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Orange carrots are well known for their nutritional value as producers of β-carotene, a vitamin A precursor. Lesser known, is their ability to accumulate antioxidants such as chlorogenic acid. Chlorogenic acid is produced through the same biosynthetic pathway that produces lignins, anthocyanins, flavonols and isoflavonoids, the phenylpropanoid pathway. This pathway can be induced by UV-B light and wounding to produce large amounts of chlorogenic acid. We identified a number of the carrot structural genes in this pathway and their proposed regulators in carrot root slices and analyzed their expression in response to UV-B light exposure using real-time PCR. Our results indicate that not only does the expression of the structural genes correlate with their proposed regulators, including DcHY5 and others, but also correlate with chlorogenic acid production. Our results indicate that the HY5 — UVR8 signaling network may exist and function in carrot roots.

Brasenia schreberi (蓴菜 chún cài) is an invasive aquatic weed found in the USA, but the plant has economic value in Asia where it is cultivated for food. The young leaves of B. schreberi are coated with gelatinous water-insoluble mucilage. This mucilage is a polysaccharide composed of galactose, mannose, fucose, and other monosaccharides. Because some carbohydrate gels are hypocholesterolemic, we evaluated their cholesterol-lowering properties in male hamsters fed hypercholesterolemic diets containing 2% gel coat from B. schreberi (GEL), or 1% cholestyramine (CA), or 5% hydroxypropyl methylcellulose (HPMC), and compared them to 5% microcrystalline cellulose (control) for 3 weeks. We found that very-low-density lipoprotein-, low-density lipoprotein-, and total-cholesterol concentrations in plasma were significantly lowered by GEL, CA, and HPMC compared to control. High-density lipoprotein-cholesterol concentration was lowered by CA and HPMC. Body weights and abdominal adipose tissue weight of GEL and control group animals were greater than those of the CA and HPMC groups. Fecal lipid excretion was greater in the CA and HPMC groups than in the control group. Expression of hepatic CYP51 and CYP7A1 mRNA was upregulated by CA, HPMC, and GEL, indicating increased hepatic cholesterol and bile acid synthesis. Expression of low-density lipoprotein receptor mRNA was upregulated by all treatments. These results suggest that modulation of hepatic expression of cholesterol and bile acid metabolism-regulated genes contributes to the cholesterol-lowering effects of GEL., Graphical abstract

The cholesterol-lowering effects of tomato pomace (TP), tomato seed oil (TSO) and defatted tomato seed (DTS) were determined in male Golden Syrian hamsters. Hamsters fed high-fat diets containing 10% TSO or 18% DTS were compared to a diet containing 10% corn oil and 10% microcrystalline cellulose (control 1), 42% TP were compared to 25% microcrystalline cellulose (control 2). TP, TSO and DTS reduced hepatic total cholesterol (TC) content. DTS also lowered plasma TC and low-density lipoprotein cholesterol (LDL-C) concentrations. Fecal excretion of lipid, bile acid and cholesterol increased in the DTS group compared to control 1. DTS-fed hamsters had higher levels of hepatic CYP7A1, CYP51, ABCB11, and ABCG5 gene expression than control, suggesting both hepatic bile acid and cholesterol synthesis increased due to increased fecal excretion of bile acid and cholesterol. The results suggest that protein, dietary fibre or phenolic compounds in DTS may be responsible for plasma cholesterol decrease.

Background The phenolic amine synephrine is a vascoconstrictor and bronchiectatic agent and holds promise as an aid to weight management and obesity reduction. Synephrine is structurally similar to the active ingredients of several commercial cold remedies. Some Citrus contain high concentrations of synephrine. An enzyme involved in synephrine biosynthesis, tyrosine decarboxylase (TYDC), is a pyridoxal-5′-phosphate (PLP)-dependent enzyme that decarboxylates tyrosine to yield CO 2 and tyramine. We used PCR to screen, clone and sequence this gene from various synephrine producing and nonproducing Citrus species and varieties to determine if DNA sequence of this gene correlated with synephrine presence. Results PCR amplification and comparison of DNA sequence indicates DNA sequence differences that may cause production of truncated proteins to be produced in some nonsynephrine producing Citru s. Conclusion Synephrine production may be genetically determined in part by the gene for TYDC.

Cholecystokinin (CCK) and the different molecular forms of CCK are well established as biomarkers for satiety but accurate analysis has been limited by the multiple naturally occurring forms and extensive similarities to gastrin. Changes in levels of one form, CCK-8, a naturally occurring eight amino acid peptide of CCK, have been correlated with satiety responses. Endogenous CCK-8 has not been well characterized in Syrian Golden hamsters, an important model in the study of fat uptake and digestion. We have cloned and sequenced hamster CCK and identified and characterized endogenous CCK-8 from hamster plasma. Hamster CCK-8 is composed of eight amino acid residues which are highly conserved among other species. Following accurate identification and characterization of hamster CCK-8, we have developed a highly specific and sensitive immunoprecipitation based LC-MS/MS assay for its quantification. The present assay enables determination of active CCK-8 over a concentration range from 0.05 to 2.5 ng/mL in hamster plasma samples. This range covers both the basal and postprandial levels of CCK-8. Method performance validation samples were examined at three concentrations replicated over the course of 4 days. The assay accuracy (percent relative error, % RE) average was 11.3% with a precision (percent coefficient of variation, % CV) of 15.5% over all samples in this 4 day period. Additionally, the method was used to determine increases of endogenous plasma CCK-8 in hamsters challenged with a high-fat meal.

To determine whether flavonoid-rich wine grape seed flour improves insulin sensitivity and glucose metabolism in obese mice, diet-induced obese (DIO) mice were fed a HF diet supplemented with either 10% partially defatted Chardonnay grape seed flours (ChrSd) or microcrystalline cellulose (MCC, control) for 4 weeks. The 2-h insulin and glucose areas under the curve were lowered by ChrSd indicating that ChrSd enhanced insulin sensitivity and improved glucose metabolism. ChrSd intake also reduced body weight gain, liver and adipose tissue weight, and plasma total and LDL-cholesterol concentration despite a significant increase in food intake. Exon microarray analysis of hepatic gene expression revealed down-regulation of genes related to gluconeogenesis, inflammation, triglyceride synthesis, and polyunsaturated fatty acid elongation, and up-regulation of genes related to fatty acid oxidation, cholesterol and bile acid synthesis. The expression of hepatic leptin receptor was up-regulated by the ChrSd diet sup...

The phytoene desaturase gene from Rhodobacter capsulatus was expressed in Escherichia coli and the resulting protein was purified. The purification steps involved were ammonium sulfate precipitation and ion exchange chromatography, leading to a homogenous protein of 57 kDa with high specific enzymatic activity. The purified enzyme was characterized with respect to substrate specificity and product formation. In addition to phytoene, the intermediates, phytofluene and zeta-carotene, were both converted to neurosporene, the end product of the reaction. Furthermore, 1,2-epoxy phytoene was a suitable substrate whereas the C30 diapophytoene was not. The Km values for phytoene and zeta-carotene were determined to be 33.3 and 16.6 microM, respectively. The desaturation reaction is dependent on the cofactor FAD. Oxidized nicotine nucleotides or ATP had no positive effect. The Km value for FAD was 4.9 microM. Inhibition of the desaturation reaction was observed with diphenylamine.

To identify differentially expressed hepatic genes contributing to the improvement of high-fat (HF) diet-induced hepatic steatosis and insulin resistance following supplementation of partially defatted flavonoid-rich Chardonnay grape seed flour (ChrSd), diet-induced obese (DIO) mice were fed HF diets containing either ChrSd or microcrystalline cellulose (MCC, control) for 5 weeks. The 2-h insulin area under the curve was significantly lowered by ChrSd, indicating that ChrSd improved insulin sensitivity. ChrSd intake also significantly reduced body weight gain, liver and adipose tissue weight, hepatic lipid content, and plasma low-density lipoprotein (LDL)-cholesterol, despite a significant increase in food intake. Exon microarray analysis of hepatic gene expression revealed down-regulation of genes related to triglyceride and ceramide synthesis, immune response, oxidative stress, and inflammation and upregulation of genes related to fatty acid oxidation, cholesterol, and bile acid synthesis. In conclusion, the effects of ChrSd supplementation in a HF diet on weight gain, insulin resistance, and progression of hepatic steatosis in DIO mice were associated with modulation of hepatic genes related to oxidative stress, inflammation, ceramide synthesis, and lipid and cholesterol metabolism.

Consumption of diets high in soluble dietary fibers has been suggested to induce biomarkers for anti-obesity and anti-inflammation, and improve insulin resistance. To investigate the effects of hyd...

The enzyme phytoene synthase (Psy) catalyzes the formation of phytoene, an intermediate in the carotenoid biosynthesis pathway. Expression of a previously described gene (PSY1) is induced by fruit ripening in tomato (Lycopersicon esculentum). We describe here the cloning of a partial cDNA for PSY2, a gene related to PSY1. A plasmid containing the PSY2 coding region under control of a bacterial promoter complements a Rhodobacter capsulatus phytoene synthase mutant, indicating that this gene has the capacity to encode an active enzyme. We used reverse transcriptase-polymerase chain reaction followed by digestion with a restriction enzyme to determine that both PSY1 and PSY2 are expressed during tomato development. PSY1 transcripts predominate in seedlings and in late stages of fruit ripening, whereas PSY2 transcripts are relatively more abundant in mature leaves. Both genes are expressed under photooxidative conditions induced by treatment with the carotenoid biosynthesis inhibitor Norflurazon. We used polymerase chain reaction-based restriction fragment length polymorphisms and alien addition lines to map PSY2 to chromosome 2. We conclude that PSY2 is a second tomato gene encoding phytoene synthase.

Carotenoids are photoprotective and colorful pigments necessary for the survival of all photosynthetic organisms. They are vital for the protection of photosynthetic complexes and membranes from reactive oxygen species. Carotenoids also cause the red, yellow, and orange coloration in flowers and vegetables to attract pollinators and vectors for distribution of seeds. The desaturation of carotenoids is essential to impart these photochemical properties to the molecule by increasing the length of the chromophore. This review discusses carotenoid desaturation from a molecular biological perspective. With the cloning of the carotenoid isomerase, most but not all the genes involved are now in the hands of researchers. This review will give the reader knowledge of the overall reaction and up-to-date information concerning new developments in carotenoid desaturation. Keywords: ζζ-carotene; ζζ-carotene desaturase; carotenoid biosynthesis; carotenoid desaturation; carotenoid isomerase; CRTISO; 3,4-didehydrolycopene; HPPDase; hydroxyneurosporene desaturase; hydroxyphenylpyruvate dioxygenase; immutans; neurosporene; lycopene; PDS; phytoene; phytoene desaturase; phytoene synthase; phytofluene; plastid terminal oxidase; prolycopene; PSY; tetra-cis-lycopene; 3,4,3′,4′-tetradehydrolycopene; ZDS; Z-ISO

This study analyzed plasma lipid profiles, genes related to cholesterol and bile acid metabolism, and inflammation in liver as well as adipose tissue from Syrian Golden hamsters fed high-fat diets supplemented with blueberry (BB) pomace byproducts including 8% dried whole blueberry peels (BBPWHL), 2% dried extract of peels (BBPX; 95% ethanol extract), and 6% residue from extracted peel (BBPEXT) compared to a diet containing 5% (w/w) microcrystalline cellulose (control). All BB diets significantly lowered plasma very low density lipoprotein cholesterol and total cholesterol concentrations. Interestingly, BB diets increased fecal lipid excretion. Hepatic CYP7A1 expression was up-regulated by all BB diets, and the expression of CYP51 was up-regulated by BBPX and BBPEXT diets, suggesting that both bile acid and cholesterol synthesis were increased. No significant changes in adipocyte gene expression related to inflammatory markers were observed with any BB diet. These data suggest that hepatic modulation of bile acid and cholesterol synthesis primarily contributes to the cholesterol-lowering effect of BB pomace byproducts.

In the initial stages of carotenoid biosynthesis in plants the enzyme phytoene synthase converts two molecules of geranylgeranyl diphosphate into phytoene, the first carotenoid of the pathway. We show here that a tomato (Lycopersicon esculentum) cDNA for a gene (Psy1) expressed during fruit ripening directs the in vitro synthesis of a 47-kDa protein which, upon import into isolated chloroplasts, is processed to a mature 42-kDa form. The imported protein is largely associated with membranes, but it can be easily solubilized by dilution or by treatment at high pH. A plasmid construct containing prokaryotic promoter and ribosome-binding sequences fused to the Psy1 cDNA complements the carotenoidless phenotype of a Rhodobacter capsulatus crtB mutant. We conclude that Psy1 encodes phytoene synthase and that this enzyme is a peripheral plastid membrane protein.

Carotenoids are orange, yellow, or red photo-protective pigments present in all plastids. The first carotenoid of the pathway is phytoene, a colorless compound that is converted into colored carotenoids through a series of desaturation reactions. Genes coding for carotenoid desaturases have been cloned from microbes but not from plants. We report the cloning of a cDNA for pds1, a soybean (Glycine max) gene that, based on a complementation assay using the photosynthetic bacterium Rhodobacter capsulatus, codes for an enzyme that catalyzes the two desaturation reactions that convert phytoene into zeta-carotene, a yellow carotenoid. The 2281-base-pair cDNA clone analyzed contains an open reading frame with the capacity to code for a 572-residue protein of predicted Mr 63,851. Alignment of the deduced Pds1 peptide sequence with the sequences of fungal and bacterial carotenoid desaturases revealed conservation of several amino acid residues, including a dinucleotide-binding motif that could mediate binding to FAD. The Pds1 protein is synthesized in vitro as a precursor that, upon import into isolated chloroplasts, is processed to a smaller mature form. Hybridization of the pds1 cDNA to genomic blots indicated that this gene is a member of a low-copy-number gene family. One of these loci was genetically mapped using restriction fragment length polymorphisms between Glycine max and Glycine soja. We conclude that pds1 is a nuclear gene encoding a phytoene desaturase enzyme that, as its microbial counterparts, contains sequence motifs characteristic of flavoproteins.

We have expressed in Escerichia coli the enzymes geranylgeranyl diphosphate synthase and phytoene synthase, from the soil bacterium Erwinia stewartii, and the two carotene desaturases phytoene desaturase and carotene zeta-carotene desaturase from Arabidopsis thaliana. We show that pro-lycopene (7,9,7',9'-tetra-cis)-lycopene is the main end product of the plant desaturation pathway in these cells. In addition, light is required in this system. Whereas in the dark mainly zeta-carotene, the phytoene desaturase product, accumulates, illumination leads to activation of this intermediate caused by its photoisomerization. zeta-Carotene then meets the stereospecific requirements of zeta-carotene desaturase and pro-lycopene is formed. In contrast, a strain of E. coli carrying geranylgeranyl diphosphate synthase, phytoene desaturase and the bacterial carotene desaturase CrtI, which mediates lycopene formation from phytoene, does not require light, nor is a poly-cis-lycopene species formed. The stereoselectivity of the plant-type desaturation pathway expressed in E. coli is the same as previously shown with chromoplast membranes. As the phytoene desaturase and zeta-carotene desaturase used originate from a system not capable of developing chromoplasts, this indicates that the poly-cis pathway of carotene desaturation may have a wider occurrence than initially believed.

Publisher Summary This chapter discusses methods for the inducible expression of carotenoid biosynthesis genes in Rhodobacter capsulatus and Escherichia coli along with novel procedures for the purification of phytoene desaturases. Carotenoid desaturases are membrane-bound enzymes in both bacteria and plants. The functional expression of Neurospora crassa and Glycine max (soybean) cDNAs coding for carotenoid desaturases in mutants of the photosynthetic bacterium Rhodobacter capsulatus have been achieved. Thus, it is logical to expect functional expression in R. capsulatus of carotenoid desaturase cDNAs from other species. In addition, it is possible that cDNAs coding for carotenoid enzymes other than desaturases could be expressed in R. capsulatus carotenoid mutants. Phytoene desaturases from various organisms catalyze a different number of desaturations. Rhodobacter capsulatus phytoene desaturase converts phytoene to neurosporene, a three-step desaturation reaction. Neurospora crassa Al-1 catalyzes six desaturations. Soybean Pds1 converts phytoene to ζ-carotene, a two-step desaturation reaction. Expression of al-1 in a phytoene-accumulating strain of R. capsulatus leads to the accumulations of lycopene and 3,4 dehydrolycopene. Expression of pds in this bacterial strain results in the accumulation of ζ-carotene. Lycopene, 3,4-dehydrolycopene, and ζ-carotene are not normally accumulated in R. capsulatus.

Photosynthetic bacteria are currently used as model systems for the study of genetic, biochemical and physical aspects of photosynthesis. In this article we briefly review genetic methods that we developed for use in Rhodobacter capsulatus and we present recent results on the molecular biology of carotenoid biosynthesis.

Carotenoids are terpenoid pigments which are accumulated in the chloroplasts of leaves and in the chromoplasts of many flowers and fruits. Phytoene desaturase (Pds), the second dedicated enzyme in carotenoid biosynthesis, is encoded in tomato by a single copy gene. A 2 kb fragment from the tomato Pds gene, comprising 1.5 kb from the promoter and 0.5 kb from the 5' non-translated region, is able to drive developmentally regulated expression of the GUS reporter gene in transgenic tomato and tobacco plants. In tomato, high levels of Pds/GUS expression are found in organs and at stages of development where chromoplasts are formed: petals, anthers and ripening fruits. Tobacco petals and fruits, which do not contain chromoplasts, show instead low levels of Pds/GUS expression. Transgenic tobacco seedlings were subjected to treatment with a range of inhibitors of carotenoid and chlorophyll biosynthesis. The results indicate that, in green tissues, carotenoid and chlorophyll levels are tightly co-regulated and that a chemically induced arrest in pigment biosynthesis results in activation of the Pds promoter. The promoter is also induced in etiolated seedlings, which contain much lower carotenoid levels than light-grown seedlings. These data suggest that in green tissues Pds gene transcription may respond to end-product regulation.