Skip to main content

Advertisement

SpringerLink
Log in

Gene cloning and expression profile of a novel carotenoid hydroxylase (CYP97C) from the green alga Haematococcus pluvialis

  • Published:
Journal of Applied Phycology Aims and scope Submit manuscript

Abstract

A full-length complementary DNA (cDNA) sequence of ε-ring CHY (designated Haecyp97c) was cloned from the green alga Haematococcus pluvialis by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods. The Haecyp97c cDNA sequence was 1,995 base pairs (bp) in length, which contained a 1,620-bp open reading frame, a 46-bp 5′-untranslated region (UTR), and a 329-bp 3′-UTR with the characteristic of the poly (A) tail. The deduced protein had a calculated molecular mass of 58.71 kDa with an estimated isoelectric point of 7.94. Multiple alignment analysis revealed that the deduced amino acid sequence of HaeCYP97C shared high identity of 72–85 % with corresponding CYP97Cs from other eukaryotes. The catalytic motifs of cytochrome P450s were detected in the amino acid sequence of HaeCYP97C. The transcriptional levels of Haecyp97c and xanthophylls accumulation under high light (HL) stress have been examined. The results revealed that Haecyp97c transcript was strongly increased after 13–28 h under HL stress. Meanwhile, the concentrations of chlorophylls, carotenes, and lutein were decreased, and zeaxanthin and astaxanthin concentrations were increased rapidly, respectively. These facts indicated that HaeCYP97C was perhaps involved in xanthophyll biosynthesis, which plays an important role in adaption to HL for H. pluvialis.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Adl SM, Simpson AGB, Farmer MA, Andersen RA, Anderson OR, Barta JR, Bowser SS, Brugerolle GUY, Fensome RA, Fredericq S (2005) The new higher level classification of eukaryotes with emphasis on the taxonomy of protists. J Eukaryot Microbiol 52:399–451

    Article  PubMed  Google Scholar 

  • Bak S, Beisson F, Bishop G, Hamberger B, Höfer R, Paquette S, Werck-Reichhart D (2011) Cytochromes P450. Arabidopsis Book 9:e0144

    Google Scholar 

  • Bjellqvist B, Hughes GJ, Pasquali C, Paquet N, Ravier F, Sanchez JC, Frutiger S, Hochstrasser D (1993) The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences. Electrophoresis 14:1023–1031

    Article  PubMed  CAS  Google Scholar 

  • Boussiba S (2000) Carotenogenesis in the green alga Haematococcus pluvialis: cellular physiology and stress response. Physiol Plant 108:111–117

    Article  CAS  Google Scholar 

  • Chevenet F, Brun C, Bañuls AL, Jacq B, Christen R (2006) TreeDyn: towards dynamic graphics and annotations for analyses of trees. BMC Bioinforma 7:439

    Article  CAS  Google Scholar 

  • Choi SK, Matsuda S, Hoshino T, Peng X, Misawa N (2006) Characterization of bacterial β-carotene 3,3′-hydroxylases, CrtZ, and P450 in astaxanthin biosynthetic pathway and adonirubin production by gene combination in Escherichia coli. Appl Microbiol Biotechnol 72:1238–1246

    Article  PubMed  CAS  Google Scholar 

  • Cserzö M, Eisenhaber F, Eisenhaber B, Simon I (2002) On filtering false positive transmembrane protein predictions. Protein Eng 15:745–752

    Article  PubMed  Google Scholar 

  • Dall’Osto L, Lico C, Alric J, Giuliano G, Havaux M, Bassi R (2006) Lutein is needed for efficient chlorophyll triplet quenching in the major LHCII antenna complex of higher plants and effective photoprotection in vivo under strong light. BMC Plant Biol 6:32

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Dall’Osto L, Fiore A, Cazzaniga S, Giuliano G, Bassi R (2007) Different roles of α- and β-branch xanthophylls in photosystem assembly and photoprotection. J Biol Chem 282:35056–35068

    Article  PubMed  CAS  Google Scholar 

  • Del Campo JA, Rodríguez H, Moreno J, Vargas MÁ, Rivas J, Guerrero MG (2004) Accumulation of astaxanthin and lutein in Chlorella zofingiensis (Chlorophyta). Appl Microbiol Biotechnol 64:848–854

    Article  PubMed  CAS  Google Scholar 

  • Emanuelsson O, Brunak S, von Heijne G, Nielsen H (2007) Locating proteins in the cell using TargetP, SignalP and related tools. Nature Protoc 2:953–971

    Article  CAS  Google Scholar 

  • Fiore A, Dall’Osto L, Cazzaniga S, Diretto G, Giuliano G, Bassi R (2012) A quadruple mutant of Arabidopsis reveals a β–carotene hydroxylation activity for LUT1/CYP97C1 and a regulatory role of xanthophylls on determination of the PSI/PSII ratio. BMC Plant Biol 12:50

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Fraser PD, Miura Y, Misawa N (1997) In vitro characterization of astaxanthin biosynthetic enzymes. J Biol Chem 272:6128–6135

    Article  PubMed  CAS  Google Scholar 

  • Graham SE, Peterson JA (1999) How similar are P450s and what can their differences teach us? Arch Biochem Biophys 369:24–29

    Article  PubMed  CAS  Google Scholar 

  • Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704

    Article  PubMed  Google Scholar 

  • Inoue K (2004) Carotenoid hydroxylation—P450 finally! Trends Plant Sci 9:515–517

    Article  PubMed  CAS  Google Scholar 

  • Katsuda T, Shimahara K, Shiraishi H, Yamagami K, Ranjbar R, Katoh S (2006) Effect of flashing light from blue light emitting diodes on cell growth and astaxanthin production of Haematococcus pluviali. J Biosci Bioeng 102:442–446

    Article  PubMed  CAS  Google Scholar 

  • Kim J, DellaPenna D (2006) Defining the primary route for lutein synthesis in plants: the role of Arabidopsis carotenoid β-ring hydroxylase CYP97A3. Proc Natl Acad Sci USA 103:3474–3479

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Kim J, Smith JJ, Tian L, DellaPenna D (2009) The evolution and function of carotenoid hydroxylases in Arabidopsis. Plant Cell Physiol 50:463–479

    Article  PubMed  CAS  Google Scholar 

  • Lababpour A, Hada K, Shimahara K, Katsuda T, Katoh S (2004) Effects of nutrient supply methods and illumination with blue light emitting diodes (LEDs) on astaxanthin production by Haematococcus pluvialis. J Biosci Bioeng 98:452–456

    Article  PubMed  CAS  Google Scholar 

  • Lagarde D, Vermaas W (1999) The zeaxanthin biosynthesis enzyme β-carotene hydroxylase is involved in myxoxanthophyll synthesis in Synechocystis sp. PCC 6803. FEBS Lett 454:247–251

    Article  PubMed  CAS  Google Scholar 

  • Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948

    Article  PubMed  CAS  Google Scholar 

  • Le SQ, Gascuel O (2008) An improved general amino acid replacement matrix. Mol Biol Evol 25:1307–1320

    Article  PubMed  CAS  Google Scholar 

  • Lemaire P, Livingstone DR (1995) Effects of the inhibitor ellipticine on cytochrome P450 reductase and cytochrome P450 (1A1) function in hepatic microsomes of flounder (Platichtys flesus). Mar Environ Res 39:73–77

    Article  CAS  Google Scholar 

  • Lemoine Y, Schoefs B (2010) Secondary ketocarotenoid astaxanthin biosynthesis in algae: a multifunctional response to stress. Photosynth Res 106:155–177

    Article  PubMed  CAS  Google Scholar 

  • Li YT, Huang JC, Sandmann G, Chen F (2009) High-light and sodium chloride stress differentially regulate the biosynthesis of astaxanthin in Chlorella zofingiensis (Chlorophyceae). J Phycol 45:635–641

    Article  CAS  Google Scholar 

  • Li YT, Sommerfeld M, Chen F, Hu Q (2010) Effect of photon flux densities on regulation of carotenogenesis and cell viability of Haematococcus pluvialis (Chlorophyceae). J Appl Phycol 22:253–263

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Linden H (1999) Carotenoid hydroxylase from Haematococcus pluvialis: cDNA sequence, regulation and functional complementation. BBA–Gene Struct Expr 1446:203–212

    Article  CAS  Google Scholar 

  • Lv MZ, Chao DY, Shan JX, Zhu MZ, Shi M, Gao JP, Lin HX (2012) Rice carotenoid β-ring hydroxylase CYP97A4 is involved in lutein biosynthesis. Plant Cell Physiol 53:987–1002

    Article  PubMed  CAS  Google Scholar 

  • Misawa N, Satomi Y, Kondo K, Yokoyama A, Kajiwara S, Saito T, Ohtani T, Miki W (1995) Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level. J Bacteriol 177:6575–6584

    PubMed Central  PubMed  CAS  Google Scholar 

  • Nelson DR (2009) The cytochrome P450 homepage. Hum Genom 4:59–65

    CAS  Google Scholar 

  • Nelson D, Werck–Reichhart D (2011) A P450-centric view of plant evolution. Plant J 66:194–211

    Article  PubMed  CAS  Google Scholar 

  • Nelson DR, Ming R, Alam M, Schuler MA (2008) Comparison of cytochrome P450 genes from six plant genomes. Trop Plant Biol 1:216–235

    Article  CAS  Google Scholar 

  • Rose TM, Henikoff JG, Henikoff S (2003) CODEHOP (COnsensus–DEgenerate hybrid oligonucleotide primer) PCR primer design. Nucleic Acids Res 31:3763–3766

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual (3‐volume set). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

  • Schoefs B, Rmiki NE, Rachidi J, Lemoine Y (2001) Astaxanthin accumulation in Haematococcus requires a cytochrome P450 hydroxylase and an active synthesis of fatty acids. FEBS Lett 500:125–128

    Article  PubMed  CAS  Google Scholar 

  • Schuler MA, Werck-Reichhart D (2003) Functional genomics of P450s. Annu Rev Plant Biol 54:629–667

    Article  PubMed  CAS  Google Scholar 

  • Schuler MA, Duan H, Bilgin M, Ali S (2006) Arabidopsis cytochrome P450s through the looking glass: a window on plant biochemistry. Phytochem Rev 5:205–237

    Article  CAS  Google Scholar 

  • Stigliani AL, Giorio G, D’Ambrosio C (2012) Characterization of P450 carotenoid β- and α-hydroxylases of tomato and transcriptional regulation of xanthophyll biosynthesis in root, leaf, petal and fruit. Plant Cell Physiol 52:851–865

    Article  CAS  Google Scholar 

  • Sun Z, Gantt E, Francis X Jr (1996) Cloning and functional analysis of the β-carotene hydroxylase of Arabidopsis thaliana. J Biol Chem 271:24349–24352

    Article  PubMed  CAS  Google Scholar 

  • Sun N, Wang Y, Li YT, Huang JC, Chen F (2008) Sugar-based growth, astaxanthin accumulation and carotenogenic transcription of heterotrophic Chlorella zofingiensis (Chlorophyta). Process Biochem 43:1288–1292

    Article  CAS  Google Scholar 

  • Takaichi S (2011) Carotenoids in algae: distributions, biosyntheses and functions. Mar Drugs 9:1101–1118

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Tan CP, Zhao FQ, Su ZL, Liang CW, Qin S (2007) Expression of β-carotene hydroxylase gene (crtR-B) from the green alga Haematococcus pluvialis in chloroplasts of Chlamydomonas reinhardtii. J Appl Phycol 19:347–355

    Article  CAS  Google Scholar 

  • Teng C, Qin S, Liu J, Yu D, Liang C, Tseng C (2002) Transient expression of lacZ in bombarded unicellular green alga Haematococcus pluvialis. J Appl Phycol 14:497–500

    Article  CAS  Google Scholar 

  • Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Tian L, DellaPenna D (2001) Characterization of a second carotenoid β-hydroxylase gene from Arabidopsis and its relationship to the LUT1 locus. Plant Mol Biol 47:379–388

    Article  PubMed  CAS  Google Scholar 

  • Tian L, DellaPenna D (2004) Progress in understanding the origin and functions of carotenoid hydroxylases in plants. Arch Biochem Biophys 430:22–29

    Article  PubMed  CAS  Google Scholar 

  • Tian L, Magallanes-Lundback M, Musetti V, DellaPenna D (2003) Functional analysis of β- and α-ring carotenoid hydroxylases in Arabidopsis. Plant Cell 15:1320–1332

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Tian L, Musetti V, Kim J, Magallanes–Lundback M, DellaPenna D (2004) The Arabidopsis LUT1 locus encodes a member of the cytochrome P450 family that is required for carotenoid α-ring hydroxylation activity. Proc Natl Acad Sci USA 101:402–407

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Wang C, Hu Z, Zhao C, Mao X (2012a) Isolation of the β-carotene ketolase gene promoter from Haematococcus pluvialis and expression of ble in transgenic Chlamydomonas. J Appl Phycol 24:1303–1310

    Article  CAS  Google Scholar 

  • Wang J, Sommerfeld M, Hu Q (2012b) Cloning and expression of isoenzymes of superoxide dismutase in Haematococcus pluvialis (Chlorophyceae) under oxidative stress. J Appl Phycol 23:995–1003

    Article  CAS  Google Scholar 

  • Werck-Reichhart D, Feyereisen R (2000) Cytochromes P450: a success story. Genome Biol 1:3003.1–3001.9

    Article  Google Scholar 

  • Williams PA, Cosme J, Sridhar V, Johnson EF, McRee DE (2000) Mammalian microsomal cytochrome P450 monooxygenase: structural adaptations for membrane binding and functional diversity. Mol Cell 5:121–131

    Article  PubMed  CAS  Google Scholar 

  • Yokoyama A, Miki W (2006) Composition and presumed biosynthetic pathway of carotenoids in the astaxanthin–producing bacterium Agrobacterium aurantiacum. FEMS Microbiol Lett 128:139–144

    Article  Google Scholar 

  • Zhang L, Fan YM, Shi F, Qin S, Liu B (2012) Molecular cloning, characterization, and expression analysis of a cytosolic HSP90 gene from Haematococcus pluvialis. J Appl Phycol 24:1601–1812

    Article  CAS  Google Scholar 

  • Zhekisheva M, Zarka A, Khozin-Goldberg I, Cohen Z, Boussiba S (2005) Inhibition of astaxanthin synthesis under high irradisnce does not abolish triacylglycerol accumulation in the green algae Haematococcus pluvialis (Chlorophyceae). J Phycol 41:819–826

    Article  CAS  Google Scholar 

  • Zheng K, Hu ZL, Li S, Lei AP, Wang CG (2007) Cloning and sequencing of β-carotene hydroxylase gene from Haematococcus pluvialis. Acta Hydrobiol Sin–Ch Ed 31:666–670

    CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Natural Science Foundation of China (40876082); International Innovation Partnership Program: Typical Environmental Process and Effects on Resources in Coastal Zone Area; Public Science and Technology Research Funds Projects of the Ocean (200905021 and 201205027); The Natural Science Foundation of Shandong Province (ZR2012DQ015); Guangdong province comprehensive strategic cooperation project of the Chinese Academy of Sciences (2011A090100040); and Science and Technology Planning Project of Yantai, China (no. 2010247).

Author information

Authors and Affiliations

  1. Key Laboratory of Coastal Biology and Biological Resources Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai, 264003, China

    Hongli Cui, Yan Wang, Yulin Cui & Song Qin

  2. University of the Chinese Academy of Sciences, Beijing, 100049, China

    Hongli Cui

  3. College of Resources and Environmental Sciences, Key Laboratory of Marine Biology, Nanjing Agricultural University, Nanjing, 210095, China

    Xiaona Yu & Zhaopu Liu

  4. Shenzhen Key Laboratory for Marine Bio-resource and Eco-environment, College of Life Sciences, Shenzhen University, Shenzhen, 518060, China

    Xueqin Li

Authors
  1. Hongli Cui
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaona Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yulin Cui
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xueqin Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Zhaopu Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Song Qin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Zhaopu Liu or Song Qin.

Additional information

H. Cui and X. Yu contributed equally.

X. Yu is the co-first author and has the same contribution to this paper.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 17 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cui, H., Yu, X., Wang, Y. et al. Gene cloning and expression profile of a novel carotenoid hydroxylase (CYP97C) from the green alga Haematococcus pluvialis . J Appl Phycol 26, 91–103 (2014). https://doi.org/10.1007/s10811-013-0055-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10811-013-0055-y

Keywords

Search

Navigation