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.     

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     

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     

Cryopreservation of sour orange (Citrus aurantium L.) shoot tips

Summary The objective of this study was to establish a cryopreservation protocol for sour orange (Citrus aurantium L.). Cryopreservation was carried out via encapsulation-dehydration, vitrification, and encapsulation-vitrification on shoot tips excised from in vitro cultures. Results indicated that a maximum of 83% survival and 47% regrowth of encapsulated-dehydrated and cryopreserved shoot tips was obtained with 0.5M sucrose in the preculture medium and further dehydration for 6 h to attain 18% moisture content. Dehydration of encapsulated shoot tips with silica gel for 2h resulted in 93% survival but only 37% regrowth of cryopreserved shoot tips. After preculturing with 0.5M sucrose, 80% of the vitrified cryopreserved shoots survived when 2M sucrose plus 10% dimethyl sulfoxide (DMSO) was used as a cryoprotectant for 20 min at 25°C. Survival and regrowth of vitrified cryopreserved shoot tips were 67% and 43%, respectively, when 0.4M sucrose plus 2M glycerol was used as a loading solution followed by application of 100% plant vitrification solution (PVS2) for 20 min. Increased duration of exposure to the loading solution up to 60 min increased survival (83%) and regrowth (47%) of cryopreserved shoot tips. With encapsulation-vitrification, dehydration with 100% PVS2 for 2 or 3 h at 0°C resulted in 50 or 57% survival and 30 or 40% regrowth, respectively, of cryopreserved shoot tips.     

Xylem network connectivity and embolism spread in grapevine(Vitis vinifera L.)

Xylem networks are vulnerable to the formation and spread of gas embolisms that reduce water transport. Embolisms spread through interconduit pits, but the three-dimensional (3D) complexity and scale of xylem networks means that the functional implications of intervessel connections are not well understood. Here, xylem networks of grapevine (Vitis vinifera L.) were reconstructed from 3D high-resolution X-ray micro-computed tomography (microCT) images. Xylem network performance was then modeled to simulate loss of hydraulic conductivity under increasingly negative xylem sap pressure simulating drought stress conditions. We also considered the sensitivity of xylem network performance to changes in key network parameters. We found that the mean pit area per intervessel connection was constant across 10 networks from three, 1.5-m stem segments, but short (0.5 cm) segments fail to capture complete network connectivity. Simulations showed that network organization imparted additional resistance to embolism spread beyond the air-seeding threshold of pit membranes. Xylem network vulnerability to embolism spread was most sensitive to variation in the number and location of vessels that were initially embolized and pit membrane vulnerability. Our results show that xylem network organization can increase stem resistance to embolism spread by 40% (0.66 MPa) and challenge the notion that a single embolism can spread rapidly throughout an entire xylem network.

A complete digital reconstruction of a grapevine xylem network reveals that network connectivity imparts greater resistance to drought-induced embolism spread than pit membrane properties suggest.     

An improved protocol for Agrobacterium-mediated transformation of grapevine (Vitis vinifera L.)

An improved protocol for efficient Agrobacterium-mediated transformation of grapevine (Vitis sp.) was developed through modification of cocultivation and subsequent washing procedures. It was determined that Agrobacterium-infected somatic embryos (SE) cocultivated on filter paper exhibited less browning and significantly higher transient GFP and GUS expression than those cultured on agar-solidified medium. Furthermore, such SE, when subjected to a prolonged washing period in liquid medium containing cefotaxime and carbenicillin, followed by another wash in similar medium with kanamycin added, exhibited significantly higher rates of stable transformation compared to previously-described procedures. Transgenic plant recovery was increased 3.5–6 Xs by careful excision of leafy cotyledons from SE that had been induced to germinate on MS medium containing 1 μM of BA. Southern blot analysis revealed the low copy number integration of transgenes in transgenic plants recovered using the improved protocol. These improved cocultivation and plant recovery procedures have been demonstrated to facilitate production of large populations of transgenic plants from V. vinifera ‘Merlot’, ‘Shiraz’ and ‘Thompson Seedless’ as well as Vitis hybrid ‘Seyval Blanc’.     

Cryopreservation of in vitro-grown shoot tips of grapevine by encapsulation-dehydration

In vitro-grown shoot tips of the LN33 hybrid (Vitis L.) and cv. Superior (Vitis vinifera L.) were successfully cryopreserved by encapsulation-dehydration. Encapsulated shoot tips were precultured stepwise on half-strength MS medium supplemented with increasing sucrose concentrations of 0.25, 0.5, 0.75 and 1.0 M for 4 days, with one day for each step. Following preculture, encapsulated shoot tips were dehydrated prior to direct immersion in liquid nitrogen for 1 h. After thawing, cryopreserved shoot tips were post-cultured on a post-culture medium for survival. An optimal survival of cryopreserved shoot tips was achieved when encapsulated shoot tips were dehydrated to 15.6 and 17.6% water content for the LN33 hybrid and cv. Superior, respectively. Comparison between the effects of dehydration with silica gel and by air drying on cryopreserved shoot tips, showed that survival was dependent on water content, not on dehydration method. The thawing method markedly affected survival of cryopreserved shoot tips, and thawing at 40 °C for 3 min was found best. No callus formation and fastest shoot elongation were obtained when cryopreserved shoot tips were post-cultured on the post-culture medium composed of half-strength MS supplemented with 1 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. With these optimized parameters, 60 and 40% survival of cryopreserved shoot tips were obtained for the LN33 hybrid and cv. Superior, respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cryopreservation of in vitro sugar beet (Beta vulgaris L.) shoot tips by a vitrification technique

 Sugar beet shoot tips from cold-acclimated plants were successfully cryopreserved using a vitrification technique. Dissected shoot tips were precultured for 1 day at 5  °C on solidified DGJ0 medium with 0.3 M sucrose. After loading for 20 min with a mixture of 2 M glycerol and 0.4 M sucrose (20  °C), shoot tips were dehydrated with PVS2 (0  °C) for 20 min prior to immersion in liquid nitrogen. Both cold acclimation and loading enhanced the dehydration tolerance of shoot tips to PVS2. After thawing, shoot tips were deloaded for 15 min in liquid DGJ0 medium with 1.2 M sucrose (20  °C). The optimal exposure time to both loading solution and PVS2 depended on the in vitro morphology of the clone. With tetraploid clones a higher sucrose concentration during cold acclimation and preculture further enhanced survival after cryopreservation. Survival rates ranged between 60% and 100% depending on the clone. Since only 10–50% of the surviving shoot tips developed into non-hyperhydric shoots, regrowth was optimized. Received: 13 September 1999 / Revision received: 2 March 2000 / Accepted: 16 March 2000     

Berry morphology and composition in irrigated and non-irrigated grapevine (Vitis vinifera L.)

The present study was carried out in a 5-year-old vineyard (Vitis vinifera L., cv. Aglianico) located in Southern Italy. Half of the plants (IRR) were fully irrigated, whereas the other half were not irrigated (NIRR). In both of the treatments, plant water status, gas exchange, photosynthetic efficiency and productive performance were determined. The arid conditions resulted in significant decreases in stem water potential in NIRR (minimum values of -1.34 and -1.52 MPa in IRR and NIRR, respectively). The values of yield per plant, cluster weight and total berry weight were significantly higher in IRR. Grape berries were separated into four weight classes, and morphometric and microscopic analyses were carried out to measure and calculate berry skin characteristics. Irrigation determined a marked shift toward heavier (+23% in the class ≥ 1.25 g) and bigger (336.35 mm3 vs 299.15 mm3) berries, and induced significant changes in other morphometric berry parameters. No differences among berry weight classes and irrigation treatments were observed for berry skin thickness. In all of the berry weight classes, total anthocyanins extracted from berry skins were significantly higher in NIRR than in IRR (12301.53 and 9585.52 mg kg?1 fresh berry skin, respectively), and appeared to be positively related to berry weight, whereas total flavonols were not significantly different between the two treatments. Qualitative changes in the levels of single anthocyanin and flavonol compounds were detected between IRR and NIRR. In addition, iron, copper and zinc, whose high concentration can negatively affect wine quality, were significantly higher in the IRR treatment. The results highlighted that the absence of irrigation did not determine decreases in grape quality. Such data can be of primary importance in environments where water availability is by far the most important limiting factor for plant growth.     

Proteomic analysis of grapevine (Vitis vinifera L.) tissues subjected to herbicide stress

Two-dimensional gel electrophoresis coupled to mass spectrometry analysis was used to examine for the first time the effect of a herbicide (flumioxazin) on a crop species (Vitis vinifera L.) at the proteome level. Examination of 2-D maps derived from chemically stressed tissues revealed the presence of 33 spots displaying a differential expression pattern. The presence of stress responsive proteins in the different plant organs analysed suggests that flumioxazin could act systemically. Among the responsive proteins, some photosynthesis-related proteins, including several fragments of the enzyme Rubisco, were identified. This effect suggests that photosynthesis could be impaired by the herbicide. The induction of several enzymatic antioxidant systems was also observed, probably as a result of an oxidative stress. Moreover, the photorespiration pathway was stimulated, as suggested by the induction of some key enzymes involved in this process. Changes in carbon metabolism-associated proteins presumably reflect altered patterns of carbon flux in response to impaired photosynthesis and an increased need for osmotic adjustment in affected tissues. Finally, plant defences were stimulated as revealed by the induction of a set of proteins belonging to the pathogenesis-related 10 class, suggesting that they could play an essential role in cell defence mechanisms against flumioxazin.     

Design of grapevine (Vitis vinifera L.) cultivar-specific SCAR primers for PCR fingerprinting

Among 34 grapevine cultivars (Vitis vinifera L.), eight putative genotype-specific RAPD markers, from ’Albariño’, ’Caíño blanco’, ’Chardonnay’, ’Folle blanche’, ’Grenache blanc’, ’Malvasía Sitges’, ’Torrontés’ and ’Treixadura’ respectively, were selected to transform into SCAR markers. Of these, seven markers were cloned and then five which showed a positive specific hybridization signal were sequenced. For these five markers, 30 sequence-specific primers ranging from 14 to 29 bases were designed to amplify genomic DNA from 64 grapevine cultivars under more-stringent PCR conditions. Only, two primer pairs, OpA11₁₁₇₅p17R/ p17F and OpD10₈₀₀p14R/p14F, still produced a specific SCAR marker, the ’Folle blanche’ ScA11₁₁₇₅ and the ’Malvasía Sitges’ ScD10₈₀₀ respectively. Moreover, the ScA11₁₁₇₅ marker was amplified only in ’Folle blanche’ among the 64 cultivars tested with a large annealing temperature range using either two different Taq DNA polymerases or two separate thermocyclers. In addition, we discuss the initial polymorphism originated by the RAPD technique and suggest a new design of SCAR primers to obtain reliable cultivar-specific SCAR markers from single PCR-based bands for identification purposes.     

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.     

Large-scale parentage analysis in an extended set of grapevine cultivars (Vitis vinifera L.)

Inheritance of nuclear microsatellite markers (nSSR) has been proved to be a powerful tool to verify or uncover the parentage of grapevine cultivars. The aim of the present study was to undertake an extended parentage analysis using a large sample of Vitis vinifera cultivars held in the INRA “Domaine de Vassal” Grape Germplasm Repository (France). A dataset of 2,344 unique genotypes (i.e. cultivars without synonyms, clones or mutants) identified using 20 nSSR was analysed with FAMOZ software. Parentages showing a logarithm of odds score higher than 18 were validated in relation to the historical data available. The analysis first revealed the full parentage of 828 cultivars resulting in: (1) 315 original full parentages uncovered for traditional cultivars, (2) 100 full parentages confirming results established with molecular markers in prior papers and 32 full parentages that invalidated prior results, (3) 255 full parentages confirming pedigrees as disclosed by the breeders and (4) 126 full parentages that invalidated breeders’ data. Second, incomplete parentages were determined in 1,087 cultivars due to the absence of complementary parents in our cultivar sample. Last, a group of 276 genotypes showed no direct relationship with any other cultivar in the collection. Compiling these results from the largest set of parentage data published so far both enlarges and clarifies our knowledge of the genetic constitution of cultivated V. vinifera germplasm. It also allows the identification of the main genitors involved in varietal assortment evolution and grapevine breeding.     

Epigenetic repressor-like genes are differentially regulated during grapevine (Vitis vinifera L.) development

Grapevine sexual reproduction involves a seasonal separation between inflorescence primordia (flowering induction) and flower development. We hypothesized that a repression mechanism implicating epigenetic changes could play a role in the seasonal separation of these two developmental processes in grapevine. Therefore, the expression of five grapevine genes with homology to the Arabidopsis epigenetic repressor genes FERTILIZATION INDEPENDENT ENDOSPERM (FIE), EMBRYONIC FLOWER 2 (EMF2), CURLY LEAF (CLF), MULTICOPY SUPPRESSOR OF IRA 1 (MSI1) and SWINGER (SWN) was analyzed during the development of buds and vegetative and reproductive organs. During bud development, the putative grapevine epigenetic repressor genes VvCLF, VvEMF2, VvMSI1, VvSWN and VvFIE are mainly expressed in latent buds at the flowering induction period, but also detected during bud burst and inflorescence/flower development. The overlapping expression patterns of grapevine PcG-like genes in buds suggest that chromatin remodeling mechanisms could be operating during grapevine bud development for controlling processes such as seasonal flowering, dormancy and bud burst. Furthermore, the expression of grapevine PcG-like genes was also detected in fruits and vegetative organs, suggesting that epigenetic changes could be at the basis of the regulation of various proliferation–differentiation cell transitions that occur during grapevine development.     

Cryopreservation of in vitro-grown shoot tips of Alnus glutinosa (L.) Gaertn.

In vitro-grown shoot tips of Alnus glutinosa (L.) Gaertn. were successfully cryopreserved by vitrification. Shoot tips (0.5–1 mm) excised from 6-week-old shoots were precultured in hormone-free Woody Plant Medium (WPM) supplemented with 0.2 M sucrose, for 2 days at 4 °C in the dark, and then treated with a mixture of 2 M glycerol plus 0.4 M sucrose, for 20 min at 25 °C. Osmoprotected shoot tips were first dehydrated with 50 % vitrification solution (PVS2), for 30 min at 0 °C, and then placed in 100 % PVS2, for 30 min at 0 °C. The solution was replaced with fresh 100 % PVS2, and the shoot tips were plunged directly into liquid nitrogen. The shoot tips were rewarmed in a water bath at 40 °C for 2 min, and then washed twice, for 10 min at 25 °C, with 1.2 M sucrose solution, before being transferred onto WPM supplemented with 0.5 mg l^?1 N ⁶-benzyladenine, 0.5 mg l^?1 indole-3-acetic acid, 0.2 mg l^?1 zeatin, 20 g l^?1 glucose and 6 g l^?1 Difco Bacto agar. The shoot tips were kept in darkness for 1 week and under dim lighting for another week, before being exposed to standard culture conditions (16 h photoperiod). This protocol was successfully applied to three alder genotypes, with recovery rates higher than 50 %.     

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.     

Monitoring the stability of Rubisco in micropropagated grapevine (Vitis vinifera L.) by two-dimensional electrophoresis

Plants cultured in vitro suffer from several physiological and biochemical impairments due to the artificial conditions of growth, namely the composition of the heterotrophic media. Upon transfer to ex vitro, the higher irradiances, compared to in vitro, can lead to oxidative stress symptoms, which can be counteracted by CO2 concentrations above atmospheric levels. Here we analyse the stability of Rubisco in in vitro grapevine plantlets, and after transfer to ex vitro under four acclimatization treatments: low irradiance (LL, 150 micromol m(-2)s(-1)) and high irradiance (HL, 300 micromol m(-2)s(-1)) in association with CO2 concentrations of 350 (LCO2) and 700 (HCO2) microL L(-1). Proteins were separated with SDS polyacrylamide gel electrophoresis and two-dimensional electrophoresis and Rubisco degradation peptides were analysed by immunoblotting with anti-LSU antibodies. These degradation products were present in the leaves of plantlets under both in vitro and ex vitro treatments. Under LCO2 they were maintained for almost all of the 28 days of the acclimatization period, while becoming scarcely detected after 14 days under HCO2 and after 7 days when HCO2 was associated with HL. These results appear to confirm the counteraction of HCO2 concentrations over the oxidative stress eventually caused by HL. The patterns of soluble sugars in acclimatizing leaves under HLHCO2 also gave an indication of a faster acquisition of autotrophic characteristics.     

Genotypic variability within Tunisian grapevine varieties (Vitis vinifera L.) facing bicarbonate-induced iron deficiency

Morpho-physiological responses to bicarbonate-induced Fe deficiency were investigated in five Vitis vinifera L. Tunisian varieties (Khamri, Blanc3, Arich Dressé, Beldi, and Balta4). One-month-old woody cuttings were cultivated for 85 days on a free calcareous soil irrigated with tap water containing increasing bicarbonate levels (0, 4, 8, 12, and 16 mM NaHCO₃). After this screening, a second experiment compared root biochemical responses of two contrasting genotypes (tolerant-sensitive) dealing with bicarbonate-induced iron deprivation (20 μM Fe ± 10 mM HCO₃⁻) for 75 days. Using morpho-physiological criteria, grapevine tolerance to HCO₃⁻-induced Fe shortage appeared to be genotype-dependent: Balta4 and Beldi varieties showed the highest leaf-chlorosis score (especially at the extreme HCO₃⁻ levels), in contrast to Khamri variety. Growth parameters (shoot height, total leaf area, leaf number, and biomass production) as well as juvenile leaf chlorophyll content were also differently affected depending on both genotype and bicarbonate dose. At 16 mM HCO₃⁻, Khamri was the less sensitive variety, contrasting with Balta4. On the other hand, chlorophyll content correlated positively with HCl-extractible Fe content of the juvenile leaves, suggesting that the grapevine response to iron deficiency may partly depend on to the plant ability to adequately supply young leaves with this element. Root biochemical responses revealed a relatively higher root acidification capacity in Khamri (tolerant) under Fe-deficiency while no significant changes occurred in Balta4 (sensitive). In addition, Fe(III)-reductase and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) activities were strongly stimulated by Fe-deficiency in Khamri, while remaining constant in Balta4. These findings suggest that biochemical parameters may constitute reliable criteria for the selection of tolerant grapevine genotypes to iron chlorosis.