Function of carotenoids
Carotenoids are isoprenoid compounds (mostly C40) with polyene chains that may contain up to 15 conjugated double bonds (Figure 4). More than 700 naturally occurring carotenoids have been identified (Britton et al., 1995, 2004). Carotenoids differ from anthocyanins and betalains in that they play essential roles in plant life, for example, photoprotective functions during photosynthesis (Green and Durnford, 1996; Niyogi, 2000) and provision of substrates for biosynthesis of the plant growth regulator abscisic acid (ABA; Nambara and Marion-Poll, 2005) and perhaps other hormones as well (Auldridge et al., 2006). Carotenoids also play an important role in human nutrition and health, providing provitamin A and having anti-cancer activities (Mayne, 1996). Some carotenoids are used as food colorants, cosmetics or pharmaceuticals.
Figure 4. Carotenoid biosynthesis pathway in plants. For the sake of simplicity, only all-trans-configurations are shown. DXPS, 1-deoxy-d-xylulose-5-phosphate synthase; DXR, 1-deoxy-d-xylulose-5-phosphate reductoisomerase; IPI, isopentenyl pyrophosphate isomerase; GGDP, geranylgeranyl diphosphate synthase; PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, ζ-carotene desaturase; LCYB, lycopene β-cyclase; LCYE, lycopene ε-cyclase; CHYB, β-ring hydroxylase; CHYE, ε-ring hydroxylase; ZEP, zeaxanthin epoxidase; VDE, violaxanthin de-epoxidase; CRTISO, carotenoid isomerase; NSY, neoxanthin synthase; NCED, 9-cis-epoxycarotenoid dioxygenase.
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Carotenoids show qualitative differences depending on the plant organs and species. For example, the green tissues of most plants show similar carotenoid profiles, accumulating both β,ε-carotenoids (with one β- and one ε-ring) and β,β-carotenoids (with two β-rings) (Goodwin and Britton, 1988). Carotenoids such as zeaxanthin, violaxanthin, antherxanthin and lutein are invariably found in leaves and stems. In contrast, carotenoids in non-green tissues show distinctive compositions that depend on the plant species. For example, tomato (Solanum lycopersicum) fruit accumulates a large amount of lycopene (Fraser et al., 1994). Capsanthin and capsorbin, ketocarotenoids that contain one and two acyl-cyclo-pentanol rings, respectively, are the typical carotenoids of red pepper (Capsicum annuum) (Hornero-Méndez et al., 2000). Bixa orellana is the only plant that accumulates bixin in its seeds (Bouvier et al., 2003a). Bixin is a dicarboxyl monomethyl ester apocarotenoid, also known as annatto, and is used in food and cosmetics as a red color additive. In general, plants do not accumulate carotenoids in their roots. The storage roots of carrot (Baranska et al., 2006) and sweet potato (Hagenimana et al., 1999) are exceptions. They accumulate a high concentration of β-carotene, and are an important source of vitamin A in the human diet. The majority of carotenoids in the petals of sandersonia (Sandersonia aurantiaca) are β,β-carotenoids, such as β-cryptoxanthin, zeaxanthin and β-carotene (Nielsen et al., 2003). On the other hand, more than 90% of the carotenoids in the petals of marigold (Tagetes sp.; Figure 1j; Moehs et al., 2001) and chrysanthemum (Kishimoto et al., 2004) are lutein and/or lutein derivatives (β,ε-carotenoids).
The main carotenoids of the flower petals of most plants are yellowish xanthophylls, which are pale to deep yellow in color (Table S1). The petals of some plants have a modified carotenoid biosynthetic capacity, accumulate unique carotenoids associated with their respective genus or even species, and are orange to red in color. Astaxanthin (3,3′-dihydroxy-4,4′-diketo-β,β-carotene) is a ketocarotenoid that is produced in a number of bacteria, fungi and algae; it furnishes an attractive orange–red color. Only a few plant species are known to produce astaxanthin. The petals of Adonis aestivalis and A. annua anomalously accumulate a large amount of astaxanthin, resulting in their blood-red color (Figure 1k; Cunningham and Gantt, 2005). The orange petals of calendula (Calendula officinalis) contain reddish carotenoids that are absent in yellow petals (Figure 1l). Some have a cis-structure at C5 or C5′, which is very rare in plants (Kishimoto et al., 2005). The red style branches of crocus (Crocus sativus), from which the spice saffron is derived, accumulate the unique apocarotenoids, crocetin glycosides, picrocrocin and safranal. They are produced by the cleavage of zeaxanthin and are responsible for the color, taste and aroma of saffron (Bouvier et al., 2003b).
Biosynthesis of carotenoids
In the past decade, the genes encoding nearly all the enzymes for carotenoid biosynthesis in plants have been identified, and their enzymatic activities have been characterized (see reviews by Cunningham and Gantt, 1998; Hirschberg, 2001; Howitt and Pogson, 2006). In plants, the entire pathway starting from isopentenyl pyrophosphate (IPP) occurs in the plastids, and it is there that the product accumulates. It is hypothesized that carotenogenic enzymes exist in complex with and associated with the plastid membranes (Cunningham and Gantt, 1998).
Figure 4 summarizes the carotenoid biosynthesis pathway in plants. Carotenoid biosynthesis starts from a C5 isoprene unit, IPP. Four IPPs are condensed to form C20 geranylgeranylpyrophosphate (GGPP). A head-to-head coupling of two GGPP molecules, catalyzed by phytoene synthase (PSY), yields the first C40 carotenoid, phytoene. In tomato, two different types of PSYs (Psy-1 and Psy-2) are expressed in an organ-specific manner (Fraser et al., 1999). Psy-1 encodes a fruit- and flower-specific isoform and is responsible for carotenogenesis in chromoplasts. In green tissues, Psy-2, which is homologous to Psy-1 but highly divergent from it, is predominantly expressed, and makes a major contribution to carotenogenesis in chloroplasts.
Conjugated double bonds are subsequently added by two structurally similar enzymes, phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS). These desaturation reactions yield the intermediates phytofluene, ζ-carotene, neurosporene and lycopene, containing 5, 7, 9 and 11 conjugated double bonds, respectively. Increasing the number of conjugated double bonds shifts the absorption towards longer wavelengths, resulting in colorless phytoene and phytofluene, pale-yellow ζ-carotene, orange–yellow neurosporene and red lycopene. During the desaturation steps, several reaction intermediates with a cis-configuration are produced. Conversion of a cis- to a trans-configuration to form all-trans-lycopene is carried out by carotenoid isomerase (CRTISO), which has been identified in tomato (Isaacson et al., 2002) and Arabidopsis (Park et al., 2002). CRTISO is specific for adjacent double bonds at the 7,9 and 7′,9′-positions, and converts 7,9,9′-tri-cis-neurosporene and 7′,9′-di-cis-lycopene, the products of ZDS, to 9′-cis-neurosporene and all-trans-lycopene, respectively. Recently, Li et al. (2007) reported a second carotenoid isomerase (termed Z-ISO) in maize that converts the 15-cis-bond in 9,15,9′-tri-cis-ζ-carotene, the product of PDS, to 9,9′-di-cis-ζ-carotene, the substrate of ZDS.
The cyclization of lycopene is a branch point in the pathway, catalyzed by lycopene β-cyclase (LCYB) and lycopene ε-cyclase (LCYE). Because LCYE in most plants adds only one ε-ring to lycopene (Cunningham and Gantt, 2001; Cunningham et al., 1996), the pathway in plants typically proceeds only along branches, leading to carotenoids with one β- and one ε-ring (α-carotene and its derivatives) or two β-rings (β-carotene and its derivatives). Lycopene ε-cyclase in romaine lettuce (Lactuca sativa) has the ability to add two ε-rings to lycopene and yields a bicyclic ε-carotene, lactucaxanthin (Cunningham and Gantt, 2001). A single amino acid residue (457th Histidine) is important to form bicyclic ε-carotene.
β- and α-carotenes are further modified by hydroxylation or epoxidation, providing a variety of structural features. The oxygenated derivatives of carotene are called xanthophylls. Hydroxylation of the β- and ε-rings is catalyzed by β-hydroxylase (CHYB) and ε-hydroxylase (CHYE), respectively. CHYB is a non-heme di-iron mono-oxygenase, while CHYE is a P450, CYP97C1 (Tian et al., 2004). CHYB is a well-studied enzyme that has been cloned and characterized from many organisms, while CHYE has been identified only in Arabidopsis. A flower-specific CHYB (CrtR-b2) was recently identified in tomato (Galpaz et al., 2006). Taken together with the existence of flower- and fruit-specific PSY, GGPS and LCYB (tomato expression database, http://ted.bti.cornell.edu/), this finding supports the hypothesis that there is a chromoplast-specific carotenoid biosynthesis pathway.
Epoxidation at positions C5,6 and C5′,6′ of the β-ring of zeaxanthin, catalyzed by zeaxanthin epoxidase (ZEP), yields violaxanthin. Violaxanthin is converted to neoxanthin by neoxanthin synthase (NSY). Both 9-cis-violaxanthin and 9-cis-neoxanthin are cleaved to xanthoxin (C15) by 9-cis-epoxycarotenoid dioxygenase (NCED), and then converted to ABA via the ABA aldehyde intermediate (Nambara and Marion-Poll, 2005).
Regulation of carotenoid biosynthesis and accumulation of carotenoids
Plant tissues, in particular flower petals and fruits, have a wide variety of carotenoid contents, ranging from little or none to large amounts even within the same plant species. There is increasing evidence that carotenogenesis in plant tissues is predominantly regulated at the transcriptional level (see review by Sandmann et al., 2006). In marigold, the differences in petal color from pale-yellow to orange–red are caused by the different levels of accumulation of yellow carotenoid lutein (Figure 1j). Moehs et al. (2001) demonstrated that a higher level of PSY and 1-deoxy-d-xylulose-5-phosphate synthase might be responsible for the color development from pale-yellow to orange. It has also been demonstrated that PSY is a rate-limiting enzyme of carotenoid biosynthesis in canola (Brassica napus) seeds (Shewmaker et al., 1999) and tomato fruits (Fraser et al., 1994).
The successful isolation of genes for carotenoid biosynthesis will allow identification of the key regulatory steps of carotenoid biosynthesis. Nevertheless, knowledge on the molecular aspects that regulate the pathway is still limited. Recently, the genes responsible for hp1 and hp2 (mutations conferring a high level of carotenoids) have been shown to encode the proteins UV-DAMAGED DNA-BINDING PROTEIN 1 (DDB1) and DEETIOLATED 1 (DET1), components that are involved in the light-signal transduction pathway (Liu et al., 2004). In addition, other light-signaling components, such as HY5 and COP1, have been shown to antagonistically regulate the carotenoid level in tomato fruits (Davuluri et al., 2005; Liu et al., 2004). In petals of the Mimulus species, a single QTL at the YUP locus controls the presence and absence of carotenoids (Bradshaw and Schemske, 2003). It is interesting to note that the observed changes of pollinator preference associated with YUP alleles in Mimulus are comparable to those associated with AN2 alleles in petunia (Hoballah et al., 2007) as described above, although the identity of the YUP locus remains to be elucidated.
The amount of carotenoids in the tissues is not attributed solely to the ability to synthesize carotenoids. Some plant tissues have the capacity to synthesize carotenoids but contain only a trace amount of carotenoids. The mechanism that controls carotenoid accumulation is largely unknown. Recently, two different regulatory mechanisms were postulated. One is focused on carotenoid degradation, and the other is focused on sink capacity. In the case of chrysanthemum petals, there was no significant difference in the expression levels of the carotenogenic genes between the white and yellow petals of chrysanthemums (Kishimoto and Ohmiya, 2006). However, a gene encoding carotenoid cleavage dioxygenase (CmCCD4a) was specifically expressed in white petals (Ohmiya et al., 2006). Suppression of CmCCD4a expression resulted in a change in the petal color from white to yellow, indicating that normally white petals synthesize carotenoids but immediately degrade them into colorless compounds. The importance of sink capacity for carotenoid accumulation was first demonstrated in cauliflower (Brassica oleracea var. botrytis) Orange (Or) mutant. Or is a gain-of-function mutation, and single-locus Or mutation confers a high level of β-carotene accumulation in tissues where accumulation of carotenoid is normally repressed (Li et al., 2001). The Or gene encodes a plastid-associated protein with a cysteine-rich domain similar to that found in DnaJ-like molecular chaperones (Lu et al., 2006). This protein plays an important role in triggering differentiation of proplastids and/or other non-colored plastids into chromoplasts, which in turn act as a metabolic sink for carotenoids. Transformation of the Or gene into wild-type cauliflower (or) converts the white colour curd tissue into an orange color with increased levels of β-carotene.