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The potential for apple peels to mitigate the deleterious effects of a high-fat diet in mice was investigated here. Mice were fed a high-fat diet supplemented with apple powders from three apple varieties or a commercial apple polyphenol. Polyphenols were characterized using colorimetric assays and high-performance liquid chromatography. Mice were tested for standard metabolic parameters. There was a dose response to dietary apple peels, with the higher intake leading to reduced weight gain and adipose tissue mass relative to the lower intake, but none of the treatments were statistically different from the control. The gene expression of liver enzyme stearoyl-CoA desaturase (Scd-1) was correlated with adipose weight, and liver enzyme cytochrome P51 (Cyp51) was downregulated by the apple diets. The feces from a subset of mice were analyzed for polyphenols and for bacteria taxa by next-generation sequencing. The results revealed that the makeup of the fecal microbiota was related to the metabolism of dietary polyphenols.

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.

The relationship between the intestinal microbiota and the hypocholesterolemic and antiobesity effects of whole grape seed flour from white and red winemaking was evaluated. Male Golden Syrian hamsters were fed a high-fat (HF) control diet or a HF diet supplemented with 10% partially defatted grape seed flours from either Chardonnay (ChrSd) or Cabernet Sauvignon (CabSd) grapes for 3 weeks. The numbers of total bacteria and relative abundances of Bifidobacterium spp., Lactobacillus spp., and Firmicutes in feces were significantly lower, while the relative abundance of Bacteroides fragilis was greater than the control from feeding the ChrSd diet. The ratio of Firmicutes/Bacteroidetes (F/B) was lower in the ChrSd diet. There were significantly positive correlations between Lactobacillus spp., ratio of F/B, and plasma total- and LDL-cholesterol and liver weight. The reduction of Lactobacillus spp. by the ChrSd diet was accompanied by inhibition of Farnesoid X receptor (FXR) signaling in the intestine as expression of intestinal fibrablast growth factor (FGF)15, positively regulated by FXR, was decreased. Expression of CYP7A1, negatively regulated by FGF15, was up-regulated in the liver, which indicates that alteration of the intestinal microbiota may regulate bile acid and lipid metabolism. These findings suggest that beneficial health effects of Chardonnay grape seed flour on HF-induced metabolic disease relate in part to modulation of intestinal microbiota and their metabolic processes.

Tomato (Solanum lycopersicum, formerly Lycopersicon esculentum) cv. VFNT Cherry sepals, when cultured in vitro between 16 degrees C and 22 degrees C, change their genetic programme to that of ripening fruit. Previously regulation of a number of transcription factors and a putative G-protein-coupled receptor that may be involved in tomato fruit ripening and cool-temperature sepal morphogenesis had been revealed. Many of those genes such as TAG1, TM4, TM6, AP2-like (LeAP2FR), YABBY2-like (LeYAB2), and LeCOR413-PM1 have not been investigated for ethylene regulation. Ethylene-independent, regulated transcripts may be part of an early signalling process induced or de-repressed by cool temperature that causes a switch in the genetic programme of the sepals. In this paper, ethylene regulation of a number of these and other putative signalling factors are investigated during cool-temperature-induced sepal morphogenesis. 1-Methylcyclopropene was used to block ethylene-induced gene expression by interrupting the ethylene signal transduction pathway that occurs in ripening tomato fruits and presumably in ripening sepals. Transcripts of several putative transcription factors previously shown to be up-regulated during cool-temperature-induced sepal morphogenesis (TAG1, TM4, LeAP2FR) were only slightly or not induced in 1-methylcyclopropene-treated sepals, indicating either direct or indirect ethylene regulation. Two genes, VAHOX1, a homeobox domain leucine-zipper-encoding gene, and LeYAB2, a putative zinc-finger transcription factor-encoding gene, increased in treated and untreated sepals indicating regulation by cool temperatures independently of ethylene.

Many sequences of genes now are contained in databases available to the public. These databases will be the foundation and starting points of research in the future. Extracting information from these databases is useful to all forms of genetic, biochemical, and molecular biological research. We are interested in improving the nutrition of the tomato (Lycopersicon esculentum) through an understanding of the fundamentals of the molecular biological and biochemical processes that occur during tomato fruit development. The TIGR Tomato Gene Index has been of much use to our laboratory in determining new areas of molecular analysis for this research. We have examined this database for genes involved in ripening by using the nucleic acid and protein sequence searching software, BLAST, and by comparing gene expression information contained within the database. In this article, we report a number of previously unobserved genes that possibly are involved in tomato fruit development. Transcripts for MADS Box genes, zinc-finger genes, homeobox genes, and polycomb genes are among the transcripts that reveal upregulation during tomato fruit ripening. We believe that the information contained in this article will be valuable in considering approaches to analyzing the developmental processes involved in tomato fruit ripening.

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 mechanisms for the hypocholesterolemic and antiobesity effects of grape seed flours derived from white and red winemaking processing were investigated using male Golden Syrian hamsters fed high-fat (HF) diets supplemented with 10% partially defatted grape seed flours from Chardonnay (ChrSd), Cabernet Sauvignon (CabSd), or Syrah (SyrSd) pomace as compared to a HF control diet for 3 weeks. Hamsters fed the ChrSd diet had significantly lowered plasma total-, VLDL-, and LDL-cholesterol concentrations compared to the CabSd, SyrSd, and control diets. The improved plasma cholesterol after ChrSd was correlated with the up-regulation of hepatic genes related to cholesterol (CYP51) and bile acid (CYP7A1) synthesis as well as LDL-cholesterol uptake (LDLR). A reduction of hepatic lipid content was associated with altered expression of the genes related to lipid metabolism. However, fecal total lipid content was not changed. Expression of ileal apical sodium bile acid transporter (ASBT) was not affected by ChrSd, indicating unchanged ileal bile acid reabsorption. The antiobesity effect of the ChrSd diet appears to be related to expression of adipogenesis- and inflammation-related genes in adipose tissue. These findings suggest that flavonoid-rich Chardonnay grape seed flour induced cholesterol-lowering, antiobesity, and anti-inflammatory health benefits and attenuation of hepatic steatosis via regulation of gene expression related to cholesterol, bile acid, and lipid metabolism in liver and adipose tissue.

Plant carotenoids are red, orange, and yellow lipid-soluble pigments found embedded in the membranes of chloroplasts and chromoplasts. Their color is masked by chlorophyll in photosynthetic tissues, but in late stages of plant development these pigments contribute to the bright colors of many flowers and fruits and the carrot root. Carotenoids protect photosynthetic organisms against potentially harmful photooxidative processes and are essential structural components of the photosynthetic antenna and reaction center complexes. In plants, some of these compounds are precursors of abscisic acid (ABA), a phytohormone that modulates developmental and stress processes (Koornneef, 1986). Carotenoids with provitamin A activity are essential components of the human diet, and there is considerable evidence that many carotenoids have anti-cancer activity. Carotenoids absorbed through the diet, and often metabolized into other compounds, are responsible for the color of familiar animals such as lobster, flamingo, and fish. Often unaware of the chemical nature of food colorants, humans consume large amounts of carotenoids added to foods primarily to please the eye (Klaui and Bauernfeind, 1981). As with other natural pigments, carotenoids attracted the attention of 19th-century organic chemists. BCarotene was isolated in 1817, and the pigments of autumn leaves were identified as xanthophylls in 1837 (Isler, 1971). Classical geneticists identified carotenoid mutants in maize, tomato, and Arabidopsis during the early part of this century. However, difficulties with the biochemical properties of the enzymes of the pathway and the generally poor viability of mutants affected in carotenoid biosynthesis during early stages of development conspired to delay development of the field. Genetic studies of carotenoid biosynthesis in bacteria (reviewed in Armstrong, 1994) have played a fundamental role in the cloning and characterization of plant carotenoid genes. Since 1991, when a bacterial probe was first used to clone a plant gene for an enzyme of the pathway, progress has been rapid. However, much remains to be learned about the environmental and developmental regulation of the pathway, and many classic mutants have not yet been characterized at the molecular level. Recent reviews (Armstrong, 1994; Bartley et al., 1994; Sandmann, 1994) and two books on methods (Packer, 1992a

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.

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.

Leaves of tomato (Lycopersicon esculentum) plants grown in soil in which moisture was lowered from field capacity to levels approaching permanent wilting point show a 10-fold increase in abscisic acid (ABA) and a 60 to 70 percent decrease in rbcS and cab steady-state mRNA levels. As indicated by transcription run-on experiments, the effect occurs primarily at the transcriptional level. Similar water deficit had only a minor effect on ABA level and on rbcS and cab expression in leaves of sitiens, an ABA mutant of tomato. Expression of rbcL, the chloroplast gene coding for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, is not affected by water stress. Application of exogenous ABA results in decreased rbcS and cab expression in both wild-type and sitiens leaves. Analysis of the expression of individual members of the rbcS gene family indicates that under water-deficit conditions, expression derives primarily from only three of the five rbcS genes. Effects of dark adaptation and water deficit are additive for cab but not for rbcS expression. These results support the hypothesis that, at least under water-deficit conditions, ABA or a derivative thereof mediates a negative regulation of rbcS and cab transcription in tomato plants.

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.

Red ripe tomatoes are the result of numerous physiological changes controlled by hormonal and developmental signals, causing maturation or differentiation of various fruit tissues simultaneously. These physiological changes affect visual, textural, flavor, and aroma characteristics, making the fruit more appealing to potential consumers for seed dispersal. Developmental regulation of tomato fruit ripening has, until recently, been lacking in rigorous investigation. We previously indicated the presence of up-regulated transcription factors in ripening tomato fruit by data mining in TIGR Tomato Gene Index. In our in-vitro system, green tomato sepals cultured at 16 to 22°C turn red and swell like ripening tomato fruit while those at 28°C remain green. Here, we have further examined regulation of putative developmental genes possibly involved in tomato fruit ripening and development. Using molecular biological methods, we have determined the relative abundance of various transcripts of genes during in vitro sepal ripening and in tomato fruit pericarp at three stages of development. A number of transcripts show similar expression in fruits to RIN and PSY1, ripening-associated genes, and others show quite different expression. Our investigation has resulted in confirmation of some of our previous database mining results and has revealed differences in gene expression that may be important for tomato cultivar variation. We present new and intriguing information on genes that should now be studied in a more focused fashion.

Phytoene synthase (Psy) and phytoene desaturase (Pds) are the first dedicated enzymes of the plant carotenoid biosynthesis pathway. We report here the organ-specific and temporal expression of PDS and PSY in tomato plants. Light increases the carotenoid content of seedlings but has little effect on PDS and PSY expression. Expression of both genes is induced in seedlings of the phytoene-accumulating mutant ghost and in wild-type seedlings treated with the Pds inhibitor norflurazon. Roots, which contain the lowest levels of carotenoids in the plant, have also the lowest levels of PDS and PSY expression. In flowers, expression of both genes and carotenoid content are higher in petals and anthers than in sepals and carpels. During flower development, expression of both PDS and PSY increases more than 10-fold immediately before anthesis. During fruit development, PSY expression increases more than 20-fold, but PDS expression increases less than threefold. We concluded that PSY and PDS are differentially regulated by stress and developmental mechanisms that control carotenoid biosynthesis in leaves, flowers, and fruits. We also report that PDS maps to chromosome 3, and thus it does not correspond to the GHOST locus, which maps to chromosome 11.

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.