Identification of Grape Laccase Genes and Their Potential Role in Secondary Metabolite Synthesis.

Laccase, a copper-containing oxidoreductase, has close links with secondary metabolite biosynthesis in plants. Its activity can affect the synthesis and accumulation of secondary metabolites, thereby influencing plant growth, development, and stress resistance. This study aims to identify the grape laccases (VviLAC) gene family members in grape (Vitis vinifera L.) and explore the transcriptional regulatory network in berry development. Here, 115 VviLACs were identified and divided into seven (Type I-VII) classes. These were distributed on 17 chromosomes and out of 47 VviLACs on chromosome 18, 34 (72.34%) were involved in tandem duplication events. VviLAC1, VviLAC2, VviLAC3, and VviLAC62 were highly expressed before fruit color development, while VviLAC4, VviLAC12, VviLAC16, VviLAC18, VviLAC20, VviLAC53, VviLAC60 and VviLAC105 were highly expressed after fruit color transformation. Notably, VviLAC105 showed a significant positive correlation with important metabolites including resveratrol, resveratrol dimer, and peonidin-3-glucoside. Analysis of the transcriptional regulatory network predicted that the 12 different transcription factors target VviLACs genes. Specifically, WRKY and ERF were identified as potential transcriptional regulatory factors for VviLAC105, while Dof and MYB were identified as potential transcriptional regulatory factors for VviLAC51. This study identifies and provides basic information on the grape LAC gene family members and, in combination with transcriptome and metabolome data, predicts the upstream transcriptional regulatory network of VviLACs.

A diel multi-tissue genome-scale metabolic model of Vitis vinifera.

Vitis vinifera, also known as grapevine, is widely cultivated and commercialized, particularly to produce wine. As wine quality is directly linked to fruit quality, studying grapevine metabolism is important to understand the processes underlying grape composition. Genome-scale metabolic models (GSMMs) have been used for the study of plant metabolism and advances have been made, allowing the integration of omics datasets with GSMMs. On the other hand, Machine learning (ML) has been used to analyze and integrate omics data, and while the combination of ML with GSMMs has shown promising results, it is still scarcely used to study plants. Here, the first GSSM of V. vinifera was reconstructed and validated, comprising 7199 genes, 5399 reactions, and 5141 metabolites across 8 compartments. Tissue-specific models for the stem, leaf, and berry of the Cabernet Sauvignon cultivar were generated from the original model, through the integration of RNA-Seq data. These models have been merged into diel multi-tissue models to study the interactions between tissues at light and dark phases. The potential of combining ML with GSMMs was explored by using ML to analyze the fluxomics data generated by green and mature grape GSMMs and provide insights regarding the metabolism of grapes at different developmental stages. Therefore, the models developed in this work are useful tools to explore different aspects of grapevine metabolism and understand the factors influencing grape quality.

Genome-wide identification of Aux/IAA gene family members in grape and functional analysis of VaIAA3 in response to cold stress.

KEY MESSAGE: Twenty-five VvIAA genes and eighteen VaIAA genes were identified from Pinot Noir and Shanputao, respectively. The overexpression of VaIAA3 in transgenic Arabidopsis increased cold tolerance by regulating auxin, ABA and ethylene signaling. Aux/IAA genes are key genes involved in regulating auxin signal transduction in plants. Although IAA genes have been characterized in various plant species, the role of IAA genes in grape cold resistance is unclear. To further explore the members of the Aux/IAA gene family in grape and their functions, in this study, using genomic data for Pinot Noir (Vitis vinifera cv. 'Pinot Noir') and Shanputao (Vitis amurensis), 25 VvIAA genes and 18 VaIAA genes were identified. The VaIAA genes presented different expression patterns at five different temperatures (28 ± 1 °C, 5 ± 1 °C, 0 ± 1 °C, -5 ± 1 °C, and -10 ± 1 °C) according to qRT­PCR results. VaIAA3 was selected as a candidate gene for further functional analysis because of its high expression level under low-temperature stress. Subcellular localization experiments revealed that VaIAA3 was localized in the nucleus. Additionally, under 4 °C treatment for 24 h, relative expression level of VaIAA3, antioxidant enzyme activity, survival rate, and cold-responsive gene expression in three transgenic lines (OE-1, OE-2, OE-3) were greater, whereas relative electrolytic conductivity (REC), malondialdehyde (MDA) content and hydrogen peroxide (H2O2) content were lower than those of the wild type (WT). Transcriptome sequencing analysis revealed that VaIAA3 regulated cold stress resistance in Arabidopsis thaliana (Arabidopsis) through pathways involving auxin, ABA, JA, or ethylene. Importantly, heterologous overexpression of VaIAA3 increased the resistance of Arabidopsis to cold stress, which provides a theoretical basis for the further use of VaIAA3 to improve cold resistance in grape.

Targeted regulation of 5-aminolevulinic acid enhances flavonoids, anthocyanins and proanthocyanidins accumulation in Vitis davidii callus.

BACKGROUND: Spine grape (Vitis davidii) is a promising source of high-quality anthocyanins, with vast potential for application in food, pharmaceutical, and cosmetic industries. However, their availability is limited by resource constraints. Plant cell culture has emerged as a valuable approach for anthocyanin production and serves as an ideal model to investigate the regulation of anthocyanin biosynthesis. Elicitors are employed to achieve targeted enhancement of anthocyanin biosynthesis. The present study investigated the impact of 5-aminolevulinic acid (ALA) as an elicitor on the accumulation of anthocyanins and flavonoids during spine grape callus growth. Specifically, we examined the effects of ALA on anthocyanin and its component accumulation in callus, and biosynthetic anthocyanin gene expression. RESULTS: ALA at 25 µg/L increased the biomass of spine grape callus. ALA induction enhanced the levels of flavonoids, anthocyanins and proanthocyanidins in callus, with maximum values reaching 911.11 mg/100 g DW, 604.60 mg/100 g DW, and 5357.00 mg/100 g DW, respectively, after callus culture for 45 days. Notably, those levels were 1.47-, 1.93- and 1.83-fold higher than controls. ALA induction modulated the flavonoid profile, and among 97 differential flavonoid metabolites differing from controls, 77 were upregulated and 20 were downregulated. Six kinds of anthocyanins, namely cyanidin (8), delphinidin (6), peonidin (5), malvidin (4), petunidin (3) and pelargonidin (3), were detected in callus, with peonidin most abundant. Compared with controls, anthocyanin components were increased in ALA-treated callus. The key genes PAL1, PAL2, PAL4, CHI, CHS3, F3'H, F3H, FLS, DFR, UFGT, MYBA1, LDOX, OMT3, GT1 and ACT involved in anthocyanin biosynthesis were upregulated following ALA treatment, resulting in anthocyanin accumulation. CONCLUSION: This study revealed a novel mode of ALA-mediated promotion of plant anthocyanin biosynthesis and accumulation at the cellular level, and a strategy for enhancing anthocyanin content in spine grape callus. The findings advance commercial-scale production of anthocyanins via spine grape callus culture. we also explored the accumulation patterns of flavonoids and anthocyanins under ALA treatment. Augmentation of anthocyanins coincided with elevated expression levels of most genes involved in anthocyanin biosynthesis within spine grape callus following ALA treatment.

VvRF2b interacts with VvTOR and influences VvTOR-regulated sugar metabolism in grape.

The production of top-quality wines is closely related to the quality of the wine grapes. In wine grapes (Vitis vinifera L., Vv), sugar is a crucial determinant of berry quality, regulated by an interplay of various transcription factors and key kinases. Many transcription factors involved in sugar metabolism remain unexplored. Target of Rapamycin (TOR) is an important protein kinase in plants, recently found to regulate sugar metabolism in grapes. However, transcription factors or other factors involved in this process are rarely reported. Here, we utilized transgenic callus tissues from 'Cabernet Sauvignon' grape fruit engineered via gene overexpression (oe) and CRISPR/Cas9-based gene knockout (ko), and discovered a bZIP transcription factor, VvRF2b, whose knockout resulted in increased accumulation of fructose and sucrose, indicating that VvRF2b is a negative regulator of sugar accumulation. Subcellular localization and transcriptional activation tests showed that VvRF2b is an activator of transcription located both in the nucleus and cell membrane. Analysis of VvRF2b and VvTOR gene levels and sugar contents (glucose, fructose, and sucrose) in 'Cabernet Sauvignon' grape fruits at 30, 70, and 90 days after bloom (DAB) revealed that VvRF2b is expressed more highly during fruit development, while VvTOR is expressed more during the sugar accumulation phase, furthermore, VvTOR gene levels in koVvRF2b transgenic calli increased significantly, suggesting a strong relationship between the knockout of VvRF2b and the overexpression of VvTOR. Additionally, bimolecular fluorescence complementation and luciferase complementation assays demonstrated the interaction between VvRF2b and VvTOR proteins. After knocking out the VvRF2b gene in oeVvTOR calli, it was found that the knockout of VvRF2b promotes VvTOR-regulated sucrose accumulation and enhances the expression of sugar metabolism-related genes regulated by VvTOR. In summary, our results suggest that VvRF2b interacts with VvTOR protein and influences VvTOR-regulated sugar metabolism.

Identification and characterisation of 'No apical meristem; Arabidopsis transcription activation factor; Cup-shape cotyledon' (NAC) family transcription factors involved in sugar accumulation and abscisic acid signalling in grape (Vitis vinifera).

The 'No apical meristem; Arabidopsis transcription activation factor; Cup-shape cotyledon' (NAC) transcription factors are pivotal in plant development and stress response. Sucrose-non-fermenting-related protein kinase 1.2 (SnRK1) is a key enzyme in glucose metabolism and ABA signalling. In this study, we used grape (Vitis vinifera ) calli to explore NAC's roles in sugar and ABA pathways and its relationship with VvSnRK1.2 . We identified 19 VvNACs highly expressed at 90days after blooming, coinciding with grape maturity and high sugar accumulation, and 11 VvNACs randomly selected from 19 were demonstrated in response to sugar and ABA treatments. VvNAC26 showed significant response to sugar and ABA treatments, and its protein, as a nucleus protein, had transcriptional activation in yeast. We obtained the overexpression (OE-VvNAC26 ) and RNA-inhibition (RNAi-VvNAC26 ) of VvNAC26 in transgenic calli by Agrobacterium tumefaciens -mediated transformation. We found that VvNAC26 negatively influenced fructose content. Under sugar and ABA treatments, VvNAC26 negatively influenced the expression of most sugar-related genes, while positively influencing the expression of most ABA pathway-related genes. Dual-luciferase reporter experiments demonstrated that VvNAC26 significantly upregulates VvSnRK1.2 promoter expression in tobacco (Nicotiana benthamiana ) leaves, although this process in grape calli requires ABA. The levels of sugar content, sugar-related genes, and ABA-related genes fluctuated significantly in OE-VvNAC26 +RNAi-VvSnRK1.2 and OE-VvSnRK1.2 +RNAi-VvNAC26 transgenic calli. These findings indicated that VvNAC26 regulates sugar metabolism and ABA pathway, displaying synergistic interactions with VvSnRK1.2 .

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.

Identification of VvAGL Genes Reveals Their Network's Involvement in the Modulation of Seed Abortion via Responding Multi-Hormone Signals in Grapevines.

The formation of seedless traits is regulated by multiple factors. AGLs, which belong to the MADS-box family, were reported to be important regulators in this process; however, the underlying mechanism remains elusive. Here, we identified the VvAGL sub-family genes during the seed abortion process in seedless grapevine cv. 'JingkeJing' and found 40 differentially expressed VvAGL members and 1069 interacting proteins in this process. Interestingly, almost all members and their interacting proteins involved in the tryptophan metabolic pathway (K14486) and participated in the phytohormone signalling (KO04075) pathway, including the growth hormone (IAA), salicylic acid (SA), abscisic acid (ABA), cytokinin (CTK), and ethylene signalling pathways. The promoters of AGL sub-family genes contain cis-elements in response to hormones such as IAA, ABA, CTK, SA, and ETH, implying that they might respond to multi-hormone signals and involve in hormone signal transductions. Further expression analysis revealed VvAGL6-2, VvAGL11, VvAGL62-11, and VvAGL15 had the highest expression at the critical period of seed abortion, and there were positive correlations between ETH-VvAGL15-VvAGL6-2, ABA-VvAGL80, and SA-VvAGL62 in promoting seed abortion but negative feedback between IAA-VvAGL15-VvAGL6-2 and CTK-VvAGL11. Furthermore, many genes in the IAA, ABA, SA, CTK, and ETH pathways had a special expressional pattern in the seed, whereby we developed a regulatory network mediated by VvAGLs by responding to multihormonal crosstalk during grape seed abortion. Our findings provide new insights into the regulatory network of VvAGLs in multi-hormone signalling to regulate grape seed abortion, which could be helpful in the molecular breeding of high-quality seedless grapes.

Transcription Factor VlbZIP14 Inhibits Postharvest Grape Berry Abscission by Directly Activating VlCOMT and Promoting Lignin Biosynthesis.

Sulfur dioxide (SO2) is the most effective preservative for table grapes as it reduces the respiratory intensity of berries and inhibits mold growth. However, excessive SO2 causes berry abscission during storage, resulting in an economic loss postharvest. In this study, grapes were exogenously treated with SO2, SO2 + 1.5% chitosan, SO2 + 1.5% eugenol, and SO2 + eugenol-loaded chitosan nanoparticles (SN). In comparison to SO2 treatment, SN treatment reduced the berries' abscission rate by 74% while maintaining the quality of the berries. Among the treatments, SN treatment most effectively inhibited berry abscission and maintained berry quality. RNA-sequencing (RNA-seq) revealed that SN treatment promoted the expression of genes related to cell wall metabolism. Among these genes, VlCOMT was detected as the central gene, playing a key role in mediating the effects of SN. Dual luciferase and yeast one-hybrid (Y1H) assays demonstrated that VlbZIP14 directly activated VlCOMT by binding to the G-box motif in the latter's promoter, which then participated in lignin synthesis. Our results provide key insights into the molecular mechanisms underlying the SN-mediated inhibition of berry abscission and could be used to improve the commercial value of SO2-treated postharvest table grapes.

Structural and Phylogenetic In Silico Characterization of Vitis vinifera PRR Protein as Potential Target for Plasmopara viticola Infection.

Fungi infection, especially derived from Plasmopara viticola, causes severe grapevine economic losses worldwide. Despite the availability of chemical treatments, looking for eco-friendly ways to control Vitis vinifera infection is gaining much more attention. When a plant is infected, multiple disease-control molecular mechanisms are activated. PRRs (Pattern Recognition Receptors) and particularly RLKs (receptor-like kinases) take part in the first barrier of the immune system, and, as a consequence, the kinase signaling cascade is activated, resulting in an immune response. In this context, discovering new lectin-RLK (LecRLK) membrane-bounded proteins has emerged as a promising strategy. The genome-wide localization of potential LecRLKs involved in disease defense was reported in two grapevine varieties of great economic impact: Chardonnay and Pinot Noir. A total of 23 potential amino acid sequences were identified, exhibiting high-sequence homology and evolution related to tandem events. Based on the domain architecture, a carbohydrate specificity ligand assay was conducted with docking, revealing two sequences as candidates for specific Vitis vinifera-Plasmopara viticola host-pathogen interaction. This study confers a starting point for designing new effective antifungal treatments directed at LecRLK targets in Vitis vinifera.

Transcriptome analysis reveals activation of detoxification and defense mechanisms in smoke-exposed Merlot grape (Vitis vinifera) berries.

A significant consequence of climate change is the rising incidence of wildfires. When wildfires occur close to wine grape (Vitis vinifera) production areas, smoke-derived volatile phenolic compounds can be taken up by the grape berries, negatively affecting the flavor and aroma profile of the resulting wine and compromising the production value of entire vineyards. Evidence for the permeation of smoke-associated compounds into grape berries has been provided through metabolomics; however, the basis for grapevines' response to smoke at the gene expression level has not been investigated in detail. To address this knowledge gap, we employed time-course RNA sequencing to observe gene expression-level changes in grape berries in response to smoke exposure. Significant increases in gene expression (and enrichment of gene ontologies) associated with detoxification of reactive compounds, maintenance of redox homeostasis, and cell wall fortification were observed in response to smoke. These findings suggest that the accumulation of volatile phenols from smoke exposure activates mechanisms that render smoke-derived compounds less reactive while simultaneously fortifying intracellular defense mechanisms. The results of this work lend a better understanding of the molecular basis for grapevines' response to smoke and provide insight into the origins of smoke-taint-associated flavor and aroma attributes in wine produced from smoke-exposed grapes.

Oxygen alters redox cofactor dynamics and induces metabolic shifts in Saccharomyces cerevisiae during alcoholic fermentation.

Environmental conditions significantly impact the metabolism of Saccharomyces cerevisiae, a Crabtree-positive yeast that maintains a fermentative metabolism in high-sugar environments even in the presence of oxygen. Although the introduction of oxygen has been reported to induce alterations in yeast metabolism, knowledge of the mechanisms behind these metabolic adaptations in relation to redox cofactor metabolism and their implications in the context of wine fermentation remains limited. This study aimed to compare the intracellular redox cofactor levels, the cofactor ratios, and primary metabolite production in S. cerevisiae under aerobic and anaerobic conditions in synthetic grape juice. The molecular mechanisms underlying these metabolic differences were explored using a transcriptomic approach. Aerobic conditions resulted in an enhanced fermentation rate and biomass yield. Total NADP(H) levels were threefold higher during aerobiosis, while a decline in the total levels of NAD(H) was observed. However, there were stark differences in the ratio of NAD+/NADH between the treatments. Despite few changes in the differential expression of genes involved in redox cofactor metabolism, anaerobiosis resulted in an increased expression of genes involved in lipid biosynthesis pathways, while the presence of oxygen increased the expression of genes associated with thiamine, methionine, and sulfur metabolism. The production of fermentation by-products was linked with differences in the redox metabolism in each treatment. This study provides valuable insights that may help steer the production of metabolites of industrial interest during alcoholic fermentation (including winemaking) by using oxygen as a lever of redox metabolism.

Ectopic and transient expression of VvDIR4 gene in Arabidopsis and grapes enhances resistance to anthracnose via affecting hormone signaling pathways and lignin production.

BACKGROUND: DIR (Dirigent) proteins play important roles in the biosynthesis of lignin and lignans and are involved in various processes such as plant growth, development, and stress responses. However, there is less information about VvDIR proteins in grapevine (Vitis vinifera L). RESULTS: In this study, we used bioinformatics methods to identify members of the DIR gene family in grapevine and identified 18 VvDIR genes in grapevine. These genes were classified into 5 subfamilies based on phylogenetic analysis. In promoter analysis, various plant hormones, stress, and light-responsive cis-elements were detected. Expression profiling of all genes following Colletotrichum gloeosporioides infection and phytohormones (salicylic acid (SA) and jasmonic acid (JA)) application suggested significant upregulation of 17 and 6 VvDIR genes, respectively. Further, we overexpressed the VvDIR4 gene in Arabidopsis thaliana and grapes for functional analysis. Ectopic expression of VvDIR4 in A. thaliana and transient expression in grapes increased resistance against C. gloeosporioides and C. higginsianum, respectively. Phenotypic observations showed small disease lesions in transgenic plants. Further, the expression patterns of genes having presumed roles in SA and JA signaling pathways were also influenced. Lignin contents were measured before and after C. higginsianum infection; the transgenic A. thaliana lines showed higher lignin content than wild-type, and a significant increase was observed after C. higginsianum infection. CONCLUSIONS: Based on the findings, we surmise that VvDIR4 is involved in hormonal and lignin synthesis pathways which regulate resistance against anthracnose. Our study provides novel insights into the function of VvDIR genes and new candidate genes for grapevine disease resistance breeding programs.

Transcriptome and metabolite profiles reveal the role of benzothiadiazole in controlling isoprenoid synthesis and berry ripening in chardonnay grapes.

Benzothiadiazole (BTH) regulates grape development, ripening, volatiles, and phenolics. This study used metabolomics and transcriptomics to understand how exogenous BTH affects Chardonnay grapes' maturation and synthesis of isoprenoids. A 0.37 mM BTH solution was sprayed during the swelling and veraison stages, and then the ripe grapes were analyzed. Our results show that BTH application significantly increased levels of important isoprenoids such as free terpinen-4-ol, bound linalool, and 8'-apo-ß-carotenal. Additionally, BTH was found to modulate several signaling pathways, including those involved in ethylene biosynthesis, salicylic acid synthesis, the abscisic acid pathway, and sugar metabolism, by regulating the expression of genes like VvACO4, VvTAR, VvPLD, VvTIP1-1, VvSTKs, VvPK, VvSUC2, VvGST4, and VvSTS. BTH also promoted grapevine resistance by up-regulating the expression of VvHSP20, VvGOLS4, VvOLP, and VvPR-10. Furthermore, BTH affected isoprenoids biosynthesis by regulating the expression of VvTPS35 and VvMYB24. Moreover, 13 hub genes in the MEgreen module were identified as crucial for the biosynthesis of isoprenoids. BTH application during the swelling stage remarkably promoted isoprenoid biosynthesis more effectively than veraison. Our study provides insights into the molecular mechanisms underlying BTH-induced regulation of grape development and offers a promising approach for enhancing the quality and resistance of grapes.

A vacuolar protein MaSCPL1 mediates anthocyanin acylation modifications in blue-flowered grape hyacinth.

The grape hyacinth is renowned for its profuse blue flowers, which confer substantial scientific and ornamental significance as well as considerable potential for industrial applications. The serine carboxypeptidase-like acyltransferases (SCPL-ATs) family is crucial for the blue flower coloration. To elucidate SCPL-ATs involved in anthocyanin modification in grape hyacinth, we performed a transcriptomic analysis of grape hyacinth SCPL-ATs. Through gene expression profiling, we identified a promising candidate gene, MaSCPL1, whose expression patterns corresponded with variations in anthocyanin content throughout petal coloration. Subsequently, the functional role of the MaSCPL1 gene was validated using the native petal regeneration system, and the silencing of MaSCPL1 led to a decreased total anthocyanin content and Dp3MG content in grape hyacinth petals. Furthermore, we employed yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), and dual-luciferase assays to explore the regulatory interactions between the anthocyanin biosynthesis transcription factor MaMybA and the MaSCPL1 promoter. Our findings indicate that MaMybA can bind to the MaSCPL1 promoter and significantly activate its expression. Furthermore, the MaMybA-RNAi resulted in a substantial multifold reduction in the expression of MaSCPL1, implying that the regulation of MaSCPL1 expression is mediated by MaMybA. This study revealed the MaSCPL1 gene has been associated with anthocyanin acylated modification in grape hyacinth and elucidated the important role of the MaMybA-MaSCPL1 module in colouration grape hyacinth.

Plasmopara viticola effector PvCRN20 represses the import of VvDEG5 into chloroplasts to suppress immunity in grapevine.

Chloroplasts play a crucial role in plant defense against pathogens, making them primary targets for pathogen effectors that suppress host immunity. This study characterizes the Plasmopara viticola CRN-like effector, PvCRN20, which interacts with DEG5 in the cytoplasm but not with its interacting protein, DEG8, which is located in the chloroplast. By transiently overexpressing in tobacco leaves, we show that PvCRN20 could inhibit INF1- and Bax-triggered cell death. Constitutive expression of PvCRN20 suppresses the accumulation of reactive oxygen species (ROS) and promotes pathogen colonization. PvCRN20 reduces DEG5 entry into chloroplasts, thereby disrupting DEG5 and DEG8 interactions in chloroplasts. Overexpression of VvDEG5 and VvDEG8 induces ROS accumulation and enhances grapevine resistance to P. viticola, whereas knockout of VvDEG8 represses ROS production and promotes P. viticola colonization. Consistently, ectopic expression of VvDEG5 and VvDEG8 in tobacco promotes chloroplast-derived ROS accumulation, whereas co-expression of PvCRN20 counteracted this promotion by VvDEG5. Therefore, DEG5 is essential for the virulence function of PvCRN20. Although PvCRN20 is located in both the nucleus and cytoplasm, only cytoplasmic PvCRN20 suppresses plant immunity and promotes pathogen infection. Our results reveal that PvCRN20 dampens plant defenses by repressing the chloroplast import of DEG5, thus reducing host ROS accumulation and facilitating pathogen colonization.

Technological affinity index for interaction between lactic acid bacteria and Saccharomyces cerevisiae strains to modulate the fruity and floreal aroma of Catarratto wines.

Microbial interactions during the fermentation process influence the sensory characteristics of wines. Alongside alcoholic fermentation, malolactic fermentation also plays a crucial role in determining the aromatic traits of wines. The time (t), rate (m) and volatile organic compounds (VOCs) of malolactic fermentation are linked to the interaction between yeasts and lactic acid bacteria. The study investigated the interactions between Lactiplantibacillus plantarum or Oenococcus oeni with Saccharomyces cerevisiae by using the Technological Affinity Index (TAIndex). The co-inoculation of L. plantarum/S. cerevisiae resulted in a higher TAIndex than the co-inoculation of O. oeni/S. cerevisiae conditions. A low TAIndex led to increased aromaticity of the wines. The time and rate of malolactic fermentation have a strong impact on the synthesis of VOCs with a high olfactory impact. Therefore, knowledge of the TAIndex could play a decisive role in improving winemaking planning to produce wines with higher fruit and floral perceptions.

Transcriptome and Metabolome Analyses of Aroma Differences between Chardonnay and a Chardonnay Bud Sport.

Chardonnay is one of the most popular white grape wine varieties in the world, but this wine lacks typical aroma, considered a sensory defect. Our research group identified a Chardonnay bud sport with typical muscat characteristics. The goal of this work was to discover the key candidate genes related to muscat characteristics in this Chardonnay bud sport to reveal the mechanism of muscat formation and guide molecular design breeding. To this end, HS-SPME-GC-MS and RNA-Seq were used to analyze volatile organic compounds and the differentially expressed genes in Chardonnay and its aromatic bud sport. Forty-nine volatiles were identified as potential biomarkers, which included mainly aldehydes and terpenes. Geraniol, linalool, and phenylacetaldehyde were identified as the main aroma components of the mutant. The GO, KEGG, GSEA, and correlation analysis revealed HMGR, TPS1, TPS2, TPS5, novel.939, and CYP450 as key genes for terpene synthesis. MAO1 and MAO2 were significantly downregulated, but there was an increased content of phenylacetaldehyde. These key candidate genes provide a reference for the development of functional markers for muscat varieties and also provide insight into the formation mechanism of muscat aroma.

Elucidating the Subcellular Localization of GLRaV-3 Proteins Encoded by the Unique Gene Block in N. benthamiana Suggests Implications on Plant Host Suppression.

Grapevine leafroll-associated virus 3 (GLRaV-3) is a formidable threat to the stability of the global grape and wine industries. It is the primary etiological agent of grapevine leafroll disease (GLD) and significantly impairs vine health, fruit quality, and yield. GLRaV-3 is a member of the genus Ampelovirus, Closteroviridae family. Viral genes within the 3' proximal unique gene blocks (UGB) remain highly variable and poorly understood. The UGBs of Closteroviridae viruses include diverse open reading frames (ORFs) that have been shown to contribute to viral functions such as the suppression of the host RNA silencing defense response and systemic viral spread. This study investigates the role of GLRaV-3 ORF8, ORF9, and ORF10, which encode the proteins p21, p20A, and p20B, respectively. These genes represent largely unexplored facets of the GLRaV-3 genome. Here, we visualize the subcellular localization of wildtype and mutagenized GLRaV-3 ORFs 8, 9, and 10, transiently expressed in Nicotiana benthamiana. Our results indicate that p21 localizes to the cytosol, p20A associates with microtubules, and p20B is trafficked into the nucleus to carry out the suppression of host RNA silencing. The findings presented herein provide a foundation for future research aimed at the characterization of the functions of these ORFs. In the long run, it would also facilitate the development of innovative strategies to understand GLRaV-3, mitigate its spread, and impacts on grapevines and the global wine industry.

The Metabolomic Profiling of the Flavonoid Compounds in Red Wine Grapes and the Impact of Training Systems in the Southern Subtropical Region of China.

Flavonoids play an important role in forming wine grapes and wine quality characteristics. The flavonoids of three winter red wine grapes, Yeniang No. 2 (YN2), Marselan (Mar), and Guipu No. 6 (GP6), were analyzed by ultra-high-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QQQ-MS). Furthermore, the flavonoids in GP6 grapevines using two types of training systems, namely, trellis (T) and espaliers (E), were also compared in this study. Overall, 196 flavonoid metabolites, including 96 flavones, 38 flavonols, 19 flavanones, 18 polyphenols, 15 anthocyanins, 7 isoflavones, and 3 proanthocyanidins, were identified. The flavonoid profiles were remarkably different among these three grape varieties, while they did not change much in the GP6 managed on trellis and espaliers. Grape varieties with different genetic backgrounds have their own unique flavonoid profiles. Compared with Mar-T, isoflavones and flavonols presented higher contents in GP6-T and YN2-T, which mainly contain glycitein, genistin, calycosin, kaempferide, isotrifoliin, and ayanin. The anthocyanin content was significantly higher in YN2-T than in the other two varieties. YN2 and GP6-T present a more stable color, with significantly more acetylated diglucosides and methylated anthocyanins in YN2-T and GP6-T than in Mar-T. Notably, GP6 had more varied flavonoids and the better characteristics to its flavonoid profile out of these three varieties, due to it containing a higher number of anthocyanins, flavone, and flavonols and the greatest number of different flavonoid metabolites (DFMs), with higher contents than YN2 and Mar. Compared with the trellis training system, the espaliers training system increased the content of flavonoids detected in GP6 grape berries; however, the composition of flavonoids strictly depends on the grape variety.