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.     

Micromorpho-Anatomical Examination of 2,4-D Phytotoxicity in Grapevine (Vitis vinifera L.) Leaves

The anatomical and micro-morphological alterations as induced by the auxinic herbicide, 2,4-D (2,4-dichlorophenoxy acetic acid) have not yet been elucidated for a commercially important fruit crop such as grapevine despite its super sensitivity to 2,4-D. Light and scanning electron microscopy techniques were employed to examine 2,4-D induced internal and external structural abnormalities in Merlot grapevines (Vitis vinifera L.). Healthy leaves were dorsiventrally flattened with well developed patterns of cellular structure and composition involving adaxial palisade parenchyma and abaxial spongy mesophyll. Dorsiventral variations in epidermal features involved large epidermal cells on the adaxial surface, and trichomes and stomata with turgid elliptical guard cells on the abaxial surface. The 2,4-D injured leaves were small and enated; the veins were fasciated with rugose bands of lamina existing between fasciated veins. The epidermal cells aggregated instead of being positioned coplanar to the epidermal plane. The adaxial elongated palisade parenchyma cells were transformed into an ovoid shape with intercellular spaces. An extensive development of replacement tissues took place on the abaxial surface wherein the stomata became roundish and were either raised or sunken with collapsed and cracked guard cells that developed abnormal outer stomatal ledges. These abnormalities are expected to severely perturb the vital functions of photosynthesis and transpiration ultimately leading to vine death attributable, at least in part, to the injured leaves.     

Effect of Grapevine Leafroll on the Photosynthesis of Field Grown Grapevine Plants (Vitis vinifera L. cv. Lagrein)

We have studied the effect of grapevine leafroll infection on some features of the thylakoids from field grown grapevine (Vitis vinifera L.) leaves. Changes in photosynthetic pigments, soluble proteins, ribulose‐1,5‐bisphosphate carboxylase (RuBP), nitrate reductase, photosynthetic activities and thylakoid membrane proteins were investigated. The level of total chlorophyll (Chl) and carotenoids were reduced in virus‐infected leaves. Similar results were also observed for soluble proteins and RuBP case activity. The in vivo nitrate reductase activity was significantly reduced in infected leaves. Virus infection considerably decreased leaf net photosynthetic rate (P_n), stomatal conductance (g_s) and transpiration rate (E) in grapevine leaves. When various photosynthetic activities were followed in isolated thylakoids, virus infection caused marked inhibition of whole chain and photosystem (PS) II activity while the inhibition of PSI activity was only marginal. The artificial exogenous electron donors, diphenyl carbazide and hydroxylamine (NH₂OH) significantly restored the loss of PSII activity in infected leaves. The same results were obtained when F_v/F_m was evaluated by Chl fluorescence measurements. The marked loss of PSII activity in infected leaves could be due to the loss of 47, 43, 33, 28–25, 23 and 17 kDa polypeptides. It is concluded that virus infection inactivates the donor side of PSII. This conclusion was confirmed by immunological studies showing that the content of the 33 kDa protein of the water‐splitting complex was diminished significantly in infected leaves.     

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.     

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.     

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.     

RAPD analysis of selected local Turkish grape cultivars (Vitis vinifera)

Turkey is very rich in local grape varieties. The solution to the problem of identifying local cultivars, which is considered an important deficiency for the region, will only be possible when they can be defined with molecular markers. Forty-nine local grapevine cultivars from ?anl?urfa (Turkey) were characterized with RAPD markers. Twenty-five decamer primers selected from 60 primers were used in this analysis. A total of 171 bands were obtained with the 25 primers, of which 112 were polymorphic; the level of DNA polymorphism was 65.49% in these local cultivars. Among the selected primers, OPA-18, OPO-07 and P-123 gave the maximum number of polymorphic bands (seven). Genetic relationships among these cultivars were determined with a similarity index and using a dendogram. Among the grape cultivars, the lowest similarity ratio (0.578) was observed among the Külahi-K?z?lbanki cultivars and the highest similarity ratio (0.908) was observed for the ?ilorut-D?külgen combination. The high similarity ratio among the grape cultivars of ?anl?urfa Province was also reflected in the dendogram. In general, no relationships were encountered between the genetic identification of the cultivars and their ampelographic properties.     

First Detection of Stolbur Phytoplasma in Grapevines (Vitis vinifera cv. Merlot) Affected with Grapevine Yellows in Bulgaria

Between 2003 and 2005, a survey was conducted throughout the grape‐growing regions of Bulgaria to identify possible infection with grapevine yellows diseases, especially Flavescence dorée (FD). The samples were checked for phytoplasmas and viruses inducing similar symptoms in the Central Laboratory for Plant Quarantine. To confirm stolbur phytoplasma infection of grapevine, a multiplex nested‐PCR assay for direct detection of FD and stolbur phytoplasmas was used. Infection of grapevine with phytoplasma was detected. The disease is very common disease in Bulgaria on tomatoes, potatoes and other crops. Monitoring is being continued. This is the first report of phytoplasma‐infected grapevine in Bulgaria.     

QTL analysis for fruit yield components in table grapes (Vitis vinifera)

A segregation population of 184 genotypes derived from a pseudo-testcross of table grapes (Vitis vinifera), together with 203 AFLP and 110 SSR markers was used to detect quantitative trait loci (QTLs) for fruit yield components. Diffferent QTLs, a low percentage of phenotypic variance explained by the QTLs detected and QTL instability over years were detected for each fruit yield component. These results confirm the complex genetic architecture of the yield components in grapevine due to the perennial nature of this species, which has to adapt to yearly variations in climate. Phenotypic correlation analyses between fruit yield components were also performed. The negative correlation between berry weight and the number of berries per cluster seems to have an indirect negative effect on cluster weight, as revealed by the path coefficient analysis; however, this negative correlation was not supported at the molecular level because no coincident QTLs were observed between these traits. Nonetheless, the possibility to select seedless genotypes with large berries without affecting cluster weight needs to be substantiated in future experiments because factors such as sample size and heritability might influence QTL identification in table grapes.     

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.     

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.     

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.     

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     

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.     

Conversion of Indole-3-butyric Acid to Indole-3-acetic Acid by Cuttings of Grapevine (Vitis vinifera) and Olive (Olea europea)

The metabolism of indolebutyric acid (IBA) in hardwood cuttingsof grapevine (Vitis vinifera cv. Perlette) and green cuttingsof olive (Olea europea cvs. Manzanillo, Kalamata and Koroneiki)was investigated. Radioactive IBA which was synthesized in ourlaboratory was used in these studies. Cuttings of both oliveand grapevine converted IBA to IAA. The identity of IAA wasconfirmed by high performance liquid chromatography and gas-liquidchromatography. The stability of IBA, its slow transport from the site of applicationat the base of the cutting and its conversion to IAA in thecutting are probably the factors which make this compound agood root promoter. ¹Contribution from the Agricultural Research Organization, theVolcani Center, Bet Dagan, Israel. No. 619-E, 1982 series. (Received April 28, 1983; Accepted April 12, 1984)     

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     

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.     

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     

葡萄基因电子表达分析平台的验证与应用

为了验证作者建立的葡萄(Vitis vinifera L.)基因电子表达分析平台在葡萄果实发育相关基因的表达预测及特定序列快速检索等方面的应用效果,利用NCBI上公布的大量葡萄EST序列及半定量PCR和RT-PCR技术,对葡萄不同器官中VvANR、VvCHI、VvCHS2和VvDFR基因的表达分析预测结果进行验证,并对该平台具有的特定序列快速检索功能进行了简介.预测结果表明:葡萄各器官中VvANR、VvCHI、VvCHS2和VvDFR基因的无冗余EST序列数量均较多,分别为33条、36条、55条和46条;4个基因的表达量有一定差异,VvANR在花序和芽中表达量较高,VvCHI在花序、果实和芽中表达量较高,VvCHS2在果实、芽、花序和花中表达量较高,VvDFR在花序、芽、花、果实和根中表达量均较高.半定量PCR和RT-PCR实验结果显示:VvANR主要在花序、花和小果中表达;VvCHI主要在小果、花、茎和花序中表达;VvCHS2主要在茎、花序、花和小果中表达;VvDFR在各组织中表达量从高至低依次排序为花、花序、茎、叶、小果、中果、大果.验证结果表明:采用葡萄基因电子表达分析平台识别表达量较高的组织时,平台的预测结果与实验结果基本一致;而在预测一些表达量较低的组织时效果较差.应用该平台、通过5个步骤,可以简便、快速检索出特定组织或特定状况下的特定cDNA文库信息.