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Cannabis plants produce a spectrum of secondary metabolites, encompassing cannabinoids and more than 300 non-cannabinoid compounds. Among these, anthocyanins have important functions in plants and also have well documented health benefits. Anthocyanins are largely responsible for the red/purple color phenotypes in plants. Although some well-known Cannabis varieties display a wide range of red/purple pigmentation, the genetic underpinnings of anthocyanin biosynthesis have not been well characterized in Cannabis. This study unveils the genetic diversity of anthocyanin biosynthesis genes found in Cannabis, and we characterize the diversity of anthocyanins and related phenolics found in four differently pigmented Cannabis varieties. Our investigation revealed that the genes 4CL , CHS , F3H , F3'H , FLS , DFR , ANS , and OMT exhibited the strongest correlation with anthocyanin accumulation in Cannabis leaves. The results of this study enhance our understanding of the anthocyanin biosynthetic pathway and shed light on the molecular mechanisms governing Cannabis leaf pigmentation., Competing Interests: M.S., K.K.G., T.M., and S.W. were employed by Willow Analytics during the data collection stage of this study. Willow Analytics was a for‐profit analytical testing and Cannabis research lab located in Burnaby, British Columbia., (© 2024 The Author(s). Plant Direct published by American Society of Plant Biologists and the Society for Experimental Biology and John Wiley & Sons Ltd.)

Monoterpenoid biosynthesis mirrors hexoses accumulation in grape berries during ripening and is affected by environmental factors such as solar radiation. However, no research has confirmed the dependency of monoterpenoid accumulation on grape maturity. Using potted 'Riesling' (Vitis vinifera L.) vines, we girdled (GD) shoots at veraison to halt leaf photoassimilate translocation to berries and also utilized light-impenetrable bags to exclude light exposure (LE) to grape clusters to <2 μmol m -2 s -1 during ripening. GD and LE were compared to an untreated control (CT), and a combined (GDxLE) treatment. GD caused leaf photosynthetic assimilation to decline from one-week post-treatment until harvest maturity due to feedback inhibition of photosynthesis. This was coupled to a decrease in hexoses and ABA accumulation in GD berries. Meanwhile, LE did not affect photosynthetic assimilation, or hexoses and ABA accumulation. We hypothesized that the accumulation of free and bound monoterpenoids in 'Riesling' berries would also be reduced by GD, if monoterpenoid accumulation was dependent on grape maturity. LE significantly decreased free monoterpenoids compared to CT. However, GD did not alter total free and bound monoterpenoid concentrations. GD decreased free linalool oxide I and II and bound linalool oxide I compared to CT, and increased citral I and II and α-terpineol. Although exogenous jasmonate applications stimulate monoterpenoid biosynthesis in grapes, we showed that endogenous jasmonate concentrations were not altered by GD or LE; therefore, any monoterpenoid modulation by GD and LE treatments was not mediated by endogenous jasmonate levels. This study indicates that free monoterpenoid biosynthesis during ripening is uncoupled from hexose accumulation and is strongly regulated by solar radiation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Legume proteins can be induced to form amyloid-like fibrils upon heating at low pH, with the exact conditions greatly impacting the fibril characteristics. The protein extraction method may also impact the resulting fibrils, although this effect has not been carefully examined. Here, the fibrillization of lentil protein prepared using various extraction methods and the corresponding fibril morphology were characterized. It was found that an acidic, rather than alkaline, protein extraction method was better suited for producing homogeneous, long, and straight fibrils from lentil proteins. During alkaline extraction, co-extracted phenolic compounds bound proteins through covalent and non-covalent interactions, contributing to the formation of heterogeneous, curly, and tangled fibrils. Recombination of isolated phenolics and proteins (from acidic extracts) at alkaline pH resulted in a distinct morphology, implicating a role for polyphenol oxidase also in modifying proteins during alkaline extraction. These results help disentangle the complex factors affecting legume protein fibrillization., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Background: Red raspberry fruit color is a key driver of consumer preference and a major target of breeding programs worldwide. Screening for fruit color typically involves the determination of anthocyanin content and/or the assessment of color through a colorimeter. However, both procedures are time-consuming when the analyses involve hundreds or thousands of samples. The objectives of this study were to develop a high-throughput method for red raspberry puree color measurement and to test the correlations between color parameters and total anthocyanin content. Color coordinates were collected with a colorimeter on 126 puree samples contained in Petri dishes and with the Tomato Analyzer Color Test (TACT) module to assess the same samples prepared in Petri dishes and in 96-well plates. An additional 425 samples were analyzed using only 96-well plates. Total anthocyanins were extracted from all 551 samples., Results: Regression models for L*, a*, b* measured with the colorimeter and TACT using Petri dishes were all significant (p < 0.001), but very consistent only for L* (R 2  = 0.94). Significant (p < 0.001) and very consistent regressions (R 2  = 0.94 for L* and b*, R 2  = 0.93 for a*) were obtained for color parameters measured with TACT using Petri dishes and TACT using plates. Of the color parameters measured with the colorimeter, only L*, a*/b*, and hue significantly correlated with total anthocyanins (p < 0.05), but, except for L* (R = - 0.79), the correlations were weak (R = - 0.23 for a*/b* and R = 0.22 for hue). Conversely, all correlations with total anthocyanins and color parameters measured with TACT were significant (p < 0.001) and moderately strong (e.g., R = - 0.69 for L* and R = 0.55 for a*/b*). These values were indicative of darker colors as total anthocyanin content increased., Conclusions: While the colorimeter and TACT-based methods were not fully interchangeable, TACT better captured color differences among raspberry genotypes than the colorimeter. The correlations between color parameters measured with TACT and total anthocyanins were not strong enough to develop prediction models, yet the use of TACT with 96-well plates instead of Petri dishes would enable the high-throughput measurement of red raspberry puree color., (© 2024. The Author(s).)

In fruits, cuticular waxes affect fruit quality traits such as surface color at harvest and water loss during postharvest storage. This study investigated the transcriptional regulation of cuticular wax deposition in northern highbush blueberries ( Vaccinium corymbosum L.) in relation to fruit water loss and surface color during ripening and postharvest storage, as well as the effects of abscisic acid (ABA)-mediated changes in cuticular wax deposition on these fruit quality traits. Total cuticular wax content (μg∙cm -2 ) decreased during fruit ripening and increased during postharvest storage. Transcriptome analysis revealed a transcript network for cuticular wax deposition in blueberries. Particularly, five OSC-Likes were identified as putative genes for triterpene alcohol production, with OSC-Like1 and OSC-Like2 encoding mixed amyrin synthases, OSC-Like3 encoding a lupeol synthase, and OSC-Like4 and OSC-Like5 encoding cycloartenol synthases. The expression of three CYP716A-like genes correlated to the accumulation of two triterpene acids oleanolic acid and ursolic acid, the major wax compounds in blueberries. Exogenous ABA application induced the expression of triterpenoid biosynthetic genes and the accumulation of β-amyrin and oleanolic acid, as well as increased the ratio of oleanolic acid to ursolic acid. These changes were associated with reduced fruit water loss. The content of β-diketones was also increased by ABA application, and this increase was associated with increased fruit lightness (measured as L* using CIELAB Color Space by a colorimeter). This study provided key insights on the molecular basis of cuticular wax deposition and its implications on fruit quality traits in blueberries., Competing Interests: No potential conflict of interest was reported by the authors., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nanjing Agricultural University.)

N-Hydroxypipecolic acid (NHP) is a signaling molecule crucial for systemic acquired resistance (SAR), a systemic immune response in plants that provides long-lasting and broad-spectrum protection against secondary pathogen infections. To identify negative regulators of NHP biosynthesis, we performed a forward genetic screen to search for mutants with elevated expression of the NHP biosynthesis gene FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1). Analysis of two constitutive expression of FMO1 (cef) and one induced expression of FMO1 (ief) mutants revealed that the AIPP3-PHD2-CPL2 protein complex, which is involved in the recognition of the histone modification H3K27me3 and transcriptional repression, contributes to the negative regulation of FMO1 expression and NHP biosynthesis. Our study suggests that epigenetic regulation plays a crucial role in controlling FMO1 expression and NHP levels in plants., (© 2023 Institute of Botany, Chinese Academy of Sciences.)

Variegation is a rare type of mosaicism not fully studied in plants, especially fruits. We examined red and white sections of grape (Vitis vinifera cv. 'Béquignol') variegated berries and found that accumulation of products from branches of the phenylpropanoid and isoprenoid pathways showed an opposite tendency. Light-responsive flavonol and monoterpene levels increased in anthocyanin-depleted areas in correlation with increasing MYB24 expression. Cistrome analysis suggested that MYB24 binds to the promoters of 22 terpene synthase (TPS) genes, as well as 32 photosynthesis/light-related genes, including carotenoid pathway members, the flavonol regulator HY5 HOMOLOGUE (HYH), and other radiation response genes. Indeed, TPS35, TPS09, the carotenoid isomerase gene CRTISO2, and HYH were activated in the presence of MYB24 and MYC2. We suggest that MYB24 modulates ultraviolet and high-intensity visible light stress responses that include terpene and flavonol synthesis and potentially affects carotenoids. The MYB24 regulatory network is developmentally triggered after the onset of berry ripening, while the absence of anthocyanin sunscreens accelerates its activation, likely in a dose-dependent manner due to increased radiation exposure. Anthocyanins and flavonols in variegated berry skins act as effective sunscreens but for different wavelength ranges. The expression patterns of stress marker genes in red and white sections of 'Béquignol' berries strongly suggest that MYB24 promotes light stress amelioration but only partly succeeds during late ripening., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background and Aims: Drought events have devasting impacts on grape berry production. The aim of this study was to investigate berry growth in the context of leaf stomatal closure under progressive drought stress., Methods: Potted grapevine plants (varieties 'Syrah' and 'Cabernet Sauvignon') were evaluated at pre-verasion (30-45 d after anthesis, DAA) and post-veraison (90-107 DAA). Berry diameter, berry absolute growth rate (AGR), leaf stomatal conductance (Gs) at midday, plant water potential at predawn and midday (ΨPD and ΨMD, respectively), and soil relative water content were measured repeatedly. The ΨPD-threshold of 90 % loss in stomatal conductance (Gs10, i.e. complete stomatal closure) was determined. Data were related to plant dehydration phases I, II and III with corresponding boundaries Θ1 and Θ2, using the water potential curve method., Key Results: At pre-veraison, berry AGR declined together with leaf Gs in response to soil drying in both varieties. Berry AGR transitioned from positive to negative (shrinkage) values when leaf Gs approached zero. The Gs10-threshold was -0.81 MPa in 'Syrah' and -0.74 MPa in 'Cabernet Sauvignon' and was linked to boundary Θ1. At post-veraison, berry AGR was negligible and negative AGR values were not intensified by increasing drought stress in either variety., Conclusion: Leaf complete stomatal closure under progressive drought stress coincides with cessation of berry growth followed by shrinkage at pre-veraison (growth stage 1)., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Soft-ripened cheeses (SRCs) are at a higher risk for the growth of the foodborne pathogen Listeria monocytogenes due to favorable moisture content and pH compared to other cheeses. L. monocytogenes growth is not consistent across SRCs, however, and may be affected by physicochemical and/or microbiome characteristics of the cheeses. Therefore, the purpose of this study was to investigate how the physicochemical and microbiome profiles of SRCs may affect L. monocytogenes growth. Forty-three SRCs produced from raw ( n  = 12) or pasteurized ( n  = 31) milk were inoculated with L. monocytogenes (10 3 CFU/g), and the pathogen growth was monitored over 12 days at 8°C. In parallel, the pH, water activity (a w ), microbial plate counts, and organic acid content of cheeses were measured, and the taxonomic profiles of the cheese microbiomes were measured using 16S rRNA gene targeted amplicon sequencing and shotgun metagenomic sequencing. L. monocytogenes growth differed significantly between cheeses (analysis of variance [ANOVA]; P < 0.001), with increases ranging from 0 to 5.4 log CFU (mean of 2.5 ± 1.2 log CFU), and was negatively correlated with a w . Raw milk cheeses showed significantly lower L. monocytogenes growth than pasteurized-milk cheeses ( t test; P = 0.008), possibly due to an increase in microbial competition. L. monocytogenes growth in cheeses was positively correlated with the relative abundance of Streptococcus thermophilus (Spearman correlation; P < 0.0001) and negatively correlated with the relative abundances of Brevibacterium aurantiacum (Spearman correlation; P = 0.0002) and two Lactococcus spp. (Spearman correlation; P < 0.01). These results suggest that the cheese microbiome may influence the food safety in SRCs. IMPORTANCE Previous studies have identified differences in L. monocytogenes growth between SRCs, but no clear mechanism has yet been elucidated. To the best of our knowledge, this is the first study to collect a wide range of SRCs from retail sources and attempt to identify key factors associated with pathogen growth. A key finding in this research was the positive correlation between the relative abundance of S. thermophilus and the growth of L. monocytogenes. The inclusion of S. thermophilus as a starter culture is more common in industrialized SRC production, suggesting that industrial production of SRC may increase the risk of L. monocytogenes growth. Overall, the results of this study further our understanding of the impact of a w and the cheese microbiome on the growth of L. monocytogenes in SRCs, hopefully leading toward the development of SRC starter/ripening cultures that can prevent L. monocytogenes growth.

Bound volatiles are odorless aroma reservoirs that modify flavor when released during food processing, and their determination is important to understand the aroma of fruit beverages. However, the generation of oxidation/degradation artifacts during analyses of glycosidically-bound volatiles has not been compared across fruit species and their dependence on diverse acidic and enzymatic hydrolytic conditions remains unclear. This work aimed to optimize and compare different hydrolytic conditions for the analysis of glycosidically-bound volatiles in blueberries, raspberries, and grapes with a solid-phase microextraction - gas chromatography/mass spectrometry (SPME-GC/MS) methodology. Enzymatic hydrolyses using AR2000® at 100 mg . mL -1 and Pectinex Ultra SPL® at 25-100 μL . mL -1 showed profiles characterized by the expected alcohols, while using AR2000® at 200-400 mg . mL -1 and citric acid at 50-100 mM resulted in profiles defined by artifacts (hydrocarbons, norisoprenoids, and aldehydes). (Z)-3-hexen-1-ol, 3-methyl-1-butanol, linalool, citronellol, and geraniol presented Odor Activity Values (OAV) > 1 for most small fruit genotypes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Pulsed light (PL) is a novel, non-thermal technology being used to control the microbial spoilage of foods and beverages. Adverse sensory changes, commonly characterized as "lightstruck", can occur in beers when exposed to the UV portion of PL due to the formation of 3-methylbut-2-ene-1-thiol (3-MBT) upon the photodegradation of iso-α-acids. This study is the first to investigate the effect of different portions of the PL spectrum on UV-sensitive beers (light-colored blonde ale and dark-colored centennial red ale) using clear and bronze-tinted UV filters. PL treatments with its entire spectrum, including the ultraviolet portion of the spectrum, resulted in up to 4.2 and 2.4 log reductions of L. brevis in the blonde ale and centennial red ale beers, respectively, but also resulted in the formation of 3-MBT and small but significant changes in physicochemical properties including color, bitterness, pH, and total soluble solids. The application of UV filters effectively maintained 3-MBT below the limit of quantification but significantly reduced microbial deactivation to 1.2 and 1.0 log reductions of L. brevis at 8.9 J/cm 2 fluence with a clear filter. Further optimization of the filter wavelengths is considered necessary to fully apply PL for beer processing and possibly other light-sensitive foods and beverages.

Terpene volatiles define the flavor of terpenic grape cultivars. However, grape terpene concentrations can vary 2- to 3-fold across seasons and vineyards, impacting vintage quality. The plant hormone methyl jasmonate (MeJA) stimulates grape terpene production but is expensive and can decrease berry weight and maturity. The synthetic jasmonate prohydrojasmon (PDJ) is cost-effective yet has not been evaluated on grape maturity and terpene production. Here, we performed in vitro (berry culture) and in vivo (vineyard) experiments using Gewürztraminer ( Vitis vinifera L.) to evaluate the time- and concentration-dependent sensitivity of maturity parameters and terpene content to MeJA and PDJ. In vitro berry weight was reduced by high MeJA and PDJ concentration across timings. Terpenes were most sensitive to low MeJA concentration at veraison (increased 24-fold) in vitro . Moderate PDJ concentration applied at veraison doubled (increased twofold) terpene concentration in vivo without impacting berry weight or maturity. In conclusion, PDJ may provide a solution to mitigate seasonal variability in terpene production in terpenic grape cultivars.

Terpenes play a formative role in grape and wine flavor, particularly for high-terpenic cultivars. Differences in terpene profiles influence grape varietal character and vintage quality. Little is known about the endogenous factors controlling terpene biosynthesis in grape. Through multiple experiments, six hormones (abscisic acid, ABA; ethylene, ETH; jasmonic acid, JA; methyl jasmonate, MeJA; indole-3-acetic acid, IAA; 1-naphthaleneacetic acid, NAA) that either promote or repress ripening were applied to Gewürztraminer clusters near veraison to gauge their effect on ripening and terpene biosynthesis. Jasmonates (JA, MeJA) increased terpene concentrations and the expression of terpene genes in grapes. Such increases were not associated to increases of other ripening-related metabolites such as sugars or anthocyanins. MeJA also affected the expression of several hormone related genes, increased IAA levels, and reduced sugar and anthocyanin concentration in grapes. This research provides novel insights into terpene regulation by ripening-related hormones and jasmonates in grapes., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Lignin, the second most abundant biopolymer, is a promising renewable energy source and chemical feedstock. A key element of lignin biosynthesis is unknown: how do lignin precursors (monolignols) get from inside the cell out to the cell wall where they are polymerized? Modeling indicates that monolignols can passively diffuse through lipid bilayers, but this has not been tested experimentally. We demonstrate significant monolignol diffusion occurs when laccases, which consume monolignols, are present on one side of the membrane. We hypothesize that lignin polymerization could deplete monomers in the wall, creating a concentration gradient driving monolignol diffusion. We developed a two-photon microscopy approach to visualize lignifying Arabidopsis thaliana root cells. Laccase mutants with reduced ability to form lignin polymer in the wall accumulated monolignols inside cells. In contrast, active transport inhibitors did not decrease lignin in the wall and scant intracellular phenolics were observed. Synthetic liposomes were engineered to encapsulate laccases, and monolignols crossed these pure lipid bilayers to form polymer within. A sink-driven diffusion mechanism explains why it has been difficult to identify genes encoding monolignol transporters and why the export of varied phenylpropanoids occurs without specificity. It also highlights an important role for cell wall oxidative enzymes in monolignol export., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Blueberry aroma is one of the most important quality traits that influences consumer purchasing decisions. This study aimed to optimize and validate a solid-phase microextraction-gas chromatography/mass spectrometry (SPME-GC/MS) method for the quantification of 73 volatile compounds in northern highbush blueberries. A SPME extraction of blueberries with water and specific proportions of sodium chloride, citric acid, and ascorbic acid, for 60 min at 50 °C using a divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber was optimal. The method was validated for sensitivity, reproducibility, linearity, and accuracy, and used to quantify volatile compounds through matrix-matched calibration curves in six blueberry cultivars ('Duke', 'Draper', 'Bluecrop', 'Calypso', 'Elliott', and 'Last Call'). Terpenes represented the most abundant volatile fraction, followed by aldehydes and alcohols. Linalool and 2-(E)-hexenal were key compounds that differentiated blueberry cultivars via Principal Component Analysis (PCA). Enantiomeric analyses revealed an excess of (-)-limonene, (-)-α-pinene, and (+)-linalool for all cultivars with potential impacts on the blueberry aroma., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The grapevine is subject to high number of fungal and viral diseases, which are responsible for important economic losses in the global wine sector every year. These pathogens deteriorate grapevine berry quality either directly via the modulation of fruit metabolic pathways and the production of endogenous compounds associated with bad taste and/or flavor, or indirectly via their impact on vine physiology. The most common and devastating fungal diseases in viticulture are gray mold, downy mildew (DM), and powdery mildew (PM), caused, respectively by Botrytis cinerea , Plasmopara viticola , and Erysiphe necator . Whereas B. cinerea mainly infects and deteriorates the ripening fruit directly, deteriorations by DM and PM are mostly indirect via a reduction of photosynthetic leaf area. Nevertheless, mildews can also infect berries at certain developmental stages and directly alter fruit quality via the biosynthesis of unpleasant flavor compounds that impair ultimate wine quality. The grapevine is furthermore host of a wide range of viruses that reduce vine longevity, productivity and berry quality in different ways. The most widespread virus-related diseases, that are known nowadays, are Grapevine Leafroll Disease (GLRD), Grapevine Fanleaf Disease (GFLD), and the more recently characterized grapevine red blotch disease (GRBD). Future climatic conditions are creating a more favorable environment for the proliferation of most virus-insect vectors, so the spread of virus-related diseases is expected to increase in most wine-growing regions. However, the impact of climate change on the evolution of fungal disease pressure will be variable and depending on region and pathogen, with mildews remaining certainly the major phytosanitary threat in most regions because their development rate is to a large extent temperature-driven. This paper aims to provide a review of published literature on most important grapevine fungal and viral pathogens and their impact on grape berry physiology and quality. Our overview of the published literature highlights gaps in our understanding of plant-pathogen interactions, which are valuable for conceiving future research programs dealing with the different pathogens and their impacts on grapevine berry quality and metabolism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rienth, Vigneron, Walker, Castellarin, Sweetman, Burbidge, Bonghi, Famiani and Darriet.)

In grapevines, as in other plants, sucrose and its constituents glucose and fructose are fundamentally important and carry out a multitude of roles. The aims of this review are three-fold. First, to provide a summary of the metabolism and transport of sucrose in grapevines, together with new insights and interpretations. Second, to stress the importance of considering the compartmentation of metabolism. Third, to outline the key role of acid invertase in osmoregulation associated with sucrose metabolism and transport in plants.

Regulated deficit irrigation (RDI) is a viticultural practice known to improve grape phenolics and color in red grapes; however, the impact of this practice on grape aromas remains largely unknown. The effects of RDI treatments applied at various berry developmental stages on canopy, yield, and free and glycoside-bound terpenes of the berry were investigated for three consecutive seasons. All RDI treatments reduced leaf photosynthesis and yield, but not when applied after veraison. Berry total soluble solids at harvest were reduced by RDI applied after veraison or throughout the season. Despite reducing berry sugars, RDI applied after veraison increased the concentration at harvest of key free terpenes for Gewürztraminer grapes such as geraniol and citronellol. RDI treatments down-regulated some terpene genes, which indicates that the observed changes in terpene concentration were not transcriptionally regulated. This study suggests that RDI applied after version can potentially improve wine aroma in Gewürztraminer., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Tartaric acid (TA) is an obscure end point to the catabolism of ascorbic acid (Asc). Here, it is proposed as a "specialized primary metabolite", originating from carbohydrate metabolism but with restricted distribution within the plant kingdom and lack of known function in primary metabolic pathways. Grapes fall into the list of high TA-accumulators, with biosynthesis occurring in both leaf and berry. Very little is known of the TA biosynthetic pathway enzymes in any plant species, although recently some progress has been made in this space. New technologies in grapevine research such as the development of global co-expression network analysis tools and genome-wide association studies, should enable more rapid progress. There is also a lack of information regarding roles for this organic acid in plant metabolism. Therefore this review aims to briefly summarize current knowledge about the key intermediates and enzymes of TA biosynthesis in grapes and the regulation of its precursor, ascorbate, followed by speculative discussion around the potential roles of TA based on current knowledge of Asc metabolism, TA biosynthetic enzymes and other aspects of fruit metabolism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Burbidge, Ford, Melino, Wong, Jia, Jenkins, Soole, Castellarin, Darriet, Rienth, Bonghi, Walker, Famiani and Sweetman.)

The abscisic acid (ABA) increase and auxin decline are both indicators of ripening initiation in grape berry, and norisoprenoid accumulation also starts at around the onset of ripening. However, the relationship between ABA, auxin, and norisoprenoids remains largely unknown, especially at the transcriptome level. To investigate the transcriptional and posttranscriptional regulation of the ABA and synthetic auxin 1-naphthaleneacetic acid (NAA) on norisoprenoid production, we performed time-series GC-MS and RNA-seq analyses on Vitis vinifera L. cv. Cabernet Sauvignon grape berries from pre-veraison to ripening. Higher levels of free norisoprenoids were found in ABA-treated mature berries in two consecutive seasons, and both free and total norisoprenoids were significantly increased by NAA in one season. The expression pattern of known norisoprenoid-associated genes in all samples and the up-regulation of specific alternative splicing isoforms of VviDXS and VviCRTISO in NAA-treated berries were predicted to contribute to the norisoprenoid accumulation in ABA and NAA-treated berries. Combined weighted gene co-expression network analysis (WGCNA) and DNA affinity purification sequencing (DAP-seq) analysis suggested that VviGATA26, and the previously identified switch genes of myb RADIALIS (VIT&#95;207s0005g02730) and MAD-box (VIT&#95;213s0158g00100) could be potential regulators of norisoprenoid accumulation. The positive effects of ABA on free norisoprenoids and NAA on total norisoprenoid accumulation were revealed in the commercially ripening berries. Since the endogenous ABA and auxin are sensitive to environmental factors, this finding provides new insights to develop viticultural practices for managing norisoprenoids in vineyards in response to changing climates.

Wine fermentation processes are driven by complex microbial systems, which comprise eukaryotic and prokaryotic microorganisms that participate in several biochemical interactions with the must and wine chemicals and modulate the organoleptic properties of wine. Among these, yeasts play a fundamental role, since they carry out the alcoholic fermentation (AF), converting sugars to ethanol and CO 2 together with a wide range of volatile organic compounds. The contribution of Saccharomyces cerevisiae , the reference organism associated with AF, has been extensively studied. However, in the last decade, selected non-Saccharomyces strains received considerable commercial and oenological interest due to their specific pro-technological aptitudes and the positive influence on sensory quality. This review aims to highlight the inter-specific variability within the heterogeneous class of non- Saccharomyces in terms of synthesis and release of volatile organic compounds during controlled AF in wine. In particular, we reported findings on the presence of model non- Saccharomyces organisms, including Torulaspora delbrueckii, Hanseniaspora spp, Lachancea thermotolerans, Metschnikowia pulcherrima, Pichia spp. and Candida zemplinina , in combination with S. cerevisiae . The evidence is discussed from both basic and applicative scientific perspective. In particular, the oenological significance in different kind of wines has been underlined.

Early leaf removal (ELR) applied in the grapevine cluster zone at bloom or pre-bloom (PB) is a vineyard practice commonly utilized to reduce fruit disease and yield. In addition, the literature reports that ELR enhances fruit quality, however, little research has deciphered the potential factors regulating this response. In this work, the objective was to understand whether the increase in fruit quality in response to manual or mechanical leaf removal is due to changes in fruit-zone microclimate, vine physiology, or ripening/stress related hormone biosynthesis. In 'Merlot' (Vitis vinifera L.) vines, 60% of leaf area was removed from shoots in three ways: 1) manual removal of 5 leaves (PB-MA), 2) mechanical removal (PB-ME), and 3) simulated mechanical removal (PB-SIM), which was implemented by removing the distal portion of leaves on the first eight nodes to understand whether PB-ME improves fruit quality via enhanced microclimate conditions or plant stress. Yield was reduced in PB-ME and PB-SIM, while total soluble solids was not different at harvest; meaning that ELR decreased the partitioning of carbohydrates to fruit. Anthocyanins and flavonols were enhanced by PB-ME, however neither ABA nor ethylene were similarly altered. Instead, the leaf area at nodes above the fruit-zone was lower in PB-ME compared to non-defoliated ones, which increased post-veraison fruit temperature (+2.8 °C). These parameters correlated with anthocyanins at harvest. In conclusion, skin phenylpropanoid concentrations were influenced by canopy density above the fruit-zone. Additionally, ripening-related phytohormones were not involved in the response of phenylpropanoid biosynthesis in vine subjected to ELR., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

This work focused on the development of starch-based (potato, corn, sweet potato, green bean and tapioca) edible packaging film incorporated with blueberry pomace powder (BPP). The optical, mechanical, thermal, and physicochemical properties were subsequently tested. The film color was not affected by the addition of BPP. BPP incorporated into corn and green bean starch films showed increased light barrier properties, indicating a beneficial effect to prevent UV radiation-induced food deterioration. Film thickness and transparency were not primarily affected by changing the starch type or the BPP concentration, although the corn starch films were the most transparent. Furthermore, all films maintained structural integrity and had a high tensile strength. The water vapor transmission rate of all the films was found to be greater than conventional polyethylene films. The average solubility of all the films made from different starch types was between 24 and 37%, which indicates the usability of these films for packaging, specifically for low to intermediate moisture foods. There were no statistical differences in Differential Scanning Calorimetry parameters with changes in the starch type and pomace levels. Migration assays showed a greater release of the active compounds from BPP into acetic acid medium (aqueous food simulant) than ethanol medium (fatty food simulant). The incorporation of BPP into starch-chitosan films resulted in the improvement of film performance, thereby suggesting the potential for applying BPP into starch-based films for active packaging.

Lignin is a key secondary cell wall chemical constituent, and is both a barrier to biomass utilization and a potential source of bioproducts. The Arabidopsis transcription factors MYB58 and MYB63 have been shown to upregulate gene expression of the general phenylpropanoid and monolignol biosynthetic pathways. The overexpression of these genes also results in dwarfism. The vascular integrity, soluble phenolic profiles, cell wall lignin, and transcriptomes associated with these MYB-overexpressing lines were characterized. Plants with high expression of MYB58 and MYB63 had increased ectopic lignin and the xylem vessels were regular and open, suggesting that the stunted growth is not associated with loss of vascular conductivity. MYB58 and MYB63 overexpression lines had characteristic soluble phenolic profiles with large amounts of monolignol glucosides and sinapoyl esters, but decreased flavonoids. Because loss of function lac4 lac17 mutants also accumulate monolignol glucosides, we hypothesized that LACCASE overexpression might decrease monolignol glucoside levels in the MYB-overexpressing plant lines. When laccases related to lignification ( LAC4 or LAC17 ) were co-overexpressed with MYB63 or MYB58 , the dwarf phenotype was rescued. Moreover, the overexpression of either LAC4 or LAC17 led to wild-type monolignol glucoside levels, as well as wild-type lignin levels in the rescued plants. Transcriptomes of the rescued double MYB63-OX/LAC17-OX overexpression lines showed elevated, but attenuated, expression of the MYB63 gene itself and the direct transcriptional targets of MYB63. Contrasting the dwarfism from overabundant monolignol production with dwarfism from lignin mutants provides insight into some of the proposed mechanisms of lignin modification-induced dwarfism., (© 2020 The Authors. Plant Direct published by American Society of Plant Biologists and the Society for Experimental Biology and John Wiley & Sons Ltd.)

Wine is a product of grape juice fermentation by yeast. Terroir is a term that encompasses all environmental factors and interactions at a specific geographical site, resulting in the development of regional-specific microbial strains and grape metabolites. In this study we determine the distribution of vineyard-associated wine yeast strains and characterize the flavonoid profile of Pinot Noir grapes among 3 sub-regions in the Okanagan Valley (OV), a major wine region in British Columbia, Canada. Pinot Noir grape samples were collected from 13 vineyards among 3 sub-regions of the OV, namely Kelowna (KE), Naramata-Penticton (NP) and Oliver-Osoyoos (OO), within a week prior to the winery harvesting date in 2016 and 2017. A total of 156 spontaneous Pinot Noir fermentations were conducted and vineyard-associated Saccharomyces strains were isolated from fermentations that reached two-thirds sugar depletion. Using microsatellite genotyping, we identified 103 Saccharomyces cerevisiae strains and 9 Saccharomyces uvarum strains. We also identified Saccharomyces paradoxus in one vineyard using ITS sequencing. We developed a microsatellite database of 160 commercial S. cerevisiae strains to determine the identity of the isolated strains and we include the database herein. Commercial strains were widely distributed across the three sub-regions. Forty-two of our 103 S. cerevisiae strains were equivalent or highly similar to commercial strains whereas the remaining 61 were considered as 'unknown' strains. Two S. uvarum strains were previously isolated in other OV studies and none matched the S. uvarum commercial strain BMV58. S. cerevisiae population structure was driven by sub-region, although S. cerevisiae populations did not differ significantly across vintages. S. uvarum and S. paradoxus were only identified in the 2017 vintage, demonstrating dynamic wine yeast populations between vintages. We found that the flavonoid profile of Pinot Noir grapes from the same 13 vineyards was also affected by sub-regional terroir . The anthocyanin content was lower and the proportion of methoxylated anthocyanins and flavonols was higher in Pinot Noir grapes from OO, the warmer sub-region as compared to KE, the cooler sub-region. Our study demonstrates that both yeast populations and metabolites associated with the Pinot Noir variety have sub-regional variation within a viticultural area., (Copyright © 2020 Cheng, Martiniuk, Hamilton, McCarthy, Castellarin and Measday.)

Water availability is arguably the most important environmental factor limiting crop growth and productivity. Erratic precipitation patterns and increased temperatures resulting from climate change will likely make drought events more frequent in many regions, increasing the demand on freshwater resources and creating major challenges for agriculture. Addressing these challenges through increased irrigation is not always a sustainable solution so there is a growing need to identify and/or breed drought-tolerant crop varieties in order to maintain sustainability in the context of climate change. Grapevine (Vitis vinifera), a major fruit crop of economic importance, has emerged as a model perennial fruit crop for the study of drought tolerance. This review synthesizes the most recent results on grapevine drought responses, the impact of water deficit on fruit yield and composition, and the identification of drought-tolerant varieties. Given the existing gaps in our knowledge of the mechanisms underlying grapevine drought responses, we aim to answer the following question: how can we move towards a more integrative definition of grapevine drought tolerance?, (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Flavonoids impart color and mouthfeel to grapes and wine and are very sensitive to environmental conditions. Growth chamber experiments were performed to investigate the effect of temperature regimes and the differences between day/night temperatures on anthocyanins and flavonols in Merlot grapes. Among the regimes tested, the ones with diurnal 20°C determined the highest levels of anthocyanins and flavonols. Higher diurnal temperatures decreased those levels but increased the proportion of methoxylated and acylated species. When regimes with the same day temperature but different night temperatures were compared, differences between day/night temperatures did not affect anthocyanins, unless a difference of 25°C between day and night temperatures was imposed. When regimes with the same night temperature but different day temperatures were compared, the regime with higher day temperature had a lower anthocyanin level. No relationships were observed between the effects of temperature regimes on anthocyanin level and the expression of key anthocyanin genes. However, the effects on anthocyanin acylation level were consistent with the effects on the acyltransferase expression, and the effects on flavonol level were consistent with the effects on the expression of key flavonol genes. This study indicates that, in Merlot grapes, anthocyanins and flavonols are mostly sensitive to day temperatures., (Copyright © 2020 Yan, Song, Falginella and Castellarin.)

Drought events are a major challenge for many horticultural crops, including grapes, which are often cultivated in dry and warm climates. It is not understood how the cuticle contributes to the grape berry response to water deficit (WD); furthermore, the cuticular waxes and the related biosynthetic pathways are poorly characterized in this fruit. In this study, we identified candidate wax-related genes from the grapevine genome by phylogenetic and transcriptomic analyses. Developmental and stress response expression patterns of these candidates were characterized across pre-existing RNA sequencing data sets and confirmed a high responsiveness of the pathway to environmental stresses. We then characterized the developmental and WD-induced changes in berry cuticular wax composition, and quantified differences in berry transpiration. Cuticular aliphatic wax content was modulated during development and an increase was observed under WD, with wax esters being strongly up-regulated. These compositional changes were related to up-regulated candidate genes of the aliphatic wax biosynthetic pathway, including CER10, CER2, CER3, CER1, CER4, and WSD1. The effect of WD on berry transpiration was not significant. This study indicates that changes in cuticular wax amount and composition are part of the metabolic response of the grape berry to WD, but these changes do not reduce berry transpiration., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Main Conclusion: The phosphorylation status of MYB75 at T-131 affects protein stability, flavonoid profiles, and patterns of gene expression. The Arabidopsis transcription factor Myeloblastosis protein 75 (MYB75, AT1G56650) is known to act as a positive transcriptional regulator of genes required for flavonoid and anthocyanin biosynthesis. MYB75 was also shown to negatively regulate lignin and other secondary cell wall biosynthetic genes (Bhargava et al. in Plant Physiol 154(3):1428-1438, 2010). While transcriptional regulation of MYB75 has been described in numerous publications, little is known about post-translational control of MYB75 protein function. In a recent publication, light-induced activation of a MAP kinase (MPK4, AT4G01370) in Arabidopsis was reported to lead to MYB75 phosphorylation at two canonical MPK target sites, threonines, T-126 and T-131. This double phosphorylation event positively influenced MYB75 protein stability (Li et al. in Plant Cell 28(11):2866-2883, 2016). We have examined this phenomenon through use of phosphomutant forms of MYB75 and found that MYB75 is phosphorylated primarily at T-131, and that the phosphorylation of MYB75 recombinant protein in vitro can be catalyzed by multiple MAP kinases, including MPK3 (AT3G45640), MPK6 (AT2G43790), MPK4 and MPK11 (AT1G01560). We also demonstrate that MYB75 can bind to a large number of Arabidopsis MPK's in vitro, suggesting it could be a target of multiple signalling pathways. The impact of MYB75 phosphorylation at T-131 on the function of this transcription factor, in terms of localization, stability, and protein-protein interactions with known binding partners was examined in transgenic lines expressing phosphomimic and phosphonull versions of MYB75, to capture the behaviour of permanently phosphorylated and unphosphorylated MYB75 protein, respectively. In addition, we describe how ectopic over-expression of different phosphovariant forms of MYB75 (MYB75 WT , MYB75 T131A , and MYB75 T131E ) affects flavonoid biochemical profiles and global changes of gene expression in the corresponding transgenic Arabidopsis plants.

The cannabis leaf is iconic, but it is the flowers of cannabis that are consumed for the psychoactive and medicinal effects of their specialized metabolites. Cannabinoid metabolites, together with terpenes, are produced in glandular trichomes. Superficially, stalked and sessile trichomes in cannabis only differ in size and whether they have a stalk. The objectives of this study were: to define each trichome type using patterns of autofluorescence and secretory cell numbers, to test the hypothesis that stalked trichomes develop from sessile-like precursors, and to test whether metabolic specialization occurs in cannabis glandular trichomes. A two-photon microscopy technique using glandular trichome intrinsic autofluorescence was developed which demonstrated that stalked glandular trichomes possessed blue autofluorescence correlated with high cannabinoid levels. These stalked trichomes had 12-16 secretory disc cells and strongly monoterpene-dominant terpene profiles. In contrast, sessile trichomes on mature flowers and vegetative leaves possessed red-shifted autofluorescence, eight secretory disc cells and less monoterpene-dominant terpene profiles. Moreover, intrinsic autofluorescence patterns and disc cell numbers supported a developmental model where stalked trichomes develop from apparently sessile trichomes. Transcriptomes of isolated floral trichomes revealed strong expression of cannabinoid and terpene biosynthetic genes, as well as uncharacterized genes highly co-expressed with CBDA synthase. Identification and characterization of two previously unknown and highly expressed monoterpene synthases highlighted the metabolic specialization of stalked trichomes for monoterpene production. These unique properties and highly expressed genes of cannabis trichomes determine the medicinal, psychoactive and sensory properties of cannabis products., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

Recent studies revealed that terpenes present in grapes determine the 'stone fruit' aroma of Viognier wines. This study analysed the evolution of free and glycosylated terpenes along with other volatile organic compounds (VOCs) during berry development in Viognier berries exposed to three irrigation regimes. These regimes (standard irrigation over the season, early deficit irrigation from 25 days after anthesis to veraison, prolonged deficit irrigation from 25 days after anthesis to harvest) were imposed to Viognier grapevines grown in the Okanagan Valley (British Columbia, Canada). Berry samples were collected over the season and analysed for determining the chemical composition, including free and glycosylated VOCs, and the expression of genes related to VOC biosynthesis. Among free VOCs, aldehydes peaked at veraison, alcohols did not vary across berry development, and terpenes increased during ripening. Among glycosylated VOCs, aldehydes and terpenes mirrored the free fractions while alcohols peaked before veraison. At harvest, a larger concentration of linalool oxides was detected in the glycosylated than in the free fraction. Increases of terpenes during ripening were modulated at the transcriptional level, particularly by two terpene synthases VviCSbOci and VviPNLinNer1. Early deficit irrigation increased the concentration of free α-terpineol and linalool, while prolonged deficit irrigation reduced the concentration of glycosylated α-terpineol. These results provide a basis for further studies on managing Viognier grape aroma in vineyards., (Copyright © 2019. Published by Elsevier Ltd.)

Abscisic acid (ABA) is a plant hormone that can mitigate heavy metal toxicity. Exogenous ABA and ABA mimic 1 (AM1) were applied to study the influence on Zn uptake and accumulation in Vitis vinifera L. cv. Merlot seedlings exposed to excess Zn. The seedlings were treated with either normal or excess levels of Zn in combination with applications of ABA and AM1. Excess Zn exposure resulted in decreased lateral root length, decreased photosynthesis, elevated uptake, and accumulation of Zn in roots, trunks, and stems, decreased jasmonic acid content in roots and leaves, and induced the expression of Zn transportation- and detoxification-related genes. Remarkably, in the presence of toxic amounts of Zn, the exogenous application of ABA, but not of AM1, reduced the uptake and accumulation of Zn in roots and induced higher expression of both ZIP genes and detoxification-related genes in root and leaf. These results indicate that exogenous ABA enhances the tolerance of grape seedlings to excess Zn and that AM1 is not a suitable ABA mimic compound for Zn stress alleviation in grapes.

Background: Grape leaves provide the biochemical substrates for berry development. Thus, understanding the regulation of grapevine leaf metabolism can aid in discerning processes fundamental to fruit development and berry quality. Here, the temporal alterations in leaf metabolism in Merlot grapevine grown under sufficient irrigation and water deficit were monitored from veraison until harvest., Results: The vines mediated water stress gradually and involving multiple strategies: osmotic adjustment, transcript-metabolite alteration and leaf shedding. Initially stomatal conductance and leaf water potential showed a steep decrease together with the induction of stress related metabolism, e.g. up-regulation of proline and GABA metabolism and stress related sugars, and the down-regulation of developmental processes. Later, progressive soil drying was associated with an incremental contribution of Ca 2+ and sucrose to the osmotic adjustment concomitant with the initiation of leaf shedding. Last, towards harvest under progressive stress conditions following leaf shedding, incremental changes in leaf water potential were measured, while the magnitude of perturbation in leaf metabolism lessened., Conclusions: The data present evidence that over time grapevine acclimation to water stress diversifies in temporal responses encompassing the alteration of central metabolism and gene expression, osmotic adjustments and reduction in leaf area. Together these processes mitigate leaf water stress and aid in maintaining the berry-ripening program.

Fruits have been traditionally classified into two categories based on their capacity to produce and respond to ethylene during ripening. Fruits whose ripening is associated to a peak of ethylene production and a respiration burst are referred to as climacteric, while those that are not are referred to as non-climacteric. However, an increasing body of literature supports an important role for ethylene in the ripening of both climacteric and non-climacteric fruits. Genome and transcriptomic data have become available across a variety of fruits and we leverage these data to compare the structure and transcriptional regulation of the ethylene receptors and related proteins. Through the analysis of four economically important fruits, two climacteric (tomato and apple), and two non-climacteric (grape and citrus), this review compares the structure and transcriptional regulation of the ethylene receptors and related proteins in both types of fruit, establishing a basis for the annotation of ethylene-related genes. This analysis reveals two interesting differences between climacteric and non-climacteric fruit: i) a higher number of ETR genes are found in climacteric fruits, and ii) non-climacteric fruits are characterized by an earlier ETR expression peak relative to sugar accumulation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Grapevine fanleaf virus (GFLV) causes grapevine fanleaf degeneration, one of the oldest known viral diseases of grapevines. The virus has been found in all winegrowing regions around the world. In the seasons 2011-12 a comparison between field grown GFLV-infected and healthy grapevines was conducted for the cultivars Schioppettino in North-Eastern Italy and Refošk in South-Western Slovenia. Our research showed that GFLV infection caused a drop of the yield due to reduction of both cluster weight and berry weight. Besides the yield, the berry composition was also affected; in detail, anthocyanin concentration increased in both varieties but significantly only in the case of Schioppettino. Upregulation of the F3'5'H gene and downregulation of F3'H gene in the berries of GFLV infected vines compared with the ones of healthy control vines resulted in modified proportions between di- and tri- hydroxylated or methylated derivatives of anthocyanins. The F3H1 gene was identified to be the most strongly regulated gene of the flavonoid biosynthetic pathway by GFLV infection, indicating its important role in increasing anthocyanin concentration in grapes of GFLV infected vines as compared with healthy controls.

Background: The major intrinsic protein (MIP) family is a family of proteins, including aquaporins, which facilitate water and small molecule transport across plasma membranes. In plants, MIPs function in a huge variety of processes including water transport, growth, stress response, and fruit development. In this study, we characterize the structure and transcriptional regulation of the MIP family in grapevine, describing the putative genome duplication events leading to the family structure and characterizing the family's tissue and developmental specific expression patterns across numerous preexisting microarray and RNAseq datasets. Gene co-expression network (GCN) analyses were carried out across these datasets and the promoters of each family member were analyzed for cis-regulatory element structure in order to provide insight into their transcriptional regulation., Results: A total of 29 Vitis vinifera MIP family members (excluding putative pseudogenes) were identified of which all but two were mapped onto Vitis vinifera chromosomes. In this study, segmental duplication events were identified for five plasma membrane intrinsic protein (PIP) and four tonoplast intrinsic protein (TIP) genes, contributing to the expansion of PIPs and TIPs in grapevine. Grapevine MIP family members have distinct tissue and developmental expression patterns and hierarchical clustering revealed two primary groups regardless of the datasets analyzed. Composite microarray and RNA-seq gene co-expression networks (GCNs) highlighted the relationships between MIP genes and functional categories involved in cell wall modification and transport, as well as with other MIPs revealing a strong co-regulation within the family itself. Some duplicated MIP family members have undergone sub-functionalization and exhibit distinct expression patterns and GCNs. Cis-regulatory element (CRE) analyses of the MIP promoters and their associated GCN members revealed enrichment for numerous CREs including AP2/ERFs and NACs., Conclusions: Combining phylogenetic analyses, gene expression profiling, gene co-expression network analyses, and cis-regulatory element enrichment, this study provides a comprehensive overview of the structure and transcriptional regulation of the grapevine MIP family. The study highlights the duplication and sub-functionalization of the family, its strong coordinated expression with genes involved in growth and transport, and the putative classes of TFs responsible for its regulation.

Postveraison water deficit is a common strategy implemented to improve fruit composition in many wine-growing regions. However, contrasting results are often reported on fruit size and composition, a challenge for generalizing the positive impact of this technique. Our research investigated the effect of water deficit (WD) imposed at veraison on Merlot grapevines, during two experimental seasons (2014-2015). In both years WD resulted in reduced carbon assimilation rates and leaf shedding. However, the treatment effect on the analyzed berry parameters varied between seasons. Modification of skin metabolites was more evident in 2015 than in 2014, despite the similar soil water content and water stress physiological parameters (gas exchange, water potential) recorded in the two experimental years. Higher solar radiation and air temperature in 2015 than in 2014 hint for the involvement of atmospheric parameters in fulfilling the potential effect of WD. Our results suggest that the interaction between water availability and weather conditions plays a crucial role in modulating the grape berry composition.

Grapes are one of the major fruit crops and they are cultivated in many dry environments. This study comprehensively characterizes the metabolic response of grape berries exposed to water deficit at different developmental stages. Increases of proline, branched-chain amino acids, phenylpropanoids, anthocyanins, and free volatile organic compounds have been previously observed in grape berries exposed to water deficit. Integrating RNA-sequencing analysis of the transcriptome with large-scale analysis of central and specialized metabolites, we reveal that these increases occur via a coordinated regulation of key structural pathway genes. Water deficit-induced up-regulation of flavonoid genes is also coordinated with the down-regulation of many stilbene synthases and a consistent decrease in stilbenoid concentration. Water deficit activated both ABA-dependent and ABA-independent signal transduction pathways by modulating the expression of several transcription factors. Gene-gene and gene-metabolite network analyses showed that water deficit-responsive transcription factors such as bZIPs, AP2/ERFs, MYBs, and NACs are implicated in the regulation of stress-responsive metabolites. Enrichment of known and novel cis -regulatory elements in the promoters of several ripening-specific/water deficit-induced modules further affirms the involvement of a transcription factor cross-talk in the berry response to water deficit. Together, our integrated approaches show that water deficit-regulated gene modules are strongly linked to key fruit-quality metabolites and multiple signal transduction pathways may be critical to achieve a balance between the regulation of the stress-response and the berry ripening program. This study constitutes an invaluable resource for future discoveries and comparative studies, in grapes and other fruits, centered on reproductive tissue metabolism under abiotic stress.

Coordinated transcriptional and metabolic reprogramming ensures a plant's continued growth and survival under adverse environmental conditions. Transcription factors (TFs) act to modulate gene expression through complex cis-regulatory element (CRE) interactions. Genome-wide analysis of known plant CREs was performed for all currently predicted protein-coding gene promoters in grapevine (Vitis vinifera L.). Many CREs such as abscisic acid (ABA)-responsive, drought-responsive, auxin-responsive, and evening elements, exhibit bona fide CRE properties such as strong position bias towards the transcription start site (TSS) and over-representation when compared with random promoters. Genes containing these CREs are enriched in a large repertoire of plant biological pathways. Large-scale transcriptome analyses also show that these CREs are highly implicated in grapevine development and stress response. Numerous CRE-driven modules in condition-specific gene co-expression networks (GCNs) were identified and many of these modules were highly enriched for plant biological functions. Several modules corroborate known roles of CREs in drought response, pathogen defense, cell wall metabolism, and fruit ripening, whereas others reveal novel functions in plants. Comparisons with Arabidopsis suggest a general conservation in promoter architecture, gene expression dynamics, and GCN structure across species. Systems analyses of CREs provide insights into the grapevine cis-regulatory code and establish a foundation for future genomic studies in grapevine., (© The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

R2R3-MYB transcription factors (TFs) belong to a large and functionally diverse protein superfamily in plants. In this study, we explore the evolution and function of this family in grapevine (Vitis vinifera L.), a high-value fruit crop. We identified and manually curated 134 genes using RNA-Seq data, and named them systematically according to the Super-Nomenclature Committee. We identified novel genes, splicing variants and grapevine/woody-specific duplicated subgroups, suggesting possible neo- and sub-functionalization events. Regulatory network analysis ascribed biological functions to uncharacterized genes and validated those of known genes (e.g. secondary cell wall biogenesis and flavonoid biosynthesis). A comprehensive analysis of different MYB binding motifs in the promoters of co-expressed genes predicted grape R2R3-MYB binding preferences and supported evidence for putative downstream targets. Enrichment of cis-regulatory motifs for diverse TFs reinforced the notion of transcriptional coordination and interaction between MYBs and other regulators. Analysis of the network of Subgroup 2 showed that the resveratrol-related VviMYB14 and VviMYB15 share common co-expressed STILBENE SYNTHASE genes with the uncharacterized VviMYB13. These regulators have distinct expression patterns within organs and in response to biotic and abiotic stresses, suggesting a pivotal role of VviMYB13 in regulating stilbene accumulation in vegetative tissues and under biotic stress conditions., (© The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

Grapevine (Vitis vinifera L.) is a species well known for its adaptation to radiation. However, photomorphogenic factors related to UV-B responses have not been molecularly characterized. We cloned and studied the role of UV-B RECEPTOR (UVR1), ELONGATED HYPOCOTYL 5 (HY5), and HY5 HOMOLOGUE (HYH) from V. vinifera We performed gene functional characterizations, generated co-expression networks, and tested them in different environmental conditions. These genes complemented the Arabidopsis uvr8 and hy5 mutants in morphological and secondary metabolic responses to radiation. We combined microarray and RNA sequencing (RNA-seq) data with promoter inspections to identify HY5 and HYH putative target genes and their DNA binding preferences. Despite sharing a large set of common co-expressed genes, we found different hierarchies for HY5 and HYH depending on the organ and stress condition, reflecting both co-operative and partially redundant roles. New candidate UV-B gene markers were supported by the presence of HY5-binding sites. These included a set of flavonol-related genes that were up-regulated in a HY5 transient expression assay. We irradiated in vitro plantlets and fruits from old potted vines with high and low UV-B exposures and followed the accumulation of flavonols and changes in gene expression in comparison with non-irradiated conditions. UVR1, HY5, and HYH expression varied with organ, developmental stage, and type of radiation. Surprisingly, UVR1 expression was modulated by shading and temperature in berries, but not by UV-B radiation. We propose that the UV-B response machinery favours berry flavonol accumulation through the activation of HY5 and HYH at different developmental stages at both high and low UV-B exposures., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The development and accumulation of secondary metabolites in grapes determine wine color, taste, and aroma. This study aimed to investigate the effect of leaf removal before flowering, a practice recently introduced to reduce cluster compactness and Botrytis rot, on anthocyanin, tannin, and methoxypyrazine concentrations in 'Merlot' grapes and wines. Leaf removal before flowering was compared with leaf removal after flowering and an untreated control. No effects on tannin and anthocyanin concentrations in grapes were observed. Both treatments reduced levels of 3-isobutyl-2-methoxypyrazine (IBMP) in the grapes and the derived wines, although the after-flowering treatment did so to a greater degree in the fruit specifically. Leaf removal before flowering can be used to reduce cluster compactness, Botrytis rot, and grape and wine IBMP concentration and to improve wine color intensity but at the expense of cluster weight and vine yield. Leaf removal after flowering accomplishes essentially the same results without loss of yield.

Background: In wine grape production, management practices have been adopted to optimize grape and wine quality attributes by producing, or screening for, berries of smaller size. Fruit size and composition are influenced by numerous factors that include both internal (e.g. berry hormone metabolism) and external (e.g. environment and cultural practices) factors. Combined physiological, biochemical, and transcriptome analyses were performed to improve our current understanding of metabolic and transcriptional pathways related to berry ripening and composition in berries of different sizes., Results: The comparison of berry physiology between small and large berries throughout development (from 31 to 121 days after anthesis, DAA) revealed significant differences in firmness, the rate of softening, and sugar accumulation at specific developmental stages. Small berries had significantly higher skin to berry weight ratio, lower number of seeds per berry, and higher anthocyanin concentration compared to large berries. RNA-sequencing analyses of berry skins at 47, 74, 103, and 121 DAA revealed a total of 3482 differentially expressed genes between small and large berries. Abscisic acid, auxin, and ethylene hormone pathway genes were differentially modulated between berry sizes. Fatty acid degradation and stilbenoid pathway genes were upregulated at 47 DAA while cell wall degrading and modification genes were downregulated at 74 DAA in small compared to large berries. In the late ripening stage, concerted upregulation of the general phenylpropanoid and stilbenoid pathway genes and downregulation of flavonoid pathway genes were observed in skins of small compared to large berries. Cis-regulatory element analysis of differentially expressed hormone, fruit texture, flavor, and aroma genes revealed an enrichment of specific regulatory motifs related to bZIP, bHLH, AP2/ERF, NAC, MYB, and MADS-box transcription factors., Conclusions: The study demonstrates that physiological and compositional differences between berries of different sizes parallel transcriptome changes that involve fruit texture, flavor, and aroma pathways. These results suggest that, in addition to direct effects brought about by differences in size, key aspects involved in the regulation of ripening likely contribute to different quality profiles between small and large berries.

Drought stress affects anthocyanin accumulation and modification in vegetative and reproductive plant tissues. Anthocyanins are the most abundant flavonoids in grape (Vitis vinifera L.) coloured berry genotypes and are essential markers of grape winemaking quality. They are mostly mono- and di-methylated, such modifications increase their stability and improve berry quality for winemaking. Anthocyanin methylation in grape berries is induced by drought stress. A few caffeoyl-CoA O-methyltransferases (CCoAOMTs) active on anthocyanins have been described in grape. However, no drought-activated O-methyltransferases have been described in grape berries yet. In this study, we characterized VvCCoAOMT, a grapevine gene known to induce methylation of CoA esters in cultured grape cells. Transcript accumulation of VvCCoAOMT was detected in berry skins, and increased during berry ripening on the plant, and in cultured berries treated with ABA, concomitantly with accumulation of methylated anthocyanins, suggesting that anthocyanins may be substrates of this enzyme. Contrary as previously observed in cell cultures, biotic stress (Botrytis cinerea inoculation) did not affect VvCCoAOMT gene expression in leaves or berries, while drought stress increased VvCCoAOMT transcript in berries. The recombinant VvCCoAOMT protein showed in vitro methylating activity on cyanidin 3-O-glucoside. We conclude that VvCCoAOMT is a multifunctional O-methyltransferase that may contribute to anthocyanin methylation activity in grape berries, in particular under drought stress conditions., (Copyright © 2016 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: Secondary metabolism contributes to the adaptation of a plant to its environment. In wine grapes, fruit secondary metabolism largely determines wine quality. Climate change is predicted to exacerbate drought events in several viticultural areas, potentially affecting the wine quality. In red grapes, water deficit modulates flavonoid accumulation, leading to major quantitative and compositional changes in the profile of the anthocyanin pigments; in white grapes, the effect of water deficit on secondary metabolism is still largely unknown., Results: In this study we investigated the impact of water deficit on the secondary metabolism of white grapes using a large scale metabolite and transcript profiling approach in a season characterized by prolonged drought. Irrigated grapevines were compared to non-irrigated grapevines that suffered from water deficit from early stages of berry development to harvest. A large effect of water deficit on fruit secondary metabolism was observed. Increased concentrations of phenylpropanoids, monoterpenes, and tocopherols were detected, while carotenoid and flavonoid accumulations were differentially modulated by water deficit according to the berry developmental stage. The RNA-sequencing analysis carried out on berries collected at three developmental stages-before, at the onset, and at late ripening-indicated that water deficit affected the expression of 4,889 genes. The Gene Ontology category secondary metabolic process was overrepresented within up-regulated genes at all the stages of fruit development considered, and within down-regulated genes before ripening. Eighteen phenylpropanoid, 16 flavonoid, 9 carotenoid, and 16 terpenoid structural genes were modulated by water deficit, indicating the transcriptional regulation of these metabolic pathways in fruit exposed to water deficit. An integrated network and promoter analyses identified a transcriptional regulatory module that encompasses terpenoid genes, transcription factors, and enriched drought-responsive elements in the promoter regions of those genes as part of the grapes response to drought., Conclusion: Our study reveals that grapevine berries respond to drought by modulating several secondary metabolic pathways, and particularly, by stimulating the production of phenylpropanoids, the carotenoid zeaxanthin, and of volatile organic compounds such as monoterpenes, with potential effects on grape and wine antioxidant potential, composition, and sensory features.

Along with sugar accumulation and colour development, softening is an important physiological change during the onset of ripening in fruits. In this work, we investigated the relationships among major events during softening in grape (Vitis vinifera L.) by quantifying elasticity in individual berries. In addition, we delayed softening and inhibited sugar accumulation using a mechanical growth-preventing treatment in order to identify processes that are sugar and/or growth dependent. Ripening processes commenced on various days after anthesis, but always at similarly low elasticity and turgor. Much of the softening occurred in the absence of other changes in berry physiology investigated here. Several genes encoding key cell wall-modifying enzymes were not up-regulated until softening was largely completed, suggesting softening may result primarily from decreases in turgor. Similarly, there was no decrease in solute potential, increase in sugar concentration, or colour development until elasticity and turgor were near minimum values, and these processes were inhibited when berry growth was prevented. Increases in abscisic acid occurred early during softening and in the absence of significant expression of the V. vinifera 9-cis-epoxycarotenoid dioxygenases. However, these increases were coincident with decreases in the abscisic acid catabolite diphasic acid, indicating that initial increases in abscisic acid may result from decreases in catabolism and/or exogenous import. These data suggest that softening, decreases in turgor, and increases in abscisic acid represent some of the earliest events during the onset of ripening. Later, physical growth, further increases in abscisic acid, and the accumulation of sugar are integral for colour development., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Plant phenolics have been for many years a theme of major scientific and applied interest. Grape berry phenolics contribute to organoleptic properties, color and protection against environmental challenges. Climate change has already caused significant warming in most grape-growing areas of the world, and the climatic conditions determine, to a large degree, the grape varieties that can be cultivated as well as wine quality. In particular, heat, drought and light/UV intensity severely affect phenolic metabolism and, thus, grape composition and development. In the variety Chardonnay, water stress increases the content of flavonols and decreases the expression of genes involved in biosynthesis of stilbene precursors. Also, polyphenolic profile is greatly dependent on genotype and environmental interactions. This review deals with the diversity and biosynthesis of phenolic compounds in the grape berry, from a general overview to a more detailed level, where the influence of environmental challenges on key phenolic metabolism pathways is approached. The full understanding of how and when specific phenolic compounds accumulate in the berry, and how the varietal grape berry metabolism responds to the environment is of utmost importance to adjust agricultural practices and thus, modify wine profile.

The mature berry of Vitis vinifera 'Alicante Bouschet' is entirely red, but anthocyanin metabolism discloses elements of histological discontinuity. This provides an experimental system amenable to studies of compartmentalised secondary metabolism in a fleshly fruit. We compared microscopy of fixed berry sections and chemical composition of anthocyanin extracts with the expression of 41 flavonoid genes in three berry tissues. In the pericarp, anthocyanins formed membrane-encased spherical coalescences that gradually enlarged and were shuttled into the vacuolar system. The size and the intensity of in situ pigmentation and of colour extracts of anthocyanin vesicles all decreased with depth beneath the epidermis. Shades of red colour, and the quantity and types of anthocyanins in skin, flesh, and seed extracts were correlated with differences in the expression of flavonoid 3',5'-hydroxylases and anthocyanin genes encoding transcription factors, enzymatic proteins, and transporters. Fine adjustments in the global transcriptional modulation of the pathway occurred distinctively in each tissue, within four groups of co-expressed genes that were more associated with either the pericarp or the seed, and with either early or late-ripening stages. All structural genes controlling early steps of the flavonoid pathway exist in the grapevine genome in multiple copies that were recruited by antagonistic branches of the pathway in the 'Alicante Bouschet' berry. Expression patterns of individual paralogs were spatiotemporally distinct from one another, in step with either anthocyanin genes or proanthocyanidin genes.