Plant and Cell Physiology Advance Access originally published online on January 15, 2009
Plant and Cell Physiology 2009 50(3):463-479; doi:10.1093/pcp/pcp005
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The Evolution and Function of Carotenoid Hydroxylases in Arabidopsis
1Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA
2Lyman Briggs College and Department of Entomology, Michigan State University, East Lansing, MI 48824, USA
3Department of Plant Sciences, University of California, Davis, CA, USA
*Corresponding author: E-mail, dellapen{at}msu.edu; Fax, +1-517-353-9334.
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Abstract |
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To gain insight into the evolution of xanthophyll synthesis in Arabidopsis thaliana, we analyzed two pairs of duplicated carotenoid hydroxylase enzymes in Arabidopsis thaliana: the cytochrome P450 enzymes CYP97A3 and CYP97C1, and non-heme di-iron enzymes, BCH1 and BCH2. Hydroxylated carotenes did not accumulate in a quadruple mutant for these four genes, demonstrating that they encode the full complement of carotenoid hydroxylases in A. thaliana. We were thus able to infer definitively the activity of each enzyme in vivo based on the phenotypes of selected double and triple mutant genotypes. The CYP97 and BCH gene pairs are primarily responsible for hydroxylation of 
- and β-carotenes, respectively, but exhibit some overlapping activities, most notably in hydroxylation of the β-ring of -carotene. Surprisingly, triple mutants containing only CYP97C1 or CYP97A3 activity produced 74 and 6% of the wild-type lutein level, indicating that CYP97C1 can efficiently hydroxylate both the β- and 
-rings of -carotene and that CYP97A3 also has low activity toward the -ring of -carotene. The modes of functional divergence for the gene pairs appear distinct, with the CYP97 duplicates being strongly co-expressed but encoding enzymes with different in vivo substrates, while the BCH duplicates encode isozymes that show significant expression divergence in reproductive organs. By integrating the evolutionary history and substrate specificities of each extant enzyme with the phenotypic responses of various mutant genotypes to high light stress, we propose two likely scenarios for the evolution of -xanthophyll biosynthesis in plants from ancestral organisms.
Keywords: Arabidopsis thaliana - Carotenoid hydroxylase - Functional divergence - Molecular evolution - Xanthophyll synthesis.
Abbreviations:
LHC, light-harvesting complex; ML, maximum likelihood; MP, maximum parsimony; MRCA, most recent common ancestor; NJ, neighbor-joining; NPQ, non-photochemical quenching; -xanthophylls, -carotene-derived xanthophylls; β-xanthophylls, β-carotene-derived xanthophylls.
4Present address Department of Chemistry and Biochemistry, Auburn University, Auburn, AL 36849, USA.
(Received October 1, 2008; Accepted January 7, 2009)
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