Received February 20, 2003
Accepted March 25, 2003
Functional Analysis of 
- and 
-Ring Carotenoid Hydroxylases in Arabidopsis
Li Tian 1, Maria Magallanes-Lundback 1, Valeria Musetti 1, and Dean DellaPenna 1*
1
Department of Biochemistry and Molecular Biology, Michigan State University, East
Lansing, Michigan 48824
* To whom correspondence should be addressed. E-mail: dellapen{at}msu.edu.
Lutein and zeaxanthin are dihydroxy xanthophylls that are produced from their corresponding
carotene precursors by the action of 
- and 
-ring carotenoid hydroxylases.
Two genes that encode -ring hydroxylases (-hydroxylases 1 and 2) have been
identified in the Arabidopsis genome and are highly active toward -rings but
only weakly active toward -rings. A third distinct activity required for -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 -hydroxylases 1 and 2 (
b1 and b2, respectively) were isolated and all possible hydroxylase
mutant combinations were generated. -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 -hydroxylase enzymes, still contains significant levels
of -carotene-derived xanthophylls, suggesting that additional -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 -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 -rings. This finding suggests that it is the presence of minimal
amounts of hydroxylated -rings, rather than minimal amounts of specific -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.
