Zeta-carotene Desaturase Research Articles

Cloning and Functional Expression of Ζ-Carotene Desaturase, A Novel Carotenoid Biosynthesis Gene From Ficus Carica

Carotene desaturation, an essential step in the carotenoid biosynthesis pathway, is catalyzed by two enzymes, phytoene desaturase (PDS) and ζ-carotene desaturase (zeta carotene desaturase, ZDS). Here we describe cloning and E. coli expression of zdsfc, a novel Ficus carica ζ-carotene desaturase catalyzing dehydrogenation of ζ-carotene into neurosporene and finally lycopene. The ζ-carotene desaturase (ZDS) gene was amplified from the fig tree by rapid amplification of cDNA ends (RACE) and spanned a 1746 bp open reading frame (ORF), encoding a protein of 582 amino acid residues with a predicted molecular weight of 64kD. The N-terminal region of this polypeptide contained a putative transit sequence for plastid targeting. By phylogenetic and sequence analyses, zdsfc showed high homology with previously described ζ-carotene desaturases from higher plant species [1-4]. Additionally, sequence analysis revealed a high degree of conservation among plant ZDSs. The deduced ZDS protein, designated zdsfc, also contains an N-terminus dinucleotide-binding, followed by a conserved region identified in other carotene desaturase sequences. These data, taken together, confirm our cloned zdsfc as an integral part of the ZDS family of proteins.

The kiwifruit lycopene beta-cyclase plays a significant role in carotenoid accumulation in fruit

The composition of carotenoids, along with anthocyanins and chlorophyll, accounts for the distinctive range of colour found in the Actinidia (kiwifruit) species. Lutein and beta-carotene are the most abundant carotenoids found during fruit development, with beta-carotene concentration increasing rapidly during fruit maturation and ripening. In addition, the accumulation of beta-carotene and lutein is influenced by the temperature at which harvested fruit are stored. Expression analysis of carotenoid biosynthetic genes among different genotypes and fruit developmental stages identified Actinidia lycopene beta-cyclase (LCY-β) as the gene whose expression pattern appeared to be associated with both total carotenoid and beta-carotene accumulation. Phytoene desaturase (PDS) expression was the least variable among the different genotypes, while zeta carotene desaturase (ZDS), beta-carotene hydroxylase (CRH-β), and epsilon carotene hydroxylase (CRH-ϵ) showed some variation in gene expression. The LCY-β gene was functionally tested in bacteria and shown to convert lycopene and delta-carotene to beta-carotene and alpha-carotene respectively. This indicates that the accumulation of beta-carotene, the major carotenoid in these kiwifruit species, appears to be controlled by the level of expression of LCY-β gene.

Cloning and Expression Analysis of Carotenogenic Genes during Ripening of Autumn Olive Fruit (Elaeagnus umbellata)

Autumn olive ( Elaeagnus umbellata Thunb.) is an extremely lycopene-rich natural source, and lycopene concentration of ripe wild autumn olive fruit was 12 times higher than that of tomato. Lycopene formation was found to increase with significant reduction of other carotenoids and chlorophylls during fruit ripening. To elucidate the molecular basis of massive lycopene accumulation in autumn olive fruit, seven cDNA fragments were cloned encoding enzymes of the main steps of carotenoid biosynthetic pathway, which were geranylgeranyl pyrophosphate synthase (EutGgps), phytoene synthase (EutPsy), phytoene desaturase (EutPds), zeta-carotene desaturase (EutZds), lycopene beta-cyclase (EutLcy-b), lycopene epsilon-cyclase (EutLcy-e), and beta-carotene hydroxylase (EutBch). The accumulation of lycopene in the fruit was concomitant with the up-regulation of upstream genes of lycopene synthesis (EutGgps, EutPsy, EutPds, and EutZds) and down-regulation of downstream genes (EutLcy-b and EutBch) and in particular with the silence of EutLcy-e throughout fruit ripening. Thus, lycopene accumulation in autumn olive fruit was highly regulated by the coordination of the expression among carotenogenic genes and by fruit ripening.

Patterns of Variation in the Evolution of Carotenoid Biosynthetic Pathway Enzymes of Higher Plants

Cellular metabolic networks depend on the products of many loci for proper functioning. These interrelationships between loci at the phenotypic level raise the question of whether they evolve independently. Previous research has demonstrated that in the anthocyanin pathway, which produces important secondary metabolites in plants, the genes encoding downstream enzymes show an increased rate of change at nonsynonymous sites when compared with upstream loci due to relaxed constraint. To test whether this pattern exists more broadly, we compared a set of 4 genes encoding enzymes of the carotenoid biosynthetic pathway, which produces a set of distinct colored secondary metabolites in plants. Comparisons between copies of phytoene desaturase, zeta-carotene desaturase, lycopene beta-cyclase, and zeaxanthin epoxidase from 6 taxa indicate that the 3 upstream enzymes (phytoene desaturase, zeta-carotene desaturase, and lycopene beta-cyclase) have similar proportions of codons under selective constraint, whereas the most downstream enzyme (zeaxanthin epoxidase) has more codons evolving under relaxed constraint. Overall, nonsynonymous substitution rates appear to be highest for zeaxanthin epoxidase, whereas synonymous substitution rates were highest for the intermediate enzyme lycopene beta-cyclase. Analysis of codon bias shows that only lycopene beta-cyclase may be under slight selection pressure for codon usage. Taken together, these results show that the enzymes of the carotenoid biosynthetic pathway are under strong selective constraint but that the most downstream enzyme is under the least constraint.

Generation of transgenic maize with enhanced provitamin A content

Vitamin A deficiency (VAD) affects over 250 million people worldwide and is one of the most prevalent nutritional deficiencies in developing countries, resulting in significant socio-economic losses. Provitamin A carotenoids such as β-carotene, are derived from plant foods and are a major source of vitamin A for the majority of the world's population. Several years of intense research has resulted in the production of ‘Golden Rice 2’ which contains sufficiently high levels of provitamin A carotenoids to combat VAD. In this report, the focus is on the generation of transgenic maize with enhanced provitamin A content in their kernels. Overexpression of the bacterial genes crtB (for phytoene synthase) and crtI (for the four desaturation steps of the carotenoid pathway catalysed by phytoene desaturase and ζ-carotene desaturase in plants), under the control of a ‘super γ-zein promoter’ for endosperm-specific expression, resulted in an increase of total carotenoids of up to 34-fold with a preferential accumulation of β-carotene in the maize endosperm. The levels attained approach those estimated to have a significant impact on the nutritional status of target populations in developing countries. The high β-carotene trait was found to be reproducible over at least four generations. Gene expression analyses suggest that increased accumulation of β-carotene is due to an up-regulation of the endogenous lycopene β-cylase. These experiments set the stage for the design of transgenic approaches to generate provitamin A-rich maize that will help alleviate VAD.

Expression of carotenoid biosynthesis genes during carrot root development

Carotenogenesis has been extensively studied in fruits and flower petals. Transcriptional regulation is thought to be the major factor in carotenoid accumulation in these organs. However, little is known about regulation in root organs. The root carotenoid content of carrot germplasm varies widely. The present study was conducted to investigate transcriptional regulation of carotenoid biosynthesis genes in relation to carotenoid accumulation during early carrot root development and up to 3 months after sowing. HPLC carotenoid content analysis and quantitative RT-PCR were compared to quantify the expression of eight genes encoding carotenoid biosynthesis enzymes during the development of white, yellow, orange, and red carrot roots. The genes chosen encode phytoene synthase (PSY1 and PSY2), phytoene desaturase (PDS), zeta-carotene desaturase (ZDS1 and ZDS2), lycopene epsilon-cyclase (LCYE), lycopene beta-cyclase (LCYB1), and zeaxanthin epoxidase (ZEP). All eight genes were expressed in the white cultivar even though it did not contain carotenoids. By contrast with fruit maturation, the expression of carotenogenic genes began during the early stages of development and then progressively increased for most of these genes during root development as the total carotenoid level increased in coloured carrots. The high expression of genes encoding LCYE and ZDS noted in yellow and red cultivars, respectively, might be consistent with the accumulation of lutein and lycopene, respectively. The results showed that the accumulation of total carotenoids during development and the accumulation of major carotenoids in the red and yellow cultivars might partially be explained by the transcriptional level of genes directing the carotenoid biosynthesis pathway.

Differential Response of Several Carotenoid Biosynthesis Inhibitors in Mixtures with Atrazine

Greenhouse studies were conducted in 2003 at the Stine–Haskell Research Center to determine whether herbicide inhibitors of six specific sites in the carotenoid biosynthesis pathway would elicit synergistic responses when applied postemergence (POST) in combination with the photosystem II (PSII) inhibitor atrazine. Based on data analysis with the Isobole method, synergistic responses were observed on red morningglory, common cocklebur, and giant foxtail when atrazine was applied in mixtures with the deoxy-D-xylulose-5-phosphate reductoisomerase (DOXP reductoisomerase) inhibitor fosmidomycin, thep-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor mesotrione, and the DuPont proprietary zeta-carotene desaturase (ZDS) inhibitor DFPC. Clomazone (its metabolite ketoclomazone is the actual enzyme inhibitor), an inhibitor of 1-deoxy-D-xylulose-5-phosphate synthatase (DOXP synthase), provided synergistic responses on red morningglory, but antagonistic responses on both common cocklebur and giant foxtail when applied in mixtures with atrazine. Combinations of the lycopene cyclase (LC) inhibitor, CPTA, with atrazine produced synergistic responses on both common cocklebur and giant foxtail but were antagonistic on red morningglory. Norflurazon, a phytoene desaturase (PDS) inhibitor, applied in mixtures with atrazine provided synergistic responses on red morningglory, antagonistic responses on giant foxtail, and independent responses on common cocklebur. Because carotenoids have been determined to play a key role in quenching singlet oxygen species in the chloroplast and also assist in the maintenance of the D1 protein in PSII, this might help explain the synergistic responses with atrazine observed in our studies.

Carotenoid Accumulation in Japanese Apricot (Prunus mume Siebold & Zucc.): Molecular Analysis of Carotenogenic Gene Expression and Ethylene Regulation

To elucidate the regulatory mechanisms of carotenogenesis in Japanese apricot (Prunus mume Siebold & Zucc.), the relationships between carotenoid accumulation and the expression of the carotenogenic genes, phytoene synthase (PmPSY-1), phytoene desaturase (PmPDS), zeta-carotene desaturase (PmZDS), lycopene beta-cyclase (PmLCYb), lycopene epsilon-cyclase (PmLCYe), beta-carotene hydroxylase (PmHYb), and zeaxanthin epoxidase (PmZEP), were analyzed in two cultivars with different ripening traits, 'Orihime' and 'Nanko.' In 'Orihime' fruits, large amounts of carotenoids accumulated on the tree, concomitant with the induction of PmPSY-1 and the downstream carotenogenic genes PmLCYb, PmHYb, and PmZEP. In 'Nanko' fruits, carotenoids accumulated mainly after harvest, correlating with an appreciable induction of PmPSY-1 expression, but the downstream genes were not notably induced, which may explain the lower total carotenoid content in 'Nanko' than in 'Orihime.' In both cultivars, a decrease in PmLCYe expression and increased or constant PmLCYb expression could cause the metabolic shift from beta,epsilon-carotenoid synthesis to beta,beta-carotenoid synthesis that occurs as ripening approaches. Next, the effects of ethylene on the expression of PmPSY-1 and carotenoid accumulation were investigated in 'Nanko' fruits treated with propylene or 1-methylcyclopropene (1-MCP). Propylene treatment induced both ethylene production and carotenoid accumulation. PmPSY-1 was constitutively expressed, but propylene treatment accelerated its induction. 1-MCP treatment caused a slight inhibition of carotenoid accumulation along with the repression, although not complete, of PmPSY-1. Collectively, although PmPSY-1 expression was not exclusively regulated by ethylene, both the notable induction of PmPSY-1 accelerated by ethylene and the subsequent induction of the downstream carotenogenic genes, especially PmLCYb, could be necessary for the massive carotenoid accumulation that occurs during ripening. Furthermore, the switch from PmLCYe expression to PmLCYb expression could cause beta,beta-carotenoid accumulation in both Japanese apricot cultivars.

GENES ASSOCIATED WITH FRUIT COLOUR IN CARICA PAPAYA: IDENTIFICATION THROUGH EXPRESSED SEQUENCE TAGS AND COLOUR COMPLEMENTATION

We have identified candidate genes that are differentially expressed during papaya fruit ripening in 'Tainung' (red-fleshed) and I B (yellow-fleshed) hybrid varieties. Genes involved in carotenoid production were initially identified by data mining of expressed sequence tags (ESTs). Putative gene function was verified by colour complementation analysis using strains of Escherichia coli engineered to synthesise carotenoids or carotenoid precursors. In all, 1022 ESTs were searched with the BlastX algorithm against existing annotated plant gene databases, identifying seven putative carotenoid and aroma biosynthesis genes. Colour complementation identified papaya cDNA clones with significant homology to three carotenoid pathway genes. Gene expression analysis of these genes in two colour-contrasted papaya cultivars identified cultivar-specific differences in patterns of mRNA accumulation during fruit development. Differential expression of the two carotene desaturase encoding genes, phytoene desaturase and zeta-carotene desaturase and a gene encoding the carotene desaturase co-factor 4-hydroxy phenylpyruvate dioxygenase were identified and may be associated with colour phenotype differences in papaya.

Isoprenoid metabolism and plastid reorganization in arbuscular mycorrhizal roots

Plant root-colonizing arbuscular mycorrhizal (AM) fungi activate the methylerythritol phosphate pathway, carotenoid biosynthesis and oxidative carotenoid cleavage in roots, leading to C13 and C14 apocarotenoids, that is, cyclohexenone and mycorradicin derivatives. Mycorradicin causes the characteristic yellow coloration of many AM roots accumulating within a complex mixture of unknown components. The accumulating C13 cyclohexenones exhibit various ring substitutions and different glycosyl moieties. Transcript levels of the first two enzymes of the MEP pathway, 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase, and of the carotenoid pathway, phytoene desaturase and zeta-carotene desaturase, along with a carotenoid-cleaving dioxygenase, are markedly increased in AM roots. This correlates with proliferation and reorganization of root plastids. These results allow at this point only speculation about the significance of apocarotenoid accumulation: participation in the production of signaling molecules and control of fungal colonization or protection against soil-borne pathogens; protection of root cells against oxidative damage of membranes by reactive oxygen species; and promotion of the symbiotic interactions between plant roots and AM fungi.

Coordinate up‐regulation of carotenoid biosynthesis as a response to light stress in Synechococcus PCC7942

Carotenoid biosynthesis is up-regulated by strong light in the cyanobacterium Synechococcus. By blocking off the pathway at the level of phytoene conversion, light-dependent accumulation of phytoene was observed. Real-time PCR studies demonstrated that four genes of the carotenogenic pathway are under transcriptional control. These were the genes encoding phytoene synthase, phytoene desaturase, zeta-carotene desaturase and beta-carotene hydroxylase. The transcript of the first three follow a similar kinetics, whereas the transcript of beta-carotene hydroxylase increased much faster upon transfer to high light. Promoter activities were determined with transcriptional fusions to chloramphenicol acyltransferase as reporter enzyme. The activity of the promoter of the phytoene desaturase/synthase operon was higher under strong light.

Biochemical and Biophysical Characterization of Photosystem I from Phytoene Desaturase and ζ-Carotene Desaturase Deletion Mutants of Synechocystis Sp. PCC 6803: EVIDENCE FOR PsaA- AND PsaB-SIDE ELECTRON TRANSPORT IN CYANOBACTERIA

In photosystem I, oxidation of reduced acceptor A(1)(-) through iron-sulfur cluster F(X) is biphasic with half-times of approximately 5-30 ns ("fast" phase) and approximately 150-300 ns ("slow" phase). Whether these biphasic kinetics reflect unidirectional electron transfer, involving only the PsaA-side phylloquinone or bi-directional electron transfer, involving both the PsaA- and PsaB-side phylloquinones, has been the source of some controversy. Brettel (Brettel, K. (1988) FEBS Lett. 239, 93-98) and Joliot and Joliot (Joliot, P., and Joliot, A. (1999) Biochemistry 38, 11130-11136) have attributed to nearby carotenoids electrochromic band shifts, accompanying A(1) reduction, centered at approximately 450 and 500-510 nm. As a test of these assignments, we separately deleted in Synechocystis sp. PCC 6803 the genes that encode phytoene desaturase (encoded by crtP (pds)) and zeta-carotene desaturase (encoded by crtQ (zds)). The pds(-) and zds(-) strains synthesize phytoene and zeta-carotene, respectively, both of which absorb to shorter wavelength than beta-carotene. Compared with wild type, the mutant A(1)(-) (FeS) - A(1)(FeS)(-) difference spectra, measured in cells and photosystem I complexes, retain the electrochromic band shift centered at 450 nm but show a complete loss of the electrochromic band shifts centered at 500-510 nm. Thus, the latter clearly arise from beta-carotene. In the wild type, the electrochromic band shift of the slow phase (centered at 500 nm) is shifted by 6 nm to the blue compared with the fast phase (centered at 506 nm). Thus, the carotenoid pigments acting as electrochromic markers during the fast and slow phases of A(1)(-) oxidation are different, indicating the involvement of both the PsaA- and the PsaB-side phylloquinones in photosystem I electron transport.

Myxol and 4-Ketomyxol 2′-Fucosides, not Rhamnosides, from Anabaena sp. PCC 7120 and Nostoc punctiforme PCC 73102, and Proposal for the Biosynthetic Pathway of Carotenoids

We identified the molecular structures of carotenoids in some Anabaena and Nostoc species. The myxoxanthophyll and ketomyxoxanthophyll in Anabaena (also known as Nostoc) sp. PCC 7120, Anabaena variabilis IAM M-3, Nostoc punctiforme PCC 73102 and Nostoc sp. HK-01 were (3R,2'S)-myxol 2'-fucoside and (3S,2'S)-4-ketomyxol 2'-fucoside, respectively. The glycoside moiety of the pigments was fucose, not rhamnose. The major carotenoids were beta-carotene and echinenone, and the minor ones were beta-cryptoxanthin, zeaxanthin, canthaxanthin and 3'-hydroxyechinenone. Based on the identification of the carotenoids and the completion of the entire nucleotide sequence of the genome in Anabaena sp. PCC 7120 and N. punctiforme PCC 73102, we proposed a biosynthetic pathway for the carotenoids and the corresponding genes and enzymes. Since only zeta-carotene desaturase (CrtQ) from Anabaena sp. PCC 7120 and beta-carotene ketolase (CrtW) from N. punctiforme PCC 73102 have been functionally identified, the other genes were searched by sequence homology only from the functionally confirmed genes. Finally, we investigated the phylogenetic relationships among some Anabaena and Nostoc species, including some newly isolated species.

?-Carotene cis isomers as products and substrates in the plant poly-cis carotenoid biosynthetic pathway to lycopene

The plant carotenoid biosynthetic pathway to cyclic carotenes proceeds via carotene precursors in cis configuration. Involvement of individual isomers was elucidated by genetic complementation of desaturations and in vitro reactions of the corresponding enzyme. Determination of substrate and product specificity of phytoene and zeta-carotene desaturase revealed that 15-cis-phytoene is converted to 9,15,9'-tricis-zeta-carotene with 15,9'-dicis-phytofluene as intermediate by the first desaturase. Prior to a subsequent conversion by zeta-carotene desaturase, the 15-cis double bond of 9,15,9'-tricis-zeta-carotene has to be (photo)isomerized to all-trans. Then, the resulting 9,9'-dicis-zeta-carotene is utilized by zeta-carotene desaturase via 7,9,9'-tricis-neurosporene to 7,9,7',9'-tetracis-lycopene. Other zeta-carotene isomers that are assumed to be spontaneous isomerization products were not converted, except for the asymmetric 9-cis-zeta-carotene. This isomer is desaturated only to 7,9-dicis-neurosporene resembling a dead-end of the pathway. Prolycopene, the product of the desaturation reactions, is finally isomerized by a specific isomerase to all-trans-lycopene, which is a prerequisite for cyclization to beta-carotene. The 5-cis-lycopene and the 9-cis-and 13-cis-beta-carotene isomers detected in leaves are thought to originate independently from cis precursors by non-enzymatic isomerization of their all-trans forms.

Biochemical and Molecular Analysis of Carotenoid Biosynthesis in Flavedo of Orange (Citrus sinensis L.) during Fruit Development and Maturation

Changes in carotenoid content and composition and expression of carotenoid biosynthetic genes were analyzed in the flavedo of sweet orange (Citrus sinensis L. Osbeck, cv. Navelate) fruit during development and maturation. Lutein and all-E-violaxanthin were the major carotenoids in chloroplast-containing tissues. During fruit coloration, phytoene, beta-cryptoxanthin, zeaxanthin, and mainly (9Z)-violaxanthin progressively accumulated, and a large proportion of apocarotenoids was also found in the flavedo of full-colored fruits. We have cloned partial and full-length cDNAs corresponding to genes involved in early condensation and desaturase reactions [phytoene synthase (PSY), phytoene desaturase (PDS), and zeta-carotene desaturase (ZDS)], coupled redox reaction (plastid terminal oxidase), cyclizations [beta-lycopene cyclase (beta-LCY) and epsilon-lycopene cyclase (epsilon-LCY)], hydroxylation [beta-carotene hydroxylase (beta-CHX)], and epoxidation [zeaxanthin epoxidase (ZEP)] and analyzed their mRNA accumulation in the flavedo of fruits during development and ripening as compared with those of leaves. Collectively, the results indicated that PDS gene expression correlated with carotenoid content in developing fruit and that up-regulation of PSY and ZDS genes at the onset of fruit coloration would enhance the production of linear carotenes and the flux into the pathway. The shift from the beta,epsilon-branch to the beta,beta-branch of the pathway that originates the changes in carotenoid composition during fruit coloration may be explained by a down-regulation of epsilon-LCY and by the increase of the beta-CHX transcript.

Glucose-induced Expression of Carotenoid Biosynthesis Genes in the Dark Is Mediated by Cytosolic pH in the Cyanobacterium Synechocystis sp. PCC 6803

The expression of carotenoid biosynthesis genes coding for phytoene synthase (crtB), phytoene desaturase (crtP), zeta-carotene desaturase (crtQ), and beta-carotene hydroxylase (crtR) is dependent upon light in the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). We have demonstrated that the expression of the above four genes was also elevated in the dark-adapted Synechocystis cells upon glucose treatment as a consequence of transcriptional activation. Treatment with glucose analogs such as l-glucose, 3-O-methylglucose, 2-deoxyglucose, and mannose, or inactivation of glucose uptake and phosphorylation by deletion mutation of glucose transporter (glcP) and glucokinase (gk), respectively, did not induce up-regulation of carotenoid genes. When respiratory electron transport or coupling to oxidative phosphorylation was inhibited, glucose induction was not observed, indicating that respiratory electron transport per se is not critical for the expression of these genes. In agreement with this view, the extent of gene expression showed a saturation curve with increasing acridine yellow fluorescence yield, without having a close correlation with the ATP contents or ATP/ADP ratio. The results indicate that glucose induction of carotenoid gene expressions is mediated by an increase in cytosolic pH rather than either redox or glucose sensing.

Accumulation of carotenoids and expression of carotenoid biosynthetic genes during maturation in citrus fruit.

The relationship between carotenoid accumulation and the expression of carotenoid biosynthetic genes during fruit maturation was investigated in three citrus varieties, Satsuma mandarin (Citrus unshiu Marc.), Valencia orange (Citrus sinensis Osbeck), and Lisbon lemon (Citrus limon Burm.f.). We cloned the cDNAs for phytoene synthase (CitPSY), phytoene desaturase (CitPDS), zeta-carotene (car) desaturase (CitZDS), carotenoid isomerase (CitCRTISO), lycopene beta-cyclase (CitLCYb), beta-ring hydroxylase (CitHYb), zeaxanthin (zea) epoxidase (CitZEP), and lycopene epsilon-cyclase (CitLCYe) from Satsuma mandarin, which shared high identities in nucleotide sequences with Valencia orange, Lisbon lemon, and other plant species. With the transition of peel color from green to orange, the change from beta,epsilon-carotenoid (alpha-car and lutein) accumulation to beta,beta-carotenoid (beta-car, beta-cryptoxanthin, zea, and violaxanthin) accumulation was observed in the flavedos of Satsuma mandarin and Valencia orange, accompanying the disappearance of CitLCYe transcripts and the increase in CitLCYb transcripts. Even in green fruit, high levels of beta,epsilon-carotenoids and CitLCYe transcripts were not observed in the juice sacs. As fruit maturation progressed in Satsuma mandarin and Valencia orange, a simultaneous increase in the expression of genes (CitPSY, CitPDS, CitZDS, CitLCYb, CitHYb, and CitZEP) led to massive beta,beta-xanthophyll (beta-cryptoxanthin, zea, and violaxanthin) accumulation in both the flavedo and juice sacs. The gene expression of CitCRTISO was kept low or decreased in the flavedo during massive beta,beta-xanthophyll accumulation. In the flavedo of Lisbon lemon and Satsuma mandarin, massive accumulation of phytoene was observed with a decrease in the transcript level for CitPDS. Thus, the carotenoid accumulation during citrus fruit maturation was highly regulated by the coordination of the expression among carotenoid biosynthetic genes. In this paper, the mechanism leading to diversity in beta,beta-xanthophyll compositions between Satsuma mandarin and Valencia orange was also discussed on the basis of the substrate specificity of beta-ring hydroxylase and the balance of expression between upstream synthesis genes (CitPSY, CitPDS, CitZDS, and CitLCYb) and downstream synthesis genes (CitHYb and CitZEP).

QTL and candidate genes phytoene synthase and zeta-carotene desaturase associated with the accumulation of carotenoids in maize.

Carotenoids are a class of fat-soluble antioxidant vitamin compounds present in maize ( Zea mays L.) that may provide health benefits to animals or humans. Four carotenoid compounds are predominant in maize grain: beta-carotene, beta-cryptoxanthin, zeaxanthin, and lutein. Although beta-carotene has the highest pro-vitamin A activity, it is present in a relatively low concentration in maize kernels. We set out to identify quantitative trait loci (QTL) affecting carotenoid accumulation in maize kernels. Two sets of segregating families were evaluated-a set of F2:3 lines derived from a cross of W64a x A632, and their testcross progeny with AE335. Molecular markers were evaluated on the F2:3 lines and a genetic linkage map created. High-performance liquid chromatography was performed to measure beta-carotene, beta-cryptoxanthin, zeaxanthin, and lutein on both sets of materials. Composite interval mapping identified chromosome regions with QTL for one or more individual carotenoids in the per se and testcross progenies. Notably QTL in the per se population map to regions with candidate genes, yellow 1 and viviparous 9, which may be responsible for quantitative variation in carotenoids. The yellow 1 gene maps to chromosome six and is associated with phytoene synthase, the enzyme catalyzing the first dedicated step in the carotenoid biosynthetic pathway. The viviparous 9 gene maps to chromosome seven and is associated with zeta-carotene desaturase, an enzyme catalyzing an early step in the carotenoid biosynthetic pathway. If the QTL identified in this study are confirmed, particularly those associated with candidates genes, they could be used in an efficient marker-assisted selection program to facilitate increasing levels of carotenoids in maize grain.

Maize phytoene desaturase and zeta-carotene desaturase catalyse a poly-Z desaturation pathway: implications for genetic engineering of carotenoid content among cereal crops.

Carotene desaturation, an essential step in the biosynthesis of coloured carotenoids, has received much attention (1) as a target of bleaching herbicide action, (2) as a determinant of geometric isomer states of carotenoids and their metabolites, and (3) as a key modulator of accumulation and structural variability of carotenoids. Having previously isolated and functionally characterized the cDNA encoding the first enzyme in maize carotene desaturation, phytoene desaturase (PDS), the isolation and functional characterization of the second desaturase, a maize endosperm cDNA (2265 bp) encoding zetacarotene (zeta-carotene) desaturase (ZDS) is reported here. Functional analysis of the concerted actions of maize PDS and ZDS ex situ showed these enzymes to mediate a poly-Z desaturation pathway to the predominate geometric isomer 7,9,7',9'-tetra-Z-lycopene (poly-Z-lycopene or prolycopene), and not the all-trans substrate required of the downstream lycopene cyclase enzymes. This finding suggests a rate-controlling isomerase associated with the carotene desaturases as a corollary of a default poly-Z carotenoid biosynthetic pathway active in planta for maize. Comparative gene analysis between maize and rice revealed that genes encoding PDS and ZDS are single copy; the Zds cDNA characterized here was mapped to maize chromosome 7S and vp9 is suggested as a candidate locus for the structural gene while y9 is ruled out. Classical genetic resources were used to dissect the desaturation steps further and hydroxyphenylpyruvate dioxygenase was linked to the vp2 locus, narrowing candidate loci for an obligate isomerase in maize to only a few. Since the first functional analysis of the paired carotene desaturases for a cereal crop is reported here, the implications for the genetic modification of the pro-vitamin A content in cereal crops such as rice and maize, are discussed.

CDNA cloning and expression of carotenogenic genes during flower development in Gentiana lutea.

All cDNAs involved in carotenoid biosynthesis leading to lycopene in yellow petals of Gentiana lutea have been cloned from a cDNA library. They encode a geranylgeranyl pyrophosphate synthase, a phytoene synthase, a phytoene desaturase and a zeta-carotene desaturase. The indicated function of all cDNAs was established by heterologous complementation in Escherichia coli. The amino acid sequences deduced from the cDNAs were between 47.5% and 78.9% identical to those reported for the corresponding enzymes from other higher plants. Southern analysis suggested that the genes for each enzyme probably represent a small multi-gene family. Tissue-specific expression of the genes and expression during flower development was investigated. The expression of the phytoene synthase gene, psy, was enhanced in flowers but transcripts were not detected in stems and leaves by northern blotting. Transcripts of the genes for geranylgeranyl pyrophosphate (ggpps), phytoene desaturase (pds) and zeta-carotene desaturase (zds) were detected in flowers and leaves but not in stems. Analysis of the expression of psy and zds in petals revealed that levels of the transcripts were lowest in young buds and highest in fully open flowers, in parallel with the formation of carotenoids. Obviously, the transcription of these genes control the accumulation of carotenoids during flower development in G. lutea. For pds only a very slight increase of mRNA was found whereas the transcripts of ggpps decreased during flower development.