An improved method for isolating high-quality DNA from<Emphasis Type="Italic">Vitis vinifera</Emphasis> nuclei

We have developed a simple and highly efficient protocol for isolating large quantities (150–400 μg/g leaf tissue) of high-quality DNA from fresh and frozenVitis vinifera leaves. Isolated DNA is essentially free of polysaccharides, polyphenols, and other major contaminants as judged by viscosity, clear color, A_260/280 ratio, digestibility by restriction enzymes for Southern blot analysis, and PCR suitability.     

Bi-directional Duplex Promoters with Duplicated Enhancers Significantly Increase Transgene Expression in Grape and Tobacco

Novel bi-directional duplex promoters (BDDP) were constructed by placing two identical core promoters divergently on both upstream and downstream sides of their duplicated enhancer elements. Estimates of promoter function were obtained by creating versions of CaMV 35S and CsVMV BDDPs that contained reporter marker genes encoding beta-glucuronidase (GUS) and enhanced green fluorescent protein (EGFP) interchangeably linked either to the upstream or downstream core promoters. GUS was used for quantitative analysis of promoter function, whereas, EGFP allowed visual qualitative evaluation. In addition, the GUS and EGFP genes placed in downstream positions were modified by translational fusion with neomycin phosphotransferase (NPTII) to allow simultaneous monitoring of promoter activity and selection of stable transformants. These versions of BDDP were compared with each other and with equivalent unidirectional constructs by evaluating their expression in grape and tobacco. For 35S promoter constructs tested in grape somatic embryos (SE), BDDP exhibited transient GUS expression 206- and 300-fold greater in downstream and upstream configurations, respectively, compared to a unidirectional 35S core promoter. Compared with a unidirectional double enhanced 35S promoter, BDDPs exhibited 0.5- and 3-fold increased GUS expression from downstream and upstream core promoters, respectively. The same differences in expression levels determined quantitatively with GUS were distinguished qualitatively with EGFP. Constructs using CsVMV core promoters yielded results relative to those obtained with 35S promoter. For example, the upstream BDDP CsVMV core promoter provided a 200-fold increase in GUS expression compared to a unidirectional core promoter. However, CsVMV promoter was found to have higher promoter activity than 35S promoter in both BDDP and unidirectional constructs. Incorporation of an additional duplicated enhancer element to BDDPs resulted in increased expression. For example, a 35S BDDP with two divergently arranged duplicated enhancer elements resulted in over a 6-fold increase in GUS expression in stably transformed tobacco plants compared to a BDDP with one duplicated enhancer element. Data demonstrate that BDDP composed of divergently-arranged core promoters separated by duplicated enhancers, all derived from a single promoter sequence, can be used to significantly enhance transgene expression and to direct synchronized expression of multiple transgenes.     

葡萄悬浮细胞长期培养过程中不同继代条件下花青素生产的不稳定性

为了深入研究植物细胞培养生产次生代谢产物不稳定性的机制,以葡萄细胞作为模式体系,研究悬浮培养过程中花青素合成的不稳定性。除了用常规的花青素总含量来表征花青素的生物合成之外,还采用HPLC测定花青素不同组分的含量。结果表明,在长期的继代培养过程中,不仅花青素的含量而且花青素的组成也表现出明显的不稳定性。首次采用了不稳定系数 (δ) 和因素得分 (Factor scores) 来表征植物细胞培养过程中次生代谢生产的不稳定性。培养条件对花青素生物合成的影响实验结果表明,继代周期和接种量均能诱发次生代谢的不稳定性表达,其中接种量的影响相对更大。在考察的 (6.5 d,2.00 g),(7 d,2.00 g),(7.5 d,2.00 g),(7 d,1.60 g) 和 (7 d,2.40 g) 五种不同的继代周期和接种量组合条件中,7 d继代周期和1.60 g接种量最有利于保持花青素的稳定生产。     

Invertase activity, grape berry development and cell compartmentation

The effect of gibberellic acid on grape (Vitis vinifera L., ev. Sultanina) growth, β-fructofuranosidase (EC 3.2.1.26) activity and carbohydrate levels was investigated throughout berry development and ripening. Although the fruits responded to hormone application with the expected increase in size, growth was not correlated with enzymic activity and hexose accumulation. This suggests that there is no direct regulatory relationship between invertase and the rate of assimilate import. However, fructose:glucose ratios changed from 0.1 in green berries to 1.0 in mature samples. The latter situation can be reconciled with the 1:1 stoichiometry of sucrolysis by invertase. It is suggested that this is attributable to a spatial separation of substrate and enzyme in green tissue. Compartmentation studies indicate that mesocarp cell integrity gradually deteriorates during ripening, which allows invertase to leak out of the vacuole into the surrounding tissue. In fact, the protein fraction retrieved from a buffered medium after incubation of ripening berry slices contained a soluble invertase of presumably vacuolar origin with an acid pH-activity profile and a pI of about 4.     

Occurrence of Fungal Pathogens Associated with Grapevine Nurseries and the Decline of Young Vines in Spain

The occurrence of fungal grapevine trunk pathogens associated with grapevine nurseries and the decline of young vines in Spain was determined in extensive surveys conducted in nurseries and vineyards with young plants. The presence of Phaeomoniella chlamydospora, Phaeoacremonium aleophilum, Cylindrocarpon spp., Botryosphaeria obtusa and Botryosphaeria spp. was detected in all the surveys that were carried out. This study provides evidence for the presence of these pathogens in Spanish grapevine nurseries. Cylindrocarpon spp., B. obtusa and Botryosphaeria spp., were isolated very early in the planting material production process, nevertheless, P. aleophilum and P. chlamydospora were not detected until the rootstock–scion combinations were planted in the field to develop shoots and roots during summer. The occurrence of trunk disease pathogens in symptomatic young vineyards was also high, suggesting that infected plant material might have been used. Phaeomoniella chlamydospora and P. aleophilum were the main species associated with the decline of young vines, followed by Botryosphaeria spp., Cylindrocarpon spp. and B. obtusa. This research confirmed the importance of fungal grapevine trunk pathogens associated with the decline of young vineyards in Spain and suggested the connection between the presence of these fungi in the nurseries and the disease in the fields.     

Phylogenetic relationships,hybridization events,and drivers of diversification of East Asian wild grapes as revealed by phylogenomic analyses

East Asian wild grapes show a high level of species diversity and have been widely recognized as important germplasm resources of wine, table grapes, and resistance to biotic and abiotic stresses for the grape industry. However, the deeper phylogenetic relationships and drivers of diversification of East Asian Vitis remain poorly understood. Hybridization and introgression events of East Asian Vitis are not well investigated, particularly at the genome-wide scale. The phylogenetic relationships of East Asian Vitis are herein explored using nuclear and plastid genome data based on target enrichment (Hyb-Seq). Seven major clades are recognized for East Asian Vitis based on the nuclear phylogenetic trees, but there is topological incongruence between concatenated and coalescent analyses. Furthermore, significant cytonuclear discordance is observed within East Asian Vitis. Species network analyses identified several hybridization events within East Asian Vitis. These interspecific hybridization events may have caused the topological discordances and relatively low support detected in our analyses. Ecological niche modeling shows that most of the diversification of East Asian Vitis species is driven by temperature and precipitation environmental variables. Sympatric parallel diversifications of major clades also may have facilitated the rich diversity in East Asian Vitis.