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.     

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.     

Fruit ripening in Vitis vinifera: light intensity before and not during ripening determines the concentration of 2-methoxy-3-isobutylpyrazine in Cabernet Sauvignon berries

The roles of light and temperature in the accumulation of the vegetal impact compound 2-methoxy-3-isobutylpyrazine (MIBP) in grape (Vitis vinifera L.) berries were determined. Individual clusters were exposed to various light intensities using neutral density shade cloth before ripening, during ripening or throughout the season in three growing seasons. A recently developed method using headspace solid-phase microextraction combined with GC-MS in the selected ion-monitoring mode was employed to measure MIBP in berries. Berry MIBP concentration increased subsequent to berry set, reached a maximum prior to onset of ripening, and then decreased thereafter until harvest. Complete shading of clusters increased the concentration of MIBP more than 100% compared to unshaded controls in 2 out of 3 years. Light increasingly inhibited MIBP concentrations up to 25-50% of ambient light intensities (1500 μmol photons m(-2) s(-1) ). However, only changes in light intensity before ripening had any effect on MIBP accumulation or final MIBP concentration. Analyses of weather data showed that the 1 year in which shading was ineffective was unusually warm, warm early in the season, and had more hot days and higher early season degree days than the other 2 years. In controlled environment experiments, warm growth conditions reduced MIBP concentrations in fruit about as much as light exposure reduced MIBP concentrations in the field experiments. The results indicate that both light and temperature significantly affect MIBP in harvested fruit, but that the light environment during ripening does not significantly affect MIBP concentrations in the berries at harvest.     

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.     

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.     

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).     

Alternaria mycotoxins associated with grape berries in vitro and in situ

The aim of this study was to assess the production of secondary metabolites of Alternaria species isolated from grape berries and determined the occurrence of Alternaria mycotoxins in vitro and in dried berries. Direct morphological examination on different media was used for identification and HPLC/MS method for quantification of Alternaria mycotoxins. We isolated A. alternata and A. tenuissima and randomly selected strains in vitro and in dried berries. In vitro we identified the production of Alternaria metabolites, included mycotoxins: alternariol, alternariol methylether, tenuazonic acid. Beside that one in dry berries were in measurable concentrations: macrosporin A, tentoxin and altenuete (I, II).     

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.     

Hexokinase from grape berries: its prokaryotic expression, polyclonal antibody preparation and biochemical property analyses

Two full-length hexokinase (HXK, EC 2.7.1.1) cDNAs, VvHXK1 with 1,413 bp and VvHXK2 with 1,458 bp were cloned from grape berries (Vitis vinifera L. Cabernet Sauvignon). VvHXK1 and VvHXK2 genes sequence from grape berries were deposited in GenBank under the accession number JN118544 and JN118545, respectively. The homology of the amino acid of VvHXK1 or VvHXK2 was very similar to ‘Pinot Noir’ grape HXK sequence, their similarties were 99.36 % and 98.97 %, respectively. More intuitive phylogenetic tree showed that the homology of amino acid sequence VvHXK1 with melon CmHXK1 was 86 %, and VvHXK2 homology with rice OsHXK3 was 83 %. The HXK proteins were successfully expressed in plasmid pET-30a (+) vectors in Escherichia coli BL21 (DE3) pLysS. The expressed proteins were purified using Ni-NTA agarose column and used to produce HXK1 antibody and conducted HXK protein blotting analysis. The results,suggested that one polypeptide band of about 51 kDa HXK protein can be detected in grape berries, HXK protein level was the highest during early grape berry development, but the lowest from 50d to 60d during development. Biochemical analysis of two hexokinase isozymes indicated that glucose was the optimal substrate of HXK, The isoelectric points of the two isozymes were 5.8 and 5.6, respectively. And the optimum pH was about 8.0. These results provide a substantial basis for the further studies of functions of grape HXKs to manipulate sugar content of grape berries.     

Novel Acyclic Analogues of Pyrimidine Nucleosides: 1-Methoxy-2, 3-Dihydroxypropyl and 1-Methoxy-3-hydroxypropyl Derivatives

Abstract

The preparation of a series of novel pyrimidine nucleoside analogues in which the N-1 ribosyl moiety is replaced by 1-methoxy-2, 3-dihydroxypropyl and 1-methoxy-3-hydroxypropyl substituents is described.     

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.     

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.     

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.     

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.     

不同颜色果袋对‘巨峰’葡萄果实中挥发性成分的影响

以‘巨峰’葡萄为试材,研究白袋、绿袋、红袋、蓝袋4种不同颜色果袋对葡萄成熟期果实中挥发性成分的影响,为葡萄专用果袋的研发提供理论依据.结果表明:不同颜色果袋可为葡萄果实的发育提供特定的光环境,不同套袋处理葡萄成熟期果实中的挥发性成分差异显著.‘巨峰’葡萄成熟期果实中检测到酯、醛、醇、酮类、萜烯类和芳香族化合物,对照、白袋、绿袋、红袋和蓝袋处理成熟期果实检测到的挥发性组分分别为33、37、38、32和34种.与对照相比,白袋处理乙酸乙酯、己酸乙酯等酯类物质含量降低,己醛、反式-2-己烯醛、癸醛含量增加;绿袋处理除3-己烯酸乙酯、反式-2-己烯酸乙酯、3-羟基丁酸乙酯、反式-2-己烯醛、(反,反)-2,4-己二烯醛、癸醛、苯乙醇外,其他共有组分的含量均出现降低;红袋处理乙酸乙酯、己酸乙酯等酯类物质含量降低,己醛、反式-2-己烯醛等的含量出现降低;蓝袋处理乙酸乙酯、己酸乙酯等主要酯类物质的含量未发生较大变化,己醛、反式-2-己烯醛等的含量出现升高.在非共有组分中,套袋处理中醇类组分的种类减少,萜烯类、芳香族化合物的种类增加.总体上,蓝袋处理果实中主要的‘果香型’酯类组分含量最高,白袋处理果实中主要的酯类组分及‘青草香型’的醛类组分含量较高,绿袋、红袋处理果实主要香气组分含量较低.     

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.     

Ascorbate metabolism and the developmental demand for tartaric and oxalic acids in ripening grape berries

Background  

Fresh fruits are well accepted as a good source of the dietary antioxidant ascorbic acid (Asc, Vitamin C). However, fruits such as grapes do not accumulate exceptionally high quantities of Asc. Grapes, unlike most other cultivated fruits do however use Asc as a precursor for the synthesis of both oxalic (OA) and tartaric acids (TA). TA is a commercially important product in the wine industry and due to its acidifying effect on crushed juice it can influence the organoleptic properties of the wine. Despite the interest in Asc accumulation in fruits, little is known about the mechanisms whereby Asc concentration is regulated. The purpose of this study was to gain insights into Asc metabolism in wine grapes (Vitis vinifera c.v. Shiraz.) and thus ascertain whether the developmental demand for TA and OA synthesis influences Asc accumulation in the berry.