Identification of the carotenoid isomerase provides insight into carotenoid biosynthesis, prolamellar body formation, and photomorphogenesis

Abstract

Carotenoids are essential photoprotective and antioxidant pigments synthesized by all photosynthetic organisms. Most carotenoid biosynthetic enzymes were thought to have evolved independently in bacteria and plants. For example, in bacteria, a single enzyme (CrtI) catalyzes the four desaturations leading from the colorless compound phytoene to the red compound lycopene, whereas plants require two desaturases (phytoene and zeta-carotene desaturases) that are unrelated to the bacterial enzyme. We have demonstrated that carotenoid desaturation in plants requires a third distinct enzyme activity, the carotenoid isomerase (CRTISO), which, unlike phytoene and zeta-carotene desaturases, apparently arose from a progenitor bacterial desaturase. The Arabidopsis CRTISO locus was identified by the partial inhibition of lutein synthesis in light-grown tissue and the accumulation of poly-cis-carotene precursors in dark-grown tissue of crtISO mutants. After positional cloning, enzymatic analysis of CRTISO expressed in Escherichia coli confirmed that the enzyme catalyzes the isomerization of poly-cis-carotenoids to all-trans-carotenoids. Etioplasts of dark-grown crtISO mutants accumulate acyclic poly-cis-carotenoids in place of cyclic all-trans-xanthophylls and also lack prolamellar bodies (PLBs), the lattice of tubular membranes that defines an etioplast. This demonstrates a requirement for carotenoid biosynthesis to form the PLB. The absence of PLBs in crtISO mutants demonstrates a function for this unique structure and carotenoids in facilitating chloroplast development during the first critical days of seedling germination and photomorphogenesis.

Figure 1.

Carotenoid Biosynthetic Pathway. The commonly held view of the carotenoid biosynthetic pathway in plants is a series of four desaturations to form all-trans-lycopene from phytoene. Lycopene is subject to two cyclization reactions to form α- or β-carotene, which are modified further to form the various xanthophylls. PDS, phytoene desaturase; ZDS, ζ-carotene desaturase; βLCY, β-cyclase; ɛLCY, ɛ-cyclase; βOH, β-hydroxylase; ɛOH, ɛ-hydroxylase; ZE, zeaxanthin epoxidase, NXS, neoxanthin synthase; VDE, violaxanthin deepoxidase.

Figure 2.

HPLC Analysis of ccr2 Pigments. (A) Extracts from etiolated ccr2 tissue accumulated prolycopene, proneurosporene, and ζ-carotene, whereas etiolated wild-type tissues accumulated lutein and violaxanthin. Insets show the absorbance spectra of prolycopene and proneurosporene and an enlargement of the HPLC trace between 26 and 28 min. (B) The amount of lutein in ccr2 leaves ranged from ∼10% after 4 days of illumination as shown here to ∼40% of wild-type levels in mature leaves. N, neoxanthin; V, violaxanthin; L, lutein; Ca, chlorophyll a; Cb, chlorophyll b; β, β-carotene; pLy, prolycopene (peak 3); pNe, proneurosporene (peak 4); mAU, milli-absorbance units. Three ζ-carotene peaks were identified and tentatively assigned as ζ-carotene (ζ; peak 5), cis-ζ-carotene (cisζ; peak 6), and pro-ζ-carotene (pζ; peak 7) based on their retention times and spectral properties. Peaks 1 and 2 correspond to a monohydroxy xanthophyll (retention time, 19.1 min) and all-trans-lycopene (23.5 min), respectively. Absorbance was at 440 nm, which underestimates the proportion of ζ-carotene. See text and Table 3 for percentages of each carotenoid and further details on carotenoid identification.

Figure 3.

Lutein Accumulation in ccr2. Lutein content of ccr2 leaves increased during development to a maximum of 30 to 40% of wild-type levels in mature leaves. Standard deviations are shown.

Figure 4.

Genetic Map of ccr1 and ccr2. Genomic DNA from recombinant inbred lines produced by crossing ccr1 and ccr2 with Landsberg erecta were used for genetic mapping. ccr1 mapped near the distal end and ccr2 mapped near the proximal end of chromosome 1. The map locations of a range of carotenoid biosynthetic genes (PDS3, phytoene desaturase; ZDS, ζ-carotene desaturase; lut2, ɛ-cyclase; lut1, ɛ-hydroxylase) and photomorphogenic loci (phyA, phyB, phyC, hy1, hy4, hy5, cop1, cop9, and det2) are shown.

Figure 5.

CRTISO Functions as an Isomerase in the Carotenoid Biosynthetic Pathway in Higher Plants. As opposed to one desaturase enzyme in the all-trans pathway of bacteria, higher plants require two desaturases and CRTISO, which can catalyze the isomerization of poly-cis-carotenes to all-trans-carotenes. See Figure 1 for abbreviations.

Figure 6.

Seedling Development and Chlorophyll Accumulation. (A) Wild-type etiolated seedlings were yellow from lutein and violaxanthin. (B) ccr2 seedlings were an orange-yellow color as a result of the presence of lycopene isomers. (C) In the absence of the PLB in ccr2, chlorophyll accumulation during photomorphogenesis was delayed markedly. wt, wild type.

Figure 7.

Diagram of the CRTISO Gene and a Phylogenetic Tree. (A) The candidate open reading frame, F4H5.10, was identified based on its linkage to ccr2. It contains a predicted chloroplast targeting sequence (arrow), a dinucleotide binding domain (closed box), and other characteristics of carotene biosynthetic enzymes. The sites of the mutations/deletions caused by the three ccr2 alleles are shown (see text for details). (B) Phylogenetic tree of isomerase genes (CRTISO) and desaturase genes from bacteria (crtI, crtN) and plants (PDS). The identity between plant and bacterial desaturases is not statistically significant.

Figure 8.

CRTISO and PLB Formation. (A) Wild-type etioplasts contained a PLB. (B) PLB diagram is based on the “wurtzite” PLB structure observed in Arabidopsis and many other species. The diagram was provided by Dr. Brian Gunning (Australian National University). (C) Model of the possible interactions between membranes, POR:Pchlide, and carotenoids in facilitating PLB formation. In vitro studies have shown that some lutein lies parallel with the membrane surface and other lutein molecules span the bilayer in a manner analogous to cholesterol (Sujak et al., 1999). It is not known which of these orientations would facilitate PLB formation. (D) All ccr2 etioplasts examined lacked a PLB, with most (29 of 34) having just a few prothylakoid membranes. (E) Some ccr2 etioplasts (15%) contained an amorphous prothylakoid aggregate. (F) A model of how the stepped structure of poly-cis-carotenes could perturb membrane curvature by increasing the spacing between fatty acids and/or by disrupting the association between the membranes and POR:Pchlide. pLy, prolycopene; pNe, proneurosporene.

Figure 9.

Content of POR and Pchlide in the Wild Type (wt) and ccr2. (A) Immunoblot of POR. POR protein levels were identical in tissue from etiolated wild type, ccr1, and ccr2. (B) Low temperature (77K) fluorescence spectra of etiolated seedlings. Fluorescence of the etiolated seedlings was measured from 600 to 700 nm with excitation at 436 nm. The total amount of Pchlide was determined by summation of peaks 650 to 657 nm and 628 to 623 nm and was slightly lower in ccr2 than in wild-type seedlings. Wild-type seedlings contained more phototransformable Pchlide (650 to 657 nm) than did nontransformable Pchlide (628 to 633 nm), whereas ccr2 had the reverse. The spectrum was normalized at 600 nm to zero. cps, counts per second.