Skip to main content
SpringerLink
Log in

Cloning and comparative analysis of carotenoid β-hydroxylase genes provides new insights into carotenoid metabolism in tetraploid (Triticum turgidum ssp. durum) and hexaploid (Triticum aestivum) wheat grains

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Carotenoid β-hydroxylases attach hydroxyl groups to the β-ionone rings (β-rings) of carotenoid substrates, resulting in modified structures and functions of carotenoid molecules. We cloned and characterized two genes (each with three homeologs), HYD1 and HYD2, which encode β-hydroxylases in wheat. The results from bioinformatic and nested degenerate PCR analyses collectively suggest that HYD1 and HYD2 may represent the entire complement of non-heme di-iron β-hydroxylases in wheat. The homeologs of wheat HYDs exhibited major β-ring and minor ε-ring hydroxylation activities in carotenoid-accumulating E. coli strains. Distinct expression patterns were observed for different HYD genes and homeologs in vegetative tissues and developing grains of tetraploid and hexaploid wheat, suggesting their functional divergence and differential regulatory control in tissue-, grain development-, and ploidy-specific manners. An intriguing observation was that the expression of HYD1, particularly HYD-B1, reached highest levels at the last stage of tetraploid and hexaploid grain development, suggesting that carotenoids (at least xanthophylls) were still actively synthesized in mature grains. This result challenges the common perception that carotenoids are simply being turned over during wheat grain development after their initial biosynthesis at the early grain development stages. Overall, this improved understanding of carotenoid biosynthetic gene expression and carotenoid metabolism in wheat grains will contribute to the improvement of the nutritional value of wheat grains for human consumption.

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
Fig. 6

Similar content being viewed by others

Abbreviations

ABA:

Abscisic acid

β-ring:

β-ionone ring

CRTISO:

Carotenoid isomerase

CYP:

Carotenoid ε-hydroxylase (cytochrome P450 type)

DAP:

Days after pollination

EST:

Expressed sequence tags

GGPP:

Geranylgeranyl diphosphate

HYD:

Carotenoid β-hydroxylase (non-heme di-iron type)

IPTG:

Isopropyl-β-d-thiogalactopyranoside

LB:

Luria–Bertani

LCY-B:

Lycopene β-cyclase

LCY-E:

Lycopene ε-cyclase

MUSCLE:

Multiple sequence comparison by log-expectation

MYA:

Million years ago

NJ:

Neighbor-joining

NXS:

Neoxanthin synthase

PDS:

Phytoene desaturase

PSY:

Phytoene synthase

RACE:

Rapid amplification of cDNA ends

TC:

Tentative contig

TLC:

Thin layer chromatography

TILLING:

Targeting Induced Local Lesions IN Genomes

VDE:

Violaxanthin de-epoxidase

ZDS:

ζ-Carotene desaturase

ZEP:

Zeaxanthin epoxidase

Z-ISO:

ζ-carotene isomerase

References

  • Applied Biosystems (2004) Guide to performing relative quantitation of gene expression using Real-Time quantitative PCR

  • Bouvier F, Keller Y, d’Harlingue A, Camara B (1998) Xanthophyll biosynthesis: molecular and functional characterization of carotenoid hydroxylases from pepper fruits (Capsicum annuum L.). Biochim Biophys Acta 1391:320–328

    Article  PubMed  CAS  Google Scholar 

  • Britton G (1995) UV/visible spectroscopy. In: Britton G, Liaaen-Jensen S, Pfander H (eds) Carotenoids, vol. 1B: Spectroscopy. Birkhauser, Basel, Switzerland pp 13–62

  • Britton G (2009) Vitamin A and vitamin A deficiency. In: Britton G, Liaaen-Jensen S, Pfander H (eds) Carotenoids. Volume 5: Nutrition and Health. Birkhäuser Verlag Basel

  • Brown C, Kim T, Ganga Z, Haynes K, De Jong D, Jahn M, Paran I, De Jong W (2006) Segregation of total carotenoid in high level potato germplasm and its relationship to beta-carotene hydroxylase polymorphism. Am J Potato Res 83:365–372

    Article  CAS  Google Scholar 

  • Cunningham FJ, Gantt E (2007) A portfolio of plasmids for identification and analysis of carotenoid pathway enzymes: Adonis aestivalis as a case study. Photosynth Res 92:245–259

    Article  PubMed  CAS  Google Scholar 

  • Cunningham F, Pogson B, Sun Z, McDonald K, DellaPenna D, Gantt E (1996) Functional analysis of the beta and epsilon lycopene cyclase enzymes of Arabidopsis reveals a mechanism for control of cyclic carotenoid formation. Plant Cell 8:1613–1626

    PubMed  CAS  Google Scholar 

  • Devos K, Dubcovsky J, Dvorak J, Chinoy C, Gale M (1995) Structural evolution of wheat chromosomes 4A, 5A, and 7B and its impact on recombination. Theor Appl Genet 91:282–288

    Article  CAS  Google Scholar 

  • Diretto G, Tavazza R, Welsch R, Pizzichini D, Mourgues F, Papacchioli V, Beyer P, Giuliano G (2006) Metabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase. BMC Plant Biol 6:13

    Article  PubMed  Google Scholar 

  • 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

    Article  PubMed  Google Scholar 

  • Du H, Wang N, Cui F, Li X, Xiao J, Xiong L (2010) Characterization of the β-carotene hydroxylase gene DSM2 conferring drought and oxidative stress resistance by increasing xanthophylls and abscisic acid synthesis in rice. Plant Physiol 154:1304–1318

    Article  PubMed  CAS  Google Scholar 

  • Dubcovsky J, Dvorak J (2007) Genome plasticity a key factor in the success of polyploid wheat under domestication. Science 316:1862–1866

    Article  PubMed  CAS  Google Scholar 

  • Dvorak J, Akhunov E (2005) Tempos of gene locus deletions and duplications and their relationship to recombination rate during piploid and polyploid evolution in the Aegilops-Triticum alliance. Genetics 171:323–332

    Article  PubMed  CAS  Google Scholar 

  • Dvorak J, McGuire P, Cassidy B (1988) Apparent sources of the A genomes of wheats inferred from polymorphism in abundance and restriction fragment length of repeated nucleotide sequences. Genome 30:680–689

    Article  CAS  Google Scholar 

  • Edgar R (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucl Acids Res 32:1792–1797

    Article  PubMed  CAS  Google Scholar 

  • Fiore A, Dall’osto L, Fraser PD, Bassi R, Giuliano G (2006) Elucidation of the β-carotene hydroxylation pathway in Arabidopsis thaliana. FEBS Lett 580:4718–4722

    Article  PubMed  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  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Harjes C, Rocheford T, Bai L, Brutnell T, Kandianis C, Sowinski S, Stapleton A, Vallabhaneni R, Williams M, Wurtzel E, Yan J, Buckler E (2008) Natural genetic variation in lycopene epsilon cyclase tapped for maize biofortification. Science 319:330–333

    Article  PubMed  CAS  Google Scholar 

  • Hentschel V, Kranl K, Hollmann J, Lindhauer M, Bohm V, Bitsch R (2002) Spectrophotometric determination of yellow pigment content and evaluation of carotenoids by high-performance liquid chromatography in durum wheat grain. J Agric Food Chem 50:6663–6668

    Article  PubMed  CAS  Google Scholar 

  • Howitt C, Cavanagh C, Bowerman A, Cazzonelli C, Rampling L, Mimica J, Pogson B (2009) Alternative splicing, activation of cryptic exons and amino acid substitutions in carotenoid biosynthetic genes are associated with lutein accumulation in wheat endosperm. Funct Integr Genomics 9:363–376

    Article  PubMed  CAS  Google Scholar 

  • Kim J, Smith J, 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 

  • Lang M, Orgogozo V (2011) Identification of homologous gene sequences by PCR with degenerate primers. Methods Mol Biol 772:245–256

    Article  PubMed  CAS  Google Scholar 

  • Laur L, Tian L (2011) Provitamin A and vitamin C content in selected California-grown cantaloupe and honeydew melons and imported melons. J Food Comp Anal 24:194–201

    Article  CAS  Google Scholar 

  • Lokstein H, Tian L, Polle JEW, DellaPenna D (2002) Xanthophyll biosynthetic mutants of Arabidopsis thaliana: altered nonphotochemical quenching of chlorophyll fluorescence is due to changes in Photosystem II antenna size and stability. Biochim Biophys Acta 1553:309–319

    Article  PubMed  CAS  Google Scholar 

  • Naqvi S, Zhu C, Farre G, Sandmann G, Capell T, Christou P (2011) Synergistic metabolism in hybrid corn indicates bottlenecks in the carotenoid pathway and leads to the accumulation of extraordinary levels of the nutritionally important carotenoid zeaxanthin. Plant Biotechnol J 9:384–393

    Article  PubMed  CAS  Google Scholar 

  • Nomura T, Ishihara A, Yanagita R, Endo T, Iwamura H (2005) Three genomes differentially contribute to the biosynthesis of benzoxazinones in hexaploid wheat. Proc Natl Acad Sci USA 102:16490–16495

    Article  PubMed  CAS  Google Scholar 

  • Panfili G, Cinquanta L, Fratianni A, Cubadda R (2003) Extraction of wheat germ oil by supercritical CO2: oil and defatted cake characterization. J Am Oil Chem Soc 80:157–161

    Article  CAS  Google Scholar 

  • Paolacci A, Tanzarella O, Porceddu E, Ciaffi M (2009) Identification and validation of reference genes for quantitative RT-PCR normalization in wheat. BMC Mol Biol 10:11

    Article  PubMed  Google Scholar 

  • Schiedt K, Liaaen-Jensen S (1995) Isolation and analysis In: Britton G, Liaaen-Jensen S, Pfander H (eds) Carotenoids, Vol. 1A: Isolation and analysis. Birkhauser, Basel, Switzerland pp 81–108

  • Schwartz S, Qin X, Zeevaart J (2001) Characterization of a novel carotenoid cleavage dioxygenase from plants. J Biol Chem 276:25208–25211

    Article  PubMed  CAS  Google Scholar 

  • Sorrells M, La Rota M, Bermudez-Kandianis C, Greene R, Kantety R, Munkvold J, Mahmoud A, Miftahudin XF, Ma X, Gustafson P, Qi L, Echalier B, Gill B, Matthews D, Lazo G, Chao S, Anderson O, Edwards H, Linkiewicz A, Dubcovsky J, Akhunov E, Dvorak J, Zhang D, Nguyen H, Peng J, Lapitan N, Gonzalez-Hernandez J, Anderson J, Hossain K, Kalavacharla V, Kianian S, Choi D, Close T, Dilbirligi M, Gill K, Steber C, Walker-Simmons M, McGuire P, Qualset C (2003) Comparative DNA sequence analysis of wheat and rice genomes. Genome Res 13:1818–1827

    PubMed  CAS  Google Scholar 

  • Sun Z, Gantt E, Cunningham FX 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 

  • Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    Article  PubMed  CAS  Google Scholar 

  • Taylor J, Raes J (2004) Duplication and divergence: the evolution of new geens and old ideas. Annu Rev Genet 38:615–643

    Article  PubMed  CAS  Google Scholar 

  • Tian L, DellaPenna D (2001) Characterization of a second carotenoid β-hydroxylase gene from Arabidopsis and its relationships to the LUT1 locus. Plant Mol Biol 45:379–388

    Article  Google Scholar 

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

    Article  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 ε-ring hydroxylation activity. Proc Natl Acad Sci USA 101:402–407

    Article  PubMed  CAS  Google Scholar 

  • Uauy C, Paraiso F, Colasuonno P, Tran R, Tsai H, Berardi S, Comai L, Dubcovsky J (2009) A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat. BMC Plant Biol 9:115

    Article  PubMed  Google Scholar 

  • Vallabhaneni R, Gallagher CE, Licciardello N, Cuttriss AJ, Quinlan RF, Wurtzel ET (2009) Metabolite sorting of a germplasm collection reveals the Hydroxylase3 locus as a new target for maize provitamin A biofortification. Plant Physiol 151:1635–1645

    Article  PubMed  CAS  Google Scholar 

  • Walker-Simmons M (1987) ABA levels and sensitivity in developing wheat embryos of sprouting resistant and susceptible cultivars. Plant Physiol 84:61–66

    Article  PubMed  CAS  Google Scholar 

  • Wan Y, Poole R, Huttly A, Toscano-Underwood C, Feeney K, Welham S, Gooding M, Mills C, Edwards K, Shewry P, Mitchell R (2008) Transcriptome analysis of grain development in hexaploid wheat. BMC Genomics 9:121

    Article  PubMed  Google Scholar 

  • Yan J, Kandianis C, Harjes C, Bai L, Kim E, Yang X, Skinner D, Fu Z, Mitchell S, Li Q, Fernandez M, Zaharieva M, Babu R, Fu Y, Palacios N, Li J, Dellapenna D, Brutnell T, Buckler E, Warburton M, Rocheford T (2010) Rare genetic variation at Zea mays crtRB1 increases beta-carotene in maize grain. Nat Genet 42:322–327

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Nadia Ono for critical reading of the manuscript, Dr. Diane Beckles for helpful discussions, Dr. Francis Cunningham for providing us the pAC-BETA, pAC-DELTA, and pAC-EPSILON plasmids, and Drs. Jan Dvorak, W. Richard McCombie, and Doreen Ware for early access to the assembly of the Ae. tauschii genome. This work was supported by the UC Davis new faculty startup fund to LT and by the Howard Hughes Medical Institute and Betty and Gordon Moore Foundation and USDA-AFRI grant 2011-68002-30029 to JD.

Author information

Authors and Affiliations

  1. Department of Plant Sciences, Mail Stop 3, University of California, Davis, Davis, CA, 95616, USA

    Xiaoqiong Qin, Wenjun Zhang, Jorge Dubcovsky & Li Tian

Authors
  1. Xiaoqiong Qin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wenjun Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jorge Dubcovsky
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Li Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Li Tian.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Qin, X., Zhang, W., Dubcovsky, J. et al. Cloning and comparative analysis of carotenoid β-hydroxylase genes provides new insights into carotenoid metabolism in tetraploid (Triticum turgidum ssp. durum) and hexaploid (Triticum aestivum) wheat grains. Plant Mol Biol 80, 631–646 (2012). https://doi.org/10.1007/s11103-012-9972-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11103-012-9972-4

Keywords

Search

Navigation