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Carotenoid biosynthesis in flowering plants Joseph Hirschberg

The general scheme of carotenoid biosynthesis has been be considered as secondary metabolites. Carotenoids in known for more than three decades. However, molecular plants are also precursors for the synthesis of the hormone description of the pathway in plants began only in the 1990s abscisic acid (ABA) [3,4]. after the genes for the carotenogenic enzymes were cloned. Recent data on the biochemistry of carotenogenesis and its Elucidation of the carotenoid biosynthesis pathway is a won- regulation in vivo present the possibility of genetically derful example of a successful interdisciplinary approach to manipulating this pathway in crop plants. studying plant biochemistry. The enzymes of this pathway exist in minute amounts and are very labile upon purifi- Addresses cation. These characteristics and the lack of genuine in vitro Department of Genetics, The Alexander Silberman Life Science assays for any of the enzymes have hindered the usage of Institute, The Hebrew University of Jerusalem, Jerusalem, 91904 Israel; conventional biochemical investigation. Because the cloning e-mail: [email protected] of the genes for these enzymes could not rely on protein Current Opinion in Plant Biology 2001, 4:210–218 purification, molecular analysis required the use of various genetic methods. Cloning of the first genes took advantage 1369-5266/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. of the fact that the pathway in plants is similar to that in cyanobacteria. Hence, the phytoene desaturase (Pds) gene was Abbreviations first isolated from mutants of Synechococcus sp. PCC7942 that ABA abscisic acid CCS capsanthin-capsorubin synthase were resistant to norflurazon, an inhibitor of PDS [5]. The CRTI bacterial phytoene desaturase gene was identified by its ability to confer herbicide resis- CYC-B chromoplast-specific lycopene cyclase tance in the wild-type background. The cyanobacterial DOXP 1-deoxyxylulose 5-phosphate genes then served as molecular probes to isolate the plant DPME 4-diphosphocytidyl-2C-methyl-D-erythritol orthologs. A similar methodology has been successfully used DXR DOXP reductoisomerase β DXS DOXP synthase to clone the gene for lycopene -cyclase [6]. GGPP geranylgeranyl diphosphate GGPS GGPP synthase Using a reverse genetics strategy, the gene for phytoene synthase Ipi IPP isomerase gene (Psy) was identified in transgenic tomato plants in which the IPP isopentenyl diphosphate ispD (ygbP) DPME synthase expression of a candidate cDNA was silenced [7]. A unique ispE DPME kinase functional complementation approach to cloning genes has LCY-B/CRTL-B lycopene β-cyclase been developed on the basis of the ability of the ε LCY-E/CRTL-E lycopene -cyclase carotenogenic enzymes to function in the bacterium Nxs neoxanthin synthase gene Pds phytoene desaturase Escherichia coli [8]. This so-called ‘color complementation’ PSY phytoene synthase technique takes advantage of E. coli engineered with bacterial VDE violaxanthin deepoxidase genes to produce a colored carotenoid that serves as a precursor ZDS ζ-carotene desaturase for the enzyme under investigation. The carotenoid accumu- Zep1 Zeaxanthin epoxidase ?1 lated in the bacteria imparts a characteristic color to the colonies that can be seen by the naked eye. The screening for Introduction a specific gene is based on the visualization of color changes Plant carotenoids are 40-carbon isoprenoids with polyene in E. coli colonies following transfection of the bacteria with chains that may contain up to 15 conjugated double bonds. plant cDNA libraries carried on expression plasmids. Because of their chemical properties carotenoids are essential components of all photosynthetic organisms. Transposon tagging was effectively used to clone the Xanthophylls, oxygenated forms of carotenes, are accessory Zeaxanthin epoxidase1 (Zep1) gene from Nicotiana pigments in the light-harvesting antennae of the chloro- plumbaginigfolia by screening for ABA-deficient phenotype plasts, which are capable of transferring energy to the [9]. An additional genetic technique that was valuable in chlorophylls. They also quench triplet excited states in obtaining carotenoid-biosynthesis genes is map-based chlorophyll molecules by dissipating the excess excitation cloning. This technique has been successfully employed energy in a non-radiative manner, a process known as non- to clone novel genes from tomato, a species with a variety photochemical quenching (NPQ) (reviewed in [1,2]). This of mutations that affect carotenoid biosynthesis and accu- function is crucial to protect against chlorophyll bleaching mulation. Advances in plant genomics offer new ways to in intense light. An additional important role of identify novel genes on the basis of their sequence simi- carotenoids in plants is to furnish flowers and fruits with larity to known genes and are expected to facilitate the distinct colors that are designed to attract animals. In cloning of novel genes in the future. Characterization of chloroplasts, carotenoids play vital roles in photosynthesis the enzymes encoded by these novel genes was often done and are indispensable, whereas in chromoplasts, they can in E. coli cells that express the cloned genes. Enzymes that Carotenoid biosynthesis in flowering plants Hirschberg 211

Figure 1

GA3P LytB POH2C DXS POH C PPO OH POH2C DXR 2 ispD ispE ispF ? Ipi O OH OH PPO + HO A B C HOOC O O DMADP NADPH NADP+ O CO2 IPP DOXP MEP + 3xIPP Ggps Pyruvate

OPP + PPO GGPP GGPP Psy

Phytoene Pds Isomerase (?) (tangerine) crtI ζ-Carotene Zds

Lycopene Cyc-B Lcy-e (CrtL-e) Lcy-b (CrtL-b)

δ-Carotene γ-Carotene Lcy-b (CrtL-b) Lcy-b (CrtL-b) Cyc-B

α-Carotene β-Carotene CrtR-b CrtR-b OH (CrtR-e) OH

Zeaxanthin Lutein HO HO Vde1 Zep1 OH OH Ccs O O Antheraxanthin Capsanthin OH HO Vde1 Zep1 Zep1 OH OH Ccs O O O O Capsorubin HO Violaxanthin Nxs VNCED OH OH (VP14) . O AO OH Neoxanthin ABA ABA-aldehyde- Xanthoxin xanthoxin HO Current Opinion in Plant Biology

The carotenoid biosynthesis pathway in plants. Enzymes are named β-ring hydroxylase, CrtR-e, ε-ring hydroxylase; DMADP, dimethylallyl according to the designation of their genes. The pathway in the box diphosphate; DOXP, 1-deoxy-D-xylulose 5-phosphate; ispF, takes place in chromoplasts of pepper fruit. A, DPME; B, 4-diphospho- 2C-methyl-D-erythritol 2,4-cyclodiphosphate; MEP, 2-C-methyl-D- cytidyl-2C-methyl-D-erythritol 2-phosphate; C, 2C-methyl-D- erythritol 4-phosphate; VNCED (VP14), 9-cis-epoxycarotenoid erythritol 2,4-cyclodiphosphate. AO, adldehyde oxidase; CrtR-b, dioxygenase.

were purified from such bacteria have been analyzed in Haematococcus pluvialis in which the last steps in the synthesis cell-free carotenogenic systems. of the ketocarotenoid astaxanthin take place in cytoplasmic lipid vesicles [10,11•]. This article reviews recent discoveries Biosynthetic pathway in carotenoid biosynthesis in higher plants. In plants, carotenoids are synthesized within the plastids by enzymes that are nuclear encoded. A unique exception to this Like all other isoprenoids, carotenoids are built from the rule has recently been discovered in the green alga 5-carbon compound isopentenyl diphosphate (IPP). In 212 Physiology and metabolism

Figure 2 GGPP. The genome of Arabidopsis contains a family of 12 genes that are similar to Ggps [24]. It is not yet clear Cyc-b (Tomato) how many of them are involved in the formation of GGPP in the plastids, but five Ggps genes have been CCS (Pepper) shown to be expressed in different tissues during plant Lcy-b (Arabidopsis) development [25]. Lcy-b (Tomato) CrtL (Cyanobacteria) The first committed step in the carotenoid pathway is the condensation of two GGPP molecules to produce 15-cis Lcy-e (Arabidopsis) phytoene, which is catalyzed by a membrane-associated Lcy-e (Tomato) enzyme, phytoene synthase (PSY) (Figure 1; [26]). PSY Current Opinion in Plant Biology shares amino-acid sequence similarity with GGPP syn- thase and other prenyl-transferases. Partial purification of The lycopene cyclase family in plants. Phylogenetic tree of the cyclase PSY from tomato indicated that the enzyme is associated genes generated on the basis of amino-acid sequence alignment. The with the isoprenoid biosynthesis enzymes IPI and GGPS data agree with the hypothesis that the gene CrtL from cyanobacteria encodes the ancestral archetype of plant cyclases, which have in a protein complex that is larger than 200 kDa [27]. In evolved, probably by gene duplication, into different types of enzymes. tomato, there are two genes for PSY: Psy-1, which encodes First, the lycopene ε-cyclase, Lcy-e, evolved from the lycopene β- a fruit- and flower-specific isoform, and Psy-2, which cyclase, Lcy-b. A duplication of Lcy-b then gave rise to Cyc-b, which in encodes an isoform that predominates in green tissues pepper was later transformed to CCS. [28,29]. PSY is a rate-limiting enzyme of carotenoid biosynthesis in ripening tomato fruits [30,31], in canola plastids, IPP is produced via in the ‘DOXP pathway’ from (Brassica napus) seeds [32••] and in marigold flowers [33•]. pyruvate and glyceraldehyde-3-phosphate (Figure 1) This rate-limiting feature makes PSY suitable to be a key [12,13]. The first enzyme in this pathway is 1-deoxyxylu- regulator of carotenogenesis. lose 5-phosphate (DOXP) synthase (DXS), which is encoded by a gene that has been cloned from pepper Two structurally and functionally similar enzymes, PDS (Capsicum annuum) [14], Mentha piperita [15], tomato and ζ-carotene desaturase (ZDS), convert phytoene to (Lycopersicon esculentum) [16•] and Arabidopsis thaliana [17]. lycopene via ζ-carotene. These FAD-containing enzymes DXS is impaired in the chilling-sensitive5 (chs5) mutant of each catalyze two symmetric dehydrogenation reactions Arabidopsis. At the restrictive temperature, chlorotic leaves that require plastoquinone [34,35] and a plastid terminal develop in young leaf tissues of this mutant but not in oxidase as electron acceptors [36••]. When co-expressed in mature leaves, indicating that DXS functions preferen- E. coli, PDS and ZDS from Arabidopsis convert phytoene to tially at an early stage of leaf development [17]. It has been 7,9,7′,9′-tetra-cis-lycopene (poly-cis lycopene, which is also suggested that DXS could potentially be a regulatory step called ‘pro-lycopene’), whereas the bacterial phytoene in carotenoid biosynthesis during early fruit ripening in desaturase (CRTI) produces all-trans lycopene [37]. The tomato [16•]. DOXP is converted to 2C-methyl-D- mechanism of carotenoid isomerization is yet unknown. It erythritol 2,4-cyclodiphosphate via 2C-methyl-D- is predicted, however, that a gene product of the tomato erythritol 4-phosphate, 4-diphosphocytidyl-2C-methyl- locus tangerine is involved in this process. In fruits of the D-erythritol (DPME) and 4-diphosphocytidyl-2C-methyl- recessive mutant tangerine, lycopene is replaced by poly-cis D-erythritol 2-phosphate. These steps are catalyzed by the lycopene. We have recently cloned this locus and iden- enzymes DOXP reductoisomerase (DXR) [18], DPME tified it in the Arabidopsis genome (T Isaacson et al., synthase (ispD [ygbP]), DPME kinase (ispE) and 2C- unpublished data). The structure of the polypeptide methyl-D-erythritol 2,4-cyclodiphosphate synthase (ispF), encoded by tangerine suggests that it belongs to the carotene respectively (reviewed in [19•]). The Dxr gene was cloned desaturase family. from A. thaliana [14] and M. piperita [15]; the IspD gene was cloned from A. thaliana [20] and the ispE gene was cloned Cyclization of lycopene marks a branching point in the from M. piperita [15] and tomato [21]. An enzyme encoded pathway: one branch leads to carotene and its derivative by the LytB gene, which was recently cloned from Adonis xanthophylls, whereas the other leads to α-carotene and aestivalis, has been hypothesized to catalyze a subsequent lutein. Lycopene β-cyclase (LCY-B/CRTL-B) catalyzes a reaction that affects the ratio of IPP to dimethylallyl two-step reaction that creates one β-ionone ring at each diphosphate [22]. IPP is isomerized to dimethylallyl end of the lycopene molecule to produce β-carotene, diphosphate by the enzyme IPP isomerase (which is whereas lycopene ε-cyclase (LCY-E/CRTL-E) creates one encoded by the Ipi genes). There are two Ipi genes in ε-ring to give δ-carotene. It is presumed that α-carotene plants and one of them is predicted to be active in the (β,ε-carotene) is synthesized by both enzymes. In view of plastids (reviewed in [23]). The sequential addition of the occurrence of a heterodimeric lycopene β-cyclase in three IPP molecules to dimethylallyl diphosphate, which is Gram-positive bacteria [38–40], it is alluring to consider catalyzed by a single enzyme, geranylgeranyl diphosphate that lycopene cyclases in plants also work as dimers. If this (GGPP) synthase (GGPS), gives the 20-carbon molecule were the case, it is possible that α-carotene is synthesized Carotenoid biosynthesis in flowering plants Hirschberg 213

by a LCY-B–LCY-E heterodimer. Interestingly, lettuce Zeaxanthin is effective in the thermal dissipation of excess (Lactuca sativa) contains a bi-cyclase CRTL-E that converts excitation energy in the light-harvesting antennae and, thus, lycopene to ε-carotene [41••]. plays a key role in protecting the photosynthetic system from damage by strong light. The inter-conversion of zeax- There is a high degree of structural resemblance, 30% anthin and violaxanthin is known as the ‘xanthophyll identity in amino-acid sequence, between LCY-B and cycle’. Lack of the xanthophyll cycle in the Arabidopsis LCY-E in both tomato and Arabidopsis. The two enzymes mutant npq1 (non-photochemical quenching1), owing to a null contain a characteristic FAD/NAD(P)-binding sequence mutation in Vde, increases the sensitivity of the plants motif at the amino termini of the mature polypeptides. In to intense light [52]. The Vde gene was originally cloned tomato, there are two lycopene β-cyclase enzymes, LCY-B from lettuce [53]. The amino-acid sequences of ZEP and (CRTL-B) [42] and CYC-B (chromoplast-specific VDE indicate that they are members of the lipocalins, a lycopene cyclase) [43••], whose amino-acid sequences are group of proteins that bind and transport small hydrophobic 53% identical. LCY-B is active in green tissues, whereas molecules [54]. CYC-B functions only in chromoplast-containing tissues. Interestingly, the amino-acid sequence of CYC-B is more Cloning of a gene for neoxanthin synthase (Nxs) from potato similar (86.1% identical) to that of capsanthin-capsorubin (Solanum tuberosum) and tomato has been recently reported synthase (CCS) from pepper, an enzyme that converts [55•,56•]. Surprisingly, the amino-acid sequence of NXS antheraxanthin and violaxanthin to the red xanthophylls from tomato is 99% identical to that of the lycopene capsanthin and capsorubin, respectively, than to that of β-cyclase CYC-B. As this sequence is unique in the tomato LCY-B [44]. A deletion mutation in the Ccs gene (locus y) genome (J Hirschberg et al., unpublished data) it is envis- that results in the accumulation of violaxanthin is respon- aged that CYC-B is a bi-functional enzyme that is capable sible for the recessive yellow fruit phenotype of pepper of converting both lycopene to β-carotene and violaxanthin [45]. CCS exhibits low lycopene β-cyclase activity when to neoxanthin. In this case, another neoxanthin synthase expressed in E. coli [46]. Similarities in function, gene must exist in tomato because neoxanthin is synthesized in structure and map position strongly suggest that the genes the mutants old-gold and old-gold-crimson, which carry null Ccs and Cyc-b are orthologs that have originated by a gene mutations in the Cyc-b (B) gene [43••]. duplication event from a common ancestor, most probably Lcy-b [43••]. In tomato, the duplicated gene has retained its Regulation in chromoplasts original catalytic function, whereas the second cyclase in Carotenogenesis in fruits and flowers is controlled by pepper acquired a new enzymatic activity of a similar bio- regulatory mechanisms that are distinct from those that chemical nature during evolution. Conservation of operate in green tissues [57]. Carotenoid biosynthesis in amino-acid sequences as well as their similar mechanisms ripening tomato fruits has been extensively studied of catalysis suggest that all plant cyclases, including CCS because of the dramatic color changes that occur during and perhaps also neoxanthin synthase, have evolved from this process and the availability of a large collection of color a common ancestor, most probably the cyanobacterial CrtL mutants. Thus, tomato fruits have become a model system (Figure 2). for other chromoplast-containing tissues.

Hydroxylation of cyclic carotenes at the 3C, 3′C positions Carotenoid composition in the green stages of fruit devel- is carried out by two types of enzymes: one specific for opment is similar to that of the green leaf. At the ‘breaker’ β-rings and the other for ε-rings [47,48]. The β-carotene stage of ripening, the color changes from green to orange hydroxylases are ferredoxin dependent and require iron, because of the accumulation of lycopene, which takes features that are characteristic of enzymes that exploit place following increased synthesis of carotenoid enzymes. iron-activated oxygen to oxygenate carbohydrates [49]. Higher levels of expression of genes encoding enzymes in Consequently, β-carotene is converted to zeaxanthin via β- the central isoprenoid pathway during early fruit develop- cryptoxanthin by these enzymes. There are two β-carotene ment has been reported [16•,58]. The mRNA levels for the hydroxylases in both Arabidopsis [24] and tomato [50]. In enzymes that produce lycopene, PSY and PDS, increase tomato, one hydroxylase is expressed in green tissues 10–20-fold at the breaker stage of ripening [59–61,62•]. At while the other is exclusively expressed in the flower the same time, the mRNAs of both lycopene cyclases, (G Ronen et al., unpublished data). The gene that encodes Lcy-b (CrtL-b) and Lcy-e (CrtL-e), disappear [42,62•]. It the ε-ring hydroxylase has not yet been identified. Zep1 has been established that the increase in Psy and Pds (ABA2) converts zeaxanthin to violaxanthin via antherax- expression during the breaker stage is controlled by tran- anthin by introducing 5,6-epoxy groups into the scriptional regulation [63,64••]. The hypothesis that 3-hydroxy-β-rings in a redox reaction that requires reduced differential gene expression is the major reason for the ferredoxin [51]. Zep1 was cloned from Nicotiana plumbagini- accumulation of lycopene is further corroborated by the folia [9] and pepper [51]. In leaves, violaxanthin can be accumulation of δ-carotene in the fruits of the Delta converted back to zeaxanthin by violaxanthin deepoxidase mutant, which results from increased transcription of (VDE), an enzyme that is activated by low pH, which is CrtL-e (Lcy-e) [62•], and by the synthesis of β-carotene in fruits generated in the chloroplast lumen under strong light. of the Beta (B) mutant, which is caused by the upregulation 214 Physiology and metabolism

of a second lycopene β-cyclase gene, Cyc-b (B) [43••]. A species that contain a β-ring can be converted to retinol similar upregulation of carotenoid gene expression during and, thus, are precursors of vitamin A. Although this is the fruit development has been found in bell pepper major value of carotenoids in human nutrition, additional [14,51,65,66], melon [67] and satsuma mandarin (Citrus health benefits are attributed to their antioxidant activity unshiu Marc) [68]. Transcriptional upregulation of in vivo [76–79]. Industrial applications of carotenoids carotenoid biosynthesis genes also appears to be the major include their use as colorants for human food and feed regulatory mechanism in carotenogenesis that takes place additives to enhance the pigmentation of fish and eggs, as in the flowers of tomato [42,43••,60,62•,64••], daffodil cosmetics and as pharmaceutical products. As natural (Narcissus pseudonarcissus) [69] and marigold (Tagetes erecta) pigments, carotenoids furnish attractive colors to fruits, [33•]. Expression of carotenogenic genes in pepper fruits is vegetables and ornamental flowers, and their composition enhanced by oxidative stress, which facilitates carotenoid in these crops has enormous economic value. The cloning synthesis during chromoplast differentiation [70,71]. of most of the carotenoid biosynthesis genes has opened the door to genetically manipulating this pathway in Although developmentally controlled transcription of plants. This promise has been fulfilled during the past carotenoid genes appears to be the major regulatory mech- couple of years during which exciting progress in the meta- anism in flowers and fruits, it is clearly not the only one. bolic engineering of carotenoids in crop plants has been Regulation at the enzymatic level is predicted to account achieved (see also reviews cited in [80,81•,82,83]). for the higher concentration of lycopene in fruits of the tomato mutants old-gold and old-gold-crimson and in fruits Because phytoene synthase catalyses the first committed of plants in which the expression of Cyc-b was silenced; for step in the carotenoid pathway, it is a preferred target for increased β-carotene in tomato fruits that express the bac- gene manipulation. Constitutive expression of the cDNA terial phytoene desaturase crtI; and for the synthesis of of Psy in transgenic tomato plants has led to dwarfism, β-carotene in rice (Oryza Sativa) seeds that are manipulated which is caused by redirecting GGPP from the gibberellin to produce lycopene. pathway into carotenoid synthesis [84]. The fruits of these plants produced lycopene earlier in development than is Regulation in chloroplasts normal, but the final concentrations of lycopene were Relatively little is known about the regulation of caroteno- lower in these plants than in the wild type. In contrast, a genesis in leaves. Although expression of carotenoid genes two-fold increase in total fruit carotenoids was achieved in does take place in etiolated plants, carotenoid biosynthesis tomato plants that expressed the bacterial phytoene syn- is stimulated upon transfer to light. A light-stimulated thase gene crtB from Erwinia in a fruit-specific manner increase in IPI activity was recorded in maize etioplasts [72]. (PD Fraser, PM Bramley, personal communication). A In developing seedlings of mustard (Sinapis alba L), the successful manipulation of phytoene synthase has been level of Psy mRNA increases in the light because of a phy- achieved in canola (Brassica napus), an important crop tochrome-mediated regulation, whereas expression of Pds species [32••]. In this species, expressing crtB from and Ggps remains constant [73]. In etioplasts,enzymatically Erwinia in a seed-specific manner, using the Brassica napin inactive PSY is localized in the prolamellar body but upon gene promoter, increased carotenoids (mainly α- and illumination this enzyme is activated and is found in the β-carotene) in the mature seeds up to 50-fold, reaching thylakoids [74•]. In many plant species, the total xantho- 1600 µg/g fresh weight. Oil from this ‘golden canola’ can phyll content increases under strong light and the ratio potentially fortify foodstuff with β-carotene and help the between lutein (L) and the xanthophyll-cycle components, fight against vitamin A deficiency. The golden canola is zeaxanthin, antheraxanthin and violaxanthin (Z+A+V), being developed further in India at the non-profit Tata decreases. Conversely, the ratio L:(Z+A+V) increases in low Energy Research Institute (TERI) in partnership with light. We have observed a five-fold increase in the ratio Monsanto Co., which donated the technology to help alle- between levels of Lcy-b and Lcy-e mRNAs in both viate vitamin A deficiency. This result is different from Arabidopsis and tomato leaves when plants were shifted that obtained in the rice system in which overexpression of from low light to strong light (I Ronin et al., unpublished the daffodil Psy in the endosperm increased only phytoene data). This result suggests that the xanthophyll composition [85]. This difference is possibly explained by the fact that of the light-harvesting complexes can be modulated by the carotenogenesis does take place in canola seeds, and carotenoid biosynthesis flux. Support for this hypothesis hence the transgenic phytoene synthase simply released a comes from the Arabidopsis mutant lutein2 (lut2), which lacks bottleneck in the pathway, whereas in rice seeds, the path- lutein in the light-harvesting antenna because of a null way is inactive. mutation in Lcy-e [48], and from the high lutein concentration caused by the transgenic overexpression of Lcy-e [75]. β-carotene in tomato fruits has been increased by various genetic manipulations. Constitutive expression of the bac- Metabolic engineering of carotenoid terial phytoene desaturase gene crtI under the regulation biosynthesis of the cauliflower mosaic virus 35S promoter tripled the There is growing interest worldwide in manipulating concentration of β-carotene in the fruit but halved the total carotenoid biosynthesis in plants. All of the carotenoid carotenoid content [86•]. Transgenic expression of the Carotenoid biosynthesis in flowering plants Hirschberg 215

native Lcy-b gene in tomato fruit resulted in a 3.8-fold Conclusions increase in the concentration of β-carotene, while the total Significant progress has been made in our understanding carotene concentration was unchanged or slightly elevated of carotenoid biosynthesis in plants. Nevertheless, we [87•]. A greater boost in β-carotene concentration was still lack fundamental knowledge on various aspects of obtained by the transgenic expression of the alternative this process. More information is needed to answer a lycopene β-cyclase, Cyc-b, in a manner that resembled the number of questions. Where exactly within the plastids situation in the Beta mutant [43••]. do the different enzymes operate? Do the enzymes of the pathway function in protein complexes? Which A breakthrough in the metabolic engineering of carotenoids metabolic regulations take place at the enzyme level? for improved nutritional value of a major crop has been What are the interactions between the carotenoid path- achieved in rice [88••]. Mature rice endosperm is capable of way and other metabolic pathways? What are the synthesizing GGPP but completely lacks carotenoids. The molecular components that control the expression of engineering of rice grains to produce β-carotene could carotenoid genes? Answering these and other questions potentially alleviate the problem of vitamin A deficiency will shed light on an important metabolic pathway and that is common in Asia, Africa and South America. To improve our capability to manipulate carotenoids in crop achieve β-carotene biosynthesis in the seeds, the daffodil plants. genes Psy and Lcyb (under the regulation of the endosperm- specific promoter of the glutelin gene) together with the Acknowledgements bacterial phytoene desaturase gene crtI (under the control of I thank Dr Peter M Bramley and Dr Paul D Fraser for communicating the cauliflower mosaic virus 35S promoter) were transferred unpublished results and Dr V Mann for valuable comments on the manuscript. Work in my laboratory is carried out under the auspices of the Avron Even-Ari to a japonica rice cultivar. Both Psy and Lcy were present on Minerva Center and is supported by Grant 578/97 from the Israel Science the same DNA construct. Surprisingly, seeds that expressed Foundation and by the Israel Ministry of Science. Psy and crtI were yellow and contained β-carotene, zeaxan- thin and lutein, rather than just lycopene. Thus, the References and recommended reading lycopene β-cyclase and the β-carotene hydroxylase are Papers of particular interest, published within the annual period of review, either constitutively expressed in normal rice endosperm or have been highlighted as: induced upon lycopene formation. Only expression of all • of special interest three genes, however, gave a significant concentration of •• of outstanding interest β-carotene. The maximum level of carotenoids in the 1. Niyogi KK: Photoprotection revisited. Annu Rev Plant Physiol Plant Mol Biol 1999, 50:391-417. endosperm of plants that were heterozygous for the trans- 2. Niyogi KK: Safety valves for photosynthesis. Curr Opin Plant Biol genes was 1.6 µg/g, which would provide 10–20% of the 2000, 3:455-460. recommended daily allowance of β-carotene in 300 g of rice. 3. Rock CD, Zeevaart JAD: The aba mutant of Arabidopsis thaliana is Golden rice has been donated by its developers, and by impaired in epoxy-carotenoid biosynthesis. Proc Natl Acad Sci other owners of intellectual property rights that were used to USA 1991, 88:7496-7499. develop it, to the Philippine-based International Rice 4. Chernys JT, Zeevaart JA: Characterization of the 9-cis- Research Institute (IRRI) where β-carotene-rich varieties epoxycarotenoid dioxygenase gene family and the regulation of abscisic acid biosynthesis in avocado. Plant Physiol 2000, will be developed and distributed to poorer farmers. 124:343-354. 5. Chamovitz D, Pecker I, Sandmann G, Boeger P, Hirschberg J: Cloning The gene crtO, which encodes β-C-4-oxygenase, a key a gene for norflurazon resistance in cyanobacteria. Z Naturforsch enzyme in ketocarotenoids synthesis, was cloned from the 1990, 45c:482-486. alga Haematococcus pluvialis [89]. Tobacco plants that 6. Cunningham FX Jr, Chamovitz D, Misawa N, Gantt E, Hirschberg J: Cloning and functional expression in Escherichia coli of a expressed CrtO in a regulated manner accumulated a high cyanobacterial gene for lycopene cyclase, the enzyme that concentration of ketocarotenoids, including astaxanthin, catalyzes the biosynthesis of β-carotene. FEBS Lett 1993, in the chromoplasts of the nectary tissue in their flowers, 328:130-138. changing the flower color from yellow to red [64••]. 7. Bird CR, Ray JA, Fletcher JD, Boniwell JM, Bird AS, Teulieres C, Blain I, Bramley PM, Schuch W: Using antisense RNA to study Astaxanthin provides a characteristic pink color to gene function: inhibition of carotenoid biosynthesis in transgenic salmonids, trout and shrimps. In nature, it is synthesized tomatoes. Bio/Technology 1991, 9:635-639. by marine bacteria and microalgae and then passed on to 8. Lotan T, Hirschberg J: Cloning and expression in Escherichia coli of fish through the food chain. Fish grown in aquaculture, the gene encoding b-C-4-oxygenase, that converts β-carotene to the ketocarotenoid canthaxanthin in Haematococcus pluvialis. however, are separated from their natural food chain and, FEBS Lett 1995, 364:125-128. thus, astaxanthin must be added to their feed in order for 9. Marin E, Nussaume L, Quesada A, Gonneau M, Sotta B, Hugueney P, them to acquire the typical pink color of their flesh. It is Frey A, Marionpoll A: Molecular identification of zeaxanthin important to note that the astaxanthin produced in the epoxidase of Nicotiana plumbaginifolia, a gene involved in abscisic acid biosynthesis and corresponding to the transgenic plants had the same chirality (3S3′S) as the natural ABA locus of Arabidopsis thaliana. EMBO J 1996, astaxanthin that is found in marine organisms. This result 15:2331-2342. demonstrates the feasibility of genetically manipulating 10. 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