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Characterization of a β-carotene hydroxylase of Adonis aestivalis and its expression in Arabidopsis thaliana

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Abstract

Carotenoids are plant secondary metabolites that comprise two main groups: carotenes and xanthophylls. The latter group includes zeaxanthin which is synthesized by β-carotene hydroxylase catalyzing the hydroxylation of the β-rings of β-carotene molecules. To develop tools to alter carotenoid biosynthesis in plants, we isolated a cDNA clone encoding a candidate β-carotene hydroxylase, CrtH1, from the flower petals of Adonis aestivalis. CrtH1 protein has homology to β-carotene hydroxylases from other organisms, and possesses the four histidine motifs conserved in this family of enzymes. Sequence analysis predicted the presence of a putative plastid transit peptide at the amino terminus and four transmembrane helical regions. Southern-blot analysis showed CrtH1 to be encoded by a multicopy gene family with at least three members in A. aestivalis. Analysis of CrtH1 transcript abundance by Northern blotting indicates it is highly expressed in flower petals, roots and stems, with relatively low expression in leaves and developing seeds. CrtH1 was able to catalyze the formation of zeaxanthin and its intermediate precursor β-cryptoxanthin from β-carotene in functional assays conducted in E. coli. Expression of CrtH1 in Arabidopsis thaliana wild type and a mutant deficient for endogenous β-carotene hydroxylases enhanced the biosynthesis of violaxanthin in the seeds.

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Abbreviations

BHT:

Butylated hydroxytoluene

DEPC:

Diethylpyrocarbonate

IPTG:

Isopropyl-β-d-thiogalactopyranoside

ORF:

Open-reading frame

TAG:

Triacylglyceride

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Acknowledgments

We thank Dr. Norihiko Misawa (Kirin Brewery Co. Ltd, Kanagawa, Japan) and Dr. Hartmut Linden (Universitat Konstanz, Germany) for the kind gift of plasmid pACCAR16ΔcrtX and Dr. Dean DellaPenna (Michigan State University, Michigan, USA) for generously providing the seeds of Arabidopsis thaliana b1b2 double knockout mutant. We are grateful to Drs. Isobel Parkin and Kevin Rozwadowski for critical reading of the manuscript, and to Delwin Epp for technical help with HPLC analysis. Funding for this project was provided by the Saskatchewan Agriculture Development Fund.

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Authors and Affiliations

  1. Agriculture and Agri-Food Canada, Saskatoon Research Centre, 107 Science Place, Saskatoon, SK, S7N 0X2, Canada

    Bianyun Yu, Derek J. Lydiate, Ulrike A. Schäfer & Abdelali Hannoufa

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  1. Bianyun Yu
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  2. Derek J. Lydiate
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  3. Ulrike A. Schäfer
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  4. Abdelali Hannoufa
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Corresponding author

Correspondence to Abdelali Hannoufa.

Additional information

The sequence of the β-carotene hydroxylase, CrtH1, has been submitted to Gene Bank, accession #EF120636.

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Yu, B., Lydiate, D.J., Schäfer, U.A. et al. Characterization of a β-carotene hydroxylase of Adonis aestivalis and its expression in Arabidopsis thaliana . Planta 226, 181–192 (2007). https://doi.org/10.1007/s00425-006-0455-1

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  • DOI: https://doi.org/10.1007/s00425-006-0455-1

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