Advertisement

Isolation and characterization of a stress-inducible Dunaliella salina Lcy-β gene encoding a functional lycopene β-cyclase

  • Ana Ramos
  • Sacha Coesel
  • Ana Marques
  • Marta Rodrigues
  • Alexandra Baumgartner
  • João Noronha
  • Amélia Rauter
  • Bertram Brenig
  • João VarelaEmail author
Applied Genetics and Molecular Biotechnology

Abstract

The halotolerant green alga Dunaliella salina accumulates large amounts of β-carotene when exposed to various stress conditions. Although several studies concerning accumulation and biotechnological production of β-carotene have been published, the molecular basis and regulation of the genes involved in carotenoid biosynthesis in D. salina are still poorly known. In this paper, we report the isolation and regulation of the lycopene β-cyclase (Lcy-β) gene by abiotic stress. The function of this gene was determined by heterologous genetic complementation in E. coli. Gene expression and physiological analyses revealed that D. salina Lcy-β steady-state transcript and carotenoid levels were up-regulated in response to all stress conditions tested (salt, light and nutrient depletion). The results presented here suggest that nutrient availability is a key factor influencing carotenogenesis as well as carotenoid biosynthesis-related gene expression in D. salina.

Keywords

Carotenoid biosynthesis Dunaliella salina Lycopene β-cyclase Gene expression Stress response regulation 

Notes

Acknowledgements

We thank Dr. F. X. Cunningham, University of Maryland, for providing the complementation plasmid pAC-LCY. Ana Ramos and Sacha Coesel were supported by the Fundação para a Ciência e a Tecnologia, Portugal, with the studentships SFRH/BD/13937/2003 and SFRH/BD/4839/2001, respectively. This work was financed by OVERCAROTEN POCTI/MAR/15237/99 and INTERREG 159-SAL—Atlantic Salt Ponds.

References

  1. Almeida ERA, Cerdá-Olmedo E (2008) Gene expression in the regulation of carotene biosynthesis in Phycomyces. Curr Genet 53:129–137CrossRefGoogle Scholar
  2. Ben-Amotz A (1987) Effect of irradiance and nutrient deficiency on the chemical composition of Dunaliella bardawil Ben-Amotz and Avron (Volvocales, Chlorophyta). J Plant Physiol 131:479–487Google Scholar
  3. Ben-Amotz A, Avron M (1983) On the factors which determine massive β-carotene accumulation in the halotolerant alga Dunaliella bardawil. Plant Physiol 72:593–597Google Scholar
  4. Ben-Amotz A, Katz A, Avron M (1982) Accumulation of β-carotene in halotolerant algae: purification and characterization of β-carotene-rich globules from Dunaliella bardawil (Chlorophyceae). J Phycol 18:529–537CrossRefGoogle Scholar
  5. Bohne F, Linden H (2002) Regulation of carotenoid biosynthesis genes in response to light in Chlamydomonas reinhardtii. Biochim Biophys Acta 1579:26–34Google Scholar
  6. Borowitzka MA, Borowitzka LJ, Kessly D (1990) Effects of salinity increase on carotenoid accumulation in the green alga Dunaliella salina. J Appl Phycol 2:111–119CrossRefGoogle Scholar
  7. Bouvier F, Backhaus RA, Camara B (1998) Induction and control of chromoplast-specific carotenoid genes by oxidative stress. J Biol Chem 273:30651–30659CrossRefGoogle Scholar
  8. Bramley PM (2002) Regulation of carotenoid formation during tomato fruit ripening and development. J Exp Bot 53:2107–2113CrossRefGoogle Scholar
  9. Brown JS (1986) A catalogue of splice junction and putative branch point sequences from plant introns. Nucleic Acids Res 14:9549–9559CrossRefGoogle Scholar
  10. Cifuentes AS, González MA, Parra OO (1996) The effect of salinity on the growth and carotenogenesis in two Chilean strains of Dunaliella salina Teodoresco. Biol Res 29:227–236Google Scholar
  11. Coesel SN, Baumgartner AC, Teles LM, Ramos AA, Henriques NM, Cancela L, Varela JCS (2008) Nutrient limitation is the main regulatory factor for carotenoid accumulation and for Psy and Pds steady state transcript levels in Dunaliella salina (Chlorophyta) exposed to high light and salt stress. Marine Biotechnol. DOI  10.1007/s10126-008-9100-2
  12. Cunningham FX Jr (2002) Regulation of carotenoid synthesis and accumulation in plants. Pure Appl Chem 74:1409–1417CrossRefGoogle Scholar
  13. Cunningham FX, Sun Z, Chamovitz D, Hirschberg J, Gantt E (1994) Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942. Plant Cell 6:1107–1121CrossRefGoogle Scholar
  14. Del Campo JA, García-González M, Guerrero MG (2007) Outdoor cultivation of microalgae for carotenoid production: current state and perspectives. Appl Microbiol Biotechnol 74:1163–1174CrossRefGoogle Scholar
  15. Diretto G, Welsch R, Tavazza R, Mourgues F, Pizzichini D, Beyer P, Giuliano G (2007) Silencing of beta-carotene hydroxylase increases total carotenoid and beta-carotene levels in potato tubers. BMC Plant Biol 7:11CrossRefGoogle Scholar
  16. Emanuelsson O, Nielsen H, von Heijne G (1999) ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci 8:978–984Google Scholar
  17. Giuliano G, Bartley GE, Scolnik PA (1993) Regulation of carotenoid biosynthesis during tomato development. Plant Cell 5:379–387CrossRefGoogle Scholar
  18. Goodwin TW (1980) The biochemistry of the carotenoids. Plants, vol. 1. Chapman and Hall, LondonGoogle Scholar
  19. Grünewald K, Eckert M, Hirschberg J, Hagen C (2000) Phytoene desaturase is localized exclusively in the chloroplast and up-regulated at the mRNA level during accumulation of secondary carotenoids in Haematococcus pluvialis (Volvocales, Chlorophyceae). Plant Physiol 122:1261–1268CrossRefGoogle Scholar
  20. Krubasik P, Sandmann G (2000) Molecular evolution of lycopene cyclases involved in the formation of carotenoids with ionone end groups. Biochem Soc Trans 28:806–810CrossRefGoogle Scholar
  21. León-Bañares R, González-Ballester D, Galván A, Fernández E (2004) Transgenic microalgae as green cell-factories. Trends Biotechnol 22:45–52CrossRefGoogle Scholar
  22. Lers A, Biener Y, Zamir A (1990) Photoinduction of massive beta-carotene accumulation by the alga Dunaliella bardawil: kinetics and dependence on gene activation. Plant Physiol 93:389–395Google Scholar
  23. Loeblich LA (1982) Photosynthesis and pigments influenced by light intensity and salinity in the halophile Dunaliella salina (Chlorophyta). J Mar Biol Assoc UK 62:493–508Google Scholar
  24. Lohr M, Im C, Grossman AR (2005) Genome-based examination of chlorophyll and carotenoid biosynthesis in Chlamydomonas reinhardtii. Plant Physiol 138:490–515CrossRefGoogle Scholar
  25. Lois LM, Rodríguez-Concepción M, Gallego F, Campos N, Boronat A (2000) Carotenoid biosynthesis during tomato fruit development: regulatory role of 1-deoxy-D-xylulose 5-phosphate synthase. Plant J 22:503–513CrossRefGoogle Scholar
  26. Long Z, Shue-Yuan W, Nelson N (1989) Cloning and nucleotide sequence analysis of genes coding for the major chlorophyll-binding protein of the moss Physcomitrella patens and the halotolerant alga Dunaliella salina. Gene 76:299–312CrossRefGoogle Scholar
  27. Moehs CP, Tian L, Osteryoung KW, DellaPenna D (2001) Analysis of carotenoid biosynthetic gene expression during marigold petal development. Plant Mol Biol 45:281–293CrossRefGoogle Scholar
  28. Mount SM (1982) A catalogue of splice junction sequences. Nucleic Acids Res 10:459–472CrossRefGoogle Scholar
  29. Navalho J (1997) Biotechnology of Dunaliella salina for beta-carotene production. M.Sc. thesis, University of Algarve, PortugalGoogle Scholar
  30. Nkondjock A, Ghadirian P, Johnson KC, Krewski D, The Canadian Cancer Registries Epidemiology Research Group (2005) Dietary intake of lycopene is associated with reduced pancreatic cancer risk. J Nutr 135:592–597Google Scholar
  31. Oren A (2005) A hundred years of Dunaliella research: 1905–2005. Saline Syst 1:2CrossRefGoogle Scholar
  32. Peñuelas J, Munné-Bosch S (2005) Isoprenoids: an evolutionary photoprotection. Trends Plant Sci 10:166–169CrossRefGoogle Scholar
  33. Raja R, Hemaiswarya S, Rengasamy R (2007) Exploitation of Dunaliella for β-carotene production. Appl Microbiol Biotechnol 74:517–523CrossRefGoogle Scholar
  34. Rodríguez-Sáiz M, Sánchez-Porro C, De La Fuente JL, Mellado E, Barredo JL (2007) Engineering the halophilic bacterium Halomonas elongata to produce β-carotene. Appl Microbiol Biotechnol 77:637–643CrossRefGoogle Scholar
  35. Römer S, Fraser PD (2005) Recent advances in carotenoid biosynthesis, regulation and manipulation. Planta 221:305–308CrossRefGoogle Scholar
  36. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–25Google Scholar
  37. Sánchez-Estudillo L, Freile-Pelegrin Y, Riviera-Madrid R, Robledo D, Narváez-Zapata JA (2006) Regulation of two photosynthetic pigment-related genes during stress-induced pigment formation in the green alga, Dunaliella salina. Biotechnol Lett 28:787–791CrossRefGoogle Scholar
  38. Sandmann G (1994) Carotenoid biosynthesis in microorganisms and plants. Eur J Biochem 223:7–24CrossRefGoogle Scholar
  39. Sandmann G (2001) Carotenoid biosynthesis and biotechnological application. Arch Biochem Biophys 385:4–12CrossRefGoogle Scholar
  40. Sandmann G (2002) Molecular evolution of carotenoid biosynthesis from bacteria to plants. Physiol Plant 116:431–440CrossRefGoogle Scholar
  41. Steinbrenner J, Linden H (2001) Regulation of two carotenoid biosynthesis genes coding for phytoene synthase and carotenoid hydroxylase during stress-induced astaxanthin formation in the green alga Haematococcus pluvialis. Plant Physiol 125:810–817CrossRefGoogle Scholar
  42. Steinbrenner J, Linden H (2003) Light induction of carotenoid biosynthesis genes in the green alga Haematococcus pluvialis: regulation by photosynthetic redox control. Plant Mol Biol 52:343–356CrossRefGoogle Scholar
  43. Tan C, Qin S, Zhang Q, Jiang P, Zhao F (2005) Establishment of a micro-particle bombardment transformation system for Dunaliella salina. J Microbiology 43:361–365Google Scholar
  44. Tao L, Picataggio S, Rouvière PE, Cheng Q (2004) Asymmetrically acting lycopene β-cyclases (CrtLm) from non-photosynthetic bacteria. Mol Genet Genomics 271:180–188CrossRefGoogle Scholar
  45. Vorst P, Baard RL, Mur LR, Korthals HJ, Van den End H (1994) Effect of growth arrest on carotene accumulation and photosynthesis in Dunaliella. Microbiology 140:1411–1417CrossRefGoogle Scholar
  46. Walne PR (1974) Culture of bivalve molluscs. Fishing News, EnglandGoogle Scholar
  47. Welsch R, Medina J, Giuliano G, Beyer P, von Lintig J (2003) Structural and functional characterization of the phytoene synthase promoter from Arabidopsis thaliana. Planta 216:523–534Google Scholar
  48. Wheelan SJ, Church DM, Ostell JM (2001) Spidey: a tool for mRNA-to genomic alignments. Genome Res 11:1952–1957Google Scholar
  49. Woitsch S, Römer S (2003) Expression of xanthophyll biosynthetic genes during light-dependent chloroplast differentiation. Plant Physiol 132:1508–1517CrossRefGoogle Scholar
  50. Yan Y, Zhu Y-H, Jiang J-G, Song D-L (2005) Cloning and sequence analysis of the phytoene synthase gene from a unicellular chlorophyte, Dunaliella salina. J Agric Food Chem 53:1466–1469CrossRefGoogle Scholar
  51. Zhu CF, 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 48:277–285CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Ana Ramos
    • 1
  • Sacha Coesel
    • 1
  • Ana Marques
    • 1
  • Marta Rodrigues
    • 1
  • Alexandra Baumgartner
    • 2
  • João Noronha
    • 3
  • Amélia Rauter
    • 4
  • Bertram Brenig
    • 2
  • João Varela
    • 1
    Email author
  1. 1.Centre of Marine Sciences (CCMAR)University of AlgarveFaroPortugal
  2. 2.Institute of Veterinary MedicineUniversity of GöttingenGöttingenGermany
  3. 3.REQUIMTE, ChemistryDQ–FCT–UNLCaparicaPortugal
  4. 4.CQB–DQB, Faculty of SciencesUniversity of LisbonLisbonPortugal

Personalised recommendations