Biochemical and in silico characterization of glycosyltransferases from red sweet cherry (Prunus avium L.) reveals their broad specificity toward phenolic substrates.

Polyphenolic compounds are a class of phytonutrients that play important roles in plants and contribute to human health when incorporated into our diet through fruit consumption. A large proportion occur as glycoconjugates but the enzymes responsible for their glycosylation are poorly characterized. Here, we report the biochemical and structural characterization of two glycosyltransferases from sweet cherry named PaUGT1 and PaUGT2. Both are promiscuous glucosyltransferases active on diverse anthocyanidins and flavonols, as well as phenolic acids in the case of PaUGT1. They also exhibit weaker galactosyltransferase activity. The expression of the gene encoding PaUGT1, the most active of the two proteins, follows anthocyanin accumulation during fruit ripening, suggesting that this enzyme is the primary glycosyltransferase involved in flavonoid glycosylation in sweet cherry. It can potentially be used to synthesize diverse glycoconjugates of flavonoids for integration into bioactive formulations, and for generating new fruit cultivars with enhanced health-promoting properties using breeding methods.

Loss of Diel Circadian Clock Gene Cycling Is a Part of Grape Berry Ripening.

Diel cycles of gene expression are thought to adapt plants to 24-h changes in environmental conditions. The circadian clock contributes to this process, but less is known about circadian programs in developing reproductive organs. While model plants and controlled conditions have contributed greatly to our knowledge of circadian clock function, there is a need to better understand its role in crop plants under field conditions with fluctuating light and temperature. In this study, we investigated changes in the circadian clock during the development of grape berries of Vitis vinifera L. We found that the transcripts of circadian clock homologs had high-amplitude oscillations prior to, but not during, ripening. As ripening progressed, the amplitude and rhythmicity of the diel oscillations decreased until most transcripts tested had no significant fluctuation over the 24-h cycle. Despite this loss of rhythmicity, the majority of circadian clock genes investigated were expressed at or near their abundance at the nadir of their pre-ripening oscillation although the berries remained transcriptionally active. From this, it can be concluded that cycling of the canonical circadian clock appears unnecessary for berry ripening. Our data suggest that changes in circadian clock dynamics during reproductive organ development may have important functional consequences.

Modifications of Grapevine Berry Composition Induced by Main Viral and Fungal Pathogens in a Climate Change Scenario.

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.

Sucrose Metabolism and Transport in Grapevines, with Emphasis on Berries and Leaves, and Insights Gained from a Cross-Species Comparison.

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.

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.

An aldo-keto reductase with 2-keto-l-gulonate reductase activity functions in l-tartaric acid biosynthesis from vitamin C in Vitis vinifera.

Tartaric acid has high economic value as an antioxidant and flavorant in food and wine industries. l-Tartaric acid biosynthesis in wine grape (Vitis vinifera) uses ascorbic acid (vitamin C) as precursor, representing an unusual metabolic fate for ascorbic acid degradation. Reduction of the ascorbate breakdown product 2-keto-l-gulonic acid to l-idonic acid constitutes a critical step in this l-tartaric acid biosynthetic pathway. However, the underlying enzymatic mechanisms remain obscure. Here, we identified a V. vinifera aldo-keto reductase, Vv2KGR, with 2-keto-l-gulonic acid reductase activity. Vv2KGR belongs to the d-isomer-specific 2-hydroxyacid dehydrogenase superfamily and displayed the highest similarity to the hydroxyl pyruvate reductase isoform 2 in Arabidopsis thaliana Enzymatic analyses revealed that Vv2KGR efficiently reduces 2-keto-l-gulonic acid to l-idonic acid and uses NADPH as preferred coenzyme. Moreover, Vv2KGR exhibited broad substrate specificity toward glyoxylate, pyruvate, and hydroxypyruvate, having the highest catalytic efficiency for glyoxylate. We further determined the X-ray crystal structure of Vv2KGR at 1.58 Å resolution. Comparison of the Vv2KGR structure with those of d-isomer-specific 2-hydroxyacid dehydrogenases from animals and microorganisms revealed several unique structural features of this plant hydroxyl pyruvate reductase. Substrate structural analysis indicated that Vv2KGR uses two modes (A and B) to bind different substrates. 2-Keto-l-gulonic acid displayed the lowest predicted free-energy binding to Vv2KGR among all docked substrates. Hence, we propose that Vv2KGR functions in l-tartaric acid biosynthesis. To the best of our knowledge, this is the first report of a d-isomer-specific 2-hydroxyacid dehydrogenase that reduces 2-keto-l-gulonic acid to l-idonic acid in plants.

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.

Shiraz wines made from grape berries (Vitis vinifera) delayed in ripening by plant growth regulator treatment have elevated rotundone concentrations and "pepper" flavor and aroma.

Preveraison treatment of Shiraz berries with either 1-naphthaleneacetic acid (NAA) or Ethrel delayed the onset of ripening and harvest. NAA was more effective than Ethrel, delaying harvest by 23 days, compared to 6 days for Ethrel. Sensory analysis of wines from NAA-treated fruit showed significant differences in 10 attributes, including higher "pepper" flavor and aroma compared to those of the control wines. A nontargeted analysis of headspace volatiles revealed modest differences between wines made from control and NAA- or Ethrel-treated berries. However, the concentration of rotundone, the metabolite responsible for the pepper character, was below the level of detection by solid phase microextraction-gas chromatography-mass spectrometry in control wines, low in Ethrel wines (2 ng/L), and much higher in NAA wines (29 ng/L). Thus, NAA, and to a lesser extent Ethrel, treatment of grapes during the preveraison period can delay ripening and enhance rotundone concentrations in Shiraz fruit, thereby enhancing wine "peppery" attributes.

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.

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.