Stilbene Synthase Expression Research Articles

Effects of Water Stress on Resveratrol Accumulation and Synthesis in ‘Cabernet Sauvignon’ Grape Berries

Resveratrol (3, 4′, 5 trihydroxy stilbene) is a natural phytoalexin produced by plants in response to biotic and abiotic stresses. It is well known for its cardio-protective, anticarcinogenic, and antioxidant properties. This study characterized physiological and molecular changes in resveratrol synthesis exposed to two levels of water stress at distinct grape berry developmental stages. Physiological data were measured to assess the berry quality. We used high-performance liquid chromatography to study enzyme activity and qRT-PCR to assess the expression levels of genes involved in resveratrol synthesis. The berry development was suppressed under water stress, while the content of total polyphenol, especially resveratrol was enhanced. Related enzymes and genes regulate the changes in resveratrol in plants. Water stress improved the enzyme activities of PAL (phenylalanine ammonia-lyase) and STS (stilbene synthase) after veraison. Moreover, the transcription factors VvMYB14, VvMYB15 and resveratrol synthesis-related genes are also changed. Expression of Vv4CL and VvMYB15 were upregulated during the pre-reversion stage, whereas VvPAL and VvSTS increased throughout development. As the co-substrate of VvSTS, VvCHS decreased during the berry development. Our study demonstrates that water stress regulates resveratrol synthesis through enzymatic activities and the gene expression of PAL and STS.

Metabolic Engineering Strategy Enables a Hundred-Fold Increase in Viniferin Levels in Vitis vinifera cv. Gamay Red Cell Culture.

Stilbenes are phytoalexins with health-promoting benefits for humans. Here, we boost stilbenes' production, and in particular the resveratrol dehydrodimer viniferin, with significant pharmacological properties, by overexpressing stilbene synthase (STS) under unlimited phenylalanine (Phe) supply. Vitis vinifera cell cultures were co-transformed with a feedback-insensitive E. coli DAHP synthase (AroG*) and STS genes, under constitutive promoters. All transgenic lines had increased levels of Phe and stilbenes (74-fold higher viniferin reaching 0.74 mg/g DW). External Phe feeding of AroG* + STS lines caused a synergistic effect on resveratrol and viniferin accumulation, achieving a 26-fold (1.33 mg/g DW) increase in resveratrol and a 620-fold increase (6.2 mg/g DW) in viniferin, which to date is the highest viniferin accumulation reported in plant cultures. We suggest that this strategy of combining higher Phe availability and STS expression generates grape cell cultures as potential factories for sustainable production of stilbenes with a minor effect on the levels of flavonoids.

The comparative analysis of stilbene production in cell cultures of two East Asian grape species Vitis amurensis Rupr. and Vitis coignetiae Pulliat

Two East Asian grape species Vitis amurensis and Vitis coignetiae have been investigated for their increased cold and drought tolerance and high level of resistance to infections. Nowadays, the grape species attract attention due to their capability to produce a group of valuable plant secondary metabolites named stilbenes. The basic stilbene, t-resveratrol, is known to demonstrate a high bioactivity and is synthesized by stilbene synthase (STS) in grape cells. In order to find the suitable biological model for t-resveratrol production, 12 cell culture lines derived from the leaf, petiole and stem explants of the East Asian grape species were tested for their ability to produce t-resveratrol and other stilbenes. The comparative analysis showed that the V. coignetiae cell culture lines were the most productive in terms of stilbene content. This observation was confirmed by the increased level of STS expression in V. coignetiae cells in comparison to V. amurensis. The expression analysis of the genes encoding transcription factors potentially involved in regulation of STS genes revealed the significant species-specific differences, suggesting the molecular basis underlying the observed differences in stilbene biosynthetic potential of V. amurensis and V. coignetiae. The comparative analysis revealed the greater stilbene production potential for cells of V. coignetiae due to enhanced STS expression determinated by Myb1/15, Myb40 and Myb60 transcription factors.

The effect ofSaccharomyces cerevisiaeon antioxidant system of grape leaves infected by downy mildew

In this article, results of the comparative analysis and assessment of resistance of two grape cultivars Vostorg and Muscat blanc against downy mildew disease (Plasmopara viticola) with or without the symbioteSaccharomyces cerevisiae(vine yeast) were shown. The highly resistant cultivar Vostorg with yeast pre-treatment demonstrated a high defensive capability to the pathogen due to the fast immune response. On the first day after inoculation with downy downy mildew the rapid increase in the hydrogen peroxide, which is involved the first step of the grape’s defense system induction, was observed. At the same time, the upregulation of the relative expression of the PR2 protein (β-1,3-gluconase), a key gene involved in the plant’s resistance to pathogens. The oxidative burst was not detected in the susceptible cultivar Muscat blanc for the couple of hours after inoculation with downy mildew pathogen. The significant increase of the total phenols content and expression of stilbene synthase, which is an enzyme involved in the synthesis of phytoalexins, was observed in leaves of Muscat blanc. It was shown that pre-treatment of grape leaves with natural symbioteS. cerevisiaeenhanced the immune response of the resistant cultivar Vostorg and inducted phytoalexins synthesis in the susceptible cultivar Muscat blanc.

Expression of stilbene synthase VqSTS6 from wild Chinese Vitis quinquangularis in grapevine enhances resveratrol production and powdery mildew resistance.

In grape (Vitis), stilbene phytoalexins can either be in situ synthesized or transported to the site of response during powdery mildew infection, enhancing disease resistance. Resveratrol is a phytoprotective stilbenoid compound that is synthesized by stilbene synthase (STS) in response to biotic and abiotic stresses, and is also known to have health benefits in the human diet. We have previously shown that transgenic Vitis vinifera cv. Thompson Seedless plants overexpressing a stilbene synthase gene, VqSTS6, from wild Chinese Vitis quinquangularis had a higher stilbenoid content, leading to an enhanced resistance to powdery mildew (Uncinula necator (Schw.) Burr). However, the biosynthesis and transportation in the plant tissue under powdery mildew infection are still unclear. Here, inhibitor and micro-grafting technologies were used to study the accumulation of resveratrol following powdery mildew infection. We observed that the levels of STS expression and stilbenoids increased in response to powdery mildew infection. Powdery mildew and inhibitor treatment on detached grape branches showed that resveratrol was in situ synthesized. Experiments with grafted plantlets showed that the abundance of stilbenoid compounds increased in the shoot during VqSTS6 overexpression in the root, while VqSTS6-Flag fusion was not tranported to the scions and only expressedin thetransgenic rootstocks. Compared with wild-type Thompson Seedless plants, the non-transgenic/VqSTS6 transgenic (scion/rootstock) grafted Thompson Seedless plantlets exhibited increased resistance to powdery mildew. In addition, overexpression of VqSTS6 in roots led to increased levels of stilbenoid compounds in five other European grape varieties (V. vinifera cvs. Chardonnay, Perlette, Cabernet Sauvignon, Riesling and Muscat Hamburg). In conclusion, stilbenoid compounds can be either in situ synthesized or transported to the site of powdery mildew infection, and overexpression of VqSTS6 in the root promotes stilbenoids accumulation and disease resistance in European grapevine varieties.

Transcription factor VqERF114 regulates stilbene synthesis in Chinese wild Vitis quinquangularis by interacting with VqMYB35.

VqERF114 regulates stilbene synthesis by interacting with VqMYB35. Resveratrol is a stilbene, an important class of secondary metabolites that accumulates in some plant species, including grapevine. In the plant, these are involved in the response to attack by plant pathogens and, as a component of the human diet, they offer a range of significant health benefits. Stilbene synthase (STS), the key enzyme responsible for resveratrol synthesis, has been characterised in a small number of plant species. However, the regulatory mechanisms for stilbene synthesis are uncertain. Here, an ERF family transcription factor from Chinese wild Vitis quinquangularis, VqERF114, was characterised as an indirect regulator of stilbene synthesis. A transient overexpression assay of VqERF114 in grapevine leaves led to increased STS expression and stilbene accumulation. However, VqERF114 did not bind to the promoters of VqSTSs but the MYB transcription factor, VqMYB35, did interact with VqERF114. This interaction was confirmed by a yeast two-hybrid assay and bimolecular fluorescence complementation. Furthermore, VqMYB35 showed activation effects on the expressions of VqSTS15, VqSTS28, VqSTS42 and VqSTS46 by binding directly to the MBS elements in their promoters. Co-overexpression of VqERF114 and VqMYB35 resulted in higher VqSTSs expression and more stilbene synthesis. These results demonstrate that VqERF114 regulates stilbene synthesis by interacting with VqMYB35.

Study of defense-related gene expression in grapevine infested by Colomerus vitis (Acari: Eriophyidae)

Real-time quantitative polymerase chain reaction was used to study the expression of some marker genes involved in the interaction between grape (Vitis vinifera L.) and the erineum mite Colomerus vitis Pagenstecher (Acari: Eriophyidae). Potted vines of cultivars Atabaki (resistant to C. vitis), Ghalati (susceptible to C. vitis) and Muscat Gordo (moderately resistant to C. vitis) were infested at the six-leaf stage. The expression of protease inhibitor (PIN), beta-1,3-glucanase (GLU), polygalacturonase inhibitor (PGIP), Vitis vinifera proline-rich protein 1 (PRP1), stilbene synthase (STS), and lipoxygenase (LOX) genes was assessed on young leaves collected 96, 120 and 144h after mite infestation (hami). As a control, non-infested leaves collected 24h before mite infestations were used. Differences were detected in expression of the selected genes during the C. vitis-grapevine interaction. The resistant cultivar Atabaki increased the expression of LOX, STS, GLU, PGIP and PRP1 genes during the first 120 hami. On the contrary, in the susceptible Ghalati, all selected genes showed an expression level similar or lower than non-infested leaves. Muscat Gordo increased the expression of all selected genes in comparison with non-infested leaves, but it was lower than in Atabaki. Significant transcript accumulation of PIN gene was detected for Muscat Gordo whereas it was slightly up-regulated in Ghalati and Atabaki. LOX, STS, PIN, GLU, PGIP and PRP1 genes were clearly expressed in response to C. vitis infestation. We therefore infer that expression of PGIP, PIN and PRP1 genes could represent a defense strategy against C. vitis infestations in grapevine leaves.

Combined elicitation of chitosan and ultraviolet C enhanced stilbene production and expression of chitinase and β-1,3-glucanase in Vitis vinifera cell suspension cultures

Cell suspension cultures of Vitis vinifera were treated with chitosan and ultraviolet C (UV-C) irradiation, alone or in combination, to investigate the relationship between the production of stilbene and the transcript profiles of genes encoding the enzymes involved in stilbene synthesis. Treatment at the proper concentration or dosage enhanced both stilbene production inside the cells and trans-resveratrol accumulation in the culture medium without loss of biomass, and the combined treatment was the most efficient. Total intracellular stilbene content was markedly increased by the joint use of chitosan and UV-C, reaching the maximum at 60 h (1945.91 ± 70.73 μg g−1 DW; 207.23 % higher than in untreated cells) after elicitation. When cell cultures were supplemented with both elicitors, we observed a synergistic effect on the accumulation of trans-resveratrol release into the culture medium with the maximal concentration of 3.05 ± 0.17 mg l−1 at 60 h. Total phenolics and total flavonoids contents were also highly increased after elicitations. The expression levels of genes associated with stilbene biosynthesis were significantly up-regulated in response to the two elicitors, and the combined elicitation showed a synergistic effect on the expression of stilbene synthase. Furthermore, the expression levels and enzyme activities of two pathogenesis-related proteins, chitinase and β-1,3-glucanase, increased after treatment. The results suggest that the combined treatment of chitosan and UV-C irradiation can significantly enhance the production of stilbene in V. vinifera cell cultures and may induce defense response by increasing the expression and activity levels of chitinase and β-1,3-glucanase.

Alternations in VaSTS gene cytosine methylation and t-resveratrol production in response to UV-C irradiation in Vitis amurensis Rupr. cells

Ultraviolet (UV) emerging with the sun light plays crucial role in plants ontogenesis as the one of the main environmental stresses. Prolonged exposure to UV causes damage to DNA, proteins and membranes, and the inhibition of protein synthesis and photosynthetic reactions. Moreover, UV radiance is known to induce metabolic modifications in plants, particularly to secondary metabolite biosynthesis. Multitude of the studies published to date considering exposure of grape cells to UV revealed enhancement in stilbene compounds production, and many of which possess unique biological and pharmacological properties. However, the epigenetic mechanisms regulating expression of the genes involved in stilbene biosynthesis in response to UV exposure were not studied. In this study as model objects were used grape Vitis amurensis plant cell cultures capable to produce trans-resveratrol (t-resveratrol). T-resveratrol, a naturally occurring plant phenol, has been reported to exhibit a wide range of valuable biological and pharmacological properties. T-resveratrol synthesized via the phenylpropanoid pathway, where phenylalanine ammonia-lyase (PAL) is the first enzyme in this pathway and stilbene synthase (STS) is the enzyme that directly catalyzes the reaction of t-resveratrol formation. Exposure of V. amurensis cells to UV of C type significantly increased t-resveratrol production and certain STS and PAL expression. Using bisulfite sequencing, we demonstrated that methylation level of the VaSTS2, VaSTS6 and VaSTS10 genes significantly decreased, while methylation level of constitutively expressed VaSTS1 gene did not changed significantly.

Screening Carica papaya native promoters driving stilbene synthase expression in Arabidopsis thaliana for resveratrol glucoside (piceid) synthesis

The use of promoters for downstream metabolic engineering in plants requires efficiently screening their transcriptional strength and the resulting levels of the desired metabolite. Carica papaya is a complex slow-growing tropical tree, which is difficult to rapidly screen for promoter activity. Therefore, we used the simple model system, Arabidopsis thaliana (Columbia), to compare the effectiveness of two native papaya promoters (Cp9 and Cp29) with a positive control (CaMV35S promoter), in driving transcription of the stilbene synthase gene (Vst1) and production of the phytoalexin trans-resveratrol glucoside (piceid). Single transgene copy numbers were verified via Southern analysis of at least three independent transgenic lines for each promoter construct. The Cp29 and control CaMV35S promoters consistently produced high Vst1 mRNA levels, whereas Vst1 mRNA was 50–60 % less in the Cp9:Vst1 lines. The evolutionarily related chalcone synthase (CHS) gene produced 20 and 40 % less mRNAs relative to WT in the Cp29:Vst1 and 35S:Vst1 lines, respectively, but was unchanged in the Cp9:Vst1 lines. The transgenic lines accumulated piceid as identified through RP-HPLC and tandem mass spectrometry. Piceid levels were the highest in the 35S:Vst1 followed by Cp29:Vst1 and Cp9:Vst1 in the studied tissues. No overt deleterious phenotypes were observed in the Cp9:Vst1 and Cp29:Vst1 lines; however, 35S:Vst1 produced smaller plants. The levels of secondary metabolites (anthocyanins) and seed pigments (tannins) decreased in the CaMV35S:Vst1 and Cp29:Vst1 lines, likely due to competition between CHS and stilbene synthase for precursors p-coumaroyl- and malonyl-CoA. The Cp29:Vst1 expression in Arabidopsis produced adequate levels of piceid for future disease-resistance studies.

Expression of genes related to flavonoid and stilbene synthesis as affected by signaling chemicals and Botrytis cinerea in grapevines

Recent studies have shown that the expression of genes related to the synthesis of flavonoids, such as the phenylalanineammonia lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and stilbene synthase (STS) genes were induced in response to different signaling molecules and Botrytis cinerea inoculation in grapevine leaves. Therefore, in the present study, the nucleotide and deduced amino acid sequences of STS genes from cultivars Campbell Early and Kyoho were compared. The deduced amino acid sequences of VlKSTS12, VlKSTS11, VICESTS13, and VlKSTS1 showed 100 % homology to VlCESTS12, VlCESTS11, VICESTS24, and VlKSTS13, respectively, in Campbell Early and Kyoho. In addition, the maximum transcription was observed 6 h after chemical treatments. In Campbell Early, the transcription of PAL, CHS, and CHI was higher in leaves treated with ethephon than in those treated with hydrogen peroxide, methyl jasmonate, and salicylic acid. The PAL, CHS, and CHI genes were more induced in Campbell Early than in Kyoho. The mRNA content of STS genes started to increase at 6 h and peaked at 48 h after the treatments. In Kyoho leaves, the expression of STSs was highly up-regulated at 1 h and peaked at 6 h after the treatments. The expression of the STS genes was induced in both the cultivars in leaves inoculated with B. cinerea. STS11 and STS12 showed differential expression patterns from STS1, STS24, and STS13 in Campbell Early leaves inoculated with B. cinerea.

Myb14, a direct activator of STS, is associated with resveratrol content variation in berry skin in two grape cultivars.

High and low resveratrol (Res) contents in two cultivars are correlated with the expression abundance of Myb14 , which could directly activate transcriptional expression of stilbene synthase gene ( STS ). Resveratrol (3,5,4'-trihydroxystilbene) is one of the natural polyphenols produced by secondary metabolism in some plants. Stilbene synthase (STS) is the key enzyme for the final step of precursor formation of resveratrol (Res) in grapevines. In this study, we found that Res contents in ripe berry skin were completely different in two grape cultivars, namely, 'Z168' (Vitis monticola × Vitis riparia) with high-Res and 'Jingzaojing' (Vitis vinifera) with low-Res. Moreover, the level of expression of STS gene was higher in the ripe berry skin of 'Z168' than in that of 'Jingzaojing'. To further investigate the underlying mechanisms, we conducted a co-expression analysis through transcriptomic data. We confirmed that Myb14, an R2R3 Myb transcription factor, is the direct regulator of STS by binding to Box-L5 motif. Moreover, the expression pattern of Myb14 is associated with the variation of Res content. To test this prediction, we conducted a number of experiments in vivo and in vitro. The expression patterns of Myb14 and STS in grapevine leaves were identical under a series of stimulus. Myb14 showed higher expression in the ripe berry skin of 'Z168' than in that of 'Jingzaojing'. Yeast one-hybrid assay indicated that grapevine Myb14 could interact with the promoter of STS in vitro, and the transient overexpression of Myb14 promoted the expression of STS. Furthermore, co-expressing 35S::Myb14 in transgenic Arabidopsis could activate GUS expression promoted by STS promoter. Thus, Myb14 is the direct activator of STS, and its expression pattern is associated with Res content variation in grapes.

Differences in the methylation patterns of the VaSTS1 and VaSTS10 genes of Vitis amurensis Rupr.

Resveratrol is a plant-derived phenol but the mechanism that regulates its biosynthesis remains unidentified. Stilbene synthase (STS) catalyzes resveratrol formation in vivo and we have proposed that inducers of resveratrol production affect STS expression through an unidentified epigenetic mechanism. To investigate the role of DNA methylation in resveratrol biosynthesis, we treated both rolB transgenic and empty vector control Vitis amurensis cell cultures with the DNA demethylation agent, 5-azacytidine. Treated cells had increased resveratrol production through activation of VaSTS10 expression. The lowest levels of cytosine methylation were at the 5'- and 3'-ends of the VaSTS1 protein-coding sequence. Cytosine methylation decreased mostly at the 5'- and 3'-ends of VaSTS10 after azaC treatment with an intriguing regularity in the number of cytosine nucleotides within the 5'- and 3'- ends of the protein-coding sequences. Thus, cytosine methylation is crucial for the regulation of the resveratrol biosynthetic pathway.

Influence of calcium influx induced by the calcium ionophore, A23187, on resveratrol content and the expression of CDPK and STS genes in the cell cultures of Vitis amurensis

The present study examines the effect of cal- cium influx induced by the calcium ionophore (CI) on the biosynthesis of resveratrol and the expression of stilbene synthase (STS) and calcium-dependent protein kinase (CDPK) genes in cell cultures of Vitis amurensis, which have different levels of resveratrol production. The present study utilized the control cell culture V2 of V. amurensis, which contains no more than 0.02 % dry weight (DW) of resveratrol, in addition to rolB transgenic cell cultures VB1 and VB2, which have increased resveratrol contents (0.1-0.8 % DW). Treatment with the CI at a 1 lM con- centration significantly increased STS gene expression (6 of 10 analyzed STS genes) and resveratrol production in the control V2 cell culture by fourfold; however, use of the CI at 10 lM significantly decreased resveratrol production by 2-4 fold in all cell cultures tested. In the control V2 grape cell culture, treatment with the CI increased expression of all of the CDPK genes except VaCDPK1a and VaCDPK3a. In the rolB transgenic VB2 grape cell culture treated with the CI, we detected alterations in expression of several CDPK genes, but these changes in gene expression were not significant. Our results indicated that treatment with 1 lM of the CI increased resveratrol content and production in control grape cells by selectively increasing the expres- sion of STS genes. Conversely, the CI treatment did not significantly increase resveratrol content and production, or the expression of CDPK or STS genes in the rolB transgenic cells. Likely, untreated VB2 cells have increased concen- trations of cytoplasmic calcium, and therefore, treatment with the CI did not significantly change CDPK expression. These results suggest that the rolB gene has an important role in the regulation of calcium-dependent transduction pathways in transformed cells.

Over-expression of a grape stilbene synthase gene in tomato induces parthenocarpy and causes abnormal pollen development

A novel strategy to induce parthenocarpy in tomato fruits by the induction of resveratrol biosynthesis in flower tissues was exploited. Two transgenic tomato lines were considered: a higher resveratrol-producing (35SS) line, constitutively expressing a grape stilbene synthase cDNA, and a lower resveratrol-producing (LoxS) line, expressing stilbene synthase under a fruit-specific promoter. The expression of the stilbene synthase gene affected flavonoid metabolism in a different manner in the transgenic lines, and in one of these, the 35SS line, resulted in complete male sterility. Resveratrol was synthesised either in 35SS or LoxS tomato flowers, at an even higher extent (about 8-10 times) in the former line. We further investigated whether stilbene synthase expression may have resulted in impaired naringenin accumulation during flower development. In the 35SS flowers, naringenin was significantly impaired by about 50%, probably due to metabolic competition. Conversely, the amount of glycosylated flavonols increased in transgenic flowers, thereby excluding the diminished production of flavonols as a reason for parthenocarpy in tomato. We further investigated whether resveratrol synthesis may have resulted changes to pollen structure. Microscopic observations revealed the presence of few and abnormal flake-like pollen grains in 35SS flowers with no germination capability. Finally, the analysis of coumaric and ferulic acids, the precursors of lignin and sporopollenin biosynthesis, revealed significant depletion of these compounds, therefore suggesting an impairment in structural compounds as a reason for pollen ablation. These overall outcomes, to the best of our knowledge, reveal for the first time the major role displayed by resveratrol synthesis on parthenocarpy in tomato fruits.

Expression pattern, genomic structure, and promoter analysis of the gene encoding stilbene synthase from Chinese wild Vitis pseudoreticulata

The gene encoding stilbene synthase (STS) plays a central role in many biochemical and physiological actions, and its metabolite resveratrol possesses broad-spectrum resistance to pathogens, as well as diverse pharmacological properties, notably an anticancer effect. Here, we report the expression analysis of the gene encoding STS and its promoter function from a powdery mildew (PM)-resistant Chinese wild Vitis pseudoreticulata, and compare it with two PM-susceptible cultivated grapevines, Vitis vinifera cvs. Carignane and Thompson Seedless. We show an unusual expression pattern of STS in V. pseudoreticulata, which differs markedly from that of the cultivated species. Sequence comparisons reveal that the genomic DNA sequences encoding STS in the three grapevines are highly conserved, but a novel residue mutation within the key motif of STS is solely present in V. pseudoreticulata. Moreover, the STS promoter in V. pseudoreticulata displays a significantly different structure from that found in the two V. vinifera. The three promoter-driven GUS differential expression patterns in transformed tobacco plants induced with Alternaria alternata, methyl jasmonate, and wounding indicated that the structurally different STS promoter of V. pseudoreticulata is responsible for its specific regulatory function. We also demonstrate that the expression of STS genes from their native promoters are functional in transformed tobacco and retain pathogen inducibility. Importantly, the genomic DNA-2 of V. pseudoreticulata under its native promoter shows good induction and the maximum level of resveratrol content. These findings further our understanding of the regulation of STS expression in a resistant grapevine and provide a new pathogen-inducible promoter system for the genetic improvement of plant disease resistance.

Low responsiveness of grapevine flowers and berries at fruit set to UV-C irradiation

In grapevine, stimulation of defence responses was evidenced in response to various types of abiotic stresses in both leaves and berries, as revealed by the increasing expression of genes encoding defence-related proteins or the stimulation of their corresponding activities. However, the capability of inflorescences to respond to abiotic stresses has never been investigated. Therefore, plant defence reactions in response to UV-C irradiation were followed in inflorescences and young clusters focusing on both bunchstems (peduncle and pedicels) and developing flowers/berries from separated floral buds stage [Biologische Bundesanstalt, Bundessortenamt and CHemical industry (BBCH) stage 57] to groat-sized berries stage (BBCH 73). For this purpose, the expression of various genes coding for pathogenesis-related (PR) proteins (class I and III chitinases, Chi1b and CH3; beta-1,3-glucanase, GLUC), an enzyme of the phenylpropanoid pathway (phenylalanine ammonia-lyase, PAL), and stilbene synthase (STS) was analysed in parallel with variations of chitinase activity and the accumulation of the phytoalexin resveratrol. Multiple defence responses were induced in bunchstems of both inflorescences and clusters following UV-C treatment. First, expression of genes encoding PR proteins was stimulated and chitinase activity was enhanced. Secondly, PAL and STS expression increased in association with resveratrol accumulation. Amazingly, none of the tested defence processes was induced in grapevine flowers following UV-C exposure, whatever the stage analysed. Similarly, in berries at fruit set, induction of gene expression was weak and neither an increase in chitinase activity nor resveratrol synthesis was noticed. However, in groat-sized berries, responsiveness to UV-C increased, as revealed by the induction of CH3, PAL, and STS expression, together with resveratrol accumulation. The differential responsiveness between bunchstems, flowers, and berries is discussed.

Molecular engineering of resveratrol in plants

The grapevine phytoalexin resveratrol, the synthesis of which is achieved by stilbene synthase (STS), displays a wide range of biological effects. Most interest has centred, in recent years, on STS gene transfer experiments from grapevine to the genome of numerous plants. This work presents a comprehensive review on plant molecular engineering with the STS gene. Gene and promoter options are discussed, namely the different promoters used to drive the transgene, as well as the enhancer elements and/or heterologous promoters used to improve transcriptional activity in the transformed lines. Factors modifying transgene expression and epigenetic modifications, for instance transgene copy number, are also presented. Resveratrol synthesis in plants, together with that of its glucoside as a result of STS expression, is described, as is the incidence of these compounds on plant metabolism and development. The ectopic production of resveratrol can lead to broad-spectrum resistance against fungi in transgenic lines, and to the enhancement of the antioxidant activities of several fruits, highlighting the potential role of this compound in health promotion and plant disease control.

Prokaryotic expression, polyclonal antibody preparation of the stilbene synthase gene from grape berry and its different expression in fruit development and under heat acclimation

Stilbene synthase (STS, EC 2.3.1.95) leads to the production of resveratrol compounds, which are major components of the phytoalexin response against fungal pathogens of the plant and are highly bioactive substances of pharmaceutical interest. STS expression and regulation are important. Temperature is one of the main external factors affecting phytoalexin accumulation in plant tissues, the effect of temperature on resveratrol synthesis and stilbene synthase expression in grape berries has not been reported before. Here we cloned the full-length sts cDNA with 1179bp from grape berry via PCR, and then introduced into an expressed plasmid pET-30a(+) vector at the EcoRI and XhoI restriction sites. With the isopropyl-beta-d-thiogalactoside (IPTG) induced, the pET-sts was highly expressed in Escherichia coli BL21 (DE3) pLysS cells. A fusion protein with the His-Tag was purified by Ni-NTA His.Bind Resin and then used as the antigen to immunize a New Zealand rabbit. Furthermore, the antiserum was precipitated by 50% saturated ammonium sulfate and DEAE-Sephadex A-50 chromatography to obtain the immunoglobulin G (IgG) fraction. These results provide a substantial basis for the further studies of the STS in grape berry as well as in other species of plants. The sts expression in fruit development and in response to heat acclimation was then assayed. The results indicated STS was regulated in fruits depending on the developmental stage and significantly accumulation of STS mRNA and synthesis of new STS protein during the early of heat acclimation, this work offers an important basis for further investigating the mechanism of post-harvest fruit adaptation to environmental stresses.

High content of resveratrol in lettuce transformed with a stilbene synthase gene of Parthenocissus henryana.

Resveratrol (trans-3,5,4'-trihydroxystilbene) is a plant phytoalexin which has positive effects on human health. Stilbene synthase (STS) is a key enzyme involved in resveratrol biosynthesis. To construct a vector for STS expression in lettuce plant, a cDNA-encoding STS of Parthenocissus henryana was fused to the Cauliflower mosaic virus (CaMV) 35S promoter, and the bar gene was used as a selective marker gene. To increase the expression of STS, the expression cassette was flanked by MARs. In transgenic lettuce plants, an additional compound was identified as resveratrol by HPLC and ESI-MS. Quantitative analysis showed that the average content of resveratrol reached 56.40 +/- 5.52 microg/g leaf fresh weight, which was comparable to the amount in grape skin. Anticancer assay in HeLa cells revealed that apoptosis was induced by 200 microM of resveratrol extracted from transgenic lettuce.