Photoinhibition of Photosynthesis in Sun and Shade Grown Leaves of Grapevine (Vitis vinifera L.)

The degree of photoinhibition of sun and shade grown leaves of grapevine was determined by means of the ratio of variable to maximum chlorophyll (Chl) fluorescence (F_v/F_m) and electron transport measurements. The potential efficiency of photosystem 2 (PS2), F_v/F_m, markedly declined under high irradiance (HI) in shade leaves with less than 10 % of F₀ level. In contrast, F_v/F_m ratio declined with about 20 % increase of F₀ level in sun leaves. In isolated thylakoids, the rate of whole chain and PS2 activity in HI shade and sun leaves was decreased by about 60 and 40 %, respectively. A smaller inhibition of photosystem 1 (PS1) activity was also observed in both leaf types. In the subsequent dark incubation, fast recovery was observed in both leaf types that reached maximum PS2 efficiencies similar to non-photoinhibited control leaves. The artificial exogenous electron donors DPC, NH₂OH, and Mn²⁺ failed to restore the HI-induced loss of PS2 activity in sun leaves, while DPC and NH₂OH were significantly restored in shade leaves. Hence HI in shade leaves inactivates on the donor side of PS2 whereas it does at the acceptor side in sun leaves, respectively. Quantification of the PS2 reaction centre protein D1 and the 33 kDa protein of water splitting complex following HI-treatment of leaves showed pronounced differences between shade and sun leaves. The marked loss of PS2 activity in HI leaves was due to the marked loss of D1 protein of the PS2 reaction centre protein and the 33 kDa protein of the water splitting complex in sun and shade leaves, respectively.     

Chloroplast Genome Diversity in Portuguese Grapevine (Vitis vinifera L.) Cultivars

Grapevine chloroplast (cp) DNA diversity was analysed for the first time through amplification and digestion of fragments of the large single copy (LSC) region by polymerase chain reaction–restriction fragment length polymorphism methodology and also by amplification of three microsatellite loci, previously described as polymorphic in grapevine. Thirty-eight grapevine cultivars collected mainly in the North of Portugal, including some neglected cultivars, four international cultivars (Chasselas, Muscat of Alexandria, Muscat of Hamburg and Pinot) and Vitis riparia and Vitis rupestris, were used in this study with the main goal of finding out their cpDNA diversity and compare the obtained results with previously published data on cultivars from other regions to ascertain their possible origin. Two different alleles were found in each of the three cpSSR loci. Allele variants of the three loci combined in a total of three different haplotypes (A, B and D). The most frequent haplotype, A, was previously reported as the most frequent in Iberian Peninsula and Occidental Europe. Haplotype B was unique to Rabigato, Muscat of Alexandria, V. riparia and V. rupestris. This haplotype was previously proposed to be an ancestral haplotype. Twenty-seven fragments of the LSC region of Vitis vinifera cpDNA were amplified and then digested with HinfI and TaqI restriction enzymes. Polymorphisms were found in the trnT-psbC (TC) and orf184-petA (OA) fragments. In the TC fragment, the polymorphism corresponds to a point mutation in a restriction site of TaqI and is only present in all cultivars with cpSSR haplotype D. In the OA fragment, a short deletion exclusive to the Rabigato cultivar was found. In this case, one sequence tagged site-based marker was developed and will be very useful in future phylogenetic and fingerprinting studies in a broader number of cultivars and in wild grapevine populations. Inference about the progenitors of the Touriga Franca cultivar is done. The present work supports and completes its origin as a descendent of the female and male parents, Marufo and Touriga Nacional.     

Photoinhibition and Recovery of photosystem 2 in Grapevine (Vitis vinifera L.) Leaves Grown Under Field Conditions

Photoinhibition of photosynthesis was investigated in grapevine (Vitis vinifera L.) exposed to 2 or 4h of high irradiance (HI) (1 700–1 800 mol m^–2 s^–1) leaves under field conditions at different sampling time in a day. The degree of photoinhibition was determined by means of the ratio of variable to maximum chlorophyll fluorescence (F_v/F_m) and photosynthetic electron transport measurements. When the photochemical efficiency of photosystem 2 (PS2), F_v/F_m, markedly declined, F₀ increased in both 2 (HI2) and 4 h (HI4) HI leaves sampled at midday. When various photosynthetic activities were followed on isolated thylakoids, HI4 leaves showed significantly higher inhibition of whole chain and PS2 activity than the HI2 leaves sampled at midday. Later, the leaves reached maximum PS2 efficiencies similar to those observed early in the morning during sampling at evening. The artificial exogenous electron donor Mn²⁺ failed to restore PS2 activity in both variants of leaves, while DPC and NH²OH significantly restored PS2 activity in HI4 midday leaf samples. Quantification of the PS2 reaction centre protein D1 and 33 kDa protein of water splitting complex following midday exposure of leaves showed pronounced differences between HI2 and HI4 leaves. The marked loss of PS2 activity noticed in midday samples was mainly due to the marked loss of D1 protein in HI2, while in HI4 it was mainly 33-kDa protein.     

Pedigree Reconstruction of the Italian Grapevine Aglianico (Vitis vinifera L.) from Campania

A total of 41 accessions of Aglianico belonging to three different biotypes (Taburno, Taurasi, and Vulture) and 9 accessions of Sirica grapes were sampled from diverse areas of Campania (Italy). All accessions were first genotyped using 21 microsatellite markers (SSR) to evaluate possible homonymies, synonymies, and the genetic structure of each group. A larger dataset was then constructed adding Italian and International cultivars. On the basis of results obtained analyzing the first dataset, further investigations were carried out enlarging the number of investigated loci (up to 43). The addition of 22 SSRs was useful in the definition of likely genetic relationships linking Aglianico biotypes, Sirica and Syrah. According to their SSR allelic profiles, the monophyletic origin of the three Aglianico biotypes was confirmed. Among Aglianico Taburno accessions, eight samples (called Aglianico like-to-type) performed a different SSR allelic profile from Aglianico true-to-type. Sirica and Syrah proved to be synonyms. This work allowed to determine the genetic relationship between Aglianico and the cultivars supposed to be related. The parentage analysis was investigated. The most likely pedigree has been reconstructed; revealing a second-degree relationship between the worldwide cultivated Syrah from the Rhone Valley and Aglianico. Aglianico like-to-type appeared related to Aglianico in a parent-offspring fashion.     

The Effect of Downy and Powdery Mildew on Grapevine (Vitis vinifera L.) Leaf Gas Exchange

The impact of powdery (Uncinula necator) and downy mildew (Plasmopara viticola) on grapevine leaf gas exchange was analysed. Gas exchange measurements (assimilation A, transpiration E, stomatal conductance g_s, intercellular concentration of CO₂C_i) were made on three different leaf materials: (i) healthy tissue of diseased leaves, (ii) infected tissue of diseased leaves, (iii) healthy tissue of healthy leaves (control treatment). Using the same source of leaf tissue, photosynthetic pigment concentration (chlorophyll a, b) and fluorescence levels (minimal fluorescence F₀, maximal fluorescence F_m and the optimal quantum yield [F_m ? F₀]/F_m) were determined to explain the mechanism of action of the two diseases on leaf assimilation. The results indicated that powdery and downy mildew reduced the assimilation rates, not only through a reduction in green leaf area (visual lesions), but also through an influence on gas exchange of the remaining green leaf tissues, determining a ‘virtual lesion’. The ratios between virtual and visual lesions were higher in powdery mildewed leaves than in the downy mildewed leaves. The photosynthetic fluorescence level (F_v/F_m) was affected by neither of the two pathogens. The reduction in intercellular concentration of CO₂ and photosynthetic pigment may explain the lower assimilation rates in the healthy tissues of powdery and downy mildewed leaves respectively.     

Dynamic Relation between Expansion and Cellular Turgor in Growing Grape (Vitis vinifera L.) Leaves

Measurements of the growth and water relations of expanding grape (Vitis vinifera L.) leaves have been used to determine the relationship between leaf expansion rate and leaf cell turgor. Direct measurement of turgor on the small (approximately 15 micrometer diameter) epidermal cells over the midvein of expanding grape leaves was made possible by improvements in the pressure probe technique. Leaf expansion rate and leaf water status were perturbed by environmentally induced changes in plant transpiration. After establishing a steady state growth rate, a step decrease in plant transpiration resulted in a rapid and large increase in leaf cell turgor (0.25 megapascal in 5 minutes), and leaf expansion rate. Subsequently, leaf expansion rate returned to the original steady state rate with no change in cell turgor. These results indicate that the expansion rate of leaves may not be strongly related to the turgor of the leaf cells, and that substantial control of leaf expansion rate, despite changes in turgor, may be part of normal plant function. It is suggested that a strictly physical interpretation of the parameters most commonly used to describe the relationship between turgor and growth in plant cells (cell wall extensibility and yield threshold) may be inappropriate when considering the process of plant cell expansion.     

Growth and Development of Roots of Grapevine (Vitis vinifera L.) in Relation to Water Uptake from Soil

Developmental patterns of lateral roots and their vascular differentiationwere investigated for Vitis vinifera L. cv. Shiraz to assessthe likely contribution of lateral roots to total water uptakeof plants subjected to different irrigation regimes. Correlationanalyses showed a significant positive correlation between mainroot diameter and the diameter of first order lateral rootsof well-watered plants, but in water-stressed plants the twowere not significantly correlated. The correlations betweendiameters of first order lateral roots and the diameters ofmain roots were greater than correlations between the lengthsof first order laterals and the diameters of main roots. Thesuberised surface area of well-watered main roots increasedfrom 4% of total surface area at 0·25 cm to 100% at 10cm from the tip, whereas that of stressed plants increased from15% at 0·25 cm to 100% at 5 cm from the tip. In all treatmentsthe highest linear density of first order laterals was about7 laterals cm^-1 of main root. More than 50% of first order lateralshad diameters less than 0·05 cm, and more than 90% ofthem had lengths less than 5 cm. Calculations of axial resistancesbased on xylem diameter measurements suggest that the axialresistances of root segments may not be uniform along rootsas is often assumed in models of water uptake. Water flow intothe main roots via the lateral root pathway is likely to bemuch smaller than that via the direct radial flow pathway asonly about 1% of surface area of main roots is directly occupiedby lateral roots, leaving the other 99% of main root surfacearea available for the direct radial flow pathway.Copyright1994, 1999 Academic Press Axial resistance, grapevine (Vitis vinifera L. cv. Shiraz) roots, root diameter, root length, xylem vessels     

Ontogenesis, Differentiation and Precocious Germination in Anther-derived Somatic Embryos of Grapevine (Vitis vinifera L.): Proembryogenesis

Histological steps of callogenesis and proembryogenesis in anthercultures ofVitis vinifera L. ‘Grenache noir’ aredescribed. Embryogenic calli were obtained on Nitsch and Nitschmedium supplemented with 1mgl^-12,4-dichlorophenoxyacetic acid(2,4-D) and 0.25mgl^-1benzylaminopurine (BAP). Calli were initiatedfrom anther connective cells only and no division of microsporesoccurred. The embryos were hence of somatic origin. Proembryosdeveloped either directly (i.e. without intervening callus)from the endothecium, or indirectly from the connective-derivedcallus. In both cases, proembryos originated from single cells.They developed from starchy differentiated cells of a predeterminedtype. The polarity of the somatic proembryo was establishedfrom the first divisions and it was marked by precocious developmentof an easily recognizable suspensor. Other analogies with thedevelopment of the zygote are also emphasised. Vitis vinifera L.; grapevine; somatic embryogenesis; proembryogenesis; histology     

Control of Flowering in the Grapevine (Vitis vinifera L.): Formation of Inflorescences in Vitro by Isolated Tendrils

Photosynthesis, primary productivity, N content, and N₂ fixation were determined as a function of applied NH₄⁺ in peas (Pisum sativum L. cv. Alaska) which were inoculated or not inoculated with Rhizobium leguminosarum. Cabon dioxide exchange rate (CER) increased 10-fold, total N content 7-fold, and total dry weight 3-fold in 26-day-old uninoculated plants as applied NH₄⁺ was increased from 0 to 16 millimolar. In inoculated plants of the same age CER and dry weight were maximal at 2 millimolar NH₄⁺, and total N content increased between 0 and 2 millimolar NH₄⁺ but did not change significantly with higher NH₄⁺ applications. Per cent N content of uninoculated plants was significantly lower than that of inoculated plants except at the highest NH₄⁺ concentration (16 millimolar). Symbiotic N₂ fixation by inoculated plants was maximal in peas grown with 2 millimolar NH₄⁺; and apparent relative efficiency of N₂ fixation, calculated from C₂H₂ reduction and H₂ evolution, was maximal in the 2 to 4 millimolar NH₄⁺ concentration range. The capacity to fix N₂ through the Rhizobium-legume symbiosis significantly enhanced the rate and efficiency of photosynthesis and plant N content when NH₄⁺ concentration in the nutrient solution was below 8 millimolar. Above 8 millimolar NH₄⁺ concentration uninoculated plants had greater CER, N content, and dry weight.     

Ontogenesis, Differentiation and Precocious Germination in Anther-derived Somatic Embryos of Grapevine (Vitis vinifera L.): Embryonic Organogenesis

Somatic embryos ofVitis vinifera L. ‘Grenache noir’develop abnormally and form viable plantlets at very low frequencies.They grow continuously and, after the torpedo stage, they formgiant structures which do not undergo further organogenesis.Morphological, histological and cytochemical data were usedto study development from the globular to the giant-embryo stage.Histological organization of somatic embryos until the torpedostage was similar to that of zygotic embryos. Somatic embryosformed bipolar axes, which differentiated precociously and simultaneouslya root and a shoot meristem. However, they differed from theirzygotic homologues by forming a cotyledonary crown or multiplecotyledons and by their rapid cellular differentiation. At theend of the torpedo stage and up to the giant-embryo stage, somaticembryos underwent some characteristic events of germination:the radical grew, tannins accumulated, and protodermal cellssuberized. However the shoot apex was rapidly disorganized anddisappeared. This peculiar behaviour is discussed in comparisonwith the phenomenon of precocious germination often observedfor immature zygotic embryos inin vitro culture. Vitis vinifera ; grapevine; somatic embryo development; precocious germination; histology     

Sugars and flowering in the grapevine (Vitis vinifera L.)

Sugars play an important role in grapevine flowering. This complex process from inflorescence initiation to fruit maturity takes two growing seasons. Currently, most of the available data concern the involvement of sugars as energy sources during the formation of reproductive structures from initiation of inflorescences during the summer of the first year, until flower opening during the following spring. Sugars devoted to the development of reproductive structures are supplied either by wood reserves or by photosynthesis in leaves or inflorescences, depending on the stage of development. Female meiosis appears to be a key point in the success of flower formation because (i) flowers are vulnerable at this stage and (ii) it corresponds in the whole plant to the transition between reserve mobilization from perennial organs (roots, trunk, and canes) towards efficient leaf photosynthesis. The perturbation of reserve replenishment during the previous year provokes perturbation in the development of inflorescences, whereas altering the photosynthetic sources affects the formation of flowers during the same year. In particular, a lack of sugar availability in flowers at female meiosis caused by various environmental or physiological fluctuations may lead to drastic flower abortion. Apart from energy, sugars also play roles as regulators of gene expression and as signal molecules that may be involved in stress responses. In the future, these two topics should be further investigated in the grapevine considering the sensitivity of flowers to environmental stresses at meiosis.     

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     

Transport and accumulation of flavonoids in grapevine (Vitis vinifera L.)

Flavonoids are a group of secondary metabolites widely distributed in plants that represent a huge portion of the soluble phenolics present in grapevine (Vitis vinifera L.). These compounds play different physiological roles and are often involved in protection against biotic and abiotic stress. Even if the flavonoid biosynthetic pathways have been largely characterized, the mechanisms of their transport and accumulation in cell wall and vacuole are still not completely understood. This review analyses the known mechanisms of flavonoid uptake and accumulation in grapevine, with reference to the transport models and membrane carrier proteins described in other plant species. The effect of different environmental factors on flavonoid biosynthesis and transporters is also discussed.Key words: ABC proteins, active transport, bilitranslocase, biotic and abiotic stress, flavonoid, secondary metabolites     

葡萄体细胞胚的保存

研究干化对酿酒葡萄品种'神索'体细胞胚保存效应的结果表明,葡萄体细胞胚失水量在40%～50%之间的萌发率较高,达58%左右,比未经干化的萌发率高9.4%;在较高相对湿度的情况下,体细胞胚失水速度变慢,干化处理时间延长,可以提高体细胞胚的存活率和萌发率;相对湿度70%的情况下干化15 d的效果最好,其萌发率从未经干化处理的33.5%提高到56.2%.     

Agrobacterium rhizogenes mediated transformation of grapevine (Vitis vinifera L.)

Genetically transformed grapevine (Vitis vinifera L.) roots were obtained after inocultation of in vitro grown whole plants (cv. Grenache) with Agrobacterium rhizogenes. The strain used contains two plasmids: the wild-type Ri plasmid pRi 15834 and a Ti-derived plasmid which carries a chimaeric neomycin phosphotrans-ferase gene (NPT II) and the nopaline synthase gene. Expression of the NPT II gene can confer kanamycin resistance to transformed plant cells. Slowly growing axenic root cultures derived from single root tips were obtained. Opine analysis indicated the presence of agropine and/or nopaline in established root cultures. For one culture, the presence of T-DNA was confirmed by dot-blot hybridization with pRi 15834 TL-DNA. Callogenesis was induced by subculturing root fragments on medium supplemented with benzylaminopurine and indoleacetic acid.Transformation of in vitro cultured grapevine cells has recently been reported (baribault T.J. et al., Plant Cell Rep (1989) 8: 137–140). In contrast with the results presented here, expession of the NPT II gene Conferred kanamycin resistance to Vitis vinifera calli that was sufficient for selection of trasformed cells.Abbreviations BAP benzylaminopurine - IAA indoleacetic acid - NAA naphtaleneacetic acid - NPT II neomycin phosphostransferase II - EDTA ethylenediaminetetraacetic acid     

Changes in the Activity of Catalase (EC 1.11.1.6) in Relation to the Dormancy of Grapevine (Vitis vinifera L.) Buds

Catalase activity in grapevine (Vitis vinifera L.) buds cv. `Perlette.' increased to a maximum in October and thereafter decreased within 3 months to less than half its maximal rate. The decrease in catalase activity coincided with the decline in temperature during winter. The rate of sprouting of buds forced at 23°C was negatively related to the activity of catalase. Artificial chilling of grapevine canes at 5°C resulted in a 25% decrease of catalase activity in the buds after 3 days and 31% after 17 days. The activity of catalase increased to the control level only 96 hours after removing canes from 5°C to room temperature. Efficient buddormancy breaking agents, such as thiourea and cyanamide decreased catalase activity to 64 and 50% of the controls respectively, while the activity of peroxidase remained the same under those conditions. A less efficient dormancy breaking agent dinitro-ortho-cresol, did not decrease catalase activity.     

Cryopreservation of grapevine (Vitis vinifera L.) in vitro shoot tips

In this work, we compared the efficiency of encapsulation-dehydration and droplet-vitrification techniques for cryopreserving grapevine (Vitis vinifera L.) cv. Portan shoot tips. Recovery of cryopreserved samples was achieved with both techniques; however, droplet-vitrification, which was used for the first time with grapevine shoot tips, produced higher regrowth. With encapsulationdehydration, encapsulated shoot tips were precultured in liquid medium with progressively increasing sucrose concentrations over a 2-day period (12 h in medium with 0.25, 0.5, 0.75 and 1.0 M sucrose), then dehydrated to 22.28% moisture content (fresh weight). After liquid nitrogen exposure 37.1% regrowth was achieved using 1 mm-long shoot tips and only 16.0% with 2 mm-long shoot tips. With droplet-vitrification, 50% regrowth was obtained following treatment of shoot tips with a loading solution containing 2 M glycerol + 0.4 M sucrose for 20 min, dehydration with half-strength PVS2 vitrification solution (30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% dimethylsulfoxide and 0.4 M sucrose in basal medium) at room temperature, then with full strength PVS2 solution at 0°C for 50 min before direct immersion in liquid nitrogen. No regrowth was achieved after cryopreservation when shoot tips were dehydrated with PVS3 vitrification solution (50% (w/v) glycerol and 50% (w/v) sucrose in basal medium).