (BIOFORCE) to PC; Aciones complementarias, BIO2007-30738-E MICINN, Spain, grants from the National Natural Science Foundation of China (grant nos. 31070269, 30370123, 39970069) and the Programme for Introducing Talents to Universities (B07017).

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Correspondence to Changfu Zhu.

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Qingjie Yang and Dawei Yuan contributed equally to this work.

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Yang, Q., Yuan, D., Shi, L. et al. Functional characterization of the Gentiana lutea zeaxanthin epoxidase (GlZEP) promoter in transgenic tomato plants. Transgenic Res 21, 1043–1056 (2012). https://doi.org/10.1007/s11248-012-9591-5

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  • Gentiana lutea
  • Zeaxanthin epoxidase
  • Promoter
  • β-Glucuronidase
  • Transgenic tomato
  • Carotenoid
  • Chromoplast