Defining the primary route for lutein synthesis in plants: the role of Arabidopsis carotenoid beta-ring hydroxylase CYP97A3

Abstract

Lutein, a dihydroxy derivative of alpha-carotene (beta,epsilon-carotene), is the most abundant carotenoid in photosynthetic plant tissues where it plays important roles in light-harvesting complex-II structure and function. The synthesis of lutein from lycopene requires at least four distinct enzymatic reactions: beta- and epsilon-ring cyclizations and hydroxylation of each ring at the C-3 position. Three carotenoid hydroxylases have already been identified in Arabidopsis, two nonheme diiron beta-ring monooxygenases (the B1 and B2 loci) that primarily catalyze hydroxylation of the beta-ring of beta,beta-carotenoids and one heme-containing monooxygenase (CYP97C1, the LUT1 locus) that catalyzes hydroxylation of the epsilon-ring of beta,epsilon-carotenoids. In this study, we demonstrate that Arabidopsis CYP97A3 (the LUT5 locus) encodes a fourth carotenoid hydroxylase with major in vivo activity toward the beta-ring of alpha-carotene (beta,epsilon-carotene) and minor activity on the beta-rings of beta-carotene (beta,beta-carotene). A cyp97a3-null allele, lut5-1, causes an accumulation of alpha-carotene at a level equivalent to beta-carotene in wild type, which is stably incorporated into photosystems, and a 35% reduction in beta-carotene-derived xanthophylls. That lut5-1 still produces 80% of wild-type lutein levels, indicating at least one of the other carotene hydroxylases, can partially compensate for the loss of CYP97A3 activity. From these data, we propose a model for the preferred pathway for lutein synthesis in plants: ring cyclizations to form alpha-carotene, beta-ring hydroxylation of alpha-carotene by CYP97A3 to produce zeinoxanthin, followed by epsilon-ring hydroxylation of zeinoxanthin by CYP97C1 to produce lutein.

Fig. 1.

Pathway showing all possible routes to xanthophyll synthesis in Arabidopsis. Enzymatic reactions are indicated by numbers: , ε-cyclization; , β-cyclization; , β-ring hydroxylation of β,ε- and β,ψ-carotenoids; , ε-ring hydroxylation;, , β-ring hydroxylation of β,β-carotenoids. Reactions blocked by mutation of the indicated loci are shown: lut1 (ε-ring hydroxylase), lut2 (ε-cyclase), lut5, b1, and b2 (three β-ring hydroxylases). Solid gray arrows indicate a reaction sequence that is supported by mutant phenotypes and/or enzyme activity assays in E. coli, whereas dashed gray arrows are not. Solid black arrows, compounds, and mutant loci indicate major biosynthetic routes.

Fig. 2.

Identification and characterization of two unknown peaks in lut5-1. (Left) HPLC analyses (440-nm absorption) of leaf extracts of the indicated genotypes. N, neoxanthin; V, violaxanthin; A, antheraxanthin; L, lutein; Z, zeaxanthin; chl a, chlorophyll a; chl b, chlorophyll b; zei, zeinoxanthin; β-car, β-carotene. (Right) An overlay of sections of the lut5-1 (black) and lut1-4 (gray) HPLC chromatograms containing unknown peaks 1 and 2 and UV-visible absorption spectra of zeinoxanthin, α-carotene, and unknown peaks 1 and 2.

Fig. 3.

Nondenaturing gel electrophoretic separation of pigment–protein complexes from thylakoid membranes of the indicated genotypes. PS, photosystem I holocomplex and photosystem II core complex; LHCT, trimeric form of LHC; LHCM, monomeric form of LHC; FP, free-pigment zone.

Fig. 4.

Expression of carotenoid biosynthetic genes in lut1-4, lut5-1, and lut5-1lut1-4 relative to WT. The dotted line refers to the expression level of each gene in WT. Transcripts were quantified by real-time PCR by using elongation factor-1α as a reference control.