Carotenoids in Algae: Distributions, Biosyntheses and Functions

For photosynthesis, phototrophic organisms necessarily synthesize not only chlorophylls but also carotenoids. Many kinds of carotenoids are found in algae and, recently, taxonomic studies of algae have been developed. In this review, the relationship between the distribution of carotenoids and the phylogeny of oxygenic phototrophs in sea and fresh water, including cyanobacteria, red algae, brown algae and green algae, is summarized. These phototrophs contain division- or class-specific carotenoids, such as fucoxanthin, peridinin and siphonaxanthin. The distribution of α-carotene and its derivatives, such as lutein, loroxanthin and siphonaxanthin, are limited to divisions of Rhodophyta (macrophytic type), Cryptophyta, Euglenophyta, Chlorarachniophyta and Chlorophyta. In addition, carotenogenesis pathways are discussed based on the chemical structures of carotenoids and known characteristics of carotenogenesis enzymes in other organisms; genes and enzymes for carotenogenesis in algae are not yet known. Most carotenoids bind to membrane-bound pigment-protein complexes, such as reaction center, light-harvesting and cytochrome b6f complexes. Water-soluble peridinin-chlorophyll a-protein (PCP) and orange carotenoid protein (OCP) are also established. Some functions of carotenoids in photosynthesis are also briefly summarized.


Introduction
Algae are classified throughout many divisions of the Kingdom Plantae. Their sizes range from single cells of picophytoplankton-the smallest of which are less than 1 µm-to seaweeds, the largest of which are more than 50 m. Attempts have been made to cultivate single-cell algae for a long time,

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but numbers were limited. With the recent development of culture techniques, some single-cell species can be cultured, and their characteristics, including pigments, can be studied. With the development of taxonomic technology, including DNA base sequences of 16S or 18S rRNA and some genes, algae phylogenetics has been developed.
More than 750 structurally defined carotenoids are reported from nature; land plants, algae, bacteria including cyanobacteria and photosynthetic bacteria, archaea, fungus and animals [1]. Except for animals, these organisms can synthesize many kinds of carotenoids, which are synthesized from diverse carotenogenesis pathways. These carotenoids and carotenogenesis pathways can be used as chemotaxonomic markers [2][3][4][5][6][7]. In addition, characteristics of carotenogenesis enzymes and genes are investigated. Some carotenogenesis genes have high similarity from bacteria to land plants, but some have low similarity. Some homologous genes have been proposed [8,9], but some carotenogenesis enzymes and genes, especially algae-specific ones, are not found.
In this review, the term algae refers to an oxygenic phototroph found in both seawater and fresh water, including cyanobacteria but excluding land plants. Distribution of carotenoids, carotenogenesis enzymes and pathways, and function of carotenoids in photosynthesis in algae are summarized.

Distribution of Carotenoids
Many different kinds of carotenoids were found from the algal species studied. Structures of some important carotenoids in algae are illustrated in Figure 1. Among them, approximately 30 types may have functions in photosynthesis, and others may be intermediates of carotenogenesis or accumulated carotenoids. Some carotenoids are found only in some algal divisions or classes; therefore, these carotenoids and also chlorophylls can be used as chemotaxonomic markers, and their distribution in algae is summarized in Table 1 [2][3][4][5][6].
Many cyanobacteria contain β-carotene, zeaxanthin, echinenone and myxol pentosides (myxoxanthophyll), while some species lack part of these and some contain additional carotenoids, such as nostoxanthin, canthaxanthin and oscillol dipentoside (Table 1, Figure 1) [13]. In addition, the carotenoid compositions of cyanobacteria are very different from those of chloroplasts in algae; consequently, during symbiosis of cyanobacteria to eukaryotic cells, carotenoids might be considerably restructured [13]. Note that since the name of myxoxanthophyll cannot specify the glycoside moieties, we have proposed the name of myxol glycosides to specify the glycosides, such as myxol 2′-α-L-fucoside, 4-ketomyxol 2′-rhamnoside and oscillol dichinovoside [13,15]. Rhodophyta (red algae) can be divided into two groups based on carotenoid composition; the unicellular type contains only β-carotene and zeaxanthin, and the macrophytic type contains additional α-carotene and lutein (Table 1, Figure 1) [16]. The relationship between phylogenetics of red algae and carotenoid composition is not clear [14]. Cryptophyta also contains α-carotene and its acetylenic derivatives, crocoxanthin and monadoxanthin, which are only found in this division.  Heterokontophyta, Haptophyta and Dinophyta contain β-carotene and its derivatives as well as chlorophyll c (Table 1, Figure 1). These divisions, except for Eustigmatophyceae, which lacks chlorophylls c, contain unique acetylenic carotenoids of diadinoxanthin and diatoxanthin. Fucoxanthin and its derivatives are found in only four classes of Heterokontophyta (Chrysophyceae, Raphidophyceae, Bacillariophyceae and Phaeophyceae), Haptophyta and Dinophyta. Peridinin and its derivatives are found only in Dinophyta. Fucoxanthin and peridinin have unique structures ( Figure 1) and are class-specific carotenoids (Table 1).
Euglenophyta, Chlorarachniophyta and Chlorophyta contain the same carotenoids, such as β-carotene, violaxanthin, 9′-cis neoxanthin [11] and lutein, as well as chlorophyll a and b with land plants (Table 1, Figure 1). Some classes contain additional carotenoids, such as loroxanthin, siphonaxanthin and prasinoxanthin, which are derivatives of lutein, and are class specific.
Note that identifications of some carotenoids were lacking because of insufficient analysis, and that some algae names were changed because of new developments in taxonomic technology and phylogenetic classification.

Carotenogenesis Pathways, Enzymes and Genes
Carotenogenesis pathways and their enzymes are mainly investigated in cyanobacteria [13] and land plants among oxygenic phototrophs [17]. Especially in land plants, carotenogenesis pathways and characteristics of enzymes are studied in detail ( Figure 2). On the other hand, algae have common pathways with land plants and also additional algae-specific pathways, which are solely proposed based on the chemical structures of carotenoids ( Figure 2). Some common carotenogenesis genes in algae are suggested from homology of the known genes [8,9], but most genes and enzymes for algae-specific pathways are still unknown ( Figure 2). In cyanobacteria, since carotenoid compositions are different from those in land plants and algae, the pathways and enzymes are also different from those in Figure 2, and they are shown in Figure 3. In addition, carotenogenesis enzymes and genes, whose functions are confirmed in algae, including cyanobacteria, are summarized in Table 2. Unfortunately, these enzymes are mostly from cyanobacteria and green algae ( Table 2).

Isopentenyl Pyrophosphate to Phytoene Synthesis
Isopentenyl pyrophosphate (IPP), a C 5 -compound, is the source of isoprenoids, terpenes, quinones, sterols, phytol of chlorophylls, and carotenoids. There are two known independent pathways of IPP synthesis: the classical mevalonate (MVA) pathway and the alternative, non-mevalonate, 1-deoxy-D-xylulose-5-phosphate (DOXP) pathway [18,19]. In the MVA pathway, acetyl-Coenzyme A is converted to IPP through mevalonate, and the enzymes and genes are well studied [20]. The pathway is found in plant cytoplasm, animals and some bacteria [18,20]. The DOXP pathway was found in the 1990s, and in this pathway, pyruvate and glycelaldehyde are converted to IPP. The DOXP pathway is found in cyanobacteria, the plastids of algae and land plants, and some bacteria [18]. Carotenoids are synthesized in plastids. Exceptionally among oxygenic phototrophs, Euglenophyceae has only the MVA pathway, and Chlorophyceae has only the DOXP pathway [18].   Red, genes and enzymes related to α-carotene.
Most carotenoids consist of eight IPP units. Farnesyl pyrophosphate (C 15 ) is synthesized from three IPPs, after which one IPP is added to farnesyl pyrophosphate by geranylgeranyl pyrophosphate synthase (CrtE, GGPS) to yield geranylgeranyl pyrophosphate (C 20 ). In a head-to-head condensation of the two C 20 compounds, the first carotene, phytoene (C 40 ), is formed by phytoene synthase (CrtB, Pys, Psy) using ATP [57,58]. This pathway has been confirmed by cloning genes from two species of Rhodobacter (purple bacteria) and two species of Pantoea (previously Erwinia) [57][58][59]. Among oxygenic phototrophs, the functions of CrtE of Thermosynechococcus elongatus BP-1 [21], and CrtB of three species of cyanobacteria [22][23][24] and two species of green algae [25,26] have also been confirmed ( Table 2). The crtE and crtB genes have high sequence similarity from bacteria to land plants, respectively.
In contrast, the bacterial type uses only one enzyme, phytoene desaturase (CrtI), to convert from phytoene to lycopene, and the primitive cyanobacterium of Gloeobacter violaceus PCC 7421 uses this type of CrtI, and the homologous genes of crtP, crtQ and crtH are not found in the genome [22,27]; therefore, G. violaceus is the first oxygenic phototroph that has been shown to use this type (Table 2). These observations suggest the following evolutionary scheme for this step in the reaction: the desaturation of phytoene was initially carried out by CrtI in ancestral cyanobacteria, crtP and related desaturase genes were acquired, and ultimately, there was replacement of crtI by crtP [27]. Among anoxygenic phototrophs, purple bacteria, green filamentous bacteria and heliobacteria use CrtI, whereas green sulfur bacteria use CrtP, CrtQ and CrtH [61].
Some cyanobacteria also contain these enzymes (Table 2). Synechococcus elongatus PCC 7942 contains a functional CrtL [36]. Prochlorococcus marinus MED4 contains two lycopene cyclases (Table 2), which have sequence homology to CrtL. CrtL-b exhibits lycopene β-cyclase activity, while CrtL-e is a bifunctional enzyme having both lycopene ε-cyclase and lycopene β-cyclase activities [37]. The combination of these two cyclases allows the production of β-carotene, α-carotene and ε-carotene. Both enzymes might have originated from the duplication of a single gene. The characteristics of this CrtL-e are somewhat different from those in land plants [66]. In addition, the β-end groups of both β-carotene and α-carotene (left half) might be hydroxylated by CrtR to zeaxanthin through β-cryptoxanthin and 3-hydroxy-α-carotene, respectively, in P. marinus. Acaryochloris marina MBIC 11017, which produces α-carotene, contains only one crtL-like gene from genome sequence [14].
Recently, a new family of functional lycopene cyclase, CruA, has been found in Chlorobaculum (previously Chlorobium) tepidum (green sulfur bacterium), and the main product is γ-carotene in Escherichia coli, which produces lycopene [68]. Homologous genes, cruA and cruP, have been found in the genome of Synechococcus sp. PCC 7002, and their main products are γ-carotene, in E. coli, which produces lycopene [63]. In addition, their homologous genes are widely distributed in cyanobacteria, such as Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120; however, these cruA-and cruP-like genes from both Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 did not show the lycopene dicyclase or monocyclase activities [14]. S. elongatus PCC 6301 and PCC 7942, and A. marina MBIC 11017 contain crtL-, cruA-and cruP-like genes; consequently, distributions of functional lycopene cyclases (CrtL-, CruA-and CruP-like) in cyanobacteria are unknown.
Since Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 lack crtL-like genes and contain non-functional cruA-like genes, there is a possibility to present a fourth new family of lycopene cyclases in these cyanobacteria. Further studies of distributions of functional lycopene cyclases (CrtL-and CruA-like, or others) in cyanobacteria are needed.
Distribution of α-carotene, that is, CrtL-e, is limited in some algae classes (Table 1). Genes and enzymes of CrtL-e are not found in algae. In some species of land plants, the characteristics of CrtL-e were investigated [66], and were shown to have sequence homology with crtL-b. Lycopene is first converted to δ-carotene by CrtL-e, and then to α-carotene by CrtL-b. γ-Carotene produced by CrtL-b is not a suitable substrate for CrtL-e.
The pathway and the enzymes to produce the right half of myxol 2′-pentoside are still unknown ( Figure 3) [13].

Land Plants
In land plants, most of the carotenogenesis pathways and the functionally confirmed enzymes are known ( Figure 2). Hydroxyl groups are introduced into β-carotene to produce zeaxanthin by β-carotene hydroxylase (CrtR, CrtR-b, BCH). Epoxy groups are introduced into zeaxanthin by zeaxanthin epoxidase (Zep, NPQ) to produce violaxanthin through antheraxanthin. Under high light conditions, violaxanthin is changed into zeaxanthin by violaxanthin de-epoxidase (Vde) for dispersion of excess energy from excited chlorophylls. One end group of violaxanthin is changed to an allene group of neoxanthin by neoxanthin synthase (Nsy). Because all neoxanthin in chloroplasts has the 9′-cis form, unknown 9′-isomerase for all trans neoxanthin to 9′-cis neoxanthin should be present [11].
Allenic carotenoids are very limited in algae. From their chemical structures, all trans neoxanthin might be changed to fucoxanthin, dinoxanthin, peridinin, vaucheriaxanthin and diadinoxanthin, but the pathways and enzymes are still unknown (Figures 1 and 2).

α-Carotene Derivatives and Their Synthesis
In Arabidopsis thaliana, β-carotene is hydroxylated mainly by the non-heme di-iron enzymes, BCH1 and BCH2 (CrtR-b), to produce zeaxanthin, while α-carotene is mainly hydroxylated by the cytochrome P450 enzymes, CYP97A3 for the β-end group and CYP97C1 for the βand ε-end groups, to produce lutein [72].
Lutein and its derivatives are found only in Rhodophyta (macrophytic type), Cryptophyta, Euglenophyta, Chlorarachniophyta and Chlorophyta (Table 1), but nothing is known for hydroxylation of α-carotene. From the chemical structures of siphonaxanthin [12], loroxanthin, prasinoxanthin and monadoxanthin, it could be considered that they are derived from lutein, but the pathways and enzymes are still unknown (Figures 1 and 2).

Function of Carotenoids
For photosynthesis, both carotenoids and chlorophylls are necessarily bound to peptides to form pigment-protein complexes in the thylakoid membrane. Five main kinds of the complexes described below are isolated from some algae, and the pigment compositions are investigated [73][74][75]. Exceptionally in cyanobacteria, myxol glycosides and some carotenoids are located in the cytoplasmic membrane for protection from high-light [76,77].
The dimeric cytochrome b 6 f complexes of the cyanobacterium Mastigocladus laminosus [78] and the green alga Chlamydomonas reinhardtii [79] contain two β-carotene and two chlorophyll a molecules, while that of the cyanobacterium Synechocystis sp. PCC 6803 contains two echinenone and two chlorophyll a molecules [80]. These carotenoids might have protective functions.
The water-soluble peripheral LHC of peridinin-chlorophyll-protein (PCP) isolated from Amphidinium carterae (Dinophyta) has a trimeric structure, and the monomer contains eight peridinin and two chlorophyll a molecules [81]. The water-soluble orange carotenoid protein (OCP) isolated from the cyanobacterium Arthrospira maima forms a homodimer with two 3′-hydroxyechinenone molecules [82]. OCP is also found in some cyanobacteria, and its function might regulate energy dissipation from phycobilisomes to PSII [83].
The keto groups at C-8 of fucoxanthin [84], siphonaxanthin [85,86] and prasinoxanthin [87], which are found only in algae, are the single-bond trans-conformation for the conjugated double bond (Figure 1). From the femtosecond time-resolved fluorescence spectroscopy of the purified carotenoids in organic solvents and the LHC in solution, these keto-carotenoids and peridinin have been found to have highly efficient energy transfer from the S 1 state, not the S 2 state, of carotenoids to chlorophylls. From the comparison of other structural carotenoids, these keto groups are essential for high efficiency [88,89]. These keto-carotenoids mainly might have light-harvesting functions.
The xanthophyll cycle, also known as the violaxanthin cycle, is the cyclical interconversion of violaxanthin, antheraxanthin and zeaxanthin in green algae and land plants (Figure 2) [90]. Zep catalyzes zeaxanthin to violaxanthin through antheraxanthin during biosynthesis. Violaxanthin is found in the peripheral LHC of PSII. Under high light conditions, Vde is activated and catalyzes de-epoxidation of violaxanthin to zeaxanthin through antheraxanthin. Zeaxanthin is used for the dissipation of excess energy from excited chlorophylls. Zep from Chlorophyceae Chlamydomonas reinhardtii [48] and Vde from Pracinophyceae Mantonilla squamata [49] are functionally confirmed (Table 2). Similarly, the diadinoxanthin cycle occurs in Heterokontophyta, Haptophyta and Dinophyta, which contain diadinoxanthin and diatoxanthin ( Figure 2). The enzymes of diadinoxanthin de-epoxidase and diatoxanthin epoxidase have not yet been found [9,91], but the characteristics of partially purified diadinoxanthin de-epoxidase from the diatom Cyclotella meneghinaina are reported [92].