The biosynthesis and nutritional uses of carotenoids.

The phytochemical resveratrol, which is found in grapes and wine, has been reported to have a variety of anti-inflammatory, anti-platelet, and anti-carcinogenic effects. Based on its structural similarity to diethylstilbestrol, a synthetic estrogen, we examined whether resveratrol might be a phytoestrogen. At concentrations (≈3–10 μM) comparable to those required for its other biological effects, resveratrol inhibited the binding of labeled estradiol to the estrogen receptor and it activated transcription of estrogen-responsive reporter genes transfected into human breast cancer cells. This transcriptional activation was estrogen receptor-dependent, required an estrogen response element in the reporter gene, and was inhibited by specific estrogen antagonists. In some cell types (e.g., MCF-7 cells), resveratrol functioned as a superagonist (i.e., produced a greater maximal transcriptional response than estradiol) whereas in others it produced activation equal to or less than that of estradiol. Resveratrol also increased the expression of native estrogen-regulated genes, and it stimulated the proliferation of estrogen-dependent T47D breast cancer cells. We conclude that resveratrol is a phytoestrogen and that it exhibits variable degrees of estrogen receptor agonism in different test systems. The estrogenic actions of resveratrol broaden the spectrum of its biological actions and may be relevant to the reported cardiovascular benefits of drinking wine.

https://www.pnas.org/content/pnas/94/25/14138.full.pdf

Resveratrol, a polyphenolic compound found in grapes and wine, is an agonist for the estrogen receptor

Production of plant secondary metabolites: a historical perspective

As an unfortunate consequence of aerobic life, active oxygen species (AOS) are formed by partial reduction of molecular oxygen. Plants possess a complex battery of enzymatic and non-enzymatic antioxidants that can protect cells from oxidative damage by scavenging AOS. It is becoming evident that AOS, which are generated during pathogen attack and abiotic stress situations, are recognized by plants as a signal for triggering defence responses. An overview of the literature is presented on the signalling role of AOS in plant defence responses, cell death, and development. Special attention is given to AOS and redox-regulated gene expression and the role of kinases and phosphatases in redox signal transduction.

Signal transduction during oxidative stress

Resveratrol (trans-3,4',5-trihydroxystilbene), a polyphenolic phytoalexin found in grapes, fruits, and root extracts of the weed Polygonum cuspidatum, exhibits anti-inflammatory, cell growth-modulatory, and anticarcinogenic effects. How this chemical produces these effects is not known, but it may work by suppressing NF-kappaB, a nuclear transcription factor that regulates the expression of various genes involved in inflammation, cytoprotection, and carcinogenesis. In this study, we investigated the effect of resveratrol on NF-kappaB activation induced by various inflammatory agents. Resveratrol blocked TNF-induced activation of NF-kappaB in a dose- and time-dependent manner. Resveratrol also suppressed TNF-induced phosphorylation and nuclear translocation of the p65 subunit of NF-kappaB, and NF-kappaB-dependent reporter gene transcription. Suppression of TNF-induced NF-kappaB activation by resveratrol was not restricted to myeloid cells (U-937); it was also observed in lymphoid (Jurkat) and epithelial (HeLa and H4) cells. Resveratrol also blocked NF-kappaB activation induced by PMA, LPS, H2O2, okadaic acid, and ceramide. The suppression of NF-kappaB coincided with suppression of AP-1. Resveratrol also inhibited the TNF-induced activation of mitogen-activated protein kinase kinase and c-Jun N-terminal kinase and abrogated TNF-induced cytotoxicity and caspase activation. Both reactive oxygen intermediate generation and lipid peroxidation induced by TNF were suppressed by resveratrol. Resveratrol's anticarcinogenic, anti-inflammatory, and growth-modulatory effects may thus be partially ascribed to the inhibition of activation of NF-kappaB and AP-1 and the associated kinases.

Resveratrol Suppresses TNF-Induced Activation of Nuclear Transcription Factors NF-κB, Activator Protein-1, and Apoptosis: Potential Role of Reactive Oxygen Intermediates and Lipid Peroxidation

A gene from groundnut (Arachis hypogaea) coding for stilbene synthase was transferred together with a chimaeric kanamycin resistance gene. It was found to be rapidly expressed after induction with UV light and elicitor in tobacco cells (Nicotiana tabacum). Comparative studies of stilbene synthase mRNA synthesis in groundnut and transgenic tobacco suspension cultures revealed the same kinetics of gene expression. Stilbene synthase specific mRNA was detectable 30 minutes after elicitor induction and 10 minutes after UV irradiation. The maximum of mRNA accumulation was between 2 and 8 hours post induction. 24 hours after induction stilbene synthase mRNA accumulation ceased. Furthermore, in transgenic tobacco plants, the gene was found to be inducible in sterile roots, stems and leaves. Stilbene synthase was demonstrated in crude protein extracts from transgenic tobacco cell cultures using specific antibodies. Resveratrol, the product of stilbene synthase, was identified by HPLC and antisera raised against resveratrol.

Expression of a stilbene synthase gene in Nicotiana tabacum results in synthesis of the phytoalexin resveratrol.

We developed a new concept of controlling plant virus infection based on the expression and secretion of full-size antibodies in plants. The neotope-specific anti-Tobacco Mosaic Virus (anti-TMV) antibody mAb24 has a high affinity towards epitopes present only on the surface of intact tobacco mosaic virions. The infectivity of the virus is inhibited almost completely if TMV is adsorbedin vitro at ratios as low as 300 antibody molecules per virion prior to inoculation. Cloned full-size cDNAs of mAb24 heavy and light chains were integrated into the plant expression vector pSS in tandem array and used for transformation ofNicotiana tabacum. The resulting transgenic tobacco plants expressed heavy- and light-chains of mAb24 which were assembled into functional antibodies and exported to the intercellular space. TMV specificity and affinity of the plant-produced antibody (mAb24-P) was not altered when compared to the original murine mAb24. F1 progenies segregated 3:1 with respect to antibody secretion and showed up to two-fold higher expression levels compared to the F0 plants. Upon infection with TMV F1 plants producing mAb24-P showed a reduction of necrotic lesion numbers which is correlated with the amount of antibody produced in transgenic plants.

Reduced virus infectivity in N. tabacum secreting a TMV-specific full-size antibody.

Resveratrol is a major phytoalexin in grapevine but its synthesis in response to phytopathogen attack decreases with grape berry ripening. A chimeric gene combining an alfalfa PR 10 promoter and Vst1 (Vitis stilbene synthase 1) gene was introduced into the genome of 41B rootstock. Transgenic plants were analysed for resveratrol production in leaves infected with Botrytis using an in vitro test. Among the 50 transgenic lines analysed, some exhibited a production lower than the non-transgenic control, but others accumulated resveratrol from 5-100-fold. Moreover, in the latter clones, symptoms were highly reduced in response to infection. These results were a good indication that the combination of a pathogen-inducible promoter and a defence gene may increase tolerance against fungi in grapevine. The efficacy of this approach should be further tested by experiments conducted in the vineyard.

/pdf/in-vitro-tolerance-to-botrytis-cinerea-of-grapevine-41b-2u0wongk4w.pdf

In vitro tolerance to Botrytis cinerea of grapevine 41B rootstock in transgenic plants expressing the stilbene synthase Vst1 gene under the control of a pathogen‐inducible PR 10 promoter

A stilbene synthase gene. More particularly, a stilbene synthase gene in which the stilbene synthase is resveratrol synthase. Also more particularly a stilbene synthase gene which is obtained from groundnut (Arachis hypogaea). Furthermore a stilbene synthase gene, corresponding to the stilbene synthase gene which is contained in the plasmid pGS 828.1, and DNA sequences acting in essentially the same say. Still further, a stilbene synthase gene unit which comprises approximately 6,700 base pairs and exhibits 3 EcoRI, 3 HindIII and 1 PstI cleavage sites, and which can be obtained by partial cleavage of the plasmid PGS 828.1 using EcoRI and HindIII, or from plasmid pGS 828.1 using SstI and PvuII.

Stilbene synthase gene

Summary
Stilbene synthase genes (STS) have previously been used to enhance disease resistance in plants. In order to study the effects of modified STS expression patterns in plants, heterologous promoters were fused to an STS gene and the chimeric genes were transferred to tobacco. Very high constitutive expression of STS mediated by a duplicated upstream region of the 35S RNA promoter from CaMV affected secondary biosynthetic pathways. STS over-expression caused altered flower pigmentation and male sterility, probably due to competition between the introduced STS and the endogenous chalcone synthase for the substrates 4-coumaroyl CoA and malonyl CoA. Furthermore, tobacco plants with tapetum-specific STS expression were male-sterile. Thus, STS genes are promising tools in strategies for engineering altered flower colours and the development of a novel hybrid seed system.

Rüdiger Hain

Papers

Expression of a stilbene synthase gene in Nicotiana tabacum results in synthesis of the phytoalexin resveratrol.

Reduced virus infectivity in N. tabacum secreting a TMV-specific full-size antibody.

In vitro tolerance to Botrytis cinerea of grapevine 41B rootstock in transgenic plants expressing the stilbene synthase Vst1 gene under the control of a pathogen‐inducible PR 10 promoter

Stilbene synthase gene

Stilbene synthase gene expression causes changes in flower colour and male sterility in tobacco