Resveratrol Synthase Research Articles

VqNAC44 enhances stilbene synthesis and disease resistance in Chinese wild grape by interacting with VqMYB15

As significant phytoalexins, stilbene compounds can improve the stress resistance of grapes under biotic and abiotic stress conditions and have biological effects such as antitumour, antioxidant, immune regulation and cardiovascular protection activities in humans. RESVERATROL SYNTHASE (RS), also known as STILBENE SYNTHASE (STS), is the critical enzyme regulating stilbene synthesis and has been identified in a few plant species. However, the regulatory mechanisms of stilbene synthesis are uncertain. In this study, an NAC family transcription factor from Vitis quinquangularis, named VqNAC44, was characterized as an indirect regulator of stilbene synthesis. It is worth noting that VqNAC44 did not bind to the STS promoter nor did it interact with the STS protein but interacted with the MYB transcription factor VqMYB15. This interaction between VqMYB15 and VqNAC44 was validated by a yeast two-hybrid assay and bimolecular fluorescence complementation. Overexpressing VqNAC44 in Arabidopsis thaliana significantly increased its tolerance to biotic and abiotic stresses. Transient overexpression of VqNAC44 and VqMYB15 in grape leaves resulted in increased expression of the STS gene and increased production of stilbene compounds. The experimental results confirmed that VqNAC44 regulated stilbene synthesis by interacting with VqMYB15, thereby enhancing the plant stress resistance.

Serendipita indica: A Biostimulant Enhancing Low-Temperature Tolerance and Active Constituent Levels in Polygonum cuspidatum

Polygonum cuspidatum is a traditional medicinal plant enriched with resveratrol and polydatin. However, low temperatures reduce the medicinal component contents of P. cuspidatum, and prolonged low temperatures also affect the growth and survival of P. cuspidatum at the seedling stage. It is unclear whether a culturable endophytic fungus Serendipita indica is able to enhance P. cuspidatum’s low-temperature tolerance and medicinal components. The objective of this study was to examine the biomass, leaf gas exchange, antioxidant enzyme activity, proline levels, medicinal constituent levels, and the expression of the resveratrol synthase (PcRS) and resveratrol-forming stilbene synthase 11 (PcRS11) genes of potted P. cuspidatum plants inoculated with S. indica at low temperatures (10 °C/6 °C, 12 h/12 h, day/night temperature). The six-week low-temperature treatment significantly reduced the root fungal colonization, biomass production, and leaf gas exchange variables, whereas S. indica inoculation significantly increased shoot and root biomass, photosynthetic rate, stomatal conductance, and transpiration rate at low temperatures. S. indica inoculation significantly increased superoxide dismutase and catalase activity as well as proline levels in leaves at low temperatures. The magnitude of root chrysophanol, emodin, polydatin, and resveratrol levels decreased by low temperatures was greater in uninoculated plants than in inoculated plants. Inoculation of S. indica, on the other hand, significantly increased the four medicinal component levels in roots at low temperatures, with a greater magnitude rise in chrysophanol, polydatin, and resveratrol at low temperatures than at suitable temperatures. The low-temperature treatment down-regulated the expression of PcRS and PcRS11 genes in roots, while S. indica up-regulated the expression of PcRS and PcRS11 genes at low temperatures. This implies that S. indica acts as a powerful microbial stimulant on P. cuspidatum to promote low-temperature resistance and medicinal component levels.

Exogenous methyl jasmonate combined with Ca2+ promote resveratrol biosynthesis and stabilize sprout growth for the production of resveratrol-rich peanut sprouts

Promoting resveratrol accumulation in plants and utilizing resveratrol-rich plants as raw materials for the development of functional foods is a promising development direction. The effects of methyl jasmonate (MeJA), in combination with CaCl2 and Ca2+ inhibitors, on physiological metabolism and resveratrol enrichment of peanut sprouts were investigated. MeJA combined with CaCl2 increased Ca2+ content, calmodulin content, and Ca2+- adenosine triphosphatase activity, as well as upregulated calcium-binding proteinase expression levels. Treatment with MeJA plus CaCl2 significantly increased peroxidase and superoxide dismutase activities and antioxidant capacities, significantly decreased the content of malondialdehyde and hydrogen peroxide, which resulted in a significantly increased in sprout length and fresh weight, and alleviated the inhibition of sprout growth. MeJA plus CaCl2 significantly increased the activities of phenylalanine ammonia-lyase and 4-coumarate coenzyme A ligase and upregulated the expression levels of phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, and resveratrol synthase, thus significantly increasing resveratrol content. However, MeJA combined with Ca2+ antagonists reversed these effects. These results indicate that MeJA interacts with Ca2+ to promote resveratrol synthesis in peanut sprouts and to improve sprout stress tolerances.

Exogenous Melatonin Regulates Physiological Responses and Active Ingredient Levels in Polygonum cuspidatum under Drought Stress.

Polygonum cuspidatum, an important medicinal plant, is rich in resveratrol and polydatin, but it frequently suffers from drought stress in the nursery stage, which inhibits the plant's growth, active components concentrations, and the price of rhizome in the later stage. The purpose of this study was to analyze how exogenous 100 mM melatonin (MT) (an indole heterocyclic compound) affected biomass production, water potential, gas exchange, antioxidant enzyme activities, active components levels, and resveratrol synthase (RS) gene expression of P. cuspidatum seedlings growing under well-watered and drought stress conditions. The 12-week drought treatment negatively affected the shoot and root biomass, leaf water potential, and leaf gas exchange parameters (photosynthetic rate, stomatal conductance, and transpiration rate), whereas the application of exogenous MT significantly increased these variables of stressed and non-stressed seedlings, accompanied by higher increases in the biomass, photosynthetic rate, and stomatal conductance under drought versus well-watered conditions. Drought treatment raised the activities of superoxide dismutase, peroxidase, and catalase in the leaves, while the MT application increased the activities of the three antioxidant enzymes regardless of soil moistures. Drought treatment reduced root chrysophanol, emodin, physcion, and resveratrol levels, while it dramatically promoted root polydatin levels. At the same time, the application of exogenous MT significantly increased the levels of the five active components, regardless of soil moistures, with the exception of no change in the emodin under well-watered conditions. The MT treatment also up-regulated the relative expression of PcRS under both soil moistures, along with a significantly positive correlation between the relative expression of PcRS and resveratrol levels. In conclusion, exogenous MT can be employed as a biostimulant to enhance plant growth, leaf gas exchange, antioxidant enzyme activities, and active components of P. cuspidatum under drought stress conditions, which provides a reference for drought-resistant cultivation of P. cuspidatum.

Study on Transformation of The Gene of Resveratrol Synthase into Cordyceps militaris

Resveratrol synthase (RS) is a key enzyme in Resveratrol (Res) synthesis pathway. RS genes have been transformed and expressed in many plants and microorganisms, and play biological roles in plant metabolism and regulation. In this study, the full length sequence of resveratrol synthase RS gene was transformed into Cordyceps militaris by Agrobacterium tumefaciens infestation with 1179 bp. The RS gene was integrated into Cordyceps militaris genome by PCR, Southern blot hybridization, Northern hybridization and other molecular biological tests. Objective To improve the content of RS gene in into Cordyceps militaris by transgenic technology, improve the resistance of Cordyceps militaris and promote the content of resveratrol downstream products, provide a theoretical basis for studying the biological function and regulation mechanism of RS gene metabolites, and provide an effective way to improve species, resistance and increase the yield of metabolites.

Study on the Resistance of ‘Cabernet Sauvignon’ Grapevine with Different Rootstocks to Colomerus vitis

In recent years, Colomerus vitis has caused serious economic losses due to reduced grape production in Xinjiang (northwest China). Several rootstock varieties have been reported to improve the resistance of Cabernet Sauvignon to Colomerus vitis. This study explored the influence of Cabernet Sauvignon with different rootstocks on the resistance to Colomerus vitis. In particular, Cabernet Sauvignon/Cabernet Sauvignon (CS/CS) was selected as the control, and Cabernet Sauvignon grafted with five resistant rootstocks (3309C, 1103P, 140R, SO4, and 5C) was employed as the treatment. The infestation rate and injury index of Colomerus vitis to grapevines was investigated, and insect-resistant types of grapevines with different rootstocks were determined. The resveratrol (Res) content, the gene expression of resveratrol synthase (RS), and the activities of peroxidase (POD), polyphenol oxidase (PPO), catalase (CAT), and superoxide dismutase (SOD) in the leaves of each rootstock grapevine were measured. The activity of the four enzymes and the content of Res were negatively correlated with the injury index. The results revealed the ability of the rootstock to improve the resistance of grapevines to Colomerus vitis by increasing the enzyme activity or Res content. In particular, 140R, SO4, and 5C rootstocks can be employed as rootstocks of the ‘Cabernet Sauvignon’ grapevine with resistance to Colomerus vitis. The contents of Res and the four resistance enzymes studied here can be used as indexes to evaluate the insect resistance of rootstock–scion combinations.

Pterostilbene production of tomato transformed with resveratrol synthase and resveratrol O-methyltransferase genes

Pterostilbene is a methylated derivative of resveratrol. It has been proved to be effective in preventing many human diseases. However, it is produced and accumulated in only small amounts in natural plant raw materials. Here, two genes coding for resveratrol synthase 3 (AhRS3) in Arachis hypogaea and resveratrol O-methyltransferase (VvROMT) in Vitis vinifera were artificially synthesized considering the codon preference of the tomato. They were linked by LP4/2A to form a fusion gene, controlled by cauliflower mosaic virus 35S promoter, and introduced into tomato via Agrobacterium-mediated transformation. This study aimed to obtain a tomato breeding material enriched with pterostilbene in fruits for a healthy food source. Two transgenic plants with high alien gene expression were selected from the regenerated plants using real-time polymerase chain reaction. High-performance liquid chromatography was used to detect the pterostilbene content in fruits. The highest content reached 146.701 ± 47.771 µg/g dry weight, which was significantly higher than natural levels in all other species tested to date. UPLC-MS/MS was used to analyze the differences in metabolites in fruits between the transgenic and wild-type plants to understand the effect of AhRS3-LP4/2A-VvROMT gene on tomato metabolism. Results showed that the synthesis pathway of stilbenes had little influence on the flavonoid metabolic pathway in tomato fruits.

Aluminum can activate grapevine defense through actin remodeling.

In the current study, we used a grapevine cell line in which actin filaments are labeled by GFP to show that aluminum causes actin remodeling through activation of NADPH oxidase in the plasma membrane, followed by activation of phytoalexin synthesis genes. Elimination of actin filaments by latrunculin B disrupts gene activation and inhibition of MAPK signaling by the inhibitor PD98059. Interestingly, aluminum also induces the transcription of ISOCHORISMATE SYNTHASE, a key enzyme for the synthesis of salicylic acid, as well as PR1, a gene that is known to be responsive to salicylic acid. However, while salicylic acid responses are usually a hallmark of the hypersensitive response, aluminum-triggered defense is not accompanied by cell death. Both actin remodeling and gene activation in response to aluminum can be suppressed by the natural auxin indole acetic acid, suggesting that the actin response is not caused by nonspecific signaling. Further evidence for the specificity of the aluminum-triggered activation of phytoalexin synthesis genes comes from experiments in which plant peptide elicitors induce significant cellular mortality but do not evoke induction of these transcription. The response in grapevine cells can be recapitulated in grapevine leaf discs from two genotypes contrasting in stilbene inducibility. Here, aluminum can induce accumulation of the central grapevine phytoalexin, the stilbene aglycone trans-resveratrol; this is preceded by a rapid induction of transcription for RESVERATROL SYNTHASE and the regulating transcription factor MYB14. The amplitude of this induction reflects the general stilbene inducibility of these genotypes, indicating that the aluminum effect is not caused by nonspecific toxicity but by activation of specific signaling pathways. The findings are discussed in relation to a model in which actin filaments activate a specific branch of defense signaling, acting in concert with calcium-dependent PAMP-triggered immunity. This pathway links the apoplastic oxidative burst through MAPK signaling with the activation of defense-related transcription.

Expression, purification and structural characterization of resveratrol synthase from Polygonum cuspidatum

Polygonum cuspidatum, an important medicinal plant in China, is a rich source of resveratrol compounds, and its synthesis related resveratrol synthase (RS) gene is highly expressed in stems. The sequence of the resveratrol synthase was amplified with specific primers. Sequence comparison showed that it was highly homologous to the STSs. The RS gene of Polygonum cuspidatum encodes 389 amino acids and has a theoretical molecular weight of 42.4 kDa, which is called PcRS1. To reveal the molecular basis of the synthesized resveratrol activity of PcRS1, we expressed the recombinant protein of full-length PcRS1 in Escherichia coli, and soluble protein products were produced. The collected products were purified by Ni-NTA chelation chromatography and appeared as a single band on SDS-PAGE. In order to obtain higher purity PcRS1, SEC was used to purify the protein and sharp single peak, and DLS detected that the aggregation state of protein molecules was homogeneous and stable. In order to verify the enzyme activity of the high-purity PcRS1, the reaction product was detected at 303 nm. By predicting the structural information of monomer PcRS1 and PcRS1 ligand complexes, we analyzed the ligand binding pocket and protein surface electrostatic potential of the complex, and compared it with the highly homologous STSs protein structures of the iso-ligand. New structural features of protein evolution are proposed. PcRS1 obtained a more complete configuration and the optimal orientation of the active site residues, thus improving its catalytic capacity in resveratrol synthesis.

Enhancing Resveratrol Bioproduction and Anti-Melanogenic Activities through Elicitation in DJ526 Cell Suspension.

Resveratrol, a secondary plant metabolite, and its derivatives, including piceid, show several potential health-related biological activities. However, resveratrol production is uncommon in plants; thus, resveratrol-enriched rice (DJ526) is produced for its nutritional and therapeutic value. Here, a DJ526 cell suspension was treated with various elicitors to determine its resveratrol-production potential and elicit its biological activity. Treatments with most elicitors produced more piceid than resveratrol; as elicitation periods increased, the average piceid levels were 75-fold higher than resveratrol levels. This increase is associated with glycosylation during growth and development. The duration of exposure and concentrations of elicitors were crucial factors affecting resveratrol synthase expression. Of all the elicitors tested, jasmonic acid and methyl jasmonate (MeJA) were strong elicitors; they increased resveratrol production to ≤115.1 μg g−1 (total resveratrol and piceid content). Moreover, 5 μM of MeJA increased total resveratrol production by >96.4% relative to the control production. In addition, the extract of cell suspension treated with 5 μM of MeJA significantly reduced melanin content and cellular tyrosinase activity (24.2% and 21.5% relative to the control, respectively) in melan-a cells without disturbing cell viability. Overall, elicitation can enhance resveratrol production and elicit the biological activity of the compound, in this case, its anti-melanogenic activities, in DJ526 cell suspension.

Global Transcriptome Profiling Identified Transcription Factors, Biological Process, and Associated Pathways for Pre-Harvest Aflatoxin Contamination in Groundnut.

Pre-harvest aflatoxin contamination (PAC) in groundnut is a serious quality concern globally, and drought stress before harvest further exacerbate its intensity, leading to the deterioration of produce quality. Understanding the host–pathogen interaction and identifying the candidate genes responsible for resistance to PAC will provide insights into the defense mechanism of the groundnut. In this context, about 971.63 million reads have been generated from 16 RNA samples under controlled and Aspergillus flavus infected conditions, from one susceptible and seven resistant genotypes. The RNA-seq analysis identified 45,336 genome-wide transcripts under control and infected conditions. This study identified 57 transcription factor (TF) families with major contributions from 6570 genes coding for bHLH (719), MYB-related (479), NAC (437), FAR1 family protein (320), and a few other families. In the host (groundnut), defense-related genes such as senescence-associated proteins, resveratrol synthase, seed linoleate, pathogenesis-related proteins, peroxidases, glutathione-S-transferases, chalcone synthase, ABA-responsive gene, and chitinases were found to be differentially expressed among resistant genotypes as compared to susceptible genotypes. This study also indicated the vital role of ABA-responsive ABR17, which co-regulates the genes of ABA responsive elements during drought stress, while providing resistance against A. flavus infection. It belongs to the PR-10 class and is also present in several plant–pathogen interactions.

Arachis hypogaea resveratrol synthase 3 alters the expression pattern of UDP-glycosyltransferase genes in developing rice seeds.

The resveratrol-producing rice (Oryza sativa L.) inbred lines, Iksan 515 (I.515) and Iksan 526 (I.526), developed by the expression of the groundnut (Arachis hypogaea) resveratrol synthase 3 (AhRS3) gene in the japonica rice cultivar Dongjin, accumulated both resveratrol and its glucoside, piceid, in seeds. Here, we investigated the effect of the AhRS3 transgene on the expression of endogenous piceid biosynthesis genes (UGTs) in the developing seeds of the resveratrol-producing rice inbred lines. Ultra-performance liquid chromatography (UPLC) analysis revealed that I.526 accumulates significantly higher resveratrol and piceid in seeds than those in I.515 seeds and, in I.526 seeds, the biosynthesis of resveratrol and piceid reached peak levels at 41 days after heading (DAH) and 20 DAH, respectively. Furthermore, RNA-seq analysis showed that the expression patterns of UGT genes differed significantly between the 20 DAH seeds of I.526 and those of Dongjin. Quantitative real-time PCR (RT-qPCR) analyses confirmed the data from RNA-seq analysis in seeds of Dongjin, I.515 and I.526, respectively, at 9 DAH, and in seeds of Dongjin and I.526, respectively, at 20 DAH. A total of 245 UGTs, classified into 31 UGT families, showed differential expression between Dongjin and I.526 seeds at 20 DAH. Of these, 43 UGTs showed more than 2-fold higher expression in I.526 seeds than in Dongjin seeds. In addition, the expression of resveratrol biosynthesis genes (PAL, C4H and 4CL) was also differentially expressed between Dongjin and I.526 developing seeds. Collectively, these data suggest that AhRS3 altered the expression pattern of UGT genes, and PAL, C4H and 4CL in developing rice seeds.

Comparative proteomics provide insights on the basis of resistance to Aspergillus flavus infection and aflatoxin production in peanut (Arachis hypogea L.)

ABSTRACT Aspergillus flavus is an opportunistic fungal pathogen that produces carcinogenic aflatoxin, a serious constraint for food safety and human health. In this study, to better understand the molecular mechanism/s of peanut resistance to A. flavus growth and aflatoxin accumulation, comparative proteomic analysis was performed in two contrasting peanut genotypes, variety JL 24 (susceptible) and its near-isogenic resistant transgenic derivative expressing an alfalfa defensin gene. Several resistance proteins associated with secondary metabolic pathways were strongly induced in the resistant genotypes including phenylalanine ammonia lyase, cinnamic acid-4-hydroxylase, chalcone synthase, resveratrol synthase, flavanone-3-hydroxylase, lipoxygenase, diacylglycerol-glycerol-3-phosphate-3-phosphatidyltransferase, β-ketoacyl-ACP-reductase, monoacylglycerol acyltransferase, and diacylglycerol acyltransferase, indicating their roles in resistance. Besides, several putative susceptibility-associated proteins were revealed providing knowledge on potential candidate target genes for precise breeding interventions for aflatoxin mitigation. This is the first study to demonstrate comparative proteomics analysis in Aspergillus–peanut interaction using contrasting near-isogenic lines to elucidate the underlying molecular mechanisms of resistance.

Presence of resveratrol in wild Arachis species adds new value to this overlooked genetic resource

Genus Arachis comprises 82 species distributed into nine taxonomic sections. Most Arachis species are wild and those from Arachis section have been evaluated for many traits, since they can be used in peanut breeding. Most of the remaining species have been neglected and understudied. Recently, resveratrol content and expression of a resveratrol synthase gene were analyzed in wild Arachis species. Our aim was to expand the knowledge about resveratrol in Arachis, analyzing species from five sections and evaluating the expression of a resveratrol synthase (RS) gene responsive to ultraviolet light (UV) along the time. In a first experiment, the resveratrol content after UV induction was analyzed on detached leaves of 12 species from five sections. Variation was observed among species and accessions of the same species. The highest contents were found in A. lignosa (843.9 μg/g) and A. triseminata (745.4 μg/g). In a second experiment, RS expression and resveratrol content in four species and one synthetic amphidiploid were analyzed at 0, 7, 15 and 24 h pos induction (hpi) with UV. In most genotypes, the highest RS expression level was at 0 hpi, whereas the highest resveratrol content was at 15 hpi. Our results suggested that resveratrol is ubiquitously present in the genus Arachis with different capacities of synthesis among species and accessions in response to ultraviolet treatment. Presence of resveratrol in wild Arachis species adds new value to these genetic resources.

De novo resveratrol production through modular engineering of an Escherichia coli\u2013Saccharomyces cerevisiae co-culture

BackgroundResveratrol is a plant secondary metabolite with diverse, potential health-promoting benefits. Due to its nutraceutical merit, bioproduction of resveratrol via microbial engineering has gained increasing attention and provides an alternative to unsustainable chemical synthesis and straight extraction from plants. However, many studies on microbial resveratrol production were implemented with the addition of water-insoluble phenylalanine or tyrosine-based precursors to the medium, limiting in the sustainable development of bioproduction.ResultsHere we present a novel coculture platform where two distinct metabolic background species were modularly engineered for the combined total and de novo biosynthesis of resveratrol. In this scenario, the upstream Escherichia coli module is capable of excreting p-coumaric acid into the surrounding culture media through constitutive overexpression of codon-optimized tyrosine ammonia lyase from Trichosporon cutaneum (TAL), feedback-inhibition-resistant 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase (aroGfbr) and chorismate mutase/prephenate dehydrogenase (tyrAfbr) in a transcriptional regulator tyrR knockout strain. Next, to enhance the precursor malonyl-CoA supply, an inactivation-resistant version of acetyl-CoA carboxylase (ACC1S659A,S1157A) was introduced into the downstream Saccharomyces cerevisiae module constitutively expressing codon-optimized 4-coumarate-CoA ligase from Arabidopsis thaliana (4CL) and resveratrol synthase from Vitis vinifera (STS), and thus further improve the conversion of p-coumaric acid-to-resveratrol. Upon optimization of the initial inoculation ratio of two populations, fermentation temperature, and culture time, this co-culture system yielded 28.5 mg/L resveratrol from glucose in flasks. In further optimization by increasing initial net cells density at a test tube scale, a final resveratrol titer of 36 mg/L was achieved.ConclusionsThis is first study that demonstrates the use of a synthetic E. coli–S. cerevisiae consortium for de novo resveratrol biosynthesis, which highlights its potential for production of other p-coumaric-acid or resveratrol derived biochemicals.

Study on the anticancer biological mechanism of Resveratrol

Trans 3, 4 ′, 5-3 hydroxy 2 styrene (resveratrol) is a kind of naturally occurring polyphenols phytoalexin. Resveratrol has significant anti-cancer activity, mainly exists in grapes, berries, and peanuts, has anti-aging, protect the heart, antioxidant, antiproliferation, promote apoptosis and immune regulation. Resveratrol has been widely concerned in the treatment of cancer and autoimmune diseases resveratrol. Resveratrol synthase (RS) is a key enzyme in the synthesis of resveratrol synthase. In this study, RS containing genes were placed under the control of fruit-specific promoter RJ39 to transform tomatoes by transgenic method. The extraction of fruits containing RS genes showed an obvious absorption peak on the HPLC chromatographic map, and it also had an obvious inhibitory effect on the growth of Hela cells.

Changes in the chlorophyll content of grape leaves could provide a physiological index for responses and adaptation to UV‐C radiation

Stilbenes are a group of phytoalexins that play an important role in grapevine (Vitis) basal immunity and can be induced by biotic and abiotic stresses. The levels of chlorophylls, the main pigments in plant cells, can also indicate the tolerance of plants to various stresses. Here, the response of different grapevine genotypes to UV‐C radiation treatment was tested and the abundance of chlorophyll in the Hoe29 and Ke53 genotypes was observed to increase significantly within 6 h of UV‐C treatment. Conversely, chlorophyll levels decreased markedly in the Augster Weiss and Müller–Thurgau genotypes. Furthermore, stilbene abundance increased substantially in the Hoe29, Ke53, Ke83 and Pinot Blanc genotypes, but increased only slightly in Augster Weiss and Müller–Thurgau. The expression of resveratrol synthase, which encodes a key enzyme in the stilbene synthesis pathway, increased in Hoe29, Ke53, Ke83 and Pinot Blanc following UV‐C treatment, in a manner consistent with stilbene accumulation. In addition, we observed that reactive oxygen species (ROS) provide a key trigger in physiological responses and changes in secondary metabolite contents. In summary, the results from this study support a link between ROS, chlorophyll levels and genetic diversity for stilbene abundance in different grape genotypes, providing insights into mechanisms for plant physiological and biochemical responses and adaptations to stress.

The transcription factor MYB15 is essential for basal immunity (PTI) in Chinese wild grape.

MYB15 promoter of Vitis quinquangularis has potential as a target for disease resistance breeding, and its involvement in PTI is associated with a range of defense mechanisms. China is a center of origin for Vitis and is home to diverse wild Vitis genotypes, some of which show superior pathogen resistance, although the underlying molecular basis for this has not yet been elucidated. In the current study, we identified a transcription factor, MYB15, from the Chinese wild grape, Vitis quinquangularis, whose promoter region (pVqMYB15) was shown to be induced by basal immunity (also called PAMP-triggered immunity, PTI) triggered by flg22, following heterologous expression in Nicotiana benthamiana and homologous expression in grapevine. By analyzing the promoter structure and activity, we identified a unique 283bp sequence that plays a key role in the activation of basal immunity. In addition, we showed that activation of the MYB15 promoter correlates with differences in the expression of MYB15 and RESVERATROL SYNTHASE (RS) induced by the flg22 elicitor. We further tested whether the MYB15 induction triggered by flg22 was consistent with MYB15 and RS expression following inoculation with Plasmopara viticola in grape (V. quinquangularis and Vitis vinifera) leaves. Mapping upstream signals, we found that calcium influx, an RboH-dependent oxidative burst, an MAPK cascade, and jasmonate and salicylic acid co-contributed to flg22-triggered pVqMYB15 activation. Our data suggest that the MYB15 promoter has potential as a target for disease resistance breeding, and its involvement in PTI is associated with a range of defense mechanisms.

A specific allele of MYB14 in grapevine correlates with high stilbene inducibility triggered by Al3+ and UV-C radiation

Key messageThe structural differences of MYB14 promoter in two grapevine genotypes affect the expression of MYB14 and stilbene synthesis in response to Al3+ and UV-C radiation.Grapevines provide an important fruit crop worldwide, but production is often limited by pathogen infection. Stilbenes, a class of secondary metabolite, represent phytoalexins that contribute to defence against pathogens in many plants, including grapevine. It is known that the transcription factors MYB14 and MYB15 are required for the activation of the promoters of resveratrol synthase to regulate stilbene biosynthesis. In the current study, we observed that stilbene levels were more highly induced by Al3+ and UV-C radiation treatments in the cultivar Vitis labrusca ‘Concord’ than in the cultivar V. vinifera ‘Cabernet Sauvignon’. We investigated whether genetic/structural variations in the MYB14 and MYB15 promoters between these two representative genotypes are responsible for the differences in stilbene accumulation. Significant differences in the structure and activity of the promoter of MYB14, but not MYB15 were identified between the two genotypes, following heterologous expression in Nicotiana benthamiana system and treatments with Al3+ and UV-C. Hydrogen peroxide (H2O2) was detected in Concord soon after the stress treatments, but after diphenyleneiodonium chloride pre-treatment, the expressing level of VlMYB14, the promoter activity of VlMYB14 and the accumulation of stilbenes was significantly reduced. A model is presented where the induction of MYB14 contributes to stilbene accumulation in Concord following Al3+ and UV-C treatments involving reactive oxygen species (ROS) production as an early signal.

Production of resveratrol in Flammulina velutipes by combinatorial expression of its biosynthetic genes

Flammulina velutipes is cultivated commercially throughout the world because of its rich nutrients and bioactive substances, especially the precursor for resveratrol biosynthesis, p-coumaric acid. In this paper, a bioconversion system was established for the production of resveratrol in F. velutipes. The expression vector pgfvs-4cl-vrs2 containing 4-coumarate: coenzyme A ligase gene (4cl) and resveratrol synthase gene (rs) from Vitis vinifera was constructed and transformed into F. velutipes. PCR, Southern blotting, and RT-PCR were used to identify positive transformants. Results indicated that the 4cl and rs genes have been successfully integrated into the genome of F. velutipes and could show normal transcription. High-Pressure Liquid Chromatography (HPLC) analysis indicated that the 4cl and rs genes were functionally expressed in three transformants, TF71, TF7H, and TF7E, which have an ability to convert p-coumaric acid into resveratrol. The contents of resveratrol for the transgenic strain TF71 were 2.77 μg/g, 1.60 μg/L, and 1.39 μg/g in the mycelia, fermentation liquid, and fruiting body (dry weight), respectively. The contents of the transgenic strain TF7H were 1.67 μg/g, 1.60 μg/L, and 1.01 μg/g, and those of the transgenic strain TF7E were 0.06 μg/g, 3.4 μg/L, and 1.13 μg/g, respectively. The present results indicated that establishing a metabolic pathway for resveratrol production in F. velutipes was feasible, thereby adding a new F. velutipes functional ingredient.