葡萄实生树阶段转变过程中的内源多胺含量变化

用高效液相色谱法测定二年生巨峰葡萄自然实生树不同节位叶片、芽、韧皮部中内源多胺含量的结果表明,随着节位的升高,在3种组织中的腐胺(Put)和亚精胺(Spd)含量增加,21～25节腐胺增加幅度最大,自21节起亚精胺含量陡增;21～25节叶片中的精胺(Spm)含量出现高峰.说明21节左右可能是童性消失进入生殖生长期的临界点.     

cDNA microarray analysis of developing grape (Vitis vinifera cv. Shiraz) berry skin

Microarray analysis of Vitis vinifera cv. Shiraz developing berries has revealed the expression patterns of several categories of genes. Microarray slides were constructed from 4,608 PCR-amplified cDNA clones derived from a ripening grape berry cDNA library. The mRNA expression levels of the genes represented by these cDNAs were measured in flowers, week 2 post-flowering whole berries, week 5, week 8, week 10 (véraison, green berries), week 12 and week 13 berry skin. In addition, a comparison of RNA expression in pigmented and unpigmented berry skin at véraison (week 10) was undertaken. Image and statistical analysis revealed four sets of genes with distinctive and similar expression profiles over the course of berry development. The first set was composed of genes which had maximum RNA expression in flowers, followed by a steady decrease in expression. The most prominent group within this set were genes which have a role in photosynthesis. The second set of cDNAs was dominated by genes involved in flavonoid biosynthesis and had a peak of expression week 2 post-flowering. The data indicate co-ordinate regulation of flavonoid biosynthetic genes which code for the enzymes 4-coumarate-CoA ligase, chalcone synthase, chalcone isomerase, flavonone hydroxylase, anthocyanidin reductase and cytochrome b5. The third set of cDNAs exhibited maximum expression week 5 post-flowering, midway between flowering and véraison, a period of rapid berry growth. This set of cDNAs is dominated by genes which code for structural cell wall proteins. The fourth set of genes was dramatically up-regulated at véraison and remained up-regulated until 13 weeks post-flowering. This set of genes was composed of a diverse range of genes, a reflection of the complexity of ripening, most with no known function.     

Potassium nutrition of Cabernet Sauvignon grapevines (Vitis vinifera L.) as affected by shoot trimming

As potassium (K) requirement of grapevine (Vitis viniferaL.) berries is high and phloem translocation from mature leaves to developing organs is well established, it was posited that shoot trimming, a widely applied technique which alters the source-sink balance and the mature-to-immature foliage ratio of a canopy, may influence K deficiency. Six-year-old Cabernet Sauvignon grapevines were grown in 0.045 m³ pots (one plant per pot) filled with a soil sampled from a vineyard previously displaying K deficiency symptoms. Two levels of K supply (K₀, no K added; K_1, 25 g K per pot added in five splits from bloom to post veraison) were tested on vines that for each level were left (a) untrimmed and trimmed at ten main leaves with (b) or without (c) maintenance of lateral shoots in a split-plot design. Potassium concentration of leaf blades, berries and canes, vegetative growth and leaf gas exchange were recorded throughout the season; yield, grape quality and must characteristics were determined at fruit maturity. While adding potassium to the soil resulted in higher K concentration in blades of both main and lateral shoots and berries at harvest, trimming with removal of lateral shoots resulted in lower blade K concentration of the main leaves at harvest and more severe K deficiency symptoms regardless of K soil availability. Berry K concentration and the fraction of whole-plant K partitioned to clusters were not significantly affected by trimming. Gas-exchange, vegetative growth, yield and grape quality were not affected by the seasonal K fertilization, whereas the latter was impaired when trimming with excision of lateral shoots was applied. The results indicate that if shoot trimming is not followed by an adequate regrowth of secondary shoots an excessive depletion of potassium from the retained old basal leaves occurs during fruit maturity and increases the risk of leaf K deficiency, particularly in the K₀ treatment.     

Ultrastructure and germination of Vitis vinifera cv. Loureiro pollen

The cultivar Loureiro of Vitis vinifera is one of the most economically important, recommended in almost the totality of the Regi?o Demarcada dos Vinhos Verdes. In vineyards, the grape productivity of this cultivar is normal while in others it is extremely low. The aim of this work was to study the morphology and germination of Vitis vinifera cv. Loureiro pollen with high and low productivity. The pollen grain was examined under light, transmission and scanning electron microscopy. Typically V. vinifera pollen present three furrows but in the cultivar Loureiro we found tricolporated and acolporated (without furrows or pores) pollen grains. Both pollen types present generative and vegetative cells with the usual aspect and a dense cytoplasm rich in organelles. In the acolporated pollen a continuous exine layer and an irregular intine layer were observed. Differences were found in the starch accumulation, since only in tricolporated pollen abundant plastids filled with numerous starch granules were observed. To determine the causes of the low productivity of this cultivar we tested pollen viability by the fluorochromatic reaction and pollen germinability by in vitro assays. We observed that the acolporated pollen grain is viable, but no germination was recorded.     

Transgenic plants of Vitis vinifera cv. Seyval blanc

Leaf discs of grapevine cv. Seyval blanc originating from in vitro cultures were transformed with Agrobacterium tumefaciens strain LBA 4404 harbouring the vector pGJ42 carrying genes for chitinase and RIP (ribosome-inactivating protein) in an attempt to improve fungal resistance. The gene for neomycin phosphotransferase II (nptII) was used as the selectable marker gene. The explants were cocultivated for 2 days with recombinant Agrobacteria and then submitted to selection on NN69 medium containing 100 mg/l kanamycin. Successful regeneration and conversion of transgenic plantlets were obtained. Stable integration of foreign DNA was confirmed by PCR and Southern blot analyses, and protein expression was detected by Western blot. The regenerated transgenic plants were adapted to the greenhouse and showed no evidence of phenotypical alterations. The foreign genes introduced into the transformed plants did not effect the expected improvement in fungal disease resistance under field conditions for the major pests Uncinula necator and Plasmopara viticola.     

QTLs for muscat flavor and monoterpenic odorant content in grapevine (Vitis vinifera L.)

Little is known about the genetic determinism of muscat flavor in grape, although this trait is of major importance for table grape breeding. We therefore performed a search for QTLs (Quantitative Trait Loci) of both muscat score and berry content in the three main free monoterpene alcohols potentially involved, linalool, nerol and geraniol, based on two years of measures. Parental and consensus framework genetic maps of the cross MTP2687-85 (Olivette × Ribol) × Muscat of Hamburg were built after genotyping the 174 offspring for 139 well-scattered SSR markers. The female, male and consensus framework maps spanned 935, 1365 and 1267 cM, respectively. For QTL detection, simple and composite interval mapping were performed, as well as non parametric Kruskal–Wallis tests. QTLs for muscat score were found on linkage groups (LGs) 1, 5 and 7. For the three ln-transformed monoterpene contents, QTLs with major effects (explaining 17–55 % of total phenotypic variance) were found to be colocated on LG 5, on the male and consensus maps in both years. One additional QTL was found for linalool on LG 2, on female and consensus maps, as well as other colocated ones for nerol and geraniol on LG 13, on male and consensus maps. These additional QTLs had lower effects (9–25%). The contribution of these results to the knowledge of muscat aroma genetic determinism is discussed, as well as their potential usefulness for marker assisted breeding of new aromatic grape varieties.     

ABA xylem concentrations determine maximum daily leaf conductance of field-grown Vitis vinifera L. plants

Differences in maximum leaf conductance in grapevine plants growing in soils with contrasting water availabilities during mid-summer in Portugal could be accounted for by differences in the concentration of ABA in xylem sap. This conclusion is reinforced by the observation that the relationship between leaf conductance and endogenous ABA concentration can be mimicked by the application of exogenous ABA to leaves detached from irrigated plants. During the day, leaf conductance decreased after a morning peak, even when the leaves remained in a constant environment at a moderate temperature and leaf-to-air vapour pressure difference. This decline in leaf conductance was not a consequence of an increase in the xylem ABA concentration or the rate of delivery of this compound by the transpiratory stream. The afternoon depression in leaf conductance was associated with an apparent limitation in stomatal opening potential, which persisted even when detached leaves were fed with water and rehydrated. The reason for this inhibition has still to be identified.     

Partitioning and mobilization of starch and N reserves in grapevine (Vitis vinifera L.)

We followed C and N reserves of grapevines grown in trenches under semi-controlled conditions over a 3-year period after planting. Temporal mobilization of stored C and N and subsequent distribution of reserve materials within the vines were described in parallel with 15N uptake, particularly during the third growing season. Storage C in the perennial tissues (roots, trunk, canes) was mainly made of starch, which accumulated in the ray parenchyma of the wood. In the permanent tissues, starch and total nitrogen contents were found to decrease early in the development (bleeding sap, budbreak) whereas, on a concentration basis, they decreased only after stage 7 (first leaf fully expanded). Starch started to accumulate again in the perennial tissues during flowering. The same observation was made with total nitrogen, although N levels were much lower than those of starch. The 15N study showed that N uptake by the roots started at budbreak and increased with vine development, becoming predominant over reserve mobilization only after the onset of flowering. Taken together, these results indicate that the spring growth period can be divided into three main phases: In the first (dormancy to budbreak), significant losses of C and N proceed mainly via root necrosis. In the second period (first leaf to the onset of bloom), a strong mobilization of starch (and, to a lower extent, of N) occurred for supporting vegetative and reproductive growth. At that point, most of the C and N reserves used on the spring flush were those of the roots, rather than those of the old wood (trunk, canes). In the third period (bloom and early berry development), the mobilization process became low and was relieved by N uptake (and CO2 assimilation) supplying nutrients to the sink structures.     

Anthocyanin production in selected cell lines of grape (Vitis vinifera L.)

Summary Anthocyanin production of two lines ofVitis vinifera cell cultures, i.e., 5.4 and 13.1, which were obtained from the same starting material after 20 and 37 mo. of clonal selection, respectively, was investigated. Cell suspension cultures of lines 5.4 and 13.1 maintained an anthocyanin content of 0.44 ± 0.15 and 1.02 ± 0.31 mg·g⁻¹ fresh weight during 50 and 32 weekly maintenance subcultures, respectively. Under anthocyanin-promoting culture conditions, both lines showed an enhancement of their anthocyanin level by approximately fourfold. While line 5.4 accumulated peonidin 3-glucoside and cyanidin 3-glucoside in decreasing order, line 13.1 accumulated primarily peonidin 3-p-coumaroylglucoside with lesser amounts of malvidin monoglucoside. Results show that while the anthocyanin content was improved during the course of repeated selections, the anthocyanin composition was modified markedly favoring the accumulation of more metabolically-advanced anthocyanins.     

Anthocyanic vacuolar inclusions (AVIs) selectively bind acylated anthocyanins in Vitis vinifera L. (grapevine) suspension culture

Anthocyanic vacuolar inclusions (AVIs) appear as dark red-to-purple spheres of various sizes in vacuoles of grapevine (Vitis vinifera L.) cell suspension culture due to their interaction with anthocyanins. AVIs were purified and the bound anthocyanins extracted and analysed by HPLC from two lines of V. vinifera isolated from the same callus accumulating anthocyanin in the dark, yet varying in their anthocyanin profiles and accumulation. An intermediate-pigmented line (FU-1) with a 1.3:1 ratio of acylated:non-acylated anthocyanins, a colour value of 0.84 units and cyanidin and peonidin as the dominant species was compared with a high-pigmented line (FU-2) with a 1.2:1 ratio of acylated:non-acylated anthocyanins, a colour value of 3.72 units and malvidin predominating. The profile of AVI-bound anthocyanins showed an increase in acylated anthocyanins in both lines of approx. 28–29%, with no apparent preference for anthocyanin species. This resulted in a ratio of acylated:non-acylated anthocyanins of 6.2:1 for FU-1 and 4.9:1 for FU-2. The reasons for the selectivity of the AVIs for acylated (specifically p-coumaroylated) species compared with the whole cell profile are discussed.     

Sap flow measurements in grapevines (Vitis vinifera L.) 2. Granier measurements

The Granier-system, a relatively simple and continuous method for measuring sap flux density, has been adapted and evaluated for its use in older, mature grapevines. The original calibration of Granier (1985, Annales Sciences Forestieres, 42, 193-200) could be extended to a sap flux density of up to 400 10^-6 m³ m^-2 s^-1 with only little error at high flux densities. A time lag of around 20 min was apparent between transpiration and calculated sap flow which was attributed to the thermal mass of the sensors themselves. The time lag and the consequently dampened response of the system caused a very low accuracy over short time periods thus reducing the value for detailed plant physiological investigations. However, when integrating over longer time intervals, much of the error cancelled out. For daily values the maximum error was within ±10% and after a period of 89 days only 1.5% error remained. This method is thus best suited for long term measurements of total water use. This revised version was published online in June 2006 with corrections to the Cover Date.     

Restriction fragment length polymorphism and molecular taxonomy in Vitis vinifera L.

Forty-six accessions of grapevine (V. vinifera L.) were compared by restriction fragment length polmorphism (RFLP) analysis, and 111 informative or unique restriction fragments were found that revealed an important level of polymorphism. RFLP patterns were compared in two ways: by calculating electrophoretic similarity degree values further analyzed by principal component analysis and by studying the distribution of rare restriction fragments. Six taxonomic groups could be defined, which partially confirmed relationships derived from ampelographical data. Our data support the existence of ecogeographical groups.     

Effects of Temperature Acclimation Pretreatment on the Ultrastructure of Mesophyll Cells in Young Grape Plants （Vitis vinifera L. cv. Jingxiu） Under Cross-Temperature Stresses

Leaves from annual young grape plants （Vitis vinifera L. cv. Jingxiu） were used as experimental materials. The ultrastructural characteristics of mesophyll cells in chilling-treated plants after heat acclimation （HA） and in heat-treated plants after cold acclimation （CA） were observed and compared using transmission electron microscopy. The results showed that slight injury appeared in the ultrastructure of mesophyll cells after either HA （38℃ for 10 h） or CA （8℃ for 2.5 d）, but the tolerance to subsequent extreme temperature stress was remarkably improved by HA or CA pretreatment. The increases in membrane permeability and malondialdehyde concentration under chilling （0℃） or heat （45℃） stress were markedly inhibited by HA or CA pretreatment. The mesophyll cells of plants not pretreated with HA were markedly damaged following chilling stress. The chloroplasts appeared irregular in shape, the arrangement of the stroma lamellae was disordered, and no starch granules were present. The cristae of the mitochondria were disrupted and became empty. The nucleus became irregular in shape and the nuclear membrane was digested. In contrast, the mesophyll cells of HA-pretreated plants maintained an intact ultrastructure under chilling stress. The mesophyll cells of control plants were also severely damaged under heat stress. The chloroplast became round in shape, the stroma lamellae became swollen, and the contents of vacuoles formed clumps. In the case of mitochondria of control plants subjected to heat stress, the outer envelope was digested and the cristae were disrupted and became many small vesicles. Compared with cellular organelles in control plants, those in CA plant cells always maintained an integrated state during whole heat stress, except for the chloroplasts, which became round in shape after 10 h heat stress. From these data, we suggest that the stability of mesophyll cells under chilling stress can be increased by HA pretreatment. Similarly, CA pretreatment can protect chloroplasts, mitochondria, and the nucleus against subsequent heat stress; thus, the thermoresistance of grape seedlings was improved. The results obtained in the present study are the first, to our knowledge, to offered cytological evidence of cross-adaptation to temperature stresses in grape plants.     

Cellular expansion and gene expression in the developing grape (Vitis vinifera L.)

Summary. Expression profiles of genes involved in cell wall metabolism and water transport were compared with changes in grape (Vitis vinifera L.) berry growth, basic chemical composition, and the shape, size, and wall thickness of cells within tissues of the berry pericarp. Expression of cell wall-modifying and aquaporin genes in berry pericarp tissues generally followed a bimodal expression profile with high levels of expression coinciding with the two periods of rapid berry growth, stages I and III, and low levels of expression corresponding to the slow-growth period, stage II. Cellular expansion was observed throughout all tissues during stage I, and only mesocarp cellular expansion was observed during stage III. Expansion of only exocarp cells was evident during transition between stages II and III. Cell wall-modifying and aquaporin gene expression profiles followed similar trends in exocarp and mesocarp tissues throughout berry development, with the exception of the up-regulation of pectin methylesterase, pectate lyase, two aquaporin genes (AQ1 and AQ2), and two expansin genes (EXP3 and EXPL) during stage II, which was delayed in the exocarp tissue compared with mesocarp tissue. Exocarp endo-(1→3)-β-glucanase and expansin-like gene expression was concurrent with increases in epidermal and hypodermal cell wall thickness. These results indicate a potential role of the grape berry skin in modulating grape berry growth. Correspondence: P. Bowen, Pacific Agri-Food Research Centre, 4200 Highway 97, Summerland, BC V0H 1Z0, Canada     

An NMR microscopic study of grape (Vitis vinifera L.)

Summary Mature healthy grape berries and berries wound-inoculated with the fungusBotrytis cinerea were examined by¹H NMR microimaging using 2D and 3D spin echo and gradient echo procedures. These NMR images were compared with representations obtained by conventional histology, where possible using the same specimens. 3D imaging datasets from excised seeds were reconstructed by surface rendering and maximum intensity projection to allow interpretation of their internal structure. T₂-weighted spin echo images revealed the major features of the pericarp, septum and loculi of whole berries. T₁-weighted images were less discriminatory of parenchyma tissues in the fruit but revealed the endosperm in seeds as a chemically shifted feature. A non-invasive study by T₁-weighted spin echo NMR imaging of infection byB. cinerea over a 6-day period showed that the disease spread throughout the exocarp but failed to spread in the mesocarp, a result confirmed by histological examination of the same specimen. Surface rendering of 3D datasets of excised seeds revealed the two ruminations of the endosperm and the distal location of the chalaza. The position of the embryonic axis was revealed in T₂-weighted maximum intensity projections. This noninvasive study revealed the need to apply a range of imaging techniques and parameters to visualise the structural features of the different parts of the grape berry.Abbrevations BF bright field - FDA fluorescein diacetate - FI field inhomogeneity - FOV field of view - NMR nuclear magnetic resonance - RF radiofrequency - T₁ spin-lattice relaxation time - T₂ spin-spin relaxation time - TE echo time - TMS tetramethylsilane - TR repeat time     

Somatic embryogenesis and plant development from anther culture of Vitis vinifera (L.)

Anthers from Frumoasa alba (White beauty), Otilia, Valerien, Mission and Siegfried Rebe (FS₄) cultivars were cultured at the uninucleate stage of the microspore on Murashige and Skoog (1962) and Nitsch and Nitsch (1969) media supplemented with 2,4-dichlorophenoxyacetic acid (4.9 M) and benzyladenine (4.4 M). The primary calli were subcultured on MS medium with 6.6 M BA and 1.1 M indolylacetic acid, in order to induce their growth and plant regeneration. After seven months, vegetative buds were obtained with Frumoasa alba (2.7%), Otilia (0.3%), Valerien (4.5%), embryogenic callus was obtained with Mission and plant regeneration with Siegfried Rebe. Long term embryogenesis was maintained in Mission cv. for four years, by selection and regular transfer of the embryogenic areas of anther-derived calli. The embryogenic calli have the ability to generate abnormal somatic embryos with one, two or three cotyledons and cup or trumpet-shaped with fused cotyledons. In parallel with the embryogenic process, organogenesis with buds, leaf and shoot differentiation was regularly observed.     

Biotransformation of C13-norisoprenoids and monoterpenes by a cell suspension culture of cv. Gamay (Vitis vinifera)

A cell suspension culture of cv. Gamay was studied for its ability to metabolize two different C₁₃-norisoprenoidic volatiles, β-ionone and dehydrovomifoliol, together with monoterpenes, geraniol and linalool, biogenetically common pathways sharing compounds. β-Ionone was totally metabolized leading to fourteen norisoprenoidic volatiles oxygenated mainly at carbons 3 or 4 of the cyclohexane ring or reduced at side chain. The biotransformation of dehydrovomifoliol was at a lesser extent, giving rise to oxygenated and reduced derivatives. The norisoprenoidic metabolites were present both under free and glycosylated forms. Geraniol and linalool were also metabolized, leading to several free and glycosylated compounds. S. Mathieu, J. Wirth contributed equally to the work and should be considered joint first authors. A short part of this paper was published at the proceedings of the 10th Weurman Flavour Research Symposium, Flavour Research at the Dawn of the Twenty-first Century, J.-L.Le Quere, P.-X.Etievant, Editors; Lavoisier,2003/Intercept Ltd, 2003.     

Sap flow measurements in grapevines (Vitis vinifera L.) 1. Stem morphology and use of the heat balance method

The heat balance method was evaluated in detail for its use in older, mature grapevines with stems of 35 – 45 mm in diameter. Dye colouring of the xylem vessels revealed that even 21 year old grapevines did not show any development of heartwood and that xylem vessels of that age still have the capacity to transport water. A comparison of weight loss of potted vines on a balance and sap flow measurements demonstrated that the heat balance system reflected rapid changes in flow rate without any time delay. However, since even 20 year old xylem vessels of grapevines have the capacity to conduct water, the heater band was not able to heat the sap in all year rings evenly. Apparently, at low flow rates this effect was small and sap flow was calculated correctly. With increasing flow rates large thermal heterogeneities developed upsetting the calculation of the heat balance and mass flow. Consequently, actual sap flow was overestimated by 50 to 100% at high flow rates. This could be attributed to thermal gradients in these relatively thick stems excluding the use of this technique for measurements of long term as well as short term water use patterns in older grapevines. This revised version was published online in June 2006 with corrections to the Cover Date.