Transcriptomic Analysis Reveals Novel Regulators of the Scots Pine Stilbene Pathway.

Stilbenes accumulate in Scots pine heartwood where they have important roles in protecting wood from decaying fungi. They are also part of active defense responses, and their production is induced by different (a)biotic stressors. The specific transcriptional regulators as well as the enzyme responsible for activating the stilbene precursor cinnamate in the pathway are still unknown. UV-C radiation was the first discovered artificial stress activator of the pathway. Here, we describe a large-scale transcriptomic analysis of pine needles in response to UV-C and treatment with translational inhibitors, both activating the transcription of stilbene pathway genes. We used the data to identify putative candidates for the missing CoA ligase and for pathway regulators. We further showed that the pathway is transcriptionally activated by phosphatase inhibitor, ethylene and jasmonate treatments, as in grapevine, and that the stilbene synthase promoter retains its inducibility in some of the tested conditions in Arabidopsis, a species that normally does not synthesize stilbenes. Shared features between gymnosperm and angiosperm regulation and partially retained inducibility in Arabidopsis suggest that pathway regulation occurs not only via ancient stress-response pathway(s) but also via species-specific regulators. Understanding which genes control the biosynthesis of stilbenes in Scots pine aids breeding of more resistant trees.

Efficient biosynthesis of pinosylvin from lignin-derived cinnamic acid by metabolic engineering of Escherichia coli.

BACKGROUND: The conversion of lignin-derived aromatic monomers into valuable chemicals has promising potential to improve the economic competitiveness of biomass biorefineries. Pinosylvin is an attractive pharmaceutical with multiple promising biological activities. RESULTS: Herein, Escherichia coli was engineered to convert the lignin-derived standard model monomer cinnamic acid into pinosylvin by introducing two novel enzymes from the wood plant: stilbene synthase from Pinus pinea (PpSTS) and 4-Coumarate-CoA ligase from Populus trichocarpa (Ptr4CL4). The expression of Ptr4CL4 drastically improved the production of pinosylvin (42.5 ± 1.1 mg/L), achieving values 15.7-fold higher than that of Ptr4CL5 (another 4-Coumarate-CoA ligase from Populus trichocarpa) in the absence of cerulenin. By adjusting the expression strategy, the optimized engineered strain produced pinosylvin at 153.7 ± 2.2 mg/L with an extremely high yield of 1.20 ± 0.02 mg/mg cinnamic acid in the presence of cerulenin, which is 83.9% ± 1.17 of the theoretical yield. This is the highest reported pinosylvin yield directly from cinnamic acid to date. CONCLUSION: Our work highlights the feasibility of microbial production of pinosylvin from cinnamic acid and paves the way for converting lignin-related aromatics to valuable chemicals.

Identification and Expression Analysis of Stilbene Synthase Genes in Arachis hypogaea in Response to Methyl Jasmonate and Salicylic Acid Induction.

Stilbene synthase is an important enzyme of the phenylpropanoid pathway, regulating the production of several biologically active stilbenoids. These compounds have antioxidant, anti-inflammatory, and anti-cancer properties. However, the detailed characterization of stilbene synthase genes in Arachis hypogaea has not yet been performed. In this study, the comprehensive characterization of stilbene synthase genes in A. hypogaea was conducted, commencing with identification, phylogenetic analysis, and study of their expression in response to exogenous hormonal treatment. We identified and isolated five AhSTSs genes and recorded their expression pattern in peanut (BARD-479) in response to methyl jasmonate (MeJA) and salicylic acid (SA) treatment. The presence of Chal_sti_synt, ACP_syn_III, and FAE1_CUT1_rppA domains in all AhSTSs indicated their role in the biosynthesis of stilbene and lipid metabolism. Cis-regulatory element analysis indicated their role in light responsiveness, defense responses, regulation of seed development, plant growth, and development. Despite close structural and functional similarities, expression and correlational analysis suggested that these genes may have a specific role in peanut, as individual AhSTS exhibited differential expression upon hormonal treatment in a genotype dependent manner. Further studies on functional characterization involving the transcriptional regulation of AhSTSs can clearly explain the differential expression of stilbene synthase genes to hormonal treatment.

Expression of a Stilbene Synthase Gene from the Vitis labrusca x Vitis vinifera L. Hybrid Increases the Resistance of Transgenic Nicotiana tabacum L. Plants to Erwinia carotovora.

'Isabel' grape (Vitis labrusca x V. vinifera L. hybrid) is one of the main grape cultivars in Russia and some other countries for processing, due to its vigor, tolerance to the main fungal diseases, high yield and potential for sugar accumulation. The stilbene synthase gene VlvSTS was isolated from the hybrid grape cv. Isabel and cloned into a pSS plant transformation vector under the control of a constitutive 35S RNA double promoter of the cauliflower mosaic virus, CaMV 35SS. VlvSTS-gene containing transgenic tobacco lines were obtained and analyzed. For the first time plants expressing the VlvSTS gene were shown to have an enhanced resistance to the bacterial pathogen Erwinia carotovora subsp. carotovora B15. Transgenic plants were tested for resistance to a number of fungal pathogens. The plants were resistant to the grey mould fungus Botrytis cinerea, but not to the fungi Fusarium oxysporum, F. sporotrichioides, or F. culmorum. According to the results of a high performance liquid chromatography-mass spectrometry analysis, the amount of trans-resveratrol in leaves of transgenic plants with the highest expression of the VlvSTS gene was in a range from 150 to 170 µg/g of raw biomass. Change in the color and a decreased anthocyanin content in the flower corollas of transgenic plants were observed in transgenic lines with the highest expression of VlvSTS. A decrease in total flavonoid content was found in the flower petals but not the leaves of these tobacco lines. High expression of the VlvSTS gene influenced pollen development and seed productivity in transgenic plants. The size of pollen grains increased, while their total number per anther decreased. A decrease in the number of fertile pollen grains resulted in a decreased average weight of a seed boll in transgenic plants.

Potential Associations among Bioactive Molecules, Antioxidant Activity and Resveratrol Production in Vitis vinifera Fruits of North America.

Grapes (Vitis vinifera L.) are rich in bioactive molecules contributing to health benefits. Consumption of grapes is linked to reduced incidence of cardiovascular diseases. Studies on table grape cultivars are limited although much attention in research was focused on the wine industry. Bioactive effects of grapes as anti-inflammatory, anticarcinogenic, cardioprotective, vasorelaxant, phytoestrogenic and neuroprotective have also been reported. For example, resveratrol is a natural food ingredient present in grapes, with high antioxidant potential. Here we conducted an exploratory study to investigate bioactive molecules, antioxidant activity and the association between constitutive stilbene synthase (STS) gene expression and the resveratrol biosynthesis in selected table grape varieties in North America. The phenolic compounds, fatty acid composition and antioxidant activity of four grape varieties were compared. Red Globe variety was rich in unsaturated fatty acids as well as phenolic compounds such as caffeic acid, quercetin and resveratrol. Meanwhile, the constitutive expression of grape stilbene synthase gene was higher in Flame and Autumn Royal where resveratrol content of these cultivars was relatively low compared to the Red Globe variety. This study shows the potential links in grape antioxidant activity and resveratrol production, but more studies are necessary to show the association.

The Influence of the Grapevine Bacterial and Fungal Endophytes on Biomass Accumulation and Stilbene Production by the In Vitro Cultivated Cells of Vitis amurensis Rupr.

Plant endophytes are known to alter the profile of secondary metabolites in plant hosts. In this study, we identified the main bacterial and fungal representatives of the wild grape Vitis amurensis Rupr. microbiome and investigated a cocultivation effect of the 14 endophytes and the V. amurensis cell suspension on biomass accumulation and stilbene biosynthesis. The cocultivation of the V. amurensis cell culture with the bacteria Agrobacterium sp., Bacillus sp., and Curtobacterium sp. for 2 weeks did not significantly affect the accumulation of cell culture fresh biomass. However, it was significantly inhibited by the bacteria Erwinia sp., Pantoea sp., Pseudomonas sp., and Xanthomonas sp. and fungi Alternaria sp., Biscogniauxia sp., Cladosporium sp., Didymella sp. 2, and Fusarium sp. Cocultivation of the grapevine cell suspension with the fungi Didymella sp. 1 and Trichoderma sp. resulted in cell death. The addition of endophytic bacteria increased the total stilbene content by 2.2-5.3 times, while the addition of endophytic fungi was more effective in inducing stilbene accumulation by 2.6-16.3 times. The highest content of stilbenes in the grapevine cells cocultured with endophytic fungi was 13.63 and 13.76 mg/g of the cell dry weight (DW) after cultivation with Biscogniauxia sp. and Didymella sp. 2, respectively. The highest content of stilbenes in the grapevine cells cocultured with endophytic bacteria was 4.49 mg/g DW after cultivation with Xanthomonas sp. The increase in stilbene production was due to a significant activation of phenylalanine ammonia lyase (PAL) and stilbene synthase (STS) gene expression. We also analyzed the sensitivity of the selected endophytes to eight antibiotics, fluconazole, and trans-resveratrol. The endophytic bacteria were sensitive to gentamicin and kanamycin, while all selected fungal strains were resistant to fluconazole with the exception of Cladosporium sp. All endophytes were tolerant of trans-resveratrol. This study showed that grape endophytes stimulate the production of stilbenes in grape cell suspension, which could further contribute to the generation of a new stimulator of stilbene biosynthesis in grapevine or grape cell cultures.

A Computational Metabolic Model for Engineered Production of Resveratrol in Escherichia coli.

Although engineered microbial production of natural compounds provides a promising alternative method to plant production and extraction, the process can be inefficient and ineffective in terms of time and cost. To render microbial systems profitable and viable, the process must be optimized to produce as much product as possible. To this end, this work illustrates the construction of a new probabilistic computational model to simulate the microbial production of a well-known cardioprotective molecule, resveratrol, and the implementation of the model to enhance the yield of the product in Escherichia coli. This model identified stilbene synthase as the limiting enzyme and informed the effects on changes in concentration and source of this enzyme. These parameters, when employed in a laboratory system, were able to improve the titer from 62.472 mg/L to 172.799 mg/L, demonstrating the model's ability to produce a useful simulation of a dynamic microbial resveratrol production system.

Impact of Environmental Factors on Stilbene Biosynthesis.

Stilbenes are a small family of polyphenolic secondary metabolites that can be found in several distantly related plant species. These compounds act as phytoalexins, playing a crucial role in plant defense against phytopathogens, as well as being involved in the adaptation of plants to abiotic environmental factors. Among stilbenes, trans-resveratrol is certainly the most popular and extensively studied for its health properties. In recent years, an increasing number of stilbene compounds were subjected to investigations concerning their bioactivity. This review presents the most updated knowledge of the stilbene biosynthetic pathway, also focusing on the role of several environmental factors in eliciting stilbenes biosynthesis. The effects of ultraviolet radiation, visible light, ultrasonication, mechanical stress, salt stress, drought, temperature, ozone, and biotic stress are reviewed in the context of enhancing stilbene biosynthesis, both in planta and in plant cell and organ cultures. This knowledge may shed some light on stilbene biological roles and represents a useful tool to increase the accumulation of these valuable compounds.

Overexpression of stilbene synthase genes to modulate the properties of plants and plant cell cultures.

Plant stilbenes have attracted special attention as they possess valuable health benefits and improve plant resistance to environmental stresses. Stilbenes are synthesized via the phenylpropanoid pathway, where stilbene synthase (STS, EC 2.3.1.95) directly catalyzes the formation of t-resveratrol (monomeric stilbene). This review discusses the features of using STS genes in genetic engineering and plant biotechnology with the purpose to increase plant resistance to environmental stresses and to modify secondary metabolite production.

In Silico Identification of Type III PKS Chalcone and Stilbene Synthase Homologs in Marine Photosynthetic Organisms.

Marine microalgae are photosynthetic microorganisms at the base of the marine food webs. They are characterized by huge taxonomic and metabolic diversity and several species have been shown to have bioactivities useful for the treatment of human pathologies. However, the compounds and the metabolic pathways responsible for bioactive compound synthesis are often still unknown. In this study, we aimed at analysing the microalgal transcriptomes available in the Marine Microbial Eukaryotic Transcriptome Sequencing Project (MMETSP) database for an in silico search of polyketide synthase type III homologs and, in particular, chalcone synthase (CHS) and stilbene synthase (STS), which are often referred to as the CHS/STS family. These enzymes were selected because they are known to produce compounds with biological properties useful for human health, such as cancer chemopreventive, anti-inflammatory, antioxidant, anti-angiogenic, anti-viral and anti-diabetic. In addition, we also searched for 4-Coumarate: CoA ligase, an upstream enzyme in the synthesis of chalcones and stilbenes. This study reports for the first time the occurrence of these enzymes in specific microalgal taxa, confirming the importance for microalgae of these pathways and giving new insights into microalgal physiology and possible biotechnological applications for the production of bioactive compounds.

Effect of high CO2 levels and low temperature on stilbene biosynthesis pathway gene expression and stilbenes production in white, red and black table grape cultivars during postharvest storage.

Phenolic compounds, such as phytoalexin resveratrol, can be induced in grapes in response to biotic and abiotic stresses and have been related in many healthy effects. Stilbene synthases (STSs) are the key enzyme responsible for resveratrol biosynthesis. They have been already isolated and characterized from several plant species, however, VviSTS is a multigene family and little is known about their modulation in response to the application of gaseous treatments that maintain table grapes quality during postharvest. In this work, we have analyzed the effect of a 3-day CO2 treatment on the modulation of 4 STSs (VviSTS6, VviSTS7, VviSTS16 and VviSTS46) and on the accumulation of different stilbene compounds (resveratrol, resveratrol-glucoside, trans-piceatannol, z-miyabenol and pallidol) during the postharvest storage at 0 °C of white (Superior Seedless, Dominga), red (Red Globe) and black (Autumn Royal) table grapes. Results indicated that the accumulation of the stilbene compounds by the application of CO2 and low temperature storage were cultivar dependent. In white Dominga fruit, accumulation of stilbene compounds increased in CO2-treated samples what seems to be modulated by VviSTS6, VviSTS7 and VviSTS46. However, in Red Globe the accumulation of compounds was mainly due to the cold storage in air and seems to be also mediated by the induction of the same VviSTSs. By contrast, in Superior Seedless and Autumn Royal table grapes the modulation of VviSTSs genes and the stilbene accumulation was independent of the atmosphere storage. Further studies would be needed to elucidate the possible role of transcription factors involved on VviSTSs modulation.

Effect of spruce PjSTS1a, PjSTS2, or PjSTS3 gene overexpression on stilbene biosynthesis in callus cultures of Vitis amurensis Rupr.

Stilbenes are natural compounds protecting plants against microbial pathogens and known to possess valuable biologically active properties. In the present study, we established transgenic grapevine callus cell cultures overexpressing three stilbene synthase (STS) genes of spruce Picea jezoensis PjSTS1a, PjSTS2, and PjSTS3. Transformation of Vitis amurensis calli with the PjSTS1a, PjSTS2, and PjSTS3 genes significantly increased total content of stilbenes in 3.6-6, 2.5-2.9, and 4.1-16.1 times, respectively, in comparison with the control calli. The most pronounced positive effect on the accumulation of stilbenes was observed for the PjSTS3-overexpressing calli where the total content of stilbenes was increased up to 3.1 mg/g DW, and the stilbene production reached 25.4 mg/L. These values were higher than those achieved for the grapevine callus cell cultures overexpressing three STS genes from V. amurensis. Thus, transformation of grapevine cell cultures with spruce STS genes with a relatively low degree of homology to the endogenous VaSTSs is a more effective strategy for induction of plant secondary metabolite biosynthesis than using the grapevine genes for the overexpression experiments.

The grapevine R2R3-type MYB transcription factor VdMYB1 positively regulates defense responses by activating the stilbene synthase gene 2 (VdSTS2).

BACKGROUND: Resveratrol is a naturally occurring plant stilbene that exhibits a wide range of valuable biological and pharmacological properties. Although the beneficial effects of trans-resveratrol to human health and plant protection against fungal pathogens and abiotic stresses are well-established, yet little is known about the molecular mechanisms regulating stilbene biosynthesis in plant defense progress. RESULTS: Here, we cloned and identified the Chinese wild grape (Vitis davidii) R2R3-MYB transcription factor VdMYB1, which activates defense responses against invading pathogen. VdMYB1 transcripts were significantly upregulated after inoculation with the grapevine powdery mildew fungus Erysiphe necator (Schw.) Burr. Transient expression analysis using onion epidermal cells and Arabidopsis thaliana protoplasts showed that VdMYB1 was localized in the nucleus. Yeast one-hybrid assays revealed that VdMYB1 acts as a transcriptional activator. Grapevine leaves transiently overexpressing VdMYB1 showed a lower number of fungal conidiophores compared with wild-type leaves. Overexpression of VdMYB1 in grapevine leaves did not alter the expression of genes in salicylic acid- and jasmonate-dependent pathways, but affected the expression of stilbene synthase (STS) genes, key regulators of flavonoid metabolism. Results of electrophoretic mobility shift assays and in vivo transcriptional activation assays showed that VdMYB1 binds to the MYB binding site (MYBBS) in the STS2 gene promoter, thus activating STS2 transcription. In heterologous expression assays using tobacco leaves, VdMYB1 activated STS2 gene expression and increased the accumulation of resveratrol. CONCLUSIONS: Our study showed that VdMYB1 activates STS2 gene expression to positively regulate defense responses, and increases the content of resveratrol in leaves.

Transcriptional regulation of stilbene synthases in grapevine germplasm differentially susceptible to downy mildew.

BACKGROUND: To limit the impact of the downy mildew disease of grapevine and reduce the need to recur to chemical treatments, an effective strategy might be recovering adaptive resistance traits in both cultivated and wild V. vinifera germplasm. Considering that stilbenes represent the most important class of phytoalexins in the Vitaceae, the constitutive expression and transcriptional activation of all the functional members of the stilbene synthase gene family were analysed in a group of nine grapevine genotypes following artificial infection with the oomycete Plasmopara viticola, the causal agent of the disease. In addition, in the same genotypes we analyzed the expression of genes encoding for two transcription factors involved in the transcriptional regulation of the stilbene synthase genes, namely VvMYB14 and VvMYB15, and of genes encoding for chalcone synthases. RESULTS: Downy mildew incidence and severity ranged from nihil to high in the grapevine genotypes considered, being low to moderate in a subgroup of V. vinifera genotypes. The constitutive expression of the stilbene synthase genes as well as the extent of their transcriptional activation following P. viticola inoculation appeared to be inversely related to the proneness to develop disease symptoms upon infection. In a specular manner, following P. viticola inoculation all the chalcone synthase genes were up-regulated in the susceptible grapevine genotypes and down-regulated in the resistant ones. The infection brought by P. viticola appeared to elicit a co-ordinated and sequential transcriptional activation of distinct stilbene synthase genes subsets, each of which may be regulated by a distinct and specific MYB transcription factor. CONCLUSIONS: The present results suggest that the induction of stilbene biosynthesis may contribute to the basal immunity against the downy mildew of grapevine, thus representing an adaptive resistance trait to recover, in both cultivated and wild V. vinifera germplasm. During the early stages of P. viticola infection, an antagonistic interaction between flavonol and stilbene biosynthesis might occur, whose outcome might determine the subsequent extent of disease symptoms. Further studies are needed to decipher the possible regulatory mechanisms involved in the antagonistic crosstalk between these two metabolic pathways in resistant and susceptible genotypes in response to P. viticola.

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

KEY MESSAGE: 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.

Evolvement of nutraceutical onion plants engineered for resveratrol biosynthetic pathway.

KEY MESSAGE: Genetically engineered onion expressing codon-optimized VvSTS1 gene accumulated stilbenes and extended life span in yeast and can serve as potential nutraceutical. Resveratrol (RV) is a natural polyphenolic compound found in certain plant species including grapes. RV is well known for its nutraceutical properties and to assuage several disease conditions. Onion is the second most consumed vegetable worldwide and contains large quantities of precursor molecules, malonyl-CoA and para-coumaroyl-CoA that are needed for RV biosynthesis. The present study reports the development of nutraceutical onion by engineering RV biosynthetic pathway. A codon-optimized grapevine synthetic stilbene synthase gene (VvSTS1) was synthesized using native grapevine sequence. Six-week-old healthy yellowish compact nodular calli were co-cultivated with Agrobacterium tumefaciens harbouring pCAMBIA1300-hpt II-CaMV35S-VvSTS1-nos. PCR analysis revealed the presence of VvSTS1 and hpt II genes in putative transgenics. Southern blot analysis confirmed the integration of VvSTS1 gene and independent nature of transformants. LC-ESI-HRMS analysis revealed the accumulation of variable quantities of RV (24.98-50.18 µg/g FW) and its glycosylated form polydatin (33.6-67.15 µg/g FW) in both leaves and bulbs, respectively, indicating the successful engineering of RV biosynthetic pathway into onion. The transgenic onion bulb extracts extended the life span in haploid yeast. The transgenic onion accumulating RV and polydatin, developed for the first of its kind, may serve as a potential nutraceutical resource.

Reactivation of rolB transgene expression in Vitis amurensis Rupr. cells upon retransformation with 2b gene from Cucumovirus isolate NK.

OBJECTIVE: Studies concerning 2b protein from Cucumovirus showed 2b to effectively repress functioning of the plant silencing complex, current study aimed whether retransformation with 2b gene able to restore silenced transgene expression in plant cells. RESULTS: A rolB-transgenic cell culture of Vitis amurensis Rupr. that was continuously subcultured during more than 10 years and exhibited decreased transcription of the rolB transgene was retransformed with the 2b gene of Cucumovirus-NK. Three cell lines retransformed with 2b showed a significant up-regulation of rolB expression accompanied with enhancements in their stilbenes content level in more than 2,7-fold compared to parental rolB-transgenic cell line. The mentioned increase in the level of stilbenes content was due to activation of certain stilbene synthase genes expression responsible for stilbenes biosynthesis in V. amurensis cells. Restoration of rolB expression upon 2b-retransformation led to increase in the expression levels of VaSTS2-VaSTS5 and VaSTS7 isoforms. CONCLUSIONS: 2b from CMV-NK can reactivate a silenced transgene expression, even after 10 years of subcultivation, nevertheless, optimization of the methods concerning 2b introduction in plant genomes is necessary to avoid undesirable silencing effects.

Dynamic translocation of stilbene synthase VpSTS29 from a Chinese wild Vitis species upon UV irradiation.

Stilbene phytoalexins derived from grapevine can be rapidly accumulated when exposed to an artificial UV-C treatment. However, the underlying mechanisms involved in this accumulation and translocation are unclear. Here, we describe an investigation of the influence of UV-C treatment on the dynamic subcellular distribution of a member of a stilbene synthase family VpSTS29 derived from Chinese wild Vitis pseudoreticulata W.T. Wang when over-expressed in V. vinifera L. cv. Thompson Seedless. Our results show that VpSTS29-GFP was accumulated at a relatively high level in roots and mature leaves of transgenic grape lines, and was predominantly distributed in the cytoplasm. When exposed to UV-C irradiation, VpSTS29 displayed UV-induced feature coupled with the accumulation of stilbene compounds. Notably, VpSTS29-GFP can be translocated from the cytoplasm into chloroplasts upon UV-irradiation. Leaves from the two VpSTS29-GFP-expressing lines displayed more serious UV damage, showing withering and marginal scorching phenotype, and decreased content of H2O2, compared to the untransformed plant. Also, overexpression of VpSTS29 altered the expression of genes related to redox regulation, stilbene biosynthesis and light stimulus. Co-expression of VpSTS29-GFP with Glycolate oxidase 1 (myc-VpGLO1) confirmed the ability of stilbenes to decrease the content of H2O2 in Arabidopsis mesophyll protoplasts. These results provide new insight into the biological functions and properties of stilbene synthase and its product in response to environmental stimulus.

Histochemical and immunohistochemical analysis of enzymes involved in phenolic metabolism during berry development in Vitis vinifera L.

Phenolics are involved in many of plants' biological functions. In particular, they play important roles in determining the quality of grape berries and the wine made from them, and can also act as antioxidants with beneficial effects for human health. Several enzymes are involved in the synthesis of phenolic compounds. Among them, stilbene synthase (STS) is a key to the biosynthesis of stilbenes, which are considered to be important secondary metabolites in plants. Other enzymes, such as polyphenol oxidase (PPO) and peroxidase (POD), are involved in the degradation of phenolics, and become activated during late stages of berry ripening. In the present study, Vitis vinifera L. berries were sampled at eight stages of development, from 10 days after anthesis to late harvest. The PPO and POD enzymatic activities were determined at each stage. The presence of STS, PPO, and POD proteins in the grape exocarp and mesocarp was detected immunohistochemically and histochemically. The amount and intensity of the immunohistochemical and histochemical signals correlate with the variations in enzyme activities throughout fruit development. Strong STS immunoreactivity was detected until the onset of ripening. Labeled tissue increased gradually from mesocarp to exocarp, showing an intense signal in epidermis. At subcellular level, STS was mainly detected in cytoplasm grains and cell walls. The amount of PPO immunoreactivity increased progressively until the end of ripening. The PPO signal was detected in hypodermal layers and, to a lesser extent, in mesocarp parenchyma cells, especially in cytoplasm grains and cell walls. Finally, POD activity was stronger at the onset of ripening, and the POD histochemical signal was mainly detected in the cell walls of both exocarp and mesocarp tissue.

Expression of a Grape VqSTS36-Increased Resistance to Powdery Mildew and Osmotic Stress in Arabidopsis but Enhanced Susceptibility to Botrytis cinerea in Arabidopsis and Tomato.

Stilbene synthase genes make a contribution to improving the tolerances of biotic and abiotic stress in plants. However, the mechanisms mediated by these STS genes remain unclear. To provide insight into the role of STS genes defense against biotic and abiotic stress, we overexpressed VqSTS36 in Arabidopsis thaliana and tomato (Micro-Tom) via Agrobacterium-mediated transformation. VqSTS36-transformed Arabidopsis lines displayed an increased resistance to powdery mildew, but both VqSTS36-transformed Arabidopsis and tomato lines showed the increased susceptibility to Botrytis cinerea. Besides, transgenic Arabidopsis lines were found to confer tolerance to salt and drought stress in seed and seedlings. When transgenic plants were treated with a different stress, qPCR assays of defense-related genes in transgenic Arabidopsis and tomato suggested that VqSTS36 played a specific role in different phytohormone-related pathways, including salicylic acid, jasmonic acid, and abscisic acid signaling pathways. All of these results provided a better understanding of the mechanism behind the role of VqSTS36 in biotic and abiotic stress.