Metabolic engineering of beta-carotene and lycopene content in tomato fruit

Ripe tomato fruits accumulate large amounts of the red linear carotene, lycopene (a dietary antioxidant) and small amounts of its orange cyclisation product, beta-carotene (pro-vitamin A). Lycopene is transformed into beta-carotene by the action of lycopene beta-cyclase (beta-Lcy). We introduced, via Agrobacterium-mediated transformation, DNA constructs aimed at up-regulating (OE construct) or down-regulating (AS construct) the expression of the beta-Lcy gene in a fruit-specific fashion. Three transformants containing the OE construct show a significant increase in fruit beta-carotene content. The fruits from these plants display different colour phenotypes, from orange to orange-red, depending on the lycopene/beta-carotene ratio. Fruits from AS transformants show up to 50% inhibition of beta-Lcy expression, accompanied by a slight increase in lycopene content. Leaf carotenoid composition is unaltered in all transformants. In most transformants, an increase in total carotenoid content is observed with respect to the parental line. This increase occurs in the absence of major variations in the expression of endogenous carotenoid genes.     

Enhancing the carotenoid content of <Emphasis Type="Italic">Brassica napus</Emphasis> seeds by downregulating lycopene epsilon cyclase

The accumulation of carotenoids in higher plants is regulated by the environment, tissue type and developmental stage. In Brassica napus leaves, beta-carotene and lutein were the main carotenoids present while petals primarily accumulated lutein and violaxanthin. Carotenoid accumulation in seeds was developmentally regulated with the highest levels detected at 35-40 days post anthesis. The carotenoid biosynthesis pathway branches after the formation of lycopene. One branch forms carotenoids with two beta rings such as beta-carotene, zeaxanthin and violaxanthin, while the other introduces both beta- and epsilon-rings in lycopene to form alpha-carotene and lutein. By reducing the expression of lycopene epsilon-cyclase (epsilon-CYC) using RNAi, we investigated altering carotenoid accumulation in seeds of B. napus. Transgenic seeds expressing this construct had increased levels of beta-carotene, zeaxanthin, violaxanthin and, unexpectedly, lutein. The higher total carotenoid content resulting from reduction of epsilon-CYC expression in seeds suggests that this gene is a rate-limiting step in the carotenoid biosynthesis pathway. epsilon-CYC activity and carotenoid production may also be related to fatty acid biosynthesis in seeds as transgenic seeds showed an overall decrease in total fatty acid content and minor changes in the proportions of various fatty acids.     

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.     

Expression profile of genes coding for carotenoid biosynthetic pathway during ripening and their association with accumulation of lycopene in tomato fruits

Fruit ripening process is associated with change in carotenoid profile and accumulation of lycopene in tomato (Solanum lycopersicum L.). In this study, we quantified the β-carotene and lycopene content at green, breaker and red-ripe stages of fruit ripening in eight tomato genotypes by using high-performance liquid chromatography. Among the genotypes, lycopene content was found highest in Pusa Rohini and lowest in VRT-32-1. To gain further insight into the regulation of lycopene biosynthesis and accumulation during fruit ripening, expression analysis of nine carotenoid pathway-related genes was carried out in the fruits of high lycopene genotype—Pusa Rohini. We found that expression of phytoene synthase and β-carotene hydroxylase-1 was four and thirty-fold higher, respectively, at breaker stage as compared to red-ripe stage of fruit ripening. Changes in the expression level of these genes were associated with a 40% increase in lycopene content at red-ripe stage as compared with breaker stage. Thus, the results from our study suggest the role of specific carotenoid pathway-related genes in accumulation of high lycopene during the fruit ripening processes.     

Contained metabolic engineering in tomatoes by expression of carotenoid biosynthesis genes from the plastid genome

Applications of chloroplast engineering in agriculture and biotechnology will depend critically on success in extending the crop range of chloroplast transformation, and on the feasibility of expressing transgenes in edible organs (such as tubers and fruits), which often are not green and thus are much less active in chloroplast gene expression. We have improved a recently developed chloroplast-transformation system for tomato plants and applied it to engineering one of the central metabolic pathways in fruits: carotenoid biosynthesis. We report that plastid expression of a bacterial lycopene beta-cyclase gene results in herbicide resistance and triggers conversion of lycopene, the main storage carotenoid of tomatoes, to beta-carotene, resulting in fourfold enhanced pro-vitamin A content of the fruits. Our results demonstrate the feasibility of engineering nutritionally important biochemical pathways in non-green plastids by transformation of the chloroplast genome.     

Why Is Golden Rice Golden (Yellow) Instead of Red?

The endosperm of Golden Rice (Oryza sativa) is yellow due to the accumulation of beta-carotene (provitamin A) and xanthophylls. The product of the two carotenoid biosynthesis transgenes used in Golden Rice, phytoene synthase (PSY) and the bacterial carotene desaturase (CRTI), is lycopene, which has a red color. The absence of lycopene in Golden Rice shows that the pathway proceeds beyond the transgenic end point and thus that the endogenous pathway must also be acting. By using TaqMan real-time PCR, we show in wild-type rice endosperm the mRNA expression of the relevant carotenoid biosynthetic enzymes encoding phytoene desaturase, zeta-carotene desaturase, carotene cis-trans-isomerase, beta-lycopene cyclase, and beta-carotene hydroxylase; only PSY mRNA was virtually absent. We show that the transgenic phenotype is not due to up-regulation of expression of the endogenous rice pathway in response to the transgenes, as was suggested to be the case in tomato (Lycopersicon esculentum) fruit, where CRTI expression resulted in a similar carotenoid phenomenon. This means that beta-carotene and xanthophyll formation in Golden Rice relies on the activity of constitutively expressed intrinsic rice genes (carotene cis-trans-isomerase, alpha/beta-lycopene cyclase, beta-carotene hydroxylase). PSY needs to be supplemented and the need for the CrtI transgene in Golden Rice is presumably due to insufficient activity of the phytoene desaturase and/or zeta-carotene desaturase enzyme in endosperm. The effect of CRTI expression was also investigated in leaves of transgenic rice and Arabidopsis (Arabidopsis thaliana). Here, again, the mRNA levels of intrinsic carotenogenic enzymes remained unaffected; nevertheless, the carotenoid pattern changed, showing a decrease in lutein, while the beta-carotene-derived xanthophylls increased. This shift correlated with CRTI-expression and is most likely governed at the enzyme level by lycopene-cis-trans-isomerism. Possible implications are discussed.     

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.     

Enhanced Levels of the Aroma and Flavor Compound S-Linalool by Metabolic Engineering of the Terpenoid Pathway in Tomato Fruits

The aromas of fruits, vegetables, and flowers are mixtures of volatile metabolites, often present in parts per billion levels or less. We show here that tomato (Lycopersicon esculentum Mill.) plants transgenic for a heterologous Clarkia breweri S-linalool synthase (LIS) gene, under the control of the tomato late-ripening-specific E8 promoter, synthesize and accumulate S-linalool and 8-hydroxylinalool in ripening fruits. Apart from the difference in volatiles, no other phenotypic alterations were noted, including the levels of other terpenoids such as gamma- and alpha-tocopherols, lycopene, beta-carotene, and lutein. Our studies indicate that it is possible to enhance the levels of monoterpenes in ripening fruits by metabolic engineering.     

Lycopenoids: are lycopene metabolites bioactive?

In vitro lycopene is the most potent antioxidant among carotenoids. While antioxidant function may be relevant to health, we hypothesize that metabolites of lycopene may be bioactive and responsible for the beneficial effects of tomato product consumption. We term these metabolites "lycopenoids," which we believe may be produced from carotenoid monooxygenase (CMO) II, paralleling the production of retinoids from beta-carotene by CMO I. We present evidence suggesting that tomato carotenoid metabolites may be responsible for the reduced risk of prostate cancer seen in men consuming high levels of tomato products. Finally, we identify gaps in knowledge in this evolving area of carotenoid research.     

Lycopene accumulation affects the biosynthesis of some carotenoid-related volatiles independent of ethylene in tomato

For elucidating the regulatory mechanism of ethylene on carotenoid-related volatiles (open chain) compounds and the relationship between lycopene and carotenoid-related volatiles,transgenic tomato fruits in which ACC synthase was suppressed were used.The transgenic tomato fruit showed a significant reduction of lycopene and aroma volatiles with low ethylene production.6-methyl-5-hepten-2-one,6-methyl-5-hepten-2-ol and geranylacetone,which were suspected to be lycopene degradation products,were lower than those in wild type tomato fruits.In order to identify whether lycopene accumulation effects the biosynthesis of some carotenoid-related volatiles independent of ethylene in tomato or not,the capability of both wild type and transgenic tomato fruits discs to convert lycopene into carotenoid-related volatiles was evaluated.The data showed that external lycopene could convert into 6-methyl-5-hepten-2-one and 6-methyl-5-hepten-2-ol in vivo,Indicating that the strong inhibition of ethylene production had no effect on enzymes in the biosynthesis pathway of some carotenoid-related volatiles.Therefore,in ACS-suppression transgenic tomato fruits,the low levels of 6-methyl-5-hepten-2-one,6-methyl-5-hepten-2-ol was due to decreased lycopene accumulation,not ethylene production.Ethylene only affected the accumulation of lycopene,and then indirectly influenceed the level of lycopene-related volatiles.     

A novel bud mutation that confers abnormal patterns of lycopene accumulation in sweet orange fruit (Citrus sinensis L. Osbeck)

A novel, pleiotropic sweet orange (Citrus sinensis L. Osbeck) mutant, 'Hong Anliu', is described. This mutation causes carotenoid accumulation, high sugar, and low acid in the fruits. Gas chromatographic analysis revealed that high sugar and low acid in the fruit were caused by the accumulation of sucrose and the deficiency of citric acid. The dominant carotenoid accumulated in albedo, segment membranes, and juice sacs is lycopene, which can reach levels that are a 1000-fold higher than those in comparable wild-type fruits. This mutation does not affect the carotenoid composition of leaves. Carotenoid concentration and biosynthetic gene expression of albedo, segment membranes, and juice sacs were dramatically altered by the mutation. Lycopene accumulation in the juice sacs was regulated by co-ordinate expression of carotenoid biosynthetic genes. However, in albedo and segment membranes, the expression of downstream carotenogenic genes seems to be feedback induced by lycopene accumulation. This implies that there must be at least two modes regulating lycopene accumulation in 'Hong Anliu' fruit. Taken together, these results suggest that massive amounts of lycopene might be synthesized in the juice sacs and then transported to the segment membrane and the albedo, which leads to lycopene accumulation there.