The Arabidopsis LUT1 locus encodes a member of the cytochrome p450 family that is required for carotenoid epsilon-ring hydroxylation activity.

Lutein, a dihydroxy xanthophyll, is the most abundant carotenoid in plant photosynthetic tissues and plays crucial structural and functional roles in the light-harvesting complexes. Carotenoid beta-and epsilon-hydroxylases catalyze the formation of lutein from alpha-carotene (beta,epsilon-carotene). In contrast to the well studied beta-hydroxylases that have been cloned and characterized from many organisms, the epsilon-hydroxylase has only been genetically defined by the lut1 mutation in Arabidopsis. We have isolated the LUT1 gene by positional cloning and found that, in contrast to all known carotenoid hydroxylases, which are the nonheme diiron monooxygenases, LUT1 encodes a cytochrome p450-type monooxygenase, CYP97C1. Introduction of a null mutant allele of LUT1, lut1-3, into the beta-hydroxylase 1/beta-hydroxylase 2 (b1 b2) double-mutant background, in which both Arabidopsis beta-hydroxylases are disrupted, yielded a genotype (lut1-3 b1 b2) in which all three known carotenoid hydroxylase activities are eliminated. Surprisingly, hydroxylated beta-rings were still produced in lut1-3 b1 b2, suggesting that a fourth unknown carotenoid beta-hydroxylase exists in vivo that is structurally unrelated to beta-hydroxylase 1 or 2. A second chloroplast-targeted member of the CYP97 family, CYP97A3, is 49% identical to LUT1 and hypothesized as a likely candidate for this additional beta-ring hydroxylation activity. Overall, LUT1 defines a class of carotenoid hydroxylases that has evolved independently from and uses a different mechanism than nonheme diiron beta-hydroxylases.

Functional analysis of beta- and epsilon-ring carotenoid hydroxylases in Arabidopsis.

Lutein and zeaxanthin are dihydroxy xanthophylls that are produced from their corresponding carotene precursors by the action of beta- and epsilon -ring carotenoid hydroxylases. Two genes that encode beta-ring hydroxylases (beta-hydroxylases 1 and 2) have been identified in the Arabidopsis genome and are highly active toward beta-rings but only weakly active toward epsilon -rings. A third distinct activity required for epsilon -ring hydroxylation has been defined by mutation of the LUTEIN1 (LUT1) locus, but LUT1 has not yet been cloned. To address the individual and overlapping functions of the three Arabidopsis carotenoid hydroxylase activities in vivo, T-DNA knockout mutants corresponding to beta-hydroxylases 1 and 2 (b1 and b2, respectively) were isolated and all possible hydroxylase mutant combinations were generated. beta-Hydroxylase single mutants do not exhibit obvious growth defects and have limited impact on carotenoid composition relative to the wild type, suggesting that the encoded proteins have a significant degree of functional redundancy in vivo. Surprisingly, the b1 b2 double mutant, which lacks both known beta-hydroxylase enzymes, still contains significant levels of beta-carotene-derived xanthophylls, suggesting that additional beta-ring hydroxylation activity exists in vivo. The phenotype of double and triple hydroxylase mutants indicates that at least a portion of this activity resides in the LUT1 gene product. Despite the severe reduction of beta-carotene-derived xanthophylls (up to 90% in the lut1 b1 b2 triple mutant), the double and triple hydroxylase mutants still contain at least 50% of the wild-type amount of hydroxylated beta-rings. This finding suggests that it is the presence of minimal amounts of hydroxylated beta-rings, rather than minimal amounts of specific beta-carotene-derived xanthophylls, that are essential for light-harvesting complex II assembly and function in vivo. The carotenoid profiles in wild-type seeds and the effect of single and multiple hydroxylase mutations are distinct from those in photosynthetic tissues, indicating that the activities of each gene product differ in the two tissues. Overall, the hydroxylase mutants provide insight into the unexpected overlapping activity of carotenoid hydroxylases in vivo.