ReviewCarotenoid biosynthesis in flowering plants
Introduction
Plant carotenoids are 40-carbon isoprenoids with polyene chains that may contain up to 15 conjugated double bonds. Because of their chemical properties carotenoids are essential components of all photosynthetic organisms. Xanthophylls, oxygenated forms of carotenes, are accessory pigments in the light-harvesting antennae of the chloroplasts, which are capable of transferring energy to the chlorophylls. They also quench triplet excited states in chlorophyll molecules by dissipating the excess excitation energy in a non-radiative manner, a process known as non-photochemical quenching (NPQ) (reviewed in 1., 2.). This function is crucial to protect against chlorophyll bleaching in intense light. An additional important role of carotenoids in plants is to furnish flowers and fruits with distinct colors that are designed to attract animals. In chloroplasts, carotenoids play vital roles in photosynthesis and are indispensable, whereas in chromoplasts, they can be considered as secondary metabolites. Carotenoids in plants are also precursors for the synthesis of the hormone abscisic acid (ABA) 3., 4..
Elucidation of the carotenoid biosynthesis pathway is a wonderful example of a successful interdisciplinary approach to studying plant biochemistry. The enzymes of this pathway exist in minute amounts and are very labile upon purifi-cation. These characteristics and the lack of genuine in vitro assays for any of the enzymes have hindered the usage of conventional biochemical investigation. Because the cloning of the genes for these enzymes could not rely on protein purification, molecular analysis required the use of various genetic methods. Cloning of the first genes took advantage of the fact that the pathway in plants is similar to that in cyanobacteria. Hence, the phytoene desaturase (Pds) gene was first isolated from mutants of Synechococcus sp. PCC7942 that were resistant to norflurazon, an inhibitor of PDS [5]. The gene was identified by its ability to confer herbicide resistance in the wild-type background. The cyanobacterial genes then served as molecular probes to isolate the plant orthologs. A similar methodology has been successfully used to clone the gene for lycopene β-cyclase [6].
Using a reverse genetics strategy, the gene for phytoene synthase (Psy) was identified in transgenic tomato plants in which the expression of a candidate cDNA was silenced [7]. A unique functional complementation approach to cloning genes has been developed on the basis of the ability of the carotenogenic enzymes to function in the bacterium Escherichia coli [8]. This so-called ‘color complementation’ technique takes advantage of E. coli engineered with bacterial genes to produce a colored carotenoid that serves as a precursor for the enzyme under investigation. The carotenoid accumulated in the bacteria imparts a characteristic color to the colonies that can be seen by the naked eye. The screening for a specific gene is based on the visualization of color changes in E. coli colonies following transfection of the bacteria with plant cDNA libraries carried on expression plasmids.
Transposon tagging was effectively used to clone the Zeaxanthin epoxidase1 (Zep1) gene from Nicotiana plumbaginigfolia by screening for ABA-deficient phenotype [9]. An additional genetic technique that was valuable in obtaining carotenoid-biosynthesis genes is map-based cloning. This technique has been successfully employed to clone novel genes from tomato, a species with a variety of mutations that affect carotenoid biosynthesis and accumulation. Advances in plant genomics offer new ways to identify novel genes on the basis of their sequence similarity to known genes and are expected to facilitate the cloning of novel genes in the future. Characterization of the enzymes encoded by these novel genes was often done in E. coli cells that express the cloned genes. Enzymes that were purified from such bacteria have been analyzed in cell-free carotenogenic systems.
Section snippets
Biosynthetic pathway
In plants, carotenoids are synthesized within the plastids by enzymes that are nuclear encoded. A unique exception to this rule has recently been discovered in the green alga Haematococcus pluvialis in which the last steps in the synthesis of the ketocarotenoid astaxanthin take place in cytoplasmic lipid vesicles 10., 11•.. This article reviews recent discoveries in carotenoid biosynthesis in higher plants.
Like all other isoprenoids, carotenoids are built from the 5-carbon compound isopentenyl
Regulation in chromoplasts
Carotenogenesis in fruits and flowers is controlled by regulatory mechanisms that are distinct from those that operate in green tissues [57]. Carotenoid biosynthesis in ripening tomato fruits has been extensively studied because of the dramatic color changes that occur during this process and the availability of a large collection of color mutants. Thus, tomato fruits have become a model system for other chromoplast-containing tissues.
Carotenoid composition in the green stages of fruit
Regulation in chloroplasts
Relatively little is known about the regulation of carotenogenesis in leaves. Although expression of carotenoid genes does take place in etiolated plants, carotenoid biosynthesis is stimulated upon transfer to light. A light-stimulated increase in IPI activity was recorded in maize etioplasts [72]. In developing seedlings of mustard (Sinapis alba L), the level of Psy mRNA increases in the light because of a phytochrome-mediated regulation, whereas expression of Pds and Ggps remains constant [73]
Metabolic engineering of carotenoid biosynthesis
There is growing interest worldwide in manipulating carotenoid biosynthesis in plants. All of the carotenoid species that contain a β-ring can be converted to retinol and, thus, are precursors of vitamin A. Although this is the major value of carotenoids in human nutrition, additional health benefits are attributed to their antioxidant activity in vivo 76., 77., 78., 79.. Industrial applications of carotenoids include their use as colorants for human food and feed additives to enhance the
Conclusions
Significant progress has been made in our understanding of carotenoid biosynthesis in plants. Nevertheless, we still lack fundamental knowledge on various aspects of this process. More information is needed to answer a number of questions. Where exactly within the plastids do the different enzymes operate? Do the enzymes of the pathway function in protein complexes? Which metabolic regulations take place at the enzyme level? What are the interactions between the carotenoid pathway and other
Acknowledgements
I thank Dr Peter M Bramley and Dr Paul D Fraser for communicating 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 Minerva Center and is supported by Grant 578/97 from the Israel Science Foundation and by the Israel Ministry of Science.
References and recommended reading
Papers of particular interest, published within the annual period of review,have been highlighted as:
•of special interest
••of outstanding interest
References (89)
Safety valves for photosynthesis
Curr Opin Plant Biol
(2000)- et al.
Cloning and functional expression in Escherichia coli of a cyanobacterial gene for lycopene cyclase, the enzyme that catalyzes the biosynthesis of β-carotene
FEBS Lett
(1993) - et al.
Cloning and expression in Escherichia coli of the gene encoding b-C-4-oxygenase, that converts β-carotene to the ketocarotenoid canthaxanthin in Haematococcus pluvialis
FEBS Lett
(1995) - et al.
Ketocarotenoid biosynthesis outside of plastids in the unicellular green alga Haematococcus pluvialis
J Biol Chem
(2001) - et al.
Biosynthesis of isoprenoids in higher plant chloroplasts proceeds via a mevalonate-independent pathway
FEBS Lett
(1997) - et al.
Isoprenoid biosynthesis via a mevalonate-independent pathway in plants: cloning and heterologous expression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase from peppermint
Arch Biochem Biophys
(1999) - et al.
Cloning and heterologous expression of a cDNA encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase of Arabidopsis thaliana
FEBS Lett
(1999) - et al.
Deoxyxylulose phosphate pathway to terpenoids
Trends Plant Sci
(2001) Plant phytoene synthase complex — component enzymes, immunology, and biogenesis
Methods Enzymol
(1993)- et al.
cDNA cloning, expression during development, and genome mapping of PSY2, a second tomato gene encoding phytoene synthase
J Biol Chem
(1993)
A plastid terminal oxidase comes to light: implications for carotenoid biosynthesis and chlororespiration
Trends Plant Sci
Cloning and functional analysis of the β-carotene hydroxylase of Arabidopsis thaliana
J Biol Chem
Xanthophyll biosynthesis: molecular and functional characterization of carotenoid hydroxylases from pepper fruits (Capsicum annuum L)
Biochim Biophys Acta
Xanthophyll biosynthesis — cloning, expression, functional reconstitution, and regulation of beta-cyclohexenyl carotenoid epoxidase from pepper (Capsicum annuum)
J Biol Chem
Plant lipocalins: violaxanthin de-epoxidase and zeaxanthin epoxidase
Biochim Biophys Acta
Identification of a novel gene coding for neoxanthin synthase from Solanum tuberosum
FEBS Lett
Expression of the genes encoding the early carotenoid biosynthetic enzymes in Capsicum annuum
Biochem Biophys Res Commun
Induction and control of chromoplast-specific carotenoid genes by oxidative stress
J Biol Chem
Antioxidant effects of carotenoids in vivo and in vitro — an overview
Methods Enzymol
Production of high-value compounds: carotenoids and vitamin E
Curr Opin Biotechnol
Metabolic engineering of plant carotenoids
Trends Plant Sci
Genetic manipulation of carotenoid biosynthesis: strategies, problems and achievements
Trends Plant Sci
Cloning and expression in Escherichia coli of the gene encoding β-C-4 oxygenase, that converts β-carotene to the ketocarotenoid canthaxanthin in Haematococcus pluvialis
FEBS Lett
Photoprotection revisited
Annu Rev Plant Physiol Plant Mol Biol
The aba mutant of Arabidopsis thaliana is impaired in epoxy-carotenoid biosynthesis
Proc Natl Acad Sci USA
Characterization of the 9-cis-epoxycarotenoid dioxygenase gene family and the regulation of abscisic acid biosynthesis in avocado
Plant Physiol
Cloning a gene for norflurazon resistance in cyanobacteria
Z Naturforsch
Using antisense RNA to study gene function: inhibition of carotenoid biosynthesis in transgenic tomatoes
Bio/Technology
Molecular identification of zeaxanthin epoxidase of Nicotiana plumbaginifolia, a gene involved in abscisic acid biosynthesis and corresponding to the ABA locus of Arabidopsis thaliana
EMBO J
β-carotene is the intermediate exported from the chloroplast during accumulation of secondary carotenoids in Haematococcus pluvialis
J Applied Phycol
Two independent biochemical pathways for isopentenyl diphosphate and isoprenoid biosynthesis in higher plants
Physiol Plant
Dedicated roles of plastid transketolases during the early onset of isoprenoid biogenesis in pepper fruits
Plant Physiol
Carotenoid biosynthesis during tomato fruit development: regulatory role of 1-deoxy-D-xylulose 5-phosphate synthase
Plant J
Temperature-sensitive Arabidopsis mutant defective in 1-deoxy-D-xylulose 5-phosphate synthase within the plastid non-mevalonate pathway of isoprenoid biosynthesis
Physiol Plant
Biosynthesis of terpenoids: 4-diphosphocytidyl-2C-methyl-D-erythritol synthase of Arabidopsis thaliana
Proc Natl Acad Sci USA
Biosynthesis of terpenoids: 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase from tomato
Proc Natl Acad Sci USA
Evidence of a role for LytB in the nonmevalonate pathway of isoprenoid biosynthesis
J Bacteriol
Genes and enzymes of carotenoid biosynthesis in plants
Annu Rev Plant Physiol Plant Mol Biol
Five geranylgeranyl diphosphate synthases expressed in different organs are localized into three subcellular compartments in Arabidopsis
Plant Physiol
Phytoene synthase from tomato (Lycopersicon esculentum) chloroplasts — partial purification and biochemical properties
Planta
Phytoene synthase-2 enzyme activity in tomato does not contribute to carotenoid synthesis in ripening fruit
Plant Mol Biol
Biochemical characterization of transgenic tomato plants in which carotenoid synthesis has been inhibited through the expression of antisense RNA to pTOM5
Plant J
Carotenoid biosynthesis during tomato fruit development
Plant Physiol
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