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Tonoplast vesicles were extracted from mature grape berries in order to study the effect of ethanol on their vacuolar proton pumps, vacuolar ATPase (V‐ATPase) and inorganic pyrophosphatase (PPase). Ethanol significantly inhibited their functioning; PPase was more sensitive than V‐ATPase. The effectiveness of alcohols with different carbon‐chain length indicated that a localised modification of the lipidic environment of the pump may be involved in this phenomenon. However, ethanol did not seem to significantly increase passive proton permeability of tonoplast vesicles. The inhibition of the hydrolytic activity of the inorganic pyrophosphatase PPase led to a decrease in the proton pumping rate into the vacuole and consequently in the generated pH gradient. PPase inhibition by ethanol was not compensated by an activation of the less sensitive V‐ATPase when both pumps operated simultaneously. The response of proton pumps to ethanol would be expected to impair cellular compartmentation during anaerobic metabolism. This phenomenon would explain the cell death in grape berries during ‘carbonic maceration’ or ‘vinification beaujolaise’.

One grapevine ( Vitis vinifera L.) Adh gene, related to grapevine ripening and referred to here as GV- Adh 1, has been completely sequenced. We report in this paper the cloning strategy and the structural characteristics of this gene. Comparative analyses of the proximal region of GV- Adh 1 promoter with other promoters from Adh genes are presented. GV- Adh 1 contains a 1140 nucleotide open reading frame encoding for a 380 amino acid polypeptide with a predicted molecular mass of about 41 kDa and a predicted isoelectric point of 6.9. Northern blot analysis detected Adh transcripts principally in ripening fruits, whereas constitutive or no expression was found in young expanding leaves, tendrils and roots. A large increase in Adh expression corresponds to the inception of berry ripening. Blot hybridization experiments show that in grapevine, Adh is coded by a small multigene family. Phylogenetic analysis is presented.

Rhamnogalacturonan II (RG-II) is a structurally complex pectic polysaccharide present in the walls of growing plant cells. We now report that RG-II, released by endopolygalacturonase treatment of the walls of suspension-cultured sycamore cells and etiolated pea stems, exists mainly as a dimer that is cross-linked by a borate ester. The borate ester is completely hydrolyzed at room temperature within 30 min at pH 1, partially hydrolyzed between pH 2 and 4, and stable above pH 4. The dimer is formed in vitro between pH 2.4 and 6.2 by treating monomeric RG-II (0.5 mM) with boric acid (1.2 mM); the dimer formed after 24 h at pH 3.4 and 5.0 accounts for ~30 and ~5%, respectively, of the RG-II. In contrast, the dimer accounts for ~80 and ~54% of the RG-II when the monomer is treated for 24 h at pH 3.4 and 5.0, respectively, with boric acid and 0.5 mM Sr2+, Pb2+, or Ba2+. The amount of dimer formed at pH 3.4 or 5.0 is not increased by addition of 0.5 mM Ca2+, Cd2+, Cu2+, Mg2+, Ni2+, and Zn2+. Steric considerations appear to regulate dimer formation since those divalent cations that enhance dimer formation have an ionic radius >1.1 A. Our data suggest that the borate ester is located on C-2 and C-3 of two of the four 3′-linked apiosyl residues of dimeric RG-II. Our results, taken together with the results of two previous studies (Kobayashi, M., Matoh, T., and Azuma, J.-I. (1996) Plant Physiol. 110, 1017-1020; Ishii, T., and Matsunaga, T. (1996) Carbohydr. Res. 284, 1-9) provide substantial evidence that this plant cell wall pectic polysaccharide is covalently cross-linked.

It has been claimed that the inorganic pyrophosphatase (PPase) of the plant vacuolar membrane transports K+ in addition to H+ in intact vacuoles (Davies, J. M., Poole, R. J., Rea, P. A., and Sanders, D.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11701-11705). Since this was not confirmed using the purified and reconstituted PPase consisting of a 75-kDa polypeptide (Sato, M. H., Kasahara, M., Ishii, N., Homareda, H., Matsui, H., and Yoshida, M. (1994) J. Biol. Chem. 269, 6725-6728), these authors proposed that K+ transport by the PPase is dependent on its association with other membrane components lost during purification. We have examined the hypothesis of K+ translocation by the PPase using native vacuolar membrane vesicles from Vitis vinifera suspension cells, multilabeled with fluorescent probes for K+, H+, and membrane potential. This material contained a high proportion of right-side-out, tightly sealed vesicles, exhibiting high PPase activity which was strongly stimulated by uncouplers and K+. Proton pumping occurred in response to pyrophosphate addition in the absence of K+. No K+ incorporation into the vesicles could be observed after PPase energization in the presence of K+, although H+ transport was highly stimulated. The hydrolytic activity was stimulated by a protonophore and by a H+/K+ exchanger but not by the K+ ionophore valinomycin. No evidence could be obtained supporting the operation of an endogenous K+/H+ exchanger capable to dissipate the putative active K+ flux generated by the PPase. We conclude that PPase in native vacuolar membrane vesicles does not transport K+.

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Fractionation of a grape (Vitis vinifera) seed extract yielded various natural procyanidin dimers and trimers isolated and identified for the first time in grapes: procyanidin dimers B5 [epicatechin-(4β → 6)-epicatechin], B6 [catechin-(4α → 6)-catechin] and B8 [catechin-(4α → 6)-epicatechin]; four procyanidin trimers [epicatechin-(4β → 6)-epicatechin-(4β → 8)-epicatechin], [epicatechin-(4β → 8)-epicatechin-(4β → 6)-catechin], [epicatechin-(4β → 8)-epicatechin-(4β → 6)-epicatechin], [epicatechin-(4β → 6)-epicatechin-(4β → 8)-catechin] along with five galloyl procyanidins: B2 3-O-gallate, B1 3-O-gallate, B4 3′-O-gallate, B2 3,3′-di-O-gallate and [epicatechin-(4β → 8)-epicatechin 3-O-gallate-(4β → 8)-catechin]. The presence of (+)-catechin, (−)-epicatechin, (−)-epicatechin 3-O-gallate, procyanidins dimers B1, B2, B3, B4, B7, B2 3′-O-gallate and procyanidin trimers C1 and [epicatechin-(4β → 8)-epicatechin-(4β → 8)-catechin] was also confirmed. The structures of all these compounds were elucidated by enzymatic hydrolysis, complete acid hydrolysis, partial acid-catalysed degradation with phloroglucinol and phenylmethanethiol, FABMS and 1H NMR. Separation of procyanidins and of their phloroglucinol or phenylmethanethiol adducts was achieved by TLC on silica plates and HPLC.

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To better describe the importance of initial assimilable nitrogen content on the kinetics of alcoholic fermentation and the effectiveness of ammonium additions, a study was done using an automatic device for fermentation monitoring. A good correlation was found between the maximum CO2 production rate, obtained early during the fermentation, and the assimilable nitrogen content of the must. So it is possible to use this kinetic parameter to detect nitrogen deficiencies. For industrial applications, a threshold value of 1.3 gCO2/l·h at 24°C (corresponding to about 140 mg N/l) is proposed. In the case of deficient must, a nitrogen addition during the fermentation rapidly increased the CO2 production rate and reduced the fermentation duration. This reduction, which may be approximately predicted from (dCO2/dt)max, is the same, provided that the addition is made before the halfway point of the fermentation.

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As in many fruits, the induction of grape berry (Vitis vinifera L.) ripening results in intense breakdown of malic acid. Using membrane fractions, we tested the hypothesis that changes in acidity resulted from malate vacuolar decompartmentation. The hydrolytic activities of the two primary vacuolar pumps inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) and vacuolar ATPase (V-ATPase; EC 3.6.1.3) increased throughout development with an acceleration during ripening, as confirmed by Western blotting and analysis of transcript expression. The ratio of V-PPase activity to V-ATPase activity was always in favour of V-PPase and reached its maximum value at véraison. The rate of anion transport strongly increased during ripening. Before ripening, tonoplast passive permeability was low, but rose during ripening. Our data indicate that tonoplast leakage dramatically increased during ripening. This leakage is probably the prime cause of malate decompartmentation, amplified by the incapacity of oxidative phosphorylation to face increased energy demand.

Alcohol dehydrogenase (ADH; alcohol: NAD oxidoreductase; EC 1.1.1.1) is a dimericzinc enzyme which catalyses the inter-conversion of acetaldehyde to ethanol, using NAD/NADH as a cofactor. It is the terminal step of glycolysis, leading to fermentative metabolism in anaerobic conditions. In this context, evolution of this enzyme’s activity, and gene expression have been widely investigated in response to anaerobiosis in the plant kingdom, especially in organs such as roots, tubers, seeds and fruit (Kadowaki et al., 1988; Matton et al., 1990; Sun Chen and Chase, 1993; Ke et al., 1994; Millar et al., 1994; Chung and Ferl, 1999). ADH activity is generally low under normal oxygen tension, but increases strongly when the absence of oxygen leads to ethanolic fermentation. It appears to play an important role in the anaerobiosis response of plants, as ADH induction was generally detected in organs adapted to this stress. In maize, Gerlach et al. (1983) showed that ADH is fundamental to anaerobic survival for Adh null mutants. Paul and Ferl (1991) described the enhanced transcription of two genes responsible for a higher ADH synthesis. During oxygen deprivation, the assumed role for ADH is to reduce toxic acetaldehyde to ethanol and to recycle NADH to NAD, thereby allowing anaerobic glycolysis to be used as energy-generating pathway. It could also be involved in cytoplasmic homeostasis, as level of ADH activity is tightly regulated by pH, and uncontrolled cytoplasmic acidosis is lethal (Roberts et al., 1984). Other abiotic factors such as wounding and elicitor treatment (Constabel et al., 1990), exposure to cold stress (Christie et al., 1991; Jarillo et al., 1993), and light (Matton et al., 1990) have been shown to increase ADH activity and/or Adh gene expression.

A membrane polypeptide involved in K+ transport in a higher plant was cloned by complementation of a yeast mutant defective in K+ uptake with a complementary DNA library from Arabidopsis thaliana. A 2.65-kilobase complementary DNA conferred ability to grow on media with K+ concentration in the micromolar range and to absorb K+ (or 86Rb+) at rates similar to those in wild-type yeast. The predicted amino acid sequence (838 amino acids) has three domains: a channel-forming region homologous to animal K+ channels, a cyclic nucleotide-binding site, and an ankyrin-like region.

Three full-length cDNAs (VvAdh1, VvAdh2, and VvAdh3) encoding alcohol dehydrogenases (EC 1.1.1.1) were obtained from grape berries (Vitis vinifera L.) by means of PCR and RACE. Pairwise comparisons at the nucleotide level showed that the three cDNAs displayed strong homology in the coding region, but were highly divergent in the 5' and 3' untranslated regions. VvAdh1 and VvAdh2 corresponded to the two previously characterised Adh genes from grapevine, but VvAdh3 was unrelated to known grapevine Adh sequences. The two first cDNAs presented a single ORF of 380 amino acids, whereas the last one has two additional residues. Moreover, the three encoded polypeptides possessed the 22 residues strictly conserved between Adh from different kingdoms. Expression pattern of the individual isogenes was investigated during fruit development. Specific primers were designed, and quantitative RT-PCR experiments were performed to increase the sensitivity of detecting isogenes with a low expression level. Results presented here revealed different developmental regulation of the three Adh isogenes during fruit ripening. VvAdh1 and VvAdh3 transcripts were temporarily accumulated in young, developing berry, whereas VvAdh2 was overexpressed later in fruit development, from the onset of ripening (veraison). Expression analysis also indicated that VvAdh2 accounted for most of the Adh mRNAs present in berries during development. The increased ADH activity detected in berries correlated with the expression pattern of VvAdh2 transcripts. The VvAdh2 and VvAdh3 encoded enzymes were purified from overexpressing E. coli cells. Comparison of kinetic properties of the two ADH enzymes showed a difference in affinity with either ethanol or acetaldehyde as substrates. Significance of multiple Adh expressed in berries is discussed.

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A large increase in hexose accumulation is typical of grape berry ripening. To analyse sugar transport processes during grape berry ( Vitis vinifera L. cv Ugni Blanc) development, a cDNA encoding for a sucrose transporter, designated VvSUT1 was cloned. Sucrose transport activity of the VvSUT1 protein was demonstrated by heterologous expression in yeast. A study of gene expression along berry development showed that VvSUT1 transcripts are present at all stages but accumulate with the onset of ripening. This work shows that sucrose transport at the plasma membrane of grape berry cells may play a role in the accumulation of sugars during maturation.

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A strain of Penicillium daleae has been isolated from a forest soil sample for its ability to degrade monomeric rhamnogalacturonan-II (mRG-II), a complex pectic polysaccharide ubiquitous in the primary plant cell wall. Monomeric RG-II, a most unusual polysaccharide in terms of composition and structure, is resistant to all known pectinolytic enzymes used in the fruit- and vegetable-processing industry. P. daleae has been cultured in a minimum mineral medium supplemented with 0.5% mRG-II as the sole carbon source. The degradation of the substrate in the culture supernatant was followed by high performance size-exclusion chromatography. P. daleae growth was supported by the degradation of 75% of the initial mRG-II. Monomeric RG-II degradation was followed by gas chromatography-mass spectrometry of the alditol acetates and trimethylsilylated derivatives of the constitutive monosaccharides at different times in the culturing process. Sequential mRG-II degradation led to a resistant core after 31 days of culture, representing 25% of the initial molecule which has been characterized. The degradation of mRG-II indicated that P. daleae is a potential source of new pectinases whose mode of action requires further elucidation. Such enzymes seemed to be exposed to the surface of fungal cell walls or were present in the periplasmic compartment.

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DNA of berries (cv. Shiraz) was extracted and quantified to determine, indirectly, the rate of cell division and enlargement in the grape pericarp. The increase of total DNA in the pericarp begins at anthesis in the ovary of grapevine flowers (day 0, 100 % of flowers at full bloom). This increase in DNA continues during the herbaceous growth period until 35 d after anthesis (day 35, 19 d before the onset of veraison). Total DNA per berry pericarp does not increase linearly during this growth period since 75 % of the DNA has already accumulated before day 20. We determined a cell enlargement index (CEI), to estimate the mean cellular volume. The pericarp cell size increases 16-fold during the whole growth of berries. Volume increase is nearly linear from berry set to the beginning of veraison and thereafter until maturity. The importance of determination of cell division and enlargement of berry pericarp based on the DNA content and its possible application in studies on the influence of environmental factors on berry growth is discussed., VITIS - Journal of Grapevine Research, Vol. 38 No. 4 (1999): Vitis

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*INRA Laboratoire de Microbiologie et Technologie des Fermentations Montpellier (FRA) Diffusion du document : INRA Laboratoire de Microbiologie et Technologie des Fermentations Montpellier (FRA)

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International audience; The Avabidopsis thaliana K+ channel AKT1 was expressed in a yeast strain defective for K+ uptake at low K+ concentrations (

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In order to gain insight into vacuolar changes that are associated with fruit specialization regarding the accumulation of organic acids, the properties of tonoplast vesicles extracted from grape cell culture and ripe berries were compared. The two primary pumps, PPase (Pyrophosphatase) and V-ATPase (V-Adenosine Triphosphatase), which were found on both fruit and cell culture tonoplast vesicles, exhibited classical behaviour for known inhibitors and activators. However, the 3-fold higher activity of the PPase in comparison to the V-ATPase was unexpected in a fruit exhibiting a vacuolar pH of 3.5. Surprisingly, the hydrolytic activities of both pumps were stimulated by temperatures up to 65 °C. Anion transport was studied by measuring simultaneously proton entry and membrane potential on the two types of tonoplast vesicles. Maximal acidification rates could be observed upon simultaneous energization by ATP and PPi, and were abolished by imidodiphosphate and bafilomycin, while vanadate had little effect. Comparison of the sequence of transport rates observed with mineral and organic anions on fruit or cultured cell tonoplast vesicles suggested that the fruit vacuole was more specialized in the transport of organic anions than the cultured cells. The transport of malate and tartrate was completely inhibited by 4,4′-diisothio-cyanato-stilbene-2,2′disulphonic acid while chloride transport was less affected. Km values for malate and tartrate transports were similar for vesicles extracted from cultured cell and fruits, around 14 mM. The transport rates of malate and tartrate were not additive, suggesting that a similar transporter was involved in the vacuolar uptake of the two major acids of grape berries.

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It is generally admitted that, due to the low levels of respiratory enzymes and mitochondrial cytochromes, classical oxidative phosphorylation does not occur to any significant extent under alcoholic fermentation conditions. Under anaerobiosis, yeast growth normally requires oxygen in order to favour the synthesis of sterols and unsaturated fatty acids. However, in such conditions, superfluous oxygen consumption by yeast cells is observed. In this study, the potential relationship between this oxygen consumption and the known respiratory pathways of S. cerevisiae was investigated during enological fermentation. Partial and limited synthesis of mitochondrial cytochromes was observed during the growth phase. The superfluous oxygen consumed by the yeast cells appeared not to be related to classical respiration, but mainly to the operation of several mitochondrial alternative respiration pathways, which were linked to the cell cytochrome contents. It was further found that oxygen-dependent ergosterol biosynthesis accounted for less than 15% of the total oxygen consumption at the beginning of stationary phase in the absence of anaerobic growth factors. No significant oxygen consumption could be clearly attributed to fatty acids desaturation in the yeast cells.

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