Stilbene Synthase Research Articles

Postharvest changes in the phenolic and free volatile compound contents in Shine Muscat grapes at room temperature

Herein, we studied changes in the contents of phenolic and free volatile compounds in Shine Muscat grapes stored at room temperature. Berry quality was maintained up to 11 d after harvest, and the levels of 35 phenolic compounds were observed to increase during storage. This increase is attributed to the upregulation of genes, including phenylalanine ammonia-lyases, 4-coumarate–CoA ligases, and stilbene synthases, in the phenylpropanoid pathway. The concentrations of total and rose-flavored volatiles, including terpenes and particularly monoterpenes, decreased in postharvest berries, which was attributed to the downregulation of genes in the mevalonate and 2-C-methyl-D-erythritol 4-phosphate pathways. By contrast, the C6 compound content increased during storage, which might have played a role in the upregulation of lipoxygenase and hydroperoxide. Additionally, the marker compounds rutin and 1-hexanol were identified during storage. Therefore, this study suggested that the health benefits and C6 compound–derived flavor increased, whereas the rose flavor decreased in postharvest berries.

The WRKY Family Transcription Factor GmWRKY72 Represses Glyceollin Phytoalexin Biosynthesis in Soybean.

Phytoalexins are plant defense metabolites that are biosynthesized transiently in response to pathogens. Despite that their biosynthesis is highly restricted in plant tissues, the transcription factors that negatively regulate phytoalexin biosynthesis remain largely unknown. Glyceollins are isoflavonoid-derived phytoalexins that have critical roles in protecting soybean crops from the oomycete pathogen Phytophthora sojae. To identify regulators of glyceollin biosynthesis, we used a transcriptomics approach to search for transcription factors that are co-expressed with glyceollin biosynthesis in soybean and stilbene synthase phytoalexin genes in grapevine. We identified and functionally characterized the WRKY family protein GmWRKY72, which is one of four WRKY72-type transcription factors of soybean. Overexpressing and RNA interference silencing of GmWRKY72 in the soybean hairy root system decreased and increased expression of glyceollin biosynthetic genes and metabolites, respectively, in response to wall glucan elicitor from P. sojae. A translational fusion with green fluorescent protein demonstrated that GFP-GmWRKY72 localizes mainly to the nucleus of soybean cells. The GmWRKY72 protein directly interacts with several glyceollin biosynthetic gene promoters and the glyceollin transcription factor proteins GmNAC42-1 and GmMYB29A1 in yeast hybrid systems. The results show that GmWRKY72 is a negative regulator of glyceollin biosynthesis that may repress biosynthetic gene expression by interacting with transcription factor proteins and the DNA of glyceollin biosynthetic genes.

CRISPR/Cas9 mediated CHS2 mutation provides a new insight into resveratrol biosynthesis by causing a metabolic pathway shift from flavonoids to stilbenoids in Vitis davidii cells

Abstract Resveratrol is an important phytoalexin that adapts to and responds to stressful conditions, plays various roles in health and medical therapies. However, it is only found in a limited number of plant species in low concentrations, which hinders its development and utilization. Chalcone synthase (CHS) and stilbene synthase (STS) catalyze the same substrates to produce flavonoids and resveratrol, respectively. However, it remains unclear how CHS and STS compete in metabolite synthesis. In this study, two CHS2 mutant cell lines (MT1 and MT2) were generated using CRISPR/Cas9 genome editing. These CHS2 mutant cell lines exhibited abundant mutations in CHS2, leading to the premature termination of protein translation and subsequent CHS2 knockout. Amplicon sequencing confirmed comprehensive CHS2 knockout in MT1, whereas the wild-type sequence remained predominant in the MT2 cell line. Transcriptome and RT-qPCR results showed a significant downregulation of genes involved in flavonoid biosynthesis, including CHS2, CHS3, F3H, F3’H, DFR, FLS, LDOX, among others, resulting in decreased flavonoid accumulation, such as anthocyanins, proanthocyanidins, quercetin, and kaempferol. Conversely, STS genes involved in stilbenoid biosynthesis were upregulated competing with the flavonoid pathway. Consequently, there was a marked increase in stilbenoids, including resveratrol, piceatannol, piceid and pterostilbene, with a 4.1-fold increase in resveratrol and a 5.3-fold increase in piceid (a derivative of resveratrol) observed in CHS2 mutant cell lines. This research demonstrates that CHS2 mutation induces a shift from flavonoid biosynthesis towards stilbenoid biosynthesis, offering new insights into metabolite biosynthesis and regulation, as well as an alternative solution for natural resveratrol production, and a novel breeding approach for eliminating non-target agronomic traits using CRISPR-Cas9.

Soil-Mulching Treatment Enhances the Content of Stilbene in Grape Berries: A Transcriptomic and Metabolomic Analysis

Soil mulching is a useful agronomic practice that promotes early fruit maturation and affects fruit quality. However, the regulatory mechanism of fruit metabolites under soil-mulching treatments remains unknown. In this study, variations in the gene sets and metabolites of grape berries after mulching (rice straw + felt + plastic film) using transcriptome and metagenomic sequencing were investigated. The results of the cluster analysis and orthogonal projection to latent structures discriminant analysis of the metabolites showed a difference between the mulching and control groups, as did the principal component analysis results for the transcriptome. In total, 36 differentially expressed metabolites were identified, of which 10 (resveratrol, ampelopsin F, piceid, 3,4′-dihydroxy-5-methoxystilbene, ε-viniferin, trans resveratrol, epsilon-viniferin, 3′-hydroxypterostilbene, 1-methyl-resveratrol, and pterostil-bene) were stilbenes. Their content increased after mulching, indicating that stilbene synthase activity increased after mulching. The weighted gene co-expression network analysis revealed that the turquoise and blue modules were positively and negatively related to stilbene compounds. The network analysis identified two seed genes (VIT_09s0054g00610, VIT_13s0156g00260) and two transcription factors (VIT_13s0156g00260, VIT_02s0025g04590). Overall, soil mulching promoted the accumulation of stilbene compounds in grapes, and the results provided key genetic information for further studies.

Tripartite interactions between grapevine, viruses, and arbuscular mycorrhizal fungi provide insights into modulation of oxidative stress responses

Arbuscular mycorrhizal fungi (AMF) can be beneficial for plants exposed to abiotic and biotic stressors. Although widely present in agroecosystems, AMF influence on crop responses to virus infection is underexplored, particularly in woody plant species such as grapevine. Here, a two-year greenhouse experiment was set up to test the hypothesis that AMF alleviate virus-induced oxidative stress in grapevine. The ‘Merlot’ cultivar was infected with three grapevine-associated viruses and subsequently colonized with two AMF inocula, containing one or three species, respectively. Five and fifteen months after AMF inoculation, lipid peroxidation - LPO as an indicator of oxidative stress and indicators of antioxidative response (proline, ascorbate - AsA, superoxide dismutase - SOD, ascorbate- APX and guaiacol peroxidases - GPOD, polyphenol oxidase - PPO, glutathione reductase - GR) were analysed. Expression of genes coding for a stilbene synthase (STS1), an enhanced disease susceptibility (EDS1) and a lipoxygenase (LOX) were determined in the second harvesting. AMF induced reduction of AsA and SOD over both years, which, combined with not AMF-triggered APX and GR, suggests decreased activation of the ascorbate-glutathione cycle. In the mature phase of the AM symbiosis establishment GPOD emerged as an important mechanism for scavenging H2O2 accumulation. These results, together with reduction in STS1 and increase in EDS1 gene expression, suggest more efficient reactive oxygen species scavenging in plants inoculated with AMF. Composition of AMF inocula was important for proline accumulation. Overall, our study improves the knowledge on ubiquitous grapevine-virus-AMF systems in the field, highlighting that established functional AM symbiosis could reduce virus-induced stress.

Metabolome and Transcriptome Joint Analysis Reveals That Different Sucrose Levels Regulate the Production of Flavonoids and Stilbenes in Grape Callus Culture.

To reveal the effect of sucrose concentration on the production of secondary metabolites, a metabolome and transcriptome joint analysis was carried out using callus induced from grape variety Mio Red cambial meristematic cells. We identified 559 metabolites-mainly flavonoids, phenolic acids, and stilbenoids-as differential content metabolites (fold change ≥2 or ≤0.5) in at least one pairwise comparison of treatments with 7.5, 15, or 30 g/L sucrose in the growing media for 15 or 30 days (d). Resveratrol, viniferin, and amurensin contents were highest at 15 d of subculture; piceid, ampelopsin, and pterostilbene had higher contents at 30 d. A transcriptome analysis identified 1310 and 498 (at 15 d) and 1696 and 2211 (at 30 d) differentially expressed genes (DEGs; log2(fold change) ≥ 1, p < 0.05) in 7.5 vs. 15 g/L and 15 vs. 30 g/L sucrose treatments, respectively. In phenylpropane and isoflavone pathways, DEGs encoding cinnamic acid 4-hydroxylase, chalcone synthase, chalcone isomerase, and flavanone 3-hydroxylase were more highly expressed at 15 d than at 30 d, while other DEGs showed different regulation patterns corresponding to sucrose concentrations and cultivation times. For all three sucrose concentrations, the stilbene synthase (STS) gene exhibited significantly higher expression at 15 vs. 30 d, while two resveratrol O-methyltransferase (ROMT) genes related to pterostilbene synthesis showed significantly higher expression at 30 vs. 15 d. In addition, a total of 481 DEGs were annotated as transcription factors in pairwise comparisons; an integrative analysis suggested MYB59, WRKY20, and MADS8 as potential regulators responding to sucrose levels in flavonoid and stilbene biosynthesis in grape callus. Our results provide valuable information for high-efficiency production of flavonoids and stilbenes using grape callus.

Unlocking the Transcriptional Reprogramming Repertoire between Variety-Dependent Responses of Grapevine Berries to Infection by Aspergillus carbonarius.

Aspergillus carbonarius causes severe decays on berries in vineyards and is among the main fungal species responsible for grape contamination by ochratoxin A (OTA), which is the foremost mycotoxin produced by this fungus. The main goal of this study was to investigate at the transcriptome level the comparative profiles between two table grape varieties (Victoria and Fraoula, the white and red variety, respectively) after their inoculation with a virulent OTA-producing A. carbonarius strain. The two varieties revealed quite different transcriptomic signatures and the expression profiles of the differential expressed genes (DEGs) highlighted distinct and variety-specific responses during the infection period. The significant enrichment of pathways related to the modulation of transcriptional dynamics towards the activation of defence responses, the triggering of the metabolic shunt for the biosynthesis of secondary metabolites, mainly phenylpropanoids, and the upregulation of DEGs encoding phytoalexins, transcription factors, and genes involved in plant-pathogen interaction and immune signaling transduction was revealed in an early time point in Fraoula, whereas, in Victoria, any transcriptional reprogramming was observed after a delay. However, both varieties, to some extent, also showed common expression dynamics for specific DEG families, such as those encoding for laccases and stilbene synthases. Jasmonate (JA) may play a critical modulator role in the defence machinery as various JA-biosynthetic DEGs were upregulated. Along with the broader modulation of the transcriptome that was observed in white grape, expression profiles of specific A. carbonarius genes related to pathogenesis, fungal sporulation, and conidiation highlight the higher susceptibility of Victoria. Furthermore, the A. carbonarius transcriptional patterns directly associated with the regulation of the pathogen OTA-biosynthesis gene cluster were more highly induced in Victoria than in Fraoula. The latter was less contaminated by OTA and showed substantially lower sporulation. These findings contribute to uncovering the interplay beyond this plant-microbe interaction.

Denovo Production of Resveratrol by Engineered Rice Wine Strain Saccharomyces cerevisiae HJ08 and Its Application in Rice Wine Brewing.

Resveratrol is a plant-derived polyphenolic compound with numerous biological activities and health-promoting properties. Rice wine is a popular traditional alcoholic beverage made from fermented rice grains, and widely consumed in Asia. To develop resveratrol-enriched rice wine, a heterologous resveratrol biosynthesis pathway was established by integrating the 4-coumaroyl-CoA ligase (Pc4CL) and the stilbene synthase (VvSTS) from Petroselinum crispum and Vitis vinifera at the δ locus sites of industrial rice wine strains Saccharomyces cerevisiae HJ. The resulting S. cerevisiae HJ01 produced a level of 0.6 ± 0.01 mg/L resveratrol. Next, the resveratrol production was increased 16.25-fold through employing the fused protein Pc4CL::VvSTS with a rigidly linked peptide (TPTP, EAAAK). Then, the strains were further modified by removing feedback inhibition of tyrosine through point mutation of ARO4 and ARO7, which integrated at the rDNA region of strain HJ03, and generated strain HJ06, HJ07, and HJ08. Subsequently, the highest resveratrol titer (34.22 ± 3.62 mg/L) was obtained by optimizing fermentation time and precursor addition amount. Finally, resveratrol content of rice wine fermented with strain HJ08 was 2.04 ± 0.08 mg/L and 1.45 ± 0.06 mg/L with or without the addition of 400 mg/L tyrosine after 7 days fermentation.

De Novo Synthesis of Resveratrol from Sucrose by Metabolically Engineered Yarrowia lipolytica.

Resveratrol, a phenylpropanoid compound, exhibits diverse pharmacological properties, making it a valuable candidate for health and disease management. However, the demand for resveratrol exceeds the capacity of plant extraction methods, necessitating alternative production strategies. Microbial synthesis offers several advantages over plant-based approaches and presents a promising alternative. Yarrowia lipolytica stands out among microbial hosts due to its safe nature, abundant acetyl-CoA and malonyl-CoA availability, and robust pentose phosphate pathway. This study aimed to engineer Y. lipolytica for resveratrol production. The resveratrol biosynthetic pathway was integrated into Y. lipolytica by adding genes encoding tyrosine ammonia lyase from Rhodotorula glutinis, 4-coumarate CoA ligase from Nicotiana tabacum, and stilbene synthase from Vitis vinifera. This resulted in the production of 14.3 mg/L resveratrol. A combination of endogenous and exogenous malonyl-CoA biosynthetic modules was introduced to enhance malonyl-CoA availability. This included genes encoding acetyl-CoA carboxylase 2 from Arabidopsis thaliana, malonyl-CoA synthase, and a malonate transporter protein from Bradyrhizobium diazoefficiens. These strategies increased resveratrol production to 51.8 mg/L. The further optimization of fermentation conditions and the utilization of sucrose as an effective carbon source in YP media enhanced the resveratrol concentration to 141 mg/L in flask fermentation. By combining these strategies, we achieved a titer of 400 mg/L resveratrol in a controlled fed-batch bioreactor. These findings demonstrate the efficacy of Y. lipolytica as a platform for the de novo production of resveratrol and highlight the importance of metabolic engineering, enhancing malonyl-CoA availability, and media optimization for improved resveratrol production.

Combined analysis of Polygonum cuspidatum transcriptome and metabolome revealed that PcMYB62, a transcription factor, responds to methyl jasmonate and inhibits resveratrol biosynthesis

A comparative transcriptomic and metabolomic analysis of Polygonum cuspidatum leaves treated with MeJA was carried out to investigate the regulatory mechanisms of its active compounds. A total of 692 metabolites and 77,198 unigenes were obtained, including 200 differentially accumulated metabolites and 6819 differentially expressed genes. We screened potential regulatory transcription factors involved in resveratrol and flavonoids biosynthesis, and successfully identified an MYB transcription factor, PcMYB62, which could significantly decrease the resveratrol content in P. cuspidatum leaves when over-expressed. PcMYB62 could directly bind to the MBS motifs in the promoter region of stilbene synthase (PcSTS) gene and repress its expression. Besides, PcMYB62 could also repress PcSTS expression and resveratrol biosynthesis in transgenic Arabidopsis thaliana. Our results provide abundant candidate genes for further investigation, and the new finding of the inhibitory role of PcMYB62 on the resveratrol biosynthesis could also potentially be used in metabolic engineering of resveratrol in P. cuspidatum.

Carbon dioxide valorization into resveratrol via lithoautotrophic fermentation using engineered Cupriavidus necator H16

BackgroundIndustrial biomanufacturing of value-added products using CO2 as a carbon source is considered more sustainable, cost-effective and resource-efficient than using common carbohydrate feedstocks. Cupriavidus necator H16 is a representative H2-oxidizing lithoautotrophic bacterium that can be utilized to valorize CO2 into valuable chemicals and has recently gained much attention as a promising platform host for versatile C1-based biomanufacturing. Since this microbial platform is genetically tractable and has a high-flux carbon storage pathway, it has been engineered to produce a variety of valuable compounds from renewable carbon sources. In this study, the bacterium was engineered to produce resveratrol autotrophically using an artificial phenylpropanoid pathway.ResultsThe heterologous genes involved in the resveratrol biosynthetic pathway—tyrosine ammonia lyase (TAL), 4-coumaroyl CoA ligase (4CL), and stilbene synthase (STS) —were implemented in C. necator H16. The overexpression of acetyl-CoA carboxylase (ACC), disruption of the PHB synthetic pathway, and an increase in the copy number of STS genes enhanced resveratrol production. In particular, the increased copies of VvSTS derived from Vitis vinifera resulted a 2-fold improvement in resveratrol synthesis from fructose. The final engineered CR-5 strain produced 1.9 mg/L of resveratrol from CO2 and tyrosine via lithoautotrophic fermentation.ConclusionsTo the best of our knowledge, this study is the first to describe the valorization of CO2 into polyphenolic compounds by engineering a phenylpropanoid pathway using the lithoautotrophic bacterium C. necator H16, demonstrating the potential of this strain a platform for sustainable chemical production.

The near‐complete genome assembly of Reynoutria multiflora reveals the genetic basis of stilbene and anthraquinone biosynthesis

AbstractReynoutria multiflora is a widely used medicinal plant in China. Its medicinal compounds are mainly stilbenes and anthraquinones which possess important pharmacological activities in anti‐aging, anti‐inflammatory and anti‐oxidation, but their biosynthetic pathways are still largely unresolved. Here, we reported a near‐complete genome assembly of R. multiflora consisting of 1.39 Gb with a contig N50 of 122.91 Mb and only one gap left. Genome evolution analysis revealed that two recent bursts of long terminal repeats (LTRs) contributed significantly to the increased genome size of R. multiflora, and numerous large chromosome rearrangements were observed between R. multiflora and Fagopyrum tataricum genomes. Comparative genomics analysis revealed that a recent whole‐genome duplication specific to Polygonaceae led to a significant expansion of gene families associated with disease tolerance and the biosynthesis of stilbenes and anthraquinones in R. multiflora. Combining transcriptomic and metabolomic analyses, we elucidated the molecular mechanisms underlying the dynamic changes in content of medicinal ingredients in R. multiflora roots across different growth years. Additionally, we identified several putative key genes responsible for anthraquinone and stilbene biosynthesis. We identified a stilbene synthase gene PM0G05131 highly expressed in roost, which may exhibit an important role in the accumulation of stilbenes in R. multiflora. These genomic data will expedite the discovery of anthraquinone and stilbenes biosynthesis pathways in medicinal plants.

Stimulation of secondary metabolism in grape berry exocarps by a nature-based strategy of foliar application of polyols

In grapes under drought stress, polyols accumulate through tight coordination at the molecular level between increased membrane transport of polyols and inhibition of polyol oxidation. Here, the effects on grape metabolism of an exogenous foliar application of polyols as a potential sustainable viticultural practice to increase grapevine performance and berry quality were thoroughly assessed. Grapevines were pulverised with a polyol solution containing 2 mM mannitol and 2 mM sorbitol, and the metabolome of grape berry exocarps and important metabolic pathways associated with berry quality and polyol metabolism were analysed at véraison and mature stages. By combining metabolomics analysis using UPLC-MS, enzyme activity assays and targeted transcriptional analyses, it was demonstrated that foliar application of polyols stimulated by 3.5-fold and 6-fold abscisic acid (ABA) biosynthesis at véraison and mature grape berries, respectively. It also stimulated the concentration of anthocyanins, stilbenes and total phenolics in exocarps through the upregulation of phenylpropanoid, stilbenoid and anthocyanin biosynthetic pathways shown by increases in phenylalanine-ammonia lyase (PAL) activity (3-fold) and VviPAL1 expression, stilbene synthase 1 (VviSTS1) transcripts (ca. 5-fold), UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) activity and VviUFGT1 expression, among other results, at the mature stage, when these changes were most noticeable. Many secondary metabolites synthesised in these pathways identified by UPLC-MS analysis were present at higher quantities in exocarps from polyol-treated plants such as fertaric acid, E-resveratrol, E-piceatannol, piceid, pallidol, E-ε-viniferin, myricetin-hexoside 1, cyanidin-3-O-glucoside and malvidin-3-O-(6-p-coumaroyl)-glucoside. Foliar application of low-concentration polyols is, therefore, a promising biostimulant-based strategy to improve grape berry quality and nutritional value in the current context of climate change.

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.

A Pinus strobus transcription factor PsbHLH1 activates the production of pinosylvin stilbenoids in transgenic Pinus koraiensis calli and tobacco leaves.

Transcription factors (TFs) play an important role in regulating the biosynthesis of secondary metabolites. In Pinus strobus, the level of methylated derivatives of pinosylvin is significantly increased upon pine wood nematode (PWN) infection, and these compounds are highly toxic to PWNs. In a previous study, we found that the expression of a basic helix-loop-helix TF gene, PsbHLH1, strongly increased in P. strobus plants after infection with PWNs. In this study, we elucidated the regulatory role of the PsbHLH1 gene in the production of methylated derivatives of pinosylvin such as pinosylvin monomethyl ether (PME) and dihydropinoylvin monomethyl ether (DPME). When PsbHLH1 was overexpressed in Pinus koraiensis calli, the production of PME and DPME was significantly increased. Overexpression of the stilbene synthase (PsSTS) and pinosylvin methyl transferase (PsPMT) genes, known as key enzymes for the biosynthesis of methylated pinosylvins, did not change PME or DPME production. Moreover, PME and DPME were not produced in tobacco leaves when the PsSTS and PsPMT genes were transiently coexpressed. However, the transient expression of three genes, PsSTS, PsPMT, and PsbHLH1, resulted in the production of PME and DPME in tobacco leaves. These results prove that PsbHLH1 is an important TF for the pinosylvin stilbene biosynthesis in pine plants and plays a regulatory role in the engineered production of PME and DPME in tobacco plants.

Tomato plants expressing a stilbene synthase gene display genotype‐depending alterations in metabolome profile and antioxidant system

AbstractIn the present work, we report a study on tomato Micro‐Tom lines expressing the Vitis vinifera StSy1 cDNA encoding stilbene synthase under the control of a CaMV 35S promoter. Transgenic lines (T1 plants) were evaluated in terms of vegetative and reproductive development, fruit production, the activity of the main antioxidant enzymes, and the metabolome profile in both leaves and fruit juices. Trans‐resveratrol and glycosylated derivates accumulated in some of the transgenic lines generated, the concentration of both compounds being higher in the leaves than in the fruit juices. Vegetative development was enhanced by transformation, whereas fruit production decreased, and fruit soluble solids content (°Bx) increased. Remarkably, trans‐resveratrol content in leaves and fruit juices showed no correlation with the expression levels of StSy1. An alteration of the metabolome profile and antioxidant enzyme activities was recorded in most of the transgenic lines. In tomato fruit juices, the variation between the wild type and transgenic lines was attributed to a major number of differentially expressed metabolites as compared to that observed in the leaves. In this sense, the major changes corresponded to an induction of phenylpropanoid pathway‐related compounds in the lines accumulating trans‐resveratrol. Transgenic lines producing resveratrol showed, overall, an imbalanced antioxidant metabolism characterized by higher superoxide dismutase (H2O2‐producing enzyme) activity and lower H2O2‐scavenging enzyme activities. This work contributes to deciphering the biochemical consequences of StSy1 introduction in tomato and opens up prospects for further characterization of the transformants.

NAC61 regulates late- and post-ripening osmotic, oxidative, and biotic stress responses in grapevine.

During late- and post-ripening stages, grape berry undergoes profound biochemical and physiological changes whose molecular control is poorly understood. Here, we report the role of NAC61, a grapevine NAC transcription factor, in regulating different processes involved in berry ripening progression. NAC61 is highly expressed during post-harvest berry dehydration and its expression pattern is closely related to sugar concentration. The ectopic expression of NAC61 in Nicotiana benthamiana leaves resulted in low stomatal conductance, high leaf temperature, tissue collapse and a higher relative water content. Transcriptome analysis of grapevine leaves transiently overexpressing NAC61 and DNA affinity purification and sequencing analyses allowed us to narrow down a list of NAC61-regulated genes. Direct regulation of the stilbene synthase regulator MYB14, the osmotic stress-related gene DHN1b, the Botrytis cinerea susceptibility gene WRKY52, and NAC61 itself was validated. We also demonstrate that NAC61 interacts with NAC60, a proposed master regulator of grapevine organ maturation, in the activation of MYB14 and NAC61 expression. Overall, our findings establish NAC61 as a key player in a regulatory network that governs stilbenoid metabolism and osmotic, oxidative, and biotic stress responses that are the hallmark of late- and post-ripening grape stages.

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.

The Stilbene Synthase Family in Arachis: A Genome-Wide Study and Functional Characterization in Response to Stress.

Peanut (Arachis hypogaea) and its wild relatives are among the few species that naturally synthesize resveratrol, a well-known stilbenoid phytoalexin that plays a crucial role in plant defense against biotic and abiotic stresses. Resveratrol has received considerable attention due to its health benefits, such as preventing and treating various human diseases and disorders. Chalcone (CHS) and Stilbene (STS) Synthases are plant-specific type III Polyketide Synthases (PKSs) that share the same substrates and are key branch enzymes in the biosynthesis of flavonoids and stilbenoids, respectively. Although resveratrol accumulation in response to external stimulus has been described in peanut, there are no comprehensive studies of the CHS and STS gene families in the genus Arachis. In the present study, we identified and characterized 6 CHS and 46 STS genes in the tetraploid peanut and an average of 4 CHS and 22 STS genes in three diploid wild species (Arachis duranensis, Arachis ipaënsis and Arachis stenosperma). The CHS and STS gene and protein structures, chromosomal distributions, phylogenetic relationships, conserved amino acid domains, and cis-acting elements in the promoter regions were described for all Arachis species studied. Based on gene expression patterns of wild A. stenosperma STS genes in response to different biotic and abiotic stresses, we selected the candidate AsSTS4 gene, which is strongly induced by ultraviolet (UV) light exposure, for further functional investigation. The AsSTS4 overexpression in peanut hairy roots significantly reduced (47%) root-knot nematode infection, confirming that stilbene synthesis activation in transgenic plants can increase resistance to pathogens. These findings contribute to understanding the role of resveratrol in stress responses in Arachis species and provide the basis for genetic engineering for improved production of valuable secondary metabolites in plants.

Denovo production of resveratrol by engineered Saccharomyces cerevisiae W303-1a using pretreated Gracilaria corticata extracts.

Assembly and construction of resveratrol production pathway in Saccharomyces cerevisiae for denovo production of resveratrol using seaweed extract as fermentation medium. Genes involved in the production of resveratrol from tyrosine pathway, tyrosine ammonia lyase (FTAL) gene from Flavobacterium johnsoniae (FjTAL), the 4-coumarate:CoA ligase gene from Arabidopsis thaliana (4CL1) and the stilbene synthase gene from Vitis vinifera (VvSTS) were introduced into low copy, high copy and integrative vector and transformed into S. cerevisiae W303-1a. The resulting strains W303-1a/pARS-res5, W303-1a/2µ-res1 and W303-1a/IntUra-res9 produced a level of 2.39 ± 0.01, 3.33 ± 0.03 and 8.34 ± 0.03mg resveratrol l-1 respectively. CRISPR mediated integration at the δ locus resulted in 17.13 ± 1.1mg resveratrol l-1. Gracilaria corticata extract was tested as a substrate for the growth of transformant to produce resveratrol. The strain produced a comparable level, 13.6 ± 0.54mg resveratrol l-1 when grown in seaweed extract medium. The strain W303-1a/IntδC-res1 utilized Gracillaria hydrolysate and produced 13.6 ± 0.54mg resveratrol l-1 and further investigations are being carried out focusing on pathway engineering and optimization of process parameters to enhance resveratrol yield.