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Leaf anatomical and ultrastructural responses of "Razegui" and "Muscat Italia" grapevine cultivars to high temperatures were studied under controlled conditions (T > 36°C), based on photonic and electron microscopy. Histological studies performed on leaves from heat-stressed and control grapevines revealed thicker leaf blades under high temperature conditions. Environmental scanning electron microscopy of leaf surfaces from both cultivars allowed observing sinuate epidermal cells on the leaves of grapevines cultivated under heat stress and irregular giant oblong pores on their adaxial surface. When observed by transmission electron microscopy, leaf cross sections in grapevines cultivated under high temperature conditions exhibited folded cuticle and cell wall on the adaxial epidermis layer. Therefore, significantly greater cell wall thicknesses were measured under heat stress than control conditions in both cultivars. Regarding chloroplasts, they were more globular in shape under heat stress compared with control conditions and had disorganized thylakoids with a reduced thickness of grana stacking. The size of starch granule decreased, while the number of plastoglobules increased with heat stress, indicating a reduced carbon metabolism and a beginning of senescence within the 3-month heat stress period. This study confirms widespread adaptive properties in two grapevine cultivars in response to high temperature stress., (Copyright © 2010 Wiley-Liss, Inc.)

An efficient protein extraction method is crucial to ensure successful separation by two-dimensional electrophoresis (2-DE) for recalcitrant plant species, in particular for grapevine (Vitis vinifera L.). Trichloroacetic acid-acetone (TCA-acetone) and phenol extraction methods were evaluated for proteome analysis of leaves and roots from the Tunisian cultivar 'Razegui'. The phenol-based protocol proved to give a higher protein yield, a greater spot resolution, and a minimal streaking on 2-DE gels for both leaf and root tissues compared with the TCA-based protocol. Furthermore, the highest numbers of detected proteins on 2-DE gels were observed using the phenol extraction from leaves and roots as compared with TCA-acetone extraction., (© 2010 Institute of Botany, Chinese Academy of Sciences.)

The effect of three alternating temperatures cycles (20/30, 20/37 and 25/42 degrees C) and photoperiod on growth and Ochratoxin A (OTA) production of six isolates of Aspergillus carbonarius on synthetic nutrient medium were investigated. The different temperature regimes affected significantly both the mycelial growth and the OTA production. The best growth and OTA production were recorded at 20/30 degrees C. The isolates from the region of Baddar produced the highest OTA yields. A 24 h light cycle generally enhanced the growth of A. carbonarius. Growth rates cycles of 11 h/13 h light/darkness and 24 h darkness were often similar for individual isolates, such conditions enhanced OTA production in two of the six isolates tested., (2010 Elsevier B.V. All rights reserved.)

This study investigated the effects of temperature (15 to 37 degrees C) and water activity (0.90 to 0.99) on the growth and production of ochratoxin A (OTA) by Aspergillus carbonarius cultured on synthetic nutrient medium (SNM) after 5 and 10 d of incubation. Total of 8 ochratoxigenic A. carbonarius, isolated from vineyards located in different regions of Tunisia, were used. Growth data were modeled by the flexible model of Baranyi and growth rates at each set of conditions were obtained. For both growth and OTA production, optimal water activity was 0.99; however, optimal temperature varied. The optimal temperature for growth was 30 degrees C. At 37 degrees C, the growth rate decreased significantly (P < 0.05). Maximum toxin production occurred at temperatures in the range of 15 to 25 degrees C with the optimum one depending on the isolate tested. Significant amounts of OTA were produced after only 5 d of incubation. Our results showed that A. carbonarius isolated from Tunisian grapes behave as those from European and Australian grapes, as reported in the literature, although some differences in trends for growth and OTA production were observed.

To identify and isolate genes related to abiotic stresses (salinity and drought) tolerance in grapevine, a candidate gene approach was developed and allowed isolating a full-length cDNA of rd22 gene from the Cabernet Sauvignon variety. The latter, named Vvrd22, is a dehydration-responsive gene that is usually induced by the application of exogenous ABA. Details of the physicochemical parameters and structural properties (molecular mass, secondary structure, conserved domains and motives, putative post-translational modification sites...) of the encoded protein have also been elucidated. The expression study of Vvrd22 was carried out at the berry growth stages and at the level of plant organs and tissues as well as under both drought and salt stresses. The results showed that Vvrd22 is constitutively expressed at a low level in all analyzed tissues. Moreover, salt stress induced Vvrd22 expression, particularly for the tolerant variety (Razegui), contrary to the sensitive one (Syrah), which did not display any expression variation during the stress, which means that Vvrd22 is involved in salt stress response and that its expression level depends on regulatory mechanisms that are efficient only for the tolerant variety. On the other hand, under drought stress, Vvrd22 is induced in an identical manner for both tolerant and sensitive varieties. In addition, stress signal molecules such as ABA (lonely applied or in combination with sucrose) induced Vvrd22 expression, even at a low level. A minimal knowledge about the role and the functionality of this gene is necessary and constitutes a prerequisite condition before starting and including Vvrd22 in any program of improvement of grapevine's abiotic stress tolerance.

In order to quickly and efficiently evaluate the grapevine's salt tolerance, salinity tests were conducted on some grapevine varieties and rootstocks under in vitro conditions. Plant materials used in the salinity test were propagated using the axillary buds culture method. Single-node shoots were subjected to seven different NaCl concentrations (0, 20, 50, 100, 150, and 200 mM NaCl) in MS medium for 45 days. The different growth parameters analysed were: survival capacity, length of shoot, bud formation, and rooting capacity. Our results showed that salinity reduced in vitro growth and development of grapevine. Proliferation, growth, rooting and viability of explants decreased due to the increase in NaCl concentration. First symptoms of stress (leaves necrosis) appeared after 10 days of treatment with 80 mM NaCl, which may lead to total desiccation. It was determined that severity of salt treatment injury varied depending on the genotype and NaCl concentration. A positive correlation was found between the vigour of plants in saline medium and their faculty to tolerate salt. The most tolerant grapevine genotypes to salt treatment were Sejnene and Asli, followed by the moderately sensitive Saouadi and Sakasly genotypes, and last Razegui, 1103P, 41B, and SO(4), which were particularity sensitive. Thus, grapevine's salt tolerance seems to be linked to the genetic background.

Salt stress is one of the major abiotic stresses in agriculture worldwide, especially in the Mediterranean area. We report here a proteomic approach to investigate the salt stress-responsive proteins in grapevine (Vitis vinifera). Two-dimensional electrophoresis (2-DE) was used to analyze the proteome of the salt-tolerant Tunisian grapevine cultivar Razegui, subjected to a supply of 100mm NaCl over 15d. Analysis of 2-DE gels derived from stressed plants revealed more than 800 reproducibly detected protein spots, with 48 proteins displaying a differential expression pattern, including 32 up-regulated, 9 down-regulated and 7 new protein spots induced after salt treatment. The presence of stress-responsive proteins in the different plant organs suggests that salt spreads systemically. Edman degradation analysis and database searching aided us in identifying a major protein GP. Database analysis revealed that this peptide has a 98% sequence similarity with a pathogenesis-related (PR) protein 10 (V. vinifera). A full-length cDNA encoding the GP protein was isolated from grapevine salt-stressed leaves and sequenced. The predicted protein contained 158 amino acids and showed 98% identity with PR10 protein of of V. vinifera (accession no. Cac16165).

We have cloned and characterized VvNHX1, a gene encoding a vacuolar cation/H(+) antiporter from Vitis vinifera cv. Cabernet Sauvignon. VvNHX1 belongs to the vacuolar NHX protein family and showed high similarity to other known vacuolar antiporters. The expression of VvNHX1 partially complements the salt- and hygromycin-sensitive phenotypes of an ena1-4 nhx1 yeast strain. Immunoblots of vacuoles of yeast expressing a VvNHX1, together with the expression of a VvNHX1-GFP (green fluorescent protein) chimera demonstrated that VvNHX1 localized to the vacuoles. VvNHX1 displayed low affinity K(+)/H(+) and Na(+)/H(+) exchange activities (12.8 and 40.2 mM, respectively). The high levels of expression of VvNHX1 during the véraison and post-véraison stages would indicate that the increase in vacuolar K(+) accumulation, mediated by VvNHX1, is needed for vacuolar expansion. This process, together with the rapid accumulation of reducing sugars, would drive water uptake to the berry and the concomitant berry size increase, typical of the post-véraison stage of growth.

The occurrence of ochratoxin A (OTA) and the identification of the ochratoxigenic microbiota in Tunisian grapes were studied for the first time. Black aspergilli were the dominant genus among the filamentous fungi isolated from grapes and were the only potential OTA-producing fungi found. The most abundant species were member of Aspergillus niger aggregate (63%) and Aspergillus carbonarius (36%). Uniseriate aspergilli were rarely present (1%). Of the A. carbonarius isolates, 97% were OTA positive but only 3% of the A. niger aggregate isolates were OTA positive. During grape maturation, the frequency of black aspergilli increased due to increase of the numbers of A. carbonarius. Musts (n=24) obtained from grapes collected at the different sampling times were analyzed for their OTA content. Up to 37% of the musts contained OTA at levels varying between 0.59 and 2.57 microg/l. The amounts of OTA in musts increased as grapes matured. These results indicate that A. carbonarius is the main cause of OTA contamination of Tunisian grapes.