Worldwide, significant postharvest losses of fruit and vegetables due to attack by microorganisms is evidence, while chemical applications is of great consumer concerns regarding food safety. Alternative sanitizers are explored, with natural compounds such as essential oils (EOs) to achieve scientific and consumer's interest for the preservation of fresh produce. In the present study, the efficacy of dittany (Origanum dictamnus L.) essential oil for the control of Botrytis cinerea, a common postharvest pathogen of three economically important vegetables, tomato, pepper and eggplant was examined. Pathogen development (vegetative or reproductive phase) in culture medium or in fruits was evaluated after treatment with dittany EO (0, 50, 100, 250 mu L L-1) in vitro and in situ when stored at 12 degrees C and 95% RH during or following exposure to EO volatiles. In vitro, fungal development was completely inhibited by the application of 100 or 250 mu L L-1 of EO volatiles. In inoculated fruits, the application of 50 mu L L-1 EO resulted in suppressed disease development by reduced lesion growth and fungal sporulation, where increasing EO concentration led to greater effects. Pre-exposure of the three fruits to volatiles, before fungal inoculation, revealed reduced lesion growth, indicating that dittany EO probably caused induced resistance of fruits against the pathogen. Moreover, EO application did not affect quality-related characteristics of fruits in general, while skin lightness and pulp lightness of eggplant fruits were improved under the presence of dittany EO volatiles. Overall, the results suggest that dittany EO volatiles may be considered as an alternative food preservative treatment, significantly reducing or eliminating B. cinerea infection during fruit storage.

Nitrogen (N) plays the most important role of all soil mineral nutrients in plant growth and development. In grapevine, nitrogen is most likely to be defi cient although it is the main fertilizer commonly applied to vineyards to increase productivity (Keller et al. 1998) and to influence grape juice composition (Ough and Bell 1980). Considerable new information has been obtained over the past twenty years on the uptake, translocation, distribution, partitioning, and storage of nitrogenous compounds in grapevines as well as new insights toward a better understanding on the regulation of the synthesis and degradation of amino acids and other nitrogenous compounds, and of the enzymes associated with these re actions.

In an effort to better understand the physiological role of the enzymes glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) in ammonia assimilation in higher plants, we have studied their protein and molecular characteristics in an economically important perennial plant species, the grapevine. Grapevine GDH has a hexameric structure consisting of two subunit polypeptides which are associated in an ordered ratio. All isoenzymes have similar anabolic and catabolic activities and substrate kinetics. Presence of ammonium ions as nitrogen source in the culture medium of grapevine calluses resulted in increased NADH-GDH specific activity due to de novo synthesis of the alpha-subunit of GDH and the assembly of the more anodic isoenzymes. Grapevine GS was analyzed into two distinct classes of isoenzymes, the cytosolic and the chloroplastic one. A full cDNA clone encoding GDH, three structurally distinct full clones encoding cytosolic GS and two partial cDNAs encoding Fd-GOGAT were isolated from a grapevine cell suspension cDNA library and characterized. The gene organization and the expression characteristics of the genes corresponding to the six cDNAs were determined by DNA and RNA blots, respectively. The transcripts corresponding to these cDNAs showed differential accumulation in the various grapevine tissues tested. Culture of cell suspensions and plantlets in media containing modified nitrogen source resulted in differential expression of the GDH, GS and Fd-GOGAT genes.

Activity of ferredoxin‐dependent glutamate synthase (Fd‐GOGAT, EC 1.4.7.1), an enzyme with a central role in the assimilation of ammonia in higher plants, was detected in all grapevine ( Vitis vinifera L.) tissues examined. Two cDNA clones, encoding the carboxy‐ and near to amino‐terminal sequences of the Fd‐GOGAT, respectively, were isolated from a grapevine cell suspension library. The deduced amino acid sequence of the grapevine enzyme is significantly identical to the respective sequences of other known plant Fd‐GOGATs and shows homology to the alfalfa nodule NADH‐dependent GOGAT and to the large subunit of the Escherichia coli NADPH‐dependent GOGAT. DNA blot analysis suggested the existence of two Fd‐GOGAT genes in grapevine. In northern blots both clones hybridized to a 5.7‐kb transcript which was abundant in leaf and root tissues. Both, the Fd‐GOGAT mRNA and the enzymic activity increased upon illumination of leaves of dark‐grown plants suggesting that grapevine Fd‐GOGAT gene expression is regulated by light. In cell cultures, Fd‐GOGAT transcript level responded differently to the external nitrogen source, showing higher levels in the presence of nitrate and lower levels in the presence of ammonium.

Southern analysis of genomic DNA indicated the presence of a multigene osmotin-like family in the grapevine genome. The expression of one of these genes that corresponds to the previously isolated pVVOSM1 cDNA is developmentally regulated showing high transcript levels in root, stem and leaf tissues of in vitro-grown plants, intermediate in calluses and berries and low in cell suspensions. Treatment of cell suspension cultures with abscisic acid (ABA), ethylene or NaCl resulted in high levels of expression of the pVVOSM1 gene suggesting regulation of the osmotin-like gene by the abiotic signals mentioned above.

Ebenus cretica , Leguminosae, an endemic perennial bush of Crete, is being studied as a potential new cut flower crop. Forty-centimeter-long spikes with two to three inflorescences and six to eight compound leaves were harvested from 5-year-old plants grown from seed at the farm of the TEI, when 1/3 of the florets had opened, and were treated with various preservatives. Flower quality was evaluated morphologically combined with measurements of chlorophyll content in leaves and anthocyanin in petals. Without any postharvest treatments, inflorescences held in a solution of 100 ppm 8-hydroxyquinone sulfate (HQS) in DI water had an average vaselife of 6.8 days. Pulsing with 0.6 mM silver thiosulfate (STS) for 2 h extended vaselife up to 8.4 days. However, when ethephon was added in the solution, vaselife was significantly reduced, causing leaf yellowing and flower senescence, which suggests sensitivity to exogenous ethylene. A solution of 0.2% Ca(NO 3 ) 2 prolonged vaselife by 2.7 days, whereas higher concentrations resulted in flower discoloration and decreased flower quality. Sucrose solutions of 0.5%, 1%, 2%, and 4% had no positive effect on flower longevity. Furthermore, the higher concentrations caused leaf yellowing and petal discoloration decreasing vaselife and quality of flowers compared to control. Samples of inflorescences were taken every second day for chlorophyll (a and b) and anthocyanin measurements. The concentrations recorded were highest in the 0.2% Ca(NO 3 ) 2 treatment and were significantly correlated to flower longevity. Results indicate that Ebenus cretica may be used as a cut flower crop; however, due to the genetic variability of the Ebenus plants, a breeding line should be developed before the crop reaches the floricultural market.

The specific activities of aminating NADH- and deaminating NAD(+)-glutamate dehydrogenase (GDH, EC 1.4.1.2) varied considerably in crude extracts of grapevine (Vitis vinifera L. cv. Sultanina) callus and were dependent on the nitrogen source of the culture medium. However, dialysis of the enzyme preparations resulted in a significant decrease in the deaminating GDH specific activity while the aminating activity was not affected. The presence of malate in the crude extract resulted in erroneous overestimation of the NAD(+)-GDH activity through the malate dehydrogenase reaction. Thus, in dialysed extracts, the ratio of the NADH-GDH/NAD(+)-GDH specific activities remained relatively constant irrespective of the nitrogen source. In view of this evidence, we now have modified methods for staining both the NADH-GDH and NAD(+)-GDH activities on gels in order to compare the aminating and deaminating activities of each of the 7 GDH isoenzymes. The results from the staining of NADH-GDH and NAD(+)-GDH activity of enzyme preparations from calluses revealed the same isoenzyme profile. Furthermore, separated leaf isoenzymes showed similar activity ratios and kinetic properties. These results may suggest that each one of the 7 isoenzymes have similar in vitro anabolic and catabolic activities.

A modified procedure for efficient isolation of intact and biologically active RNA from leaves, berries and cell cultures of grapevine (Vitis vinifera L.) is described. RNA is extracted in the presence of mild denaturants followed by sequential precipitations with potassium acetate and isopropanol. RNA is recovered free of contaminants after centrifugation through a CsCl cushion. Alternatively, RNA from grapevine callus and berry cell suspensions can be purified after the isopropanol step by high-salt precipitations, without a CsCl centrifugation step. The resulting RNA is of high-quality, as judged by agarose gel electrophoresis and northern blot analysis. Furthermore, RNA isolated by this method is biologically active as was evidenced by in vitro translation and analysis of the products by polyacrylamide gel electrophoresis and fluorography. This procedure can also be applied to other perennial woody plant species.

The activity, protein, and isoenzymic profiles of glutamate de-hydrogenase (GDH) and glutamine synthetase (GS) were studied during development and ripening of avocado (Percea americana Mill. cv Hass) fruit. During fruit development, the activity and protein content of both GDH and GS remained relatively constant. In contrast, considerable changes in these enzymes were observed during ripening of avocado fruit. The specific activity of GDH increased about 4-fold, coincident with a similar increase in GDH protein content and mRNA levels. On the other hand, GS specific activity showed a decline at the end of the ripening process. On the isoenzymic profile of GDH, changes in the prevalence of the seven isoenzymes were found, with a predominance of the more cathodal isoenzymes in the unripe and of the most anodal isoenzymes in the ripe fruit. Two-dimensional electrophoresis revealed that avocado fruit GDH consists of two subunits whose association gives rise to seven isoenzymes. The results support the view that the predominance of the more anodal isoenzymes in the overripe fruit was due to the accumulation of the [alpha]-polypeptide.

The effect of different oxygen levels on preclimacteric and ripening initiated avocado fruits was studied at total protein and gene expression levels. Low oxygen induced the accumulation of new polypeptides in both types of fruits as was evident by two dimensional PAGE (2-D) of total proteins and SDS-PAGE of in vitro translation products, whereas it suppressed the development of new polypeptides only in initiated fruits. The synthesis of cellulase was irrespective of low oxygen concentration in preclimacteric fruits and oxygen dependent in initiated fruits. Alcohol dehydrogenase isoenzymes were induced in both types of fruits correlated with elevated mRNA levels.

The structure and function of NAD(H)-glutamate dehydrogenase in plants was studied by using grapevine (Vitis vinifera L. cv Sultanina) callus grown under different nitrogen sources. The enzyme consists of two subunit-polypeptides, alpha and beta, with similar antigenic properties but with different molecular mass and charge. The two polypeptides have molecular masses of 43.0 and 42.5 kilodaltons, respectively. The holoenzyme is hexameric and is resolved into seven isoenzymes by native gel electrophoresis. Two-dimensional native/SDS-PAGE revealed that the 1 and 7 isoenzymes are homohexamers and the isoenzymes 2 through 6 are hybrids of the two polypeptides following an ordered ratio. The total quantity of alpha- and beta-polypeptides and the isoenzymic pattern was altered by the exogenous nitrogen source. The sample derived from callus grown on nitrate or glutamic acid contained a slightly greater amount of beta-polypeptide and of the more cathodal isoenzymes, whereas alpha-polypeptide and the more anodal isoenzymes predominated in callus grown in the presence of either ammonium or glutamine. The anabolic reaction was correlated with the alpha- and the catabolic reaction with the beta-polypeptide; this could suggest that each isoenzyme exhibits anabolic and catabolic function of different magnitude. The isoenzymic patterns did not obey the expected binomial distribution proportions.

actions. However, in spite of the widespread usage of nitrogen fertilization in vineyards, the physiological and biochemical effects of nitrogen on shoot and fruit growth, fruit bud initiation, flowering, fruit set, and crop yield are still poorly understood. Grapevines differ from herbaceous plants and many other woody plants in that they do not form terminal buds at the end of shoots, but they can continue to grow late into the season. The individual flower parts are formed after bud break, unlike most deciduous fruit trees. Nitrate and ammonium ions are the most common forms of nitrogen available to plants in soils. The relative importance and concentration of each of these two ions depends on the genetic, developmental, and physiological status