Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

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

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.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. Almeida ERA, Cerdá-Olmedo E (2008) Gene expression in the regulation of carotene biosynthesis in Phycomyces. Curr Genet 53:129–137

  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 131:479–487

  3. Ben-Amotz A, Avron M (1983) On the factors which determine massive β-carotene accumulation in the halotolerant alga Dunaliella bardawil. Plant Physiol 72:593–597

  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 18:529–537

  5. Bohne F, Linden H (2002) Regulation of carotenoid biosynthesis genes in response to light in Chlamydomonas reinhardtii. Biochim Biophys Acta 1579:26–34

  6. Borowitzka MA, Borowitzka LJ, Kessly D (1990) Effects of salinity increase on carotenoid accumulation in the green alga Dunaliella salina. J Appl Phycol 2:111–119

  7. Bouvier F, Backhaus RA, Camara B (1998) Induction and control of chromoplast-specific carotenoid genes by oxidative stress. J Biol Chem 273:30651–30659

  8. Bramley PM (2002) Regulation of carotenoid formation during tomato fruit ripening and development. J Exp Bot 53:2107–2113

  9. Brown JS (1986) A catalogue of splice junction and putative branch point sequences from plant introns. Nucleic Acids Res 14:9549–9559

  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 29:227–236

  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 https://doi.org/10.1007/s10126-008-9100-2

  12. Cunningham FX Jr (2002) Regulation of carotenoid synthesis and accumulation in plants. Pure Appl Chem 74:1409–1417

  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 6:1107–1121

  14. Del Campo JA, García-González M, Guerrero MG (2007) Outdoor cultivation of microalgae for carotenoid production: current state and perspectives. Appl Microbiol Biotechnol 74:1163–1174

  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 7:11

  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 8:978–984

  17. Giuliano G, Bartley GE, Scolnik PA (1993) Regulation of carotenoid biosynthesis during tomato development. Plant Cell 5:379–387

  18. Goodwin TW (1980) The biochemistry of the carotenoids. Plants, vol. 1. Chapman and Hall, London

  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 122:1261–1268

  20. Krubasik P, Sandmann G (2000) Molecular evolution of lycopene cyclases involved in the formation of carotenoids with ionone end groups. Biochem Soc Trans 28:806–810

  21. León-Bañares R, González-Ballester D, Galván A, Fernández E (2004) Transgenic microalgae as green cell-factories. Trends Biotechnol 22:45–52

  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 93:389–395

  23. Loeblich LA (1982) Photosynthesis and pigments influenced by light intensity and salinity in the halophile Dunaliella salina (Chlorophyta). J Mar Biol Assoc UK 62:493–508

  24. Lohr M, Im C, Grossman AR (2005) Genome-based examination of chlorophyll and carotenoid biosynthesis in Chlamydomonas reinhardtii. Plant Physiol 138:490–515

  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 22:503–513

  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 76:299–312

  27. Moehs CP, Tian L, Osteryoung KW, DellaPenna D (2001) Analysis of carotenoid biosynthetic gene expression during marigold petal development. Plant Mol Biol 45:281–293

  28. Mount SM (1982) A catalogue of splice junction sequences. Nucleic Acids Res 10:459–472

  29. Navalho J (1997) Biotechnology of Dunaliella salina for beta-carotene production. M.Sc. thesis, University of Algarve, Portugal

  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 135:592–597

  31. Oren A (2005) A hundred years of Dunaliella research: 1905–2005. Saline Syst 1:2

  32. Peñuelas J, Munné-Bosch S (2005) Isoprenoids: an evolutionary photoprotection. Trends Plant Sci 10:166–169

  33. Raja R, Hemaiswarya S, Rengasamy R (2007) Exploitation of Dunaliella for β-carotene production. Appl Microbiol Biotechnol 74:517–523

  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 77:637–643

  35. Römer S, Fraser PD (2005) Recent advances in carotenoid biosynthesis, regulation and manipulation. Planta 221:305–308

  36. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–25

  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 28:787–791

  38. Sandmann G (1994) Carotenoid biosynthesis in microorganisms and plants. Eur J Biochem 223:7–24

  39. Sandmann G (2001) Carotenoid biosynthesis and biotechnological application. Arch Biochem Biophys 385:4–12

  40. Sandmann G (2002) Molecular evolution of carotenoid biosynthesis from bacteria to plants. Physiol Plant 116:431–440

  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 125:810–817

  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 52:343–356

  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 43:361–365

  44. Tao L, Picataggio S, Rouvière PE, Cheng Q (2004) Asymmetrically acting lycopene β-cyclases (CrtLm) from non-photosynthetic bacteria. Mol Genet Genomics 271:180–188

  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 140:1411–1417

  46. Walne PR (1974) Culture of bivalve molluscs. Fishing News, England

  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 216:523–534

  48. Wheelan SJ, Church DM, Ostell JM (2001) Spidey: a tool for mRNA-to genomic alignments. Genome Res 11:1952–1957

  49. Woitsch S, Römer S (2003) Expression of xanthophyll biosynthetic genes during light-dependent chloroplast differentiation. Plant Physiol 132:1508–1517

  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 53:1466–1469

  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 48:277–285

Download references

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.

Author information

Correspondence to João Varela.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Ramos, A., Coesel, S., Marques, A. et al. Isolation and characterization of a stress-inducible Dunaliella salina Lcy-β gene encoding a functional lycopene β-cyclase. Appl Microbiol Biotechnol 79, 819 (2008). https://doi.org/10.1007/s00253-008-1492-4

Download citation

Keywords

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