In situ fixation of grape berries

Summary Usual immersion protocols in aldehyde solutions fail to fully preserve the fine structure of both exocarp and mesocarp cells of grape berries, especially for theveraison (onset of ripening) and post-veraison stages. In exocarp cells, fixative diffusion is hampered by the thick polysaccharide cell walls. In mesocarp cells, plasma membrane and tonoplast are disrupted before aldehyde crosslinking occurs, owing to the high osmotic pressure and cell wall texture. The fixative was therefore injected under pressure as small droplets in the outer and inner parts of the fruit, with limited changes in the steady-state organization of fruit tissues. Compared to a selective range of immersion protocols, a striking improvement in cell preservation was observed for all berry tissues, allowing new information on various compartments of grape berry cells. The preservation of organ integrity and local concentration of aldehyde molecules are the most critical parameters of improved fixation. This technique may be applicable to a large array of fleshy fruits containing mainly cells comprising a high volumetric proportion of vacuoles accumulating large amounts of organic acids and sugars and bounded by thick-walled exocarp cells.     

Monoterpene glycoside biosynthesis in detached grape berries grown in vitro

A procedure for the culture in vitro of isolated small berries of Vitis vinifera L. cv. Muscat of Alexandria in a Murashige and Skoog basal medium supplemented with N⁶-benzyladenine and indoleacetic acid is described. Berries developed well in culture during 60 days and tripled in size, but remained green and smaller than normal berries grown in vivo. Some callus formed on the distal end of the berry, and where major skin damage occurred, callus emerged from the cracked berries. In order to examine their biosynthetic competency, berries which were previously cultured in vitro for 60 days were incubated for 48 h in a Murashige and Skoog medium containing a [¹⁴C]-labelled water-soluble fraction. This fraction was isolated from grape berries located adjacent to a leaf that had been exposed to gaseous ¹⁴CO₂ in full sunlight for 5 h. The berries were then recultured for 48 h after which a glycosidic fraction was isolated on a C18 reversed phase column and further separated by thin layer chromatography (TLC). The major labelled band corresponded to the geranyl-β-rutinoside marker, indicating that grape berries have the ability to synthesize monoterpene glycosides. This band also consisted of other monoterpene glycosides as revealed by the gas chromatography-mass spectrometry (GC-MS) analysis of their aglycones (released by enzymatic hydrolysis).     

Yeasts associated to Malbec grape berries from Mendoza, Argentina

AIMS: The aims of this work were to evaluate different pre-isolation treatments applied to complete yeast extraction from grapes and to identify the yeast microflora associated to Malbec grapes from two vineyards located in Mendoza, Argentina. METHODS AND RESULTS: The pre-isolation treatments evaluated were shaking, jet streaming with pressurized water and grape blending. The overall results clearly indicated that when a more vigorous and disruptive pre-isolation treatment was applied; larger numbers of yeast species were recovered. The yeast population on healthy and ripe Malbec grapes was in the order of 10(5)-10(6) CFU g(-1). Eight different yeast species were isolated from berries, including Kloeckera apiculata, Metschnikowia pulcherrima, Pichia membranifaciens, Saccharomycodes ludwigii, Candida species (Candida stellata and Candida raghi), Issatchenkia orientalis and Rhodotorula spp. CONCLUSIONS: Grape blending gave the highest yeast counts. Rainfall near grape harvest time quantitatively and qualitatively modifies the yeast microflora. The yeast species identified on ripe grapes from the Mendoza region, partially match those previously documented in different parts of the world related. S. ludwigii has not been previously reported in grapes. SIGNIFICANCE AND IMPACT OF THE STUDY: The report is on yeast microbiota in grapes from Mendoza, Argentina. Saccharomycodes ludwigii was found in high percentage (17%), this species has not been described before on grapes surface. The importance of pre-isolation steps to the recovery of high number of yeasts was shown. Influence of climatic conditions near harvest time on microflora was confirmed.     

Phosphoenolpyruvate carboxykinase activity in grape berries

Phosphoenolpyruvate (PEP) carboxykinase activity was found in crude extracts of ;Pinot noir' grape berries. The enzyme required ATP, Mn(2+) plus Mg(2+), a pH of 6.6, and a temperature of 40 C for maximum activity. The range in concentration of oxaloacetic acid needed for maximum phosphoenolpyruvate carboxykinase activity was 5 to 10 mm, and the Km for HCO(3) (-) in the exchange of (14)CO(2) into oxaloacetic acid was 26.8 mm.Changes in the activity of PEP carboxykinase and PEP carboxylase in berries were studied at weekly intervals throughout fruit development. PEP carboxykinase had maximum activity 4 weeks after flowering, and during the following 11 weeks remained relatively constant. The activity of PEP carboxylase was 2- to 4-fold higher than PEP carboxykinase throughout fruit development, and changed little except for a sharp reduction at the onset of ripening.     

Metabolism of sugars and organic acids in immature grape berries

Individual intact excised immature Sultana berries were supplied through the cut pedicel with ¹⁴C-sugars and organic acids. When ¹⁴C-hexoses were supplied malic and tartaric acids accounted for 25% and 10% of the total activity extracted after 24 hours, and sucrose was synthesized. It is proposed that the changes in the levels of organic acids during ripening are related to changes in the ability of the berry to synthesize them. Although administration of uniformly labeled sucrose resulted in the unequal labeling of glucose and fructose, the results indicate breakdown of sucrose by invertase. It is suggested that the route of entry of the pedicel-fed sugars into the berry may be different from the route taken by sugar translocated from the leaf.     

Effect of carbon source and sucrose concentration on growth and hexose accumulation of grape berries cultured in vitro

Using in vitro culture of isolated small berries of Vitis vinifera L. cv. Sultana, it was possible to study the effect of different carbon sources and sucrose concentration on fruit growth, hexose accumulation and soluble invertase activity during the first stage of berry development by eliminating the source tissue. Berries cultured in vitro lack stage III of berry development which is characterised by massive accumulation of water and sugars, and thereby berries reached only 30% of the weight of those grown in the plant. Sucrose and glucose were both good carbon sources for berry growth, while fructose was not as good. Berry growth, hexose accumulation and invertase activity increased as sucrose concentration increased up to 15% in the medium. Furthermore, the onset of hexose accumulation in cultured berries depended on the concentration of sucrose in the medium, starting earlier at higher concentrations. This revised version was published online in June 2006 with corrections to the Cover Date.     

Permeation and metabolism of Alternaria mycotoxins with perylene quinone structure in cultured Caco-2 cells

The absorption of four Alternaria toxins with perylene quinone structures, i.e. altertoxin (ATX) I and II, alteichin (ALTCH) and stemphyltoxin (STTX) III, has been determined in the Caco-2 cell Transwell system, which represents a widely accepted in vitro model for human intestinal absorption and metabolism. The cells were incubated with the four mycotoxins on the apical side, and the concentration of the toxins in the incubation media of both chambers and in the cell lysate were determined by liquid chromatography coupled with diode array detection and mass spectrometry (LC-DAD-MS) analysis. ATX I and ALTCH were not metabolised in Caco-2 cells, but ATX II and STTX III were partly biotransformed by reductive de-epoxidation to the metabolites ATX I and ALTCH, respectively. Based on the apparent permeability coefficients (P_app), the following ranking order for the permeation into the basolateral compartment was obtained: ATX I > ALTCH >> ATX II > STTX III. Total recovery of the four toxins decreased in the same order. It is assumed that the losses of STTX III, ATX II and ALTCH in Caco-2 cells are caused by covalent binding to cell components due to the epoxide group and/or the α,β-unsaturated carbonyl group present in these toxins. We conclude from this study that ATX I and ALTCH are well absorbed from the intestinal lumen into the portal blood in vivo. For ATX II and STTX III, intestinal absorption of the parent toxins is very low, but these toxins are partly metabolised to ATX I and ALTCH, respectively, in the intestinal epithelium and absorbed as such.     

Involvement of polyamines in gibberellin-induced development of seedless grape berries

The roles of polyamines (PAs) in the development of seedless grape berries induced by gibberellin (GA₃) was investigated. The development of seedless grape berries was stimulated by the application of putrescine (Put), but not by that of spermidine (Spd) and spermine (Spm), regardless of the presence of GA₃. At harvest, the fresh weight of seedless grape berries treated with 500 ppm Put + 25 ppm GA₃ and 500 ppm Put increased to 111 and 112%, respectively, of the control. Treatment with methylglyoxal-bis (guanyl hydrazone), a potent inhibitor of S-adenosylmethionine decarboxylase that plays a role in Spd and Spm synthesis, did not affect the development of seedless grape berries induced by 100 ppm GA₃. The application of 100 ppm GA₃ significantly increased endogenous free Put levels. Levels of free Spd and Spm were not affected by GA₃. Although the levels of endogenous perchloric acid insoluble bound PAs were higher than those of free PAs, obvious changes in the levels of bound PAs were not observed. These results indicate that free Put is implicated in the development of seedless grape berries induced by GA₃.     

Solute accumulation differs in the vacuoles and apoplast of ripening grape berries

Phloem unloading is thought to switch from a symplastic route to an apoplastic route at the beginning of ripening in grape berries and some other fleshy fruits. However, it is unclear whether different solutes accumulate in both the mesocarp vacuoles and the apoplast. We modified a method developed for tomato fruit to extract apoplastic sap from grape berries and measured the changes in apoplastic and vacuolar pH, soluble sugars, organic acids, and potassium in ripening berries of Vitis vinifera ‘Merlot’ and V. labruscana ‘Concord’. Solute accumulation varied by genotype, compartment, and chemical species. The apoplast pH was substantially higher than the vacuolar pH, especially in Merlot (approximately two units). However, the vacuole–apoplast proton gradient declined during ripening and in Merlot, but not in Concord, collapsed entirely at maturity. Hexoses accumulated in both the vacuoles and apoplast but at different rates. Organic acids, especially malate, declined much more in the vacuoles than in the apoplast. Potassium accumulated in the vacuoles and apoplast of Merlot. In Concord, by contrast, potassium increased in the vacuoles but decreased in the apoplast. These results suggest that solutes in the fruit apoplast are tightly regulated and under developmental control.     

Sugars induce anthocyanin accumulation and flavanone 3-hydroxylase expression in grape berries

Flavanone 3-hydroxylase (EC 1.14.11.9, F3H) plays a key role in anthocyanin biosynthesis, and sugars enhance anthocyanin accumulation and F3H expression in some other plants. However, information about the relationship between sugars, anthocyanin accumulation and F3H expression in grape berries has been little reported. Present experiment was done with sliced grape berry system. The optimum fruit developmental stage, sugar concentration, and incubation time in sugar induction anthocyanin accumulation and F3H expression were determined. Mannose and 2-deoxyglucose, glucose analogs known to be phosphorylated by hexokinase but are poorly metabolized, obviously induced the anthocyanin accumulation and F3H expression, whereas 3-O-methylglucose and 6-deoxyglucose, glucose analogs transported inside the cell but not substrates for hexokinase, did not induce them. Glucosamine and mannoheptulose, the specific inhibitors of hexokinase, blocked the activation induced by sugar on both anthocyanin accumulation and F3H expression.     

Effect of light exposure on linalool biosynthesis and accumulation in grape berries

Linalool is an important compound that contributes to the floral aroma in wines. This study showed the effect of light exposure on linalool accumulation in berries. The grape bunches were covered with films that block the full light spectrum (Shade) and the UV spectrum (UV-block), and a transparent film (Control). The linalool content was significantly higher in juice from Control-covered berries than in juice from Shade- and UV-block-covered berries, and the expression levels of the representative genes in linalool biosynthesis in Shade- and UV-block-covered berries were markedly lower than in Control-covered berries. These findings suggest that exposing berries to light is essential for linalool biosynthesis. To reflect sunlight onto grape clusters, reflective sheets were placed on the ground of a vineyard. The linalool content in berries exposed to sunlight reflected from the reflective sheets was higher than those in the control.     

2-Methoxy-3-isobutylpyrazine in grape berries and its dependence on genotype

2-Methoxy-3-isobutylpyrazine (MIBP) contributes a bell pepper aroma to many grape cultivars and has a reported aroma threshold of ～2 ng L(-1) in water. The purpose of this study was twofold: (1) develop a procedure using headspace solid phase micro-extraction combined with GC-MS in the selected ion monitoring mode (HS-SPME-GC-MS-SIM) for analysis of MIBP in grape berries, and (2) determine the location of MIBP biosynthesis in grapevines by approach grafting clusters of Vitis vinifera L. cvs Cabernet Sauvignon and Muscat blanc onto each other. The soluble solids and pH of the grape juice/homogenate matrix from different grape berry developmental stages influenced the method precision; therefore, quantification via the method of standard addition was used. Using our developed method, the limit of detection (LOD) and limit of quantitation (LOQ) of MIBP were 0.1 ng L(-1) and 2 ng L(-1), respectively, measured in a model juice and non-MIBP containing Chardonnay juice. Spiked recoveries averaged between 91% and 112% in Cabernet Sauvignon and Pinot noir homogenates and the overall relative standard deviation was less than 10%. The method was used to analyze MIBP in 29 grape cultivars and in fruit from clusters grafted to Cabernet Sauvignon or Muscat vines. Quantifiable levels were found only in Cabernet franc, Cabernet Sauvignon, Merlot, Sauvignon blanc and Semillon, providing information on the genetic connection for the occurrence of MIBP in grapes. No MIBP was detected in the berries of Muscat blanc clusters grafted onto Cabernet Sauvignon vines when sampled at fruit maturity. MIBP was detected in all berries of Cabernet Sauvignon regardless the graft configuration. The data indicate that MIBP or its precursors originate in the berry and its formation depends upon grape genotype.     

Field evaluation of water transport in grape berries during water deficits

The net flow in vascular and transpirational components of the grape berry water budget was evaluated during water deficits imposed at different stages of fruit development. Diurnal fluctuations in berry diameter were measured on field-grown grapevines ( Vitis vinifera L. cv. Cabernet Sauvignon) by using electronic displacement transducers. Water deficits were imposed by withholding irrigation, and water potentials of mid-shoot leaves, basal stem xylem and clusters were determined with a pressure chamber. The relative net flows through pedicel xylem and phloem and through berry transpiration were estimated pre-veraison and post-veraison. The xylem functioned nearly exclusively in providing net inflow pre-veraison, while the phloem was clearly dominant post-veraison. Accordingly, the amplitude of diurnal contraction was markedly smaller post-veraison than pre-veraison. The amplitude of diurnal contraction increased dramatically with decreasing plant water status pre-veraison, yet exhibited little sensitivity to low vine water status post-veraison. Measurements of the difference in water potential between clusters and source stems did not provide evidence of a gradient that would elicit significant water movement from the cluster to the stem at any time of the day. This was true for both irrigated and non-irrigated vines, although the non-irrigated vines exhibited a smaller gradient favoring inflow throughout much of the day. The gradient for xylem water transport to the cluster was considerably smaller post-veraison than pre-veraison. The results showed that berry transpiration functioned as the primary pathway for water loss both pre- and post-veraison.     

Apoplastic sugar may be lost from grape berries and retrieved in pedicels

In ripening grape (Vitis sp.) berries, the combination of rapid sugar import, apoplastic phloem unloading, and water discharge via the xylem creates a potential risk for apoplastic sugar to be lost from the berries. We investigated the likelihood of such sugar loss and a possible sugar retrieval mechanism in the pedicels of different Vitis genotypes. Infusion of D-glucose-1-¹³C or L-glucose-1-¹³C to the stylar end of attached berries demonstrated that both sugars can be leached from the berries, but only the nontransport sugar L-glucose moved beyond the pedicels. No ¹³C enrichment was found in peduncles and leaves. Genes encoding 10 sugar transporters were expressed in the pedicels throughout grape ripening. Using an immunofluorescence technique, we localized the sucrose transporter SUC27 to pedicel xylem parenchyma cells. These results indicate that pedicels possess the molecular machinery for sugar retrieval from the apoplast. Plasmodesmata were observed between vascular parenchyma cells in pedicels, and movement of the symplastically mobile dye carboxyfluorescein demonstrated that the symplastic connection is physiologically functional. Taken together, the chemical, molecular, and anatomical evidence gathered here supports the idea that some apoplastic sugar can be leached from grape berries and is effectively retrieved in a two-step process in the pedicels. First, sugar transporters may actively retrieve leached sugar from the xylem. Second, retrieved sugar may move symplastically to the pedicel parenchyma for local use or storage, or to the phloem for recycling back to the berry.

Grape berry pedicels may retrieve sugar that is lost via the xylem following apoplastic phloem unloading in the berries.     

Extraction and characterization of pectic substances from pulp of grape berries

Pectic substances were successively extracted from the alcohol-insoluble residue (AIR) of the pulp of grape berries, by water (WSP), oxalate (OXP), hot dilute HCl (HP) and cold dilute NaOH (OHP). Pectins (WSP, OXP, HP) were purified by ion-exchange chromatography (DEAE-Sephacel) or precipitation with cupric ions (OHP). Total pectic substances represent 20·8% (w/w) of the AIR, WSP and HP being the main components (6% and 12% of the AIR, respectively). An alternative to oxalate extraction of the water-insoluble pectin was extraction with NaCl solutions of increasing concentrations, which had released small amounts of pectins. Each of the fractions contained mainly galacturonic acid, arabinose and galactose, lower amounts of rhamnose and xylose, and minor amounts of glucose and mannose. Ion-exchange chromatography was performed on DEAE-Sephacel, M_w distribution was checked by gel permeation on Sepharose CL-2B, and M_v was determined as well as degrees of methylesterification (DM), acetylation (DA), and protein content.     

Direct in situ measurement of cell turgor in grape (Vitis vinifera L.) berries during development and in response to plant water deficits

Vitis vinifera L. berries are non-climacteric fruits that exhibit a double-sigmoid growth pattern, and at the point known as 'veraison', which is just before the beginning of the second period of rapid fruit growth, these berries undergo several abrupt physiological changes. Cell pressure probe was used to examine the in situ turgor (P) of cells in the mesocarp during berry development and in response to plant water deficits. Initial tests comparing attached and detached berries demonstrated that cell P was stable for up to 48 h after detachment from the vine, provided that water loss from the berry was prevented. Cell P at pre-dawn was on the order of 0.25 MPa pre-veraison (PreV) and was reduced by an order of magnitude to 0.02 MPa post veraison (PostV). Cell P declined slightly but significantly with depth from the berry surface PreV, but not PostV. When water was withheld from potted vines, cell P declined about 0.2 Mpa, as pre-dawn vine water potential declined about 0.6 MPa over 12 d, whereas cell P was completely insensitive to a 1.10 MPa decrease in pre-dawn vine water potential after veraison. Rewatering of stressed plants also resulted in a 24 h recovery of cell P before, but not after veraison. The substantial decline in cell P around veraison is consistent with the decline in berry firmness that is known to occur at this time, and the PostV insensitivity of P to changes in vine water status is consistent with current hypotheses that the PostV berry is hydraulically isolated from the vine. The fact that a measurable P of about 0.02 MPa and typical cell hydraulic/osmotic behaviour were exhibited in PostV berries, however, indicates that cell membranes remain intact after veraison, contrary to many current hypotheses that veraison is associated with a general loss of membrane function and cellular compartmentation in the grape berry. We hypothesize that cell P is low in the PostV berry, and possibly other fleshy fruits, because of the presence of regulated quantities of apoplastic solutes.     

Changes in cell-wall polysaccharides from the mesocarp of grape berries during veraison

Softening of grape berries ( Vitis vinifera L. × V. labruscana cv. Kyoho) was evaluated by studying changes in composition and degradation of cell-wall polysaccharides. The grape berry softens at the beginning of the second growth cycle many weeks before harvest. The softening stage is called 'veraison' by viticulturists. On day 50 after full bloom, green hard berries (before veraison [BV]), softening berries (veraison [V]) and partly peel colored berries (C) were selected from the same clusters. In addition, mature berries (M) were collected on day 78 after full bloom. Mesocarp tissues at each stage were fractionated into hot water-soluble (WS), hot EDTA-soluble (pectin), alkali-soluble (hemicellulose) and residual (cellulose) fractions. Neutral and acidic sugar contents of WS and pectin fractions decreased only after the V stage, while the neutral sugar content of the hemicellulose fraction decreased from the BV to V stages. Cellulose content constantly decreased as the berry ripened, but the large decrease was found from the BV to V stages. Molecular masses of pectic and hemicellulosic polysaccharides decreased from the BV to V stages. Hemicellulosic xyloglucan was markedly depolymerized from the BV to V stages. The neutral and acidic sugar composition of each fraction changed little during the berry ripening. These data indicated that softening of berry during veraison involved the depolymerization of pectin and xyloglucan molecules and decrease in the amounts of hemicellulose and cellulose.