An enzyme complex for the dehydrogenation of phytoene in Phycomyces.

Phycomyces strain C5, carrying mutation carB10, accumulates phytoene instead of beta-carotene. Heterokaryons containing C5 nuclei and different other nuclei carrying the wild type carB allele accumulate significant amounts of phytofluene, zota-carotene and neurosporene. From quantitative analyses of carotenes and nuclear proportions in the heterokaryons we conclude that four copies of the carB gene product, assembled in an enzyme complex, act sequentially in the conversion of phytoene to lycopene.     

beta-Carotene accumulation induced by the cauliflower Or gene is not due to an increased capacity of biosynthesis

The cauliflower (Brassica oleracea L. var. botrytis) Or gene is a rare carotenoid gene mutation that confers a high level of beta-carotene accumulation in various tissues of the plant, turning them orange. To investigate the biochemical basis of Or-induced carotenogenesis, we examined the carotenoid biosynthesis by evaluating phytoene accumulation in the presence of norflurazon, an effective inhibitor of phytoene desaturase. Calli were generated from young seedlings of wild type and Or mutant plants. While the calli derived from wild type seedlings showed a pale green color, the calli derived from Or seedlings exhibited intense orange color, showing the Or mutant phenotype. Concomitantly, the Or calli accumulated significantly more carotenoids than the wild type controls. Upon treatment with norflurazon, both the wild type and Or calli synthesized significant amounts of phytoene. The phytoene accumulated at comparable levels and no major differences in carotenogenic gene expression were observed between the wild type and Or calli. These results suggest that Or-induced beta-carotene accumulation does not result from an increased capacity of carotenoid biosynthesis.     

A chromoplast-specific carotenoid biosynthesis pathway is revealed by cloning of the tomato white-flower locus

Carotenoids and their oxygenated derivatives xanthophylls play essential roles in the pigmentation of flowers and fruits. Wild-type tomato (Solanum lycopersicum) flowers are intensely yellow due to accumulation of the xanthophylls neoxanthin and violaxanthin. To study the regulation of xanthophyll biosynthesis, we analyzed the mutant white-flower (wf). It was found that the recessive wf phenotype is caused by mutations in a flower-specific beta-ring carotene hyroxylase gene (CrtR-b2). Two deletions and one exon-skipping mutation in different CrtR-b2 wf alleles abolish carotenoid biosynthesis in flowers but not leaves, where the homologous CrtR-b1 is constitutively expressed. A second beta-carotene hydroxylase enzyme as well as flower- and fruit-specific geranylgeranyl diphosphate synthase, phytoene synthase, and lycopene beta-cyclase together define a carotenoid biosynthesis pathway active in chromoplasts only, underscoring the crucial role of gene duplication in specialized plant metabolic pathways. We hypothesize that this pathway in tomato was initially selected during evolution to enhance flower coloration and only later recruited to enhance fruit pigmentation. The elimination of beta-carotene hydroxylation in wf petals results in an 80% reduction in total carotenoid concentration, possibly caused by the inability of petals to store high concentrations of carotenoids other than xanthophylls and by degradation of beta-carotene, which accumulates as a result of the wf mutation but is not due to altered expression of genes in the biosynthetic pathway.     

Isolation, sequence, and characterization of the Cercospora nicotianae phytoene dehydrogenase gene.

We have cloned and sequenced the Cercospora nicotianae gene for the carotenoid biosynthetic enzyme phytoene dehydrogenase. Analysis of the derived amino acid sequence revealed it has greater than 50% identity with its counterpart in Neurospora crassa and approximately 30% identity with prokaryotic phytoene dehydrogenases and is related, but more distantly, to phytoene dehydrogenases from plants and cyanobacteria. Our analysis confirms that phytoene dehydrogenase proteins fall into two groups: those from plants and cyanobacteria and those from eukaryotic and noncyanobacter prokaryotic microbes. Southern analysis indicated that the C. nicotianae phytoene dehydrogenase gene is present in a single copy. Extraction of beta-carotene, the sole carotenoid accumulated by C. nicotianae, showed that both light- and dark-grown cultures synthesize carotenoids, but higher levels accumulate in the light. Northern (RNA) analysis of poly(A)+ RNA, however, showed no differential accumulation of phytoene dehydrogenase mRNA between light- and dark-grown fungal cultures.     

Construction and characterization of genetically modified synechocystis sp. PCC 6803 photosystem II core complexes containing carotenoids with shorter pi-conjugation than beta-carotene

Beta-carotene has been identified as an intermediate in a secondary electron transfer pathway that oxidizes Chl(Z) and cytochrome b(559) in Photosystem II (PS II) when normal tyrosine oxidation is blocked. To test the redox function of carotenoids in this pathway, we replaced the zeta-carotene desaturase gene (zds) or both the zds and phytoene desaturase (pds) genes of Synechocystis sp. PCC 6803 with the phytoene desaturase gene (crtI) of Rhodobacter capsulatus, producing carotenoids with shorter conjugated pi-electron systems and higher reduction potentials than beta-carotene. The PS II core complexes of both mutant strains contain approximately the same number of chlorophylls and carotenoids as the wild type but have replaced beta-carotene (11 double bonds), with neurosporene (9 conjugated double bonds) and beta-zeacarotene (9 conjugated double bonds and 1 beta-ionylidene ring). The presence of the ring appears necessary for PS II assembly. Visible and near-infrared spectroscopy were used to examine the light-induced formation of chlorophyll and carotenoid radical cations in the mutant PS II core complexes at temperatures from 20 to 160 K. At 20 K, a carotenoid cation radical is formed having an absorption maximum at 898 nm, an 85 nm blue shift relative to the beta-carotene radical cation peak in the WT, and consistent with the formation of the cation radical of a carotenoid with 9 conjugated double bonds. The ratio of Chl(+)/Car(+) is higher in the mutant core complexes, consistent with the higher reduction potential for Car(+). As the temperature increases, other carotenoids become accessible to oxidation by P(680)(+).     

Elevation of the provitamin A content of transgenic tomato plants

Tomato products are the principal dietary sources of lycopene and major source of beta-carotene, both of which have been shown to benefit human health. To enhance the carotenoid content and profile of tomato fruit, we have produced transgenic lines containing a bacterial carotenoid gene (crtI) encoding the enzyme phytoene desaturase, which converts phytoene into lycopene. Expression of this gene in transgenic tomatoes did not elevate total carotenoid levels. However, the beta-carotene content increased about threefold, up to 45% of the total carotenoid content. Endogenous carotenoid genes were concurrently upregulated, except for phytoene synthase, which was repressed. The alteration in carotenoid content of these plants did not affect growth and development. Levels of noncarotenoid isoprenoids were unchanged in the transformants. The phenotype has been found to be stable and reproducible over at least four generations.     

脐橙晚熟突变体"奉晚"与原品种"奉节72-1"的果实着色差异

测定了果实成熟过程中“奉晚”和“奉节72．1”两个脐橙（Citrus sinensis L．Osbeck）品种果皮中叶绿素和总类胡萝卜素的含量,并采用RT-PCR技术研究了相关酶的基因表达。结果表明：“奉晚”脐橙果皮中叶绿素含量的显著降低发生在11月份,“奉节72．1”则发生存10月中下旬到11月上旬;“奉晚”脐橙果皮中的总类胡萝卜素含量显著积累始于12月中旬,而“奉节72.1”品种则始于11月初。另外,“奉晚”脐橙果皮中叶绿素含量在10～11月显著高于原品种,总类胡萝卜素含量在12-1月显著低于原品种。从基因表达分析结果看到,“奉晚”脐橙果皮中与类胡萝卜素合成酶相关的基因较强表达的时间也整体迟于“奉节72．1”脐橙;叶绿素水解酶基因表达在10～12月中旬表达较“奉节72—1”弱,翌年1月份则较“奉节72．1”强。     

Metabolic engineering of high carotenoid potato tubers containing enhanced levels of beta-carotene and lutein

In order to enhance the carotenoid content of potato tubers, transgenic potato plants have been produced expressing an Erwinia uredovora crtB gene encoding phytoene synthase, specifically in the tuber of Solanum tuberosum L. cultivar Desiree which normally produces tubers containing c. 5.6 microg carotenoid g(-1) DW and also in Solanum phureja L. cv. Mayan Gold which has a tuber carotenoid content of typically 20 microg carotenoid g(-1) DW. In developing tubers of transgenic crtB Desiree lines, carotenoid levels reached 35 microg carotenoid g(-1) DW and the balance of carotenoids changed radically compared with controls: beta-carotene levels in the transgenic tubers reached c. 11 microg g(-1) DW, whereas control tubers contained negligible amounts and lutein accumulated to a level 19-fold higher than empty-vector transformed controls. The crtB gene was also transformed into S. phureja (cv. Mayan Gold), again resulting in an increase in total carotenoid content to 78 microg carotenoid g(-1) DW in the most affected transgenic line. In these tubers, the major carotenoids were violaxanthin, lutein, antheraxanthin, and beta-carotene. No increases in expression levels of the major carotenoid biosynthetic genes could be detected in the transgenic tubers, despite the large increase in carotenoid accumulation. Microarray analysis was used to identify a number of genes that were consistently up- or down-regulated in transgenic crtB tubers compared with empty vector controls. The implications of these data from a nutritional standpoint and for further modifications of tuber carotenoid content are discussed.     

Metabolic engineering of the carotenoid biosynthetic pathway in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both single and double crossover events, resulting in non-carotenoid-producing transformants. In addition, the crtYB gene, linked to either its homologous or a glyceraldehyde-3-phosphate dehydrogenase promoter, was overexpressed in the wild type and a beta-carotene-accumulating mutant of X. dendrorhous. In several transformants containing multiple copies of the crtYB gene, the total carotenoid content was higher than in the control strain. This increase was mainly due to an increase of the beta-carotene and echinone content, whereas the total content of astaxanthin was unaffected or even lower. Overexpression of the phytoene synthase-encoding gene (crtI) had a large impact on the ratio between mono- and bicyclic carotenoids. Furthermore, we showed that in metabolic engineered X. dendrorhous strains, the competition between the enzymes phytoene desaturase and lycopene cyclase for lycopene governs the metabolic flux either via beta-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3'-4'-didehydro-beta-psi-caroten-4-one (HDCO). The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains.     

A carotenoid biosynthesis gene cluster in Fusarium fujikuroi: the genes carB and carRA

Phytoene synthase, phytoene dehydrogenase and carotene cyclase are three of the four enzyme activities needed to produce the acidic carotenoid neurosporaxanthin from the precursor geranylgeranyl pyrophosphate. In the filamentous fungus Fusarium fujikuroi, these three enzyme activities are encoded by two closely linked genes, carRA and carB, oriented in the same direction in the genome. The two genes are separated by 548 bp and code for two polypeptides of 612 and 541 amino acids, respectively, which are highly similar to the homologous proteins from other filamentous fungi. The ORF of carRA contains a 96-bp insertion that is absent in the other fungal homologues. The 32 additional residues are located in one of the two repeated domains responsible for the cyclase activity in the homologous fungal proteins. We have determined the function of carRA by gene disruption. The resulting mutants were albino and had lost the ability to produce phytoene, as expected from the simultaneous loss of phytoene synthase and carotene cyclase. In the same experiments, we also found transformants in which carB had been deleted. These mutants accumulate phytoene, confirming the function of the gene previously shown by gene-targeted mutagenesis. Expression of carRA and carB is strongly induced by light. Loss of carB or disruption of the carRA ORF led to enhanced expression of the carRA gene, suggesting the existence of a feedback regulatory mechanism.     

The carotenoid-deficient mutant, C-6E, of Scenedesmus obliquus is blocked at the site of phytoene synthase

In contrast to the wild type strain of Scenedesmus , mutant C-6E synthesized only trace amounts of the carotenoids violaxanthin and lutein during prolonged heterotrophic growth. All other carotenoids and carotenoid precursors, such as phytoene, were undetectable. Additionally, only reduced levels of chlorophyll a and no chlorophyll b were formed. To evaluate the potential site of inhibition in the pathway for carotenoid biosynthesis the enzymatic activities of geranylgeranyl pyrophosphate synthase and phytoene synthase were assayed in cell-free extracts. Both enzymes were highly active in extracts of the wild type but only geranylgeranyl pyrophosphate synthase was active in comparable extracts from mutant C-6E . This observation strongly indicates that the phenotype of C-6E results from either a mutation of the phytoene synthase structural gene or of a regulatory gene involved in expression of this enzyme. Other phenotypic effects on composition and structure of the photosynthetic apparatus are discussed as a secondary consequence of the carotenoid deficiency in the thylakoid membranes.