Tangerine Dreams

Carotenoids are vital components of photosynthetic cells, where they play important accessory roles in light harvesting and energy transfer to the chlorophylls, maintain structural integrity of the photosynthetic apparatus, and provide photoprotection against the damaging effects of reactive oxygen species that are by-products of photosynthesis. Violaxanthin and neoxanthin also serve as precursors for biosynthesis of the hormone abscisic acid. In higher plants, carotenoids provide the bright yellow, orange, and red colors of flowers and fruits. Carotenoids also are important in human nutrition, serving as antioxidants in protecting against certain degenerative diseases such as macular degeneration of the eye, a leading cause of age-related blindness in the United States. Additionally, β-carotene is the precursor of vitamin A. Carotenoids are long (mainly 40-carbon) isoprenoid chains that may be linear or cyclized at one or both ends of the molecule. The first committed step in carotenoid biosynthesis in plants and cyanobacteria is the production of the colorless compound phytoene via the condensation of two molecules of geranylgeranyl diphosphate by phytoene synthase. In photosynthetic organisms, phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS) catalyze successive dehydrogenation reactions of phytoene to yield the colored molecules ζ-carotene and lycopene, respectively. In nonphotosynthetic (anoxygenic) bacteria, the desaturation of phytoene to lycopene is catalyzed by a single enzyme, bacterial phytoene desaturase (CrtI). The sequence of CrtI is unrelated to that of PDS or ZDS, suggesting that some of the steps in carotenoid biosynthesis arose independently in photosynthetic organisms (higher plants and cyanobacteria) and nonphotosynthetic bacteria (Hirschberg et al., 1997). Phytoene, ζ-carotene, and lycopene are linear molecules. Cyclization of lycopene marks a branch point of the pathway to either α- or β-carotene. Hydroxylation of α-carotene yields lutein, the major xanthophyll in the light-harvesting apparatus of most higher plants, whereas hydroxylation of β-carotene leads to the formation of the xanthophylls zeaxanthin, violaxanthin, and neoxanthin. The isoprenoid chains of carotenoids contain numerous double bonds that allow the formation of a variety of cis or trans geometric isomers. The cis-trans configurations are critical features of carotenoids, because they determine the spectral quality and resulting photochemical properties and colors of these pigments. Carotenoid isomerization in plants has been something of a mystery for more than 60 years since the tangerine (t) mutant of tomato, with its orange-tinted flowers and characteristic tangerine-orange fruit, was described by Zechmeister et al. (1941)and Tomes et al. (1953). L. Zechmeister, a noted chemist who went on to publish numerous articles on the stereochemistry of carotenoids in the 1940s and 1950s, collaborated with Fritz Went and Linus Pauling to determine the carotenoid composition of the tangerine tomato. They determined that the fruit of tangerine accumulate a poly-cis-isomer of the carotenoid lycopene, named prolycopene, whereas the wild-type red tomato varieties produce mainly trans-isomers of lycopene and other carotenoids (Zechmeister et al., 1941). Like tomato, most plants appear to produce mainly trans forms of lycopene and other carotenoids (Britton, 1988). Isomerization could occur at any number of steps, and this aspect of carotenoid biosynthesis is not well understood in higher plants. CRTISO Catalyzes the Formation of the Red Pigment trans-Lycopene from Poly-cis-Lycopene. The tangerine tomato (top left) and ccr2 mutant of Arabidopsis (top right) lack a functional CRTISO enzyme and accumulate the tangerine-orange pigment poly-cis-lycopene. PDS, phytoene desaturase; ZDS, ζ-carotene desaturase. (Figure courtesy of Hyoungshin Park). Isaacson et al. identified the tomato CRTISO gene at the tangerine locus via map-based cloning using introgression lines (ILs) of a wild tomato species, Lycopersicon pennellii, in the cultivated tomato L. esculentum. Eshed and Zamir (1994)(1995) created a population of 50 ILs from crosses between L. pennellii and L. esculentum that are nearly isogenic to L. esculentum. Each of the lines contains a single homozygous restriction fragment length polymorphism–defined L. pennellii chromosome segment, and together the lines provide complete coverage of the genome. Isaacson et al. made use of this IL population by mapping the tangerine locus to a marker that overlapped with IL 10-2. Homozygous tangerine was crossed to IL 10-2, and a marker cosegregating with tangerine was identified and used to screen a bacterial artificial chromosome library developed by Budiman et al. (2000). A bacterial artificial chromosome containing the tangerine locus was identified and found to contain an open reading frame for CRTISO. Sequence analysis of wild-type and tangerine mutant alleles of CRTISO showed that the mutant alleles contained deletions that would either yield a nonfunctional CRTISO protein (tangerine mic) or alter CRTISO expression (tangerine 3183), which was consistent with the mutant phenotypes of the respective alleles. Map-based cloning using the ILs developed by Eshed and Zamir (1994)(1995) has been used successfully to characterize several other mutations that affect carotenoid biosynthesis in tomato. Ronen et al. (1999)(2000) made use of this population of IL lines to identify the Delta mutation in the gene for lycopene ε-cyclase and the Beta and old-gold mutations in the gene for lycopene β-cyclase. Lycopene ε-cyclase and β-cyclase catalyze the cyclization of lycopene to α-carotene and β-carotene, respectively. Delta mutants have orange-colored fruit caused by the hyperaccumulation of δ-carotene as a result of a dominant mutation that enhances the expression and activity of lycopene ε-cyclase (Ronen et al., 1999). The partially dominant Beta mutants have orange-colored fruit caused by the enhanced transcription of a chromoplast-specific lycopene β-cyclase and the hyperaccumulation of β-carotene, whereas the recessive old-gold mutation, which yields deep red fruit that lack β-carotene, was found to be attributable to null mutations in this lycopene β-cyclase (Ronen et al., 2000). These studies suggest that in wild-type tomato, the differential expression and relative activities of lycopene ε-cyclase and lycopene β-cyclase determine the flow of carotenoids to either α-carotene or β-carotene. The ILs appear to be an excellent resource for cloning genes of interest from tomato as well as for the introduction of yield-improving quantitative trait loci (QTLs) from exotic variation (Zamir, 2001). Because the ILs are highly polymorphic at the DNA level and nearly isogenic with L. esculentum (each IL contains, on average, 2.75% of the L. pennellii genome), the population provides high-resolution power for fine mapping of QTL and increases the ability to identify QTL with small effects (Eshed and Zamir, 1995). Park et al. (2002)isolated the CRTISO mutant in Arabidopsis as a carotenoid and chloroplast regulation mutant named ccr2. The ccr class of mutants and a similar class of lutein-deficient (lut) mutants were identified in a screen of an ethyl methane sulfonate–mutagenized population that was based on HPLC profiles of carotenoid pigments (Pogson et al., 1996), and additional alleles of ccr2 were identified in a delayed greening screen (Park et al., 2002). Analysis of the ccr2 mutations revealed that dark-grown tissue lacked all xanthophylls and produced a tangerine-like carotenoid profile; lutein deficiency was a secondary effect. The phenotype of the ccr mutants, which have altered plastid development and pigment composition, suggests that carotenoid composition is important in early plastid development. The Arabidopsis CRTISO sequence also was identified by map-based cloning and is related closely to the sequence from tomato and to an open reading frame from the cyanobacterium Synechocystis, suggesting that the enzyme may be present in all photosynthetic organisms in which phytoene desaturation is performed by the desaturases PDS and ZDS. The tomato genome appears to have a single copy of the gene (Isaacson et al., 2002). The Arabidopsis genome contains an open reading frame that is related to CRTISO (Park et al., 2002). Expression characteristics and the possible function of the CRTISO-like sequence, located on the opposite end of chromosome I from CRTISO, are unknown. The functional importance of CRTISO activity is unclear; crtISO mutant plants are late greening compared with the wild type but otherwise are completely viable and appear “normal.” Moreover, photoisomerization of carotenoids has been observed in vitro and is believed to occur in vivo (Cunningham and Schiff, 1985; Sandmann, 1991). Thus, CRTISO activity could be partially redundant in the light. crtISO Mutants of Arabidopsis and Tomato. Arabidopsis ccr2 mutant seedlings produce etioplasts (bottom right) that lack prolamellar bodies, the paracrystalline membrane lattice that is a defining characteristic of the wild-type etioplast (top left), and exhibit a carotenoid composition similar to the tangerine tomato (bottom left). (Figure courtesy of Joseph Hirschberg and Barry Pogson). Of course, an obvious function of CrtISO in tomato involves its influence on fruit and flower color. In fruit and flowers, carotenoids are synthesized in nonphotosynthetic chromoplasts. Isaacson et al. show that CRTISO ex-pression is upregulated in flowers and during fruit ripening. Thus, CRTISO appears to have a unique function in dark-grown and nonphotosynthetic tissues. Because cyanobacteria also appear to synthesize trans-lycopene via PDS, ZDS, and CRTISO and yet lack both etioplasts and chromoplasts, there may be other important functions of CRTISO in photosynthetic organisms. Park et al. (2002)and Isaacson et al. (2002)discuss the possibility that the cis configuration of prolycopene is not cyclized easily by lycopene cyclases because of steric hindrance. It may be that greater steric hindrance of the ε-cyclase by certain lycopene isomers explains the reduced lutein in mature chloroplasts. This interesting possibility deserves further investigation.