Auxin treatment of grapevine (Vitis vinifera L.) berries delays ripening onset by inhibiting cell expansion.

KEY MESSAGE: Auxin treatment of grape (Vitis vinifera L.) berries delays ripening by inducing changes in gene expression and cell wall metabolism and could combat some deleterious climate change effects. Auxins are inhibitors of grape berry ripening and their application may be useful to delay harvest to counter effects of climate change. However, little is known about how this delay occurs. The expression of 1892 genes was significantly changed compared to the control during a 48 h time-course where the auxin 1-naphthaleneacetic acid (NAA) was applied to pre-veraison grape berries. Principal component analysis showed that the control and auxin-treated samples were most different at 3 h post-treatment when approximately three times more genes were induced than repressed by NAA. There was considerable cross-talk between hormone pathways, particularly between those of auxin and ethylene. Decreased expression of genes encoding putative cell wall catabolic enzymes (including those involved with pectin) and increased expression of putative cellulose synthases indicated that auxins may preserve cell wall structure. This was confirmed by immunochemical labelling of berry sections using antibodies that detect homogalacturonan (LM19) and methyl-esterified homogalacturonan (LM20) and by labelling with the CMB3a cellulose-binding module. Comparison of the auxin-induced changes in gene expression with the pattern of these genes during berry ripening showed that the effect on transcription is a mix of changes that may specifically alter the progress of berry development in a targeted manner and others that could be considered as non-specific changes. Several lines of evidence suggest that cell wall changes and associated berry softening are the first steps in ripening and that delaying cell expansion can delay ripening providing a possible mechanism for the observed auxin effects.

Fermentation-Guided Natural Products Isolation of a Grape Berry Triacylglyceride that Enhances Ethyl Ester Production.

A full understanding of the origin, formation and degradation of volatile compounds that contribute to wine aroma is required before wine style can be effectively managed. Fractionation of grapes represents a convenient and robust method to simplify the grape matrix to enhance our understanding of the grape contribution to volatile compound production during yeast fermentation. In this study, acetone extracts of both Riesling and Cabernet Sauvignon grape berries were fractionated and model wines produced by spiking aliquots of these grape fractions into model grape juice must and fermented. Non-targeted SPME-GCMS analyses of the wines showed that several medium chain fatty acid ethyl esters were more abundant in wines made by fermenting model musts spiked with certain fractions. Further fractionation of the non-polar fractions and fermentation of model must after addition of these fractions led to the identification of a mixture of polyunsaturated triacylglycerides that, when added to fermenting model must, increase the concentration of medium chain fatty acid ethyl esters in wines. Dosage-response fermentation studies with commercially-available trilinolein revealed that the concentration of medium chain fatty acid ethyl esters can be increased by the addition of this triacylglyceride to model musts. This work suggests that grape triacylglycerides can enhance the production of fermentation-derived ethyl esters and show that this fractionation method is effective in segregating precursors or factors involved in altering the concentration of fermentation volatiles.

A Grapevine Anthocyanin Acyltransferase, Transcriptionally Regulated by VvMYBA, Can Produce Most Acylated Anthocyanins Present in Grape Skins.

Anthocyanins are flavonoid compounds responsible for red/purple colors in the leaves, fruit, and flowers of many plant species. They are produced through a multistep pathway that is controlled by MYB transcription factors. VvMYBA1 and VvMYBA2 activate anthocyanin biosynthesis in grapevine (Vitis vinifera) and are nonfunctional in white grapevine cultivars. In this study, transgenic grapevines with altered VvMYBA gene expression were developed, and transcript analysis was carried out on berries using a microarray technique. The results showed that VvMYBA is a positive regulator of the later stages of anthocyanin synthesis, modification, and transport in cv Shiraz. One up-regulated gene, ANTHOCYANIN 3-O-GLUCOSIDE-6″-O-ACYLTRANSFERASE (Vv3AT), encodes a BAHD acyltransferase protein (named after the first letter of the first four characterized proteins: BEAT [for acetyl CoA:benzylalcohol acetyltransferase], AHCT [for anthocyanin O-hydroxycinnamoyltransferase], HCBT [for anthranilate N-hydroxycinnamoyl/benzoyltransferase], and DAT [for deacetylvindoline 4-O-acetyltransferase]), belonging to a clade separate from most anthocyanin acyltransferases. Functional studies (in planta and in vitro) show that Vv3AT has a broad anthocyanin substrate specificity and can also utilize both aliphatic and aromatic acyl donors, a novel activity for this enzyme family found in nature. In cv Pinot Noir, a red-berried grapevine mutant lacking acylated anthocyanins, Vv3AT contains a nonsense mutation encoding a truncated protein that lacks two motifs required for BAHD protein activity. Promoter activation assays confirm that Vv3AT transcription is activated by VvMYBA1, which adds to the current understanding of the regulation of the BAHD gene family. The flexibility of Vv3AT to use both classes of acyl donors will be useful in the engineering of anthocyanins in planta or in vitro.

The R2R3-MYB transcription factors MYB14 and MYB15 regulate stilbene biosynthesis in Vitis vinifera.

Plant stilbenes are phytoalexins that accumulate in a small number of plant species, including grapevine (Vitis vinifera), in response to biotic and abiotic stresses and have been implicated in many beneficial effects on human health. In particular, resveratrol, the basic unit of all other complex stilbenes, has received widespread attention because of its cardio-protective, anticarcinogenic, and antioxidant properties. Although stilbene synthases (STSs), the key enzymes responsible for resveratrol biosynthesis, have been isolated and characterized from several plant species, the transcriptional regulation underlying stilbene biosynthesis is unknown. Here, we report the identification and functional characterization of two R2R3-MYB-type transcription factors (TFs) from grapevine, which regulate the stilbene biosynthetic pathway. These TFs, designated MYB14 and MYB15, strongly coexpress with STS genes, both in leaf tissues under biotic and abiotic stress and in the skin and seed of healthy developing berries during maturation. In transient gene reporter assays, MYB14 and MYB15 were demonstrated to specifically activate the promoters of STS genes, and the ectopic expression of MYB15 in grapevine hairy roots resulted in increased STS expression and in the accumulation of glycosylated stilbenes in planta. These results demonstrate the involvement of MYB14 and MYB15 in the transcriptional regulation of stilbene biosynthesis in grapevine.

Changes in transcription of cytokinin metabolism and signalling genes in grape (Vitis vinifera L.) berries are associated with the ripening-related increase in isopentenyladenine.

BACKGROUND: Cytokinins are known to play an important role in fruit set and early fruit growth, but their involvement in later stages of fruit development is less well understood. Recent reports of greatly increased cytokinin concentrations in the flesh of ripening kiwifruit (Actinidia deliciosa (A. Chev.) C.F. Liang & A.R. Ferguson) and grapes (Vitis vinifera L.) have suggested that these hormones are implicated in the control of ripening-related processes. RESULTS: A similar pattern of isopentenyladenine (iP) accumulation was observed in the ripening fruit of several grapevine cultivars, strawberry (Fragaria ananassa Duch.) and tomato (Solanum lycopersicum Mill.), suggesting a common, ripening-related role for this cytokinin. Significant differences in maximal iP concentrations between grapevine cultivars and between fruit species might reflect varying degrees of relevance or functional adaptations of this hormone in the ripening process. Grapevine orthologues of five Arabidopsis (Arabidopsis thaliana L.) gene families involved in cytokinin metabolism and signalling were identified and analysed for their expression in developing grape berries and a range of other grapevine tissues. Members of each gene family were characterised by distinct expression profiles during berry development and in different grapevine organs, suggesting a complex regulation of cellular cytokinin activities throughout the plant. The post-veraison-specific expression of a set of biosynthesis, activation, perception and signalling genes together with a lack of expression of degradation-related genes during the ripening phase were indicative of a local control of berry iP concentrations leading to the observed accumulation of iP in ripening grapes. CONCLUSIONS: The transcriptional analysis of grapevine genes involved in cytokinin production, degradation and response has provided a possible explanation for the ripening-associated accumulation of iP in grapes and other fruit. The pre- and post-veraison-specific expression of different members from each of five gene families suggests a highly complex and finely-tuned regulation of cytokinin concentrations and response to different cytokinin species at particular stages of fruit development. The same complexity and specialisation is also reflected in the distinct expression profiles of cytokinin-related genes in other grapevine organs.

Influence of fruit maturity at harvest on the intensity of smoke taint in wine.

Bushfire smoke can affect the composition and sensory properties of grapes and wines, in some cases leading to wines which exhibit undesirable "smoky", "ashy" and "medicinal" characters. This study investigated the extent to which fruit maturity (i.e., ripeness) influences the perception of smoke taint in wine. Two white grape varieties (Chardonnay and Sauvignon Blanc) and two red grape varieties (Merlot and Shiraz) were exposed to smoke under experimental conditions, at approximately seven days post-veraison. Fruit was then harvested at two levels of maturity: Harvest A, when total soluble solids were 16-20 °Brix, i.e., the berry ripeness typically required for production of sparkling or light-bodied wines; and Harvest B, when total soluble solids were 22-25 °Brix, i.e., the berry ripeness typically required for production of full-bodied wines. The intensity of smoke taint in resulting wines was found to be influenced by fruit maturity, but differed between grape varieties. Smoke-related sensory attributes were apparent in Sauvignon Blanc wine made from early-harvested fruit and in Chardonnay wine made from late-harvested fruit, only; whereas Merlot and Shiraz wines exhibited smoke taint irrespective of fruit maturity. Smoke-derived volatile phenols, and various alcohols, esters and acids, were also quantified to determine the impact of smoke exposure and fruit maturity respectively, on wine composition.

Potential grape-derived contributions to volatile ester concentrations in wine.

Grape composition affects wine flavour and aroma not only through varietal compounds, but also by influencing the production of volatile compounds by yeast. C9 and C12 compounds that potentially influence ethyl ester synthesis during fermentation were studied using a model grape juice medium. It was shown that the addition of free fatty acids, their methyl esters or acyl-carnitine and acyl-amino acid conjugates can increase ethyl ester production in fermentations. The stimulation of ethyl ester production above that of the control was apparent when lower concentrations of the C9 compounds were added to the model musts compared to the C12 compounds. Four amino acids, which are involved in CoA biosynthesis, were also added to model grape juice medium in the absence of pantothenate to test their ability to influence ethyl and acetate ester production. ß-Alanine was the only one shown to increase the production of ethyl esters, free fatty acids and acetate esters. The addition of 1 mg∙L(-1) ß-alanine was enough to stimulate production of these compounds and addition of up to 100 mg∙L(-1) ß-alanine had no greater effect. The endogenous concentrations of ß-alanine in fifty Cabernet Sauvignon grape samples exceeded the 1 mg∙L(-1) required for the stimulatory effect on ethyl and acetate ester production observed in this study.

Jasmonic acid-isoleucine formation in grapevine (Vitis vinifera L.) by two enzymes with distinct transcription profiles.

The plant hormone jasmonic acid (JA) is essential for stress responses and the formation of reproductive organs, but its role in fruit development and ripening is unclear. Conjugation of JA to isoleucine is a crucial step in the JA signaling pathway since only JA-Ile is recognized by the jasmonate receptor. The conjugation reaction is catalyzed by JA-amido synthetases, belonging to the family of Gretchen Hagen3 (GH3) proteins. Here, in vitro studies of two grapevine (Vitis vinifera L. cv Shiraz) GH3 enzymes, VvGH3-7 and VvGH3-9, demonstrated JA-conjugating activities with an overlapping range of amino acid substrates, including isoleucine. Expression studies of the corresponding genes in grape berries combined with JA and JA-Ile measurements suggested a primary role for JA signaling in fruit set and cell division and did not support an involvement of JA in the ripening process. In response to methyl JA (MeJA) treatment, and in wounded and unwounded (distal) leaves, VvGH3-9 transcripts accumulated, indicating a participation in the JA response. In contrast, VvGH3-7 was unresponsive to MeJA and local wounding, demonstrating a differential transcriptional regulation of VvGH3-7 and VvGH3-9. The transient induction of VvGH3-7 in unwounded, distal leaves was suggestive of the involvement of an unknown mobile wound signal.

A methyltransferase essential for the methoxypyrazine-derived flavour of wine.

Methoxypyrazines are a family of potent volatile compounds of diverse biological significance. They are used by insects and plants in chemical defence, are present in many vegetables and fruit and, in particular, impart herbaceous/green/vegetal sensory attributes to wines of certain varieties, including Cabernet Sauvignon. While pathways for methoxypyrazine biosynthesis have been postulated, none of the steps have been confirmed genetically. We have used the F2 progeny of a cross between a rapid flowering grapevine dwarf mutant, which does not produce 3-isobutyl-2-methoxypyrazine (IBMP), and Cabernet Sauvignon to identify the major locus responsible for accumulation of IBMP in unripe grape berries. Two candidate methyltransferase genes within the locus were identified and one was significantly associated with berry IBMP levels using association mapping. The enzyme encoded by this gene (VvOMT3) has high affinity for hydroxypyrazine precursors of methoxypyrazines. The gene is not expressed in the fruit of Pinot varieties, which lack IBMP, but is expressed in Cabernet Sauvignon at the time of accumulation of IBMP in the fruit. The results suggest that VvOMT3 is responsible for the final step in methoxypyrazine synthesis in grape berries and is the major determinant of IBMP production.

Interactions between ethylene and auxin are crucial to the control of grape (Vitis vinifera L.) berry ripening.

BACKGROUND: Fruit development is controlled by plant hormones, but the role of hormone interactions during fruit ripening is poorly understood. Interactions between ethylene and the auxin indole-3-acetic acid (IAA) are likely to be crucial during the ripening process, since both hormones have been shown to be implicated in the control of ripening in a range of different fruit species. RESULTS: Grapevine (Vitis vinifera L.) homologues of the TRYPTOPHAN AMINOTRANSFERASE RELATED (TAR) and YUCCA families, functioning in the only characterized pathway of auxin biosynthesis, were identified and the expression of several TAR genes was shown to be induced by the pre-ripening application of the ethylene-releasing compound Ethrel. The induction of TAR expression was accompanied by increased IAA and IAA-Asp concentrations, indicative of an upregulation of auxin biosynthesis and conjugation. Exposure of ex planta, pre-ripening berries to the ethylene biosynthesis inhibitor aminoethoxyvinylglycine resulted in decreased IAA and IAA-Asp concentrations. The delayed initiation of ripening observed in Ethrel-treated berries might therefore represent an indirect ethylene effect mediated by increased auxin concentrations. During berry development, the expression of three TAR genes and one YUCCA gene was upregulated at the time of ripening initiation and/or during ripening. This increase in auxin biosynthesis gene expression was preceded by high expression levels of the ethylene biosynthesis genes 1-aminocyclopropane-1-carboxylate synthase and 1-aminocyclopropane-1-carboxylate oxidase. CONCLUSIONS: In grape berries, members of both gene families involved in the two-step pathway of auxin biosynthesis are expressed, suggesting that IAA is produced through the combined action of TAR and YUCCA proteins in developing berries. The induction of TAR expression by Ethrel applications and the developmental expression patterns of auxin and ethylene biosynthesis genes indicate that elevated concentrations of ethylene prior to the initiation of ripening might lead to an increased production of IAA, suggesting a complex involvement of this auxin and its conjugates in grape berry ripening.

Use of Machine Learning with Fused Spectral Data for Prediction of Product Sensory Characteristics: The Case of Grape to Wine.

Generations of sensors have been developed for predicting food sensory profiles to circumvent the use of a human sensory panel, but a technology that can rapidly predict a suite of sensory attributes from one spectral measurement remains unavailable. Using spectra from grape extracts, this novel study aimed to address this challenge by exploring the use of a machine learning algorithm, extreme gradient boosting (XGBoost), to predict twenty-two wine sensory attribute scores from five sensory stimuli: aroma, colour, taste, flavour, and mouthfeel. Two datasets were obtained from absorbance-transmission and fluorescence excitation-emission matrix (A-TEEM) spectroscopy with different fusion methods: variable-level data fusion of absorbance and fluorescence spectral fingerprints, and feature-level data fusion of A-TEEM and CIELAB datasets. The results for externally validated models showed slightly better performance using only A-TEEM data, predicting five out of twenty-two wine sensory attributes with R2 values above 0.7 and fifteen with R2 values above 0.5. Considering the complex biotransformation involved in processing grapes to wine, the ability to predict sensory properties based on underlying chemical composition in this way suggests that the approach could be more broadly applicable to the agri-food sector and other transformed foodstuffs to predict a product's sensory characteristics from raw material spectral attributes.

Machine learning for classifying and predicting grape maturity indices using absorbance and fluorescence spectra.

Absorbance-transmission and fluorescence excitation-emission matrix (A-TEEM) spectroscopy was investigated as a rapid method for predicting maturity indices using Cabernet Sauvignon grapes produced under four viticulture treatments during two growing seasons. Machine learning models were developed with fused spectral data to predict 3-isobutyl-2-methoxypyrazine (IBMP), pH, total tannins (Tannin), total soluble solids (TSS), and malic and tartaric acids based on the results from traditional analysis methods. Extreme gradient boosting (XGB) regression yielded R2 values of 0.92-0.96 for IBMP, malic acid, pH, and TSS for externally validated (Test) models, with partial least squares regression being superior for TSS prediction (R2 = 0.97). R2 values of 0.64-0.81 were achieved with either approach for tartaric acid and Tannin predictions. Classification of grape maturity, defined by quantile ranges for red colour, IBMP, malic acid, and TSS, was investigated using XGB discriminant analysis, providing an average of 78 % correctly classified samples for the Test model.

Methoxypyrazine concentrations in the grape bunch rachis of Vitis vinifera L. Cv Shiraz: Influence of rootstock, region and light.

Vitis vinifera L. cv Shiraz appears unable to synthesise 3-alkyl-2-methoxypyrazines (MPs) in the berry, but can still produce significant concentrations in rachis. MPs are readily extracted from rachis during fermentation, producing Shiraz wines with uncharacteristic "green" flavours. Recently, rootstocks were shown to significantly alter MP concentrations in Cabernet Sauvignon rachis compared to own-rooted varieties, but whether Shiraz followed a similar trend required investigation. This study considered the effect of thirteen rootstocks on the concentrations of 3-isobutyl-2-methoxypyrazine (IBMP), 3-isopropyl-2-methoxypyrazine (IPMP), and 3-sec-butyl-2-methoxypyrazine (SBMP) in the rachis of Shiraz bunches sampled during multiple vintages across several Australian growing regions. Although IBMP was the most abundant, all measured MP concentrations were significantly affected by vintage, rootstock, and region. In addition, vine vigour showed positive correlations with IBMP, which were attributed to changes in canopy coverage impacting rachis light exposure. This hypothesis was explored with light exclusion trials, which significantly increased rachis IBMP concentrations.

Grape Heterogeneity Index: Assessment of Overall Grape Heterogeneity Using an Aggregation of Multiple Indicators.

Uniform grape maturity can be sought by producers to minimise underripe and/or overripe proportions of fruit and limit any undesirable effects on wine quality. Considering that grape heterogeneity is a multifaceted phenomenon, a composite index summarising overall grape heterogeneity was developed to benefit vineyard management and harvest date decisions. A grape heterogeneity index (GHI) was constructed by aggregating the sum of absolute residuals multiplied by the range of values from measurements of total soluble solids, pH, fresh weight, total tannins, absorbance at 520 nm (red colour), 3-isobutyl-2-methoxypyrazine, and malic acid. Management of grape heterogeneity was also studied, using Cabernet Sauvignon grapes grown under four viticultural regimes (normal/low crop load, full/deficit irrigation) during the 2019/2020 and 2020/2021 seasons. Comparisons of GHI scores showed grape variability decreased throughout ripening in both vintages, then significantly increased at the harvest time point in 2020, but plateaued on sample dates nearing the harvest date in 2021. Irrigation and crop load had no effect on grape heterogeneity by the time of harvest in both vintages. Larger vine yield, leaf area index, and pruning weight significantly increased GHI score early in ripening, but no significant relationship was found at the time of harvest. Differences in the Ravaz index, normalised difference vegetation index, and soil electrical conductivity did not significantly change the GHI score.

Insights into the Uptake, Distribution, and Metabolism of 3-Isobutyl-2-hydroxypyrazine in Grapevine Using a Stable Isotope Tracer.

Methoxypyrazines (MPs) are potent aroma compounds that have been predominately studied in grape berries but can also be detected in other vine tissues. The synthesis of MPs in berries from hydroxypyrazines by VvOMT3 is well established, but the origin of MPs in vine tissues that have negligible VvOMT3 gene expression is unknown. This research gap was addressed through the application of stable isotope tracer 3-isobutyl-2-hydroxy-[2H2]-pyrazine (d2-IBHP) to the roots of Pinot Meunier L1 microvines and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) quantification of HPs from grapevine tissues following a novel solid-phase extraction method. Four weeks post-application, d2-IBHP and its O-methylated product 3-isobutyl-2-methoxy-[2H2]-pyrazine (d2-IBMP) were present in excised cane, berry, leaf, root, and rachis material. Translocation of d2-IBHP and d2-IBMP was investigated, but results were inconclusive. Nonetheless, knowledge that d2-IBHP, and potentially d2-IBMP, are translocated from roots to other vine organs, including the berries, could provide opportunities for controlling MP accumulation in grapevine tissues pertinent to winemaking.

Influence of Kazachstania spp. on the chemical and sensory profile of red wines.

We report the fermentative traits of two Kazachstania species (K. aerobia and K. servazzii) in non-sterile red wine and the resulting chemical and sensory properties. This builds on our previous work which revealed that Kazachstania spp. increased acetate esters in sterilised white wine. In this study Kazachstania spp. were initially evaluated in laboratory-scale fermentations (500 mL) in Merlot must to assess whether similar increases in chemical/volatile compounds would occur. The impact of malolactic fermentation (MLF) by Oenococcus oeni (VP41) on aroma composition was considered and found to reduce ester profiles in Merlot wines. The sensory implications of sequential inoculation with Kazachstania spp., followed by Saccharomyces cerevisiae, were then evaluated in small-lot fermentations (7 kg) of Shiraz must. Fungal diversity was monitored during early fermentation stages and was influenced by the early implantation of Kazachstania spp., followed by the dominance of S. cerevisiae. The effect of MLF in Shiraz wines was inconclusive due to high ethanol levels providing an inhospitable environment for lactic acid bacteria. When compared to S. cerevisiae alone, Kazachstania spp. significantly increased acetate esters, particularly phenylethyl acetate and isoamyl acetate, in both Merlot and Shiraz. The Shiraz wines fermented with Kazachstania spp. had higher jammy and red fruit aroma/flavour compared to S. cerevisiae (monoculture) wines. No influence was observed on colour one-year post-bottling. Results from this study show the contribution of Kazachstania spp. to the aroma profile of red wines and demonstrate their potential as starter cultures for improving the aromatic complexity of wines.

Rootstock, Vine Vigor, and Light Mediate Methoxypyrazine Concentrations in the Grape Bunch Rachis of Vitis vinifera L. cv. Cabernet Sauvignon.

Ramsey rootstock has previously been implicated in an approximate 8-fold increase of 3-isobutyl-2-methoxypyrazine (IBMP) levels in the rachis (grape bunch stem) of Vitis vinifera L. cv. Shiraz scions over own-rooted Shiraz vines at harvest. IBMP extracted from rachis during red wine fermentation can contribute potent "green" flavors. Methoxypyrazines (MPs) are normally present in Cabernet Sauvignon grapes, rachis, and wines, but it is unknown whether rootstocks can influence the MP concentration in the rachis. This study considered the effect of eight rootstocks including Ramsey and own roots on the concentrations of IBMP, 3-isopropyl-2-methoxypyrazine (IPMP), and 3-sec-butyl-2-methoxypyrazine (SBMP) in the rachis and grapes of Cabernet Sauvignon over two seasons. IBMP predominated, and its concentration in rachis and berries at harvest was significantly affected by rootstock and growing season. In the 2020 vintage, light exclusion, vine vigor, and spatial variation in vine vigor were shown to significantly affect MP concentrations in rachis.

Transcriptional analysis of late ripening stages of grapevine berry.

BACKGROUND: The composition of grapevine berry at harvest is a major determinant of wine quality. Optimal oenological maturity of berries is characterized by a high sugar/acidity ratio, high anthocyanin content in the skin, and low astringency. However, harvest time is still mostly determined empirically, based on crude biochemical composition and berry tasting. In this context, it is interesting to identify genes that are expressed/repressed specifically at the late stages of ripening and which may be used as indicators of maturity. RESULTS: Whole bunches and berries sorted by density were collected in vineyard on Chardonnay (white cultivar) grapevines for two consecutive years at three stages of ripening (7-days before harvest (TH-7), harvest (TH), and 10-days after harvest (TH+10)). Microvinification and sensory analysis indicate that the quality of the wines made from the whole bunches collected at TH-7, TH and TH+10 differed, TH providing the highest quality wines.In parallel, gene expression was studied with Qiagen/Operon microarrays using two types of samples, i.e. whole bunches and berries sorted by density. Only 12 genes were consistently up- or down-regulated in whole bunches and density sorted berries for the two years studied in Chardonnay. 52 genes were differentially expressed between the TH-7 and TH samples. In order to determine whether these genes followed a similar pattern of expression during the late stages of berry ripening in a red cultivar, nine genes were selected for RT-PCR analysis with Cabernet Sauvignon grown under two different temperature regimes affecting the precocity of ripening. The expression profiles and their relationship to ripening were confirmed in Cabernet Sauvignon for seven genes, encoding a carotenoid cleavage dioxygenase, a galactinol synthase, a late embryogenesis abundant protein, a dirigent-like protein, a histidine kinase receptor, a valencene synthase and a putative S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase. CONCLUSIONS: This set of up- and down-regulated genes characterize the late stages of berry ripening in the two cultivars studied, and are indirectly linked to wine quality. They might be used directly or indirectly to design immunological, biochemical or molecular tools aimed at the determination of optimal ripening in these cultivars.

Acyl substrate preferences of an IAA-amido synthetase account for variations in grape (Vitis vinifera L.) berry ripening caused by different auxinic compounds indicating the importance of auxin conjugation in plant development.

Nine Gretchen Hagen (GH3) genes were identified in grapevine (Vitis vinifera L.) and six of these were predicted on the basis of protein sequence similarity to act as indole-3-acetic acid (IAA)-amido synthetases. The activity of these enzymes is thought to be important in controlling free IAA levels and one auxin-inducible grapevine GH3 protein, GH3-1, has previously been implicated in the berry ripening process. Ex planta assays showed that the expression of only one other GH3 gene, GH3-2, increased following the treatment of grape berries with auxinic compounds. One of these was the naturally occurring IAA and the other two were synthetic, α-naphthalene acetic acid (NAA) and benzothiazole-2-oxyacetic acid (BTOA). The determination of steady-state kinetic parameters for the recombinant GH3-1 and GH3-2 proteins revealed that both enzymes efficiently conjugated aspartic acid (Asp) to IAA and less well to NAA, while BTOA was a poor substrate. GH3-2 gene expression was induced by IAA treatment of pre-ripening berries with an associated increase in levels of IAA-Asp and a decrease in free IAA levels. This indicates that GH3-2 responded to excess auxin to maintain low levels of free IAA. Grape berry ripening was not affected by IAA application prior to veraison (ripening onset) but was considerably delayed by NAA and even more so by BTOA. The differential effects of the three auxinic compounds on berry ripening can therefore be explained by the induction and acyl substrate specificity of GH3-2. These results further indicate an important role for GH3 proteins in controlling auxin-related plant developmental processes.

Functional effect of grapevine 1-deoxy-D-xylulose 5-phosphate synthase substitution K284N on Muscat flavour formation.

Grape berries of Muscat cultivars (Vitis vinifera L.) contain high levels of monoterpenols and exhibit a distinct aroma related to this composition of volatiles. A structural gene of the plastidial methyl-erythritol-phosphate (MEP) pathway, 1-deoxy-D-xylulose 5-phosphate synthase (VvDXS), was recently suggested as a candidate gene for this trait, having been co-localized with a major quantitative trait locus for linalool, nerol, and geraniol concentrations in berries. In addition, a structured association study discovered a putative causal single nucleotide polymorphism (SNP) responsible for the substitution of a lysine with an asparagine at position 284 of the VvDXS protein, and this SNP was significantly associated with Muscat-flavoured varieties. The significance of this nucleotide difference was investigated by comparing the monoterpene profiles with the expression of VvDXS alleles throughout berry development in Moscato Bianco, a cultivar heterozygous for the SNP mutation. Although correlation was detected between the VvDXS transcript profile and the accumulation of free monoterpenol odorants, the modulation of VvDXS expression during berry development appears to be independent of nucleotide variation in the coding sequence. In order to assess how the non-synonymous mutation may enhance Muscat flavour, an in vitro characterization of enzyme isoforms was performed followed by in vivo overexpression of each VvDXS allele in tobacco. The results showed that the amino acid non-neutral substitution influences the enzyme kinetics by increasing the catalytic efficiency and also dramatically affects monoterpene levels in transgenic lines. These findings confirm a functional effect of the VvDXS gene polymorphism and may pave the way for metabolic engineering of terpenoid contents in grapevine.