Metabolic changes of genetically engineered grapes (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.) studied by <Superscript>1</Superscript>H-NMR,metabolite heatmaps and <Emphasis Type="Italic">i</Emphasis>PLS

Introduction

The Deficiens Homologue 9-iaaM (DefH9-iaaM) gene is an ovule-specific auxin-synthesizing gene which is expressed specifically in placenta/ovules and promotes auxin-synthesis. It was introduced into the genome of two grape cultivars Thompson Seedless and Silcora and both transgenic cultivars had an increased number of berries per bunch.

Objectives

This study investigates the down-stream metabolic changes of Silcora and Thompson seedless grape cultivars when genetically modified through the insertion of the DefH9-iaaM gene into their genome.

Methods

The effects of the genetic modification upon the grape metabolome were evaluated through ¹H-NMR and exploratory data analysis. Chemometric tools such as Interval Partial Least Squares regression and metabolite heatmaps were employed for scrutinizing the changes in the transgenic metabolome as compared to the wild type one.

Results

The results show that the pleiotropic effect on the grape metabolome as a function of the gene modifications is relatively low, although the insertion of the transgene caused a decrement in malic acid and proline and an increment in p-coumaric acid content. In addition, the concentration of malic acid was successfully correlated with the number of inserted copies of transgene in the Silcora cultivar, proving that the increased production of berries, promoted by the inserted gene, is achieved at the expense of a decrement in malic acid concentration.

Conclusion

NMR together with chemometrics is able to identify specific metabolites that were up- or down regulated in the genetically engineered plants allowing highlighting alterations in the down-stream metabolic pathways due to the up-stream genetic modifications.

     

Genetic engineering of parthenocarpic fruit development in tomato

Parthenocarpy was engineered in two genotypes of Lycopersicon esculentum Mill. by using the DefH9-iaaM chimeric gene. The parthenocarpic trait consists of fruit set and growth in the absence of fertilization. Seedless parthenocarpic fruits were obtained from emasculated flowers, and fruits with seeds from pollinated flowers. All parthenocarpic tomato plants analysed expressed the DefH9-iaaM gene during flower development. The fruit set percentage of emasculated transgenic flowers was similar to that of control plants. In 7 out of 8 independent transgenic plants, the fresh weight of fruits derived from pollinated or emasculated flowers did not significantly differ from that of fruits obtained by pollination of the control plants. The pH of the parthenocarpic fruit was generally unaffected and the soluble solid concentration was either unchanged or increased. Thus, the DefH9-iaaM gene is a genetic tool that might be used to improve tomato productivity.     

Xenia and metaxenia in grapes: differences in berry and seed characteristics of maternal grape cv. ‘Narince’ (Vitis vinifera L.) as influenced by different pollen sources

Literature investigations indicate that the grapes have quite complex fertilisation biology. This complexity necessitates extensive investigations to obtain reliable knowledge for both well‐organised hybridisation studies and maximising grape yield. Therefore, this study was conducted to investigate the influences of self‐, free‐ and cross‐pollination on berry and seed characteristics in grape. Five different pollination treatments were applied to ‘Narince’, the most widely known and popular white wine grape in Turkey. Pollen tests indicated that all the cultivars had satisfactory in vitro pollen viability percentages. Free‐pollination produced a significantly higher percentage berry set. Among the pollinizers, the use of pollen of ‘Thompson Seedless’ and ‘Cardinal’ varieties resulted in higher berry set percentage in ‘Narince’. The free‐pollination was also superior in giving the highest weight, length and width of the berry, as well as number of seeds per berry. These findings revealed that there were strong xenial and metaxenial effects in the studied grape cultivars. Among the pollinizer cultivars, the most effective pollinator was ‘Thompson Seedless’. Hence, for better berry set and quality, the use of ‘Thompson Seedless’ as a pollinizer may be an attractive option in both grape production and breeding studies.     

Abscisic Acid Sprays Significantly Increase Yield per Plant in Vineyard-Grown Wine Grape (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.) cv. Cabernet Sauvignon Through Increased Berry Set with No Negative Effects on Anthocyanin Content and Total Polyphenol Index of Both Juice and Wine

In many cultivars of Vitis vinifera periods of mild water stress during ripening are thought to increase grape quality for winemaking, even though yields may be negatively affected. Because abscisic acid (ABA) is involved in the signaling of water stress in plants, we examine the effects of the ABA signal being given without the concomitant water stress. ABA at 250 mg l⁻¹ was sprayed weekly or biweekly from bud-burst until harvest onto the leaves of vineyard-grown plants of cv. Cabernet Sauvignon. For ABA-treated plants berry yield per bunch and per plant was significantly increased (1.5- to 2.0-fold) across three consecutive harvests (2005 through 2007). Number of berries per bunch and per plant was the primary basis for the significant crop increases, although bunches per plant also tended to increase (1.1- to 1.3-fold) across all three harvests. Other parameters assessed included number of internodes, shoot length, leaf area, leaf water potential at midday, photosynthesis, and stomatal conductance. These parameters showed no significant change with ABA treatment, although shoot length tended to be reduced, as was leaf area relative to control plants. The significantly increased fruit yields were thus accomplished without accompanying increases in leaf photosynthesis and leaf areas. Juice at harvest had equal levels of sugars (Brix) and somewhat higher levels of anthocyanins and total polyphenols relative to control values. The two latter trends continued for the resultant wine across two vintage years. In conclusion, three seasons of experimental trials have demonstrated that ABA application can significantly enhance yield per plant in the field-grown grape (cv. Cabernet Sauvignon) by favoring increased berry set without diminishing the quality of the fruit for winemaking use.     

Damage Patterns caused by Lygocoris spinolae (Hemiptera: Miridae) on ‘Campbell Early’ Grapes

The phenology and damage patterns of Lygocoris spinolae (Meyer-Dür) on ‘Campbell Early’ grape were examined in different grape development stage of inflorescence formation, blooming, and berry set. Nymphs fed on newly unfolding leaves of shoot tip before inflorescence formation. As the inflorescences were clearly visible, nymphs on leaves moved to flower clusters. Sting spots with brown color occurred at sucking sites of leaves, then expanded to hole as the leaves developed, and resulted in leaf malformation. The nymphs on flower clusters sucked developing florets, which induced the drying of florets followed by defoliation. After flower caps had fallen, L. spinolae fed on young berries causing blackening of berry skin around the sting. The blackening of berry skin changed to corky-scarred tissue as the berry developed. L. spinolae feeding significantly reduced the number of berries per cluster in all grape stages. A few berries became corky-scarred or shot berries when fed at inflorescence formation period. Feeding at blooming caused shot berries with low rate of corky-scarred berry. Most of the berries were corky-scarred or became shot berries when fed at berry set. Feeding by adults caused the same damage patterns as nymphs. Heavy damages were observed where L. spinolae were introduced after inflorescence formation: reduced number of berries per cluster by 35.0 to 99.1% at blooming and by 80.0% at berry setting depending on L. spinolae densities.     

Analysis of polymorphism based on SSCP markers in gamma-irradiated (Co60) grape (Vitis vinifera) varieties

The effects of induced mutation produced by five different doses of gamma irradiation (20, 25, 30, 40, and 45 Gy) were determined using molecular approaches in Vitis vinifera cultivars, namely Thompson Seedless (Sultani ?ekirdeksiz) (progenitor of seedless vinifera variety) and Kalecik Karasi (one of the best quality wine grape variety of Turkey). Mutant candidates were selected through morphological observations of mutation-induced phenotypic changes during the first, second and third vegetation periods after radiation applications. Amplification studies started with 50 primers (expressed sequence tags) applied to the mutated individuals. Only 15 of these primers revealed polymorphic profiles. Twenty-two candidate mutants of Thompson Seedless and Kalecik Karasi, selected based on morphological observations, were analyzed with 15 single-strand conformation polymorphism (SSCP) markers, together with 46 control plants. Polymorphic bands were rarely obtained in the SSCP analysis, and they were not reproducible.     

Vascular development of the grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.) inflorescence rachis in response to flower number,plant growth regulators and defoliation

The grapevine inflorescence is a determinate panicle and as buds emerge, shoot, flower and rachis development occur simultaneously. The growth and architecture of the rachis is determined by genetic and environmental factors but here we examined the role of flower and leaf number as well as hormones on its elongation and vascular development. The consequences of rachis morphology and vascular area on berry size and composition were also assessed. One week prior to anthesis, Merlot and Cabernet Sauvignon field vines were exposed to manual flower removal, exogenous plant growth regulators or pre-bloom leaf removal. Manual removal of half the flowers along the vertical axis of the inflorescence resulted in a shorter rachis in both cultivars. Conversely, inflorescences treated with gibberellic acid (GA₃) and the synthetic cytokinin, 6-benzylaminopurine (BAP) resulted in a longer rachis while pre-bloom removal of all leaves on the inflorescence-bearing shoot did not alter rachis length relative to untreated inflorescences. Across the treatments, the cross-sectional areas of the conducting xylem and phloem in the rachis were positively correlated to rachis girth, flower number at anthesis, bunch berry number, bunch berry fresh mass and bunch sugar content at harvest. Conversely, average berry size and sugar content were not linked to rachis vascular area. These data indicate that the morphological and vascular development of the rachis was more responsive to flower number and plant growth regulators than to leaf removal.     

Physiological Response of Resistant and Susceptible Vitis vinifiera Cultivars to Meloidogyne incognita

The effect of Meloidogyne incognita on growth, general physiological response, and the concentration of reducing and nonreducing sugars at the nematode feeding sites of French Colombard (susceptible) and Thompson Seedless (moderately resistant) Vitis vinifiera cultivars was studied up to 2,100 degree-days (DD-base 10 C). Nematode stress dosage, measured as the product of cumulative number of juveniles and females and their total energy (calories) demand, accounted for up to 15 and 10% of the energy assimilated by French Colombard and Thompson Seedless plants, respectively. Total leaf area, total carbon dioxide fixed, transpiration rate, stomatal conductance, and internal leaf CO₂ concentration were not affected, but energy assimilated into plant tissue and respiration were decreased by nematode infection in both cultivars. Energy consumed by nematodes accounted for most of the difference in total energy assimilated between infected and uninfected plants on French Colombard but not on Thompson Seedless, suggesting that the resistant cultivar may be using more energy to curtail the nematode''s activity. Nematodes did not affect the concentration of reducing sugars, but the concentration of nonreducing sugars increased in French Colombard and decreased in Thompson Seedless. This indicates that there was more translocation of photosynthate to the feeding sites of the susceptible than to those of the resistant cultivar, and may explain why M. incognita causes more damage to French Colombard than to Thompson Seedless.     

Growth and Energy Demand of Meloidogyne incognita on Susceptible and Resistant Vitis vinifera Cultivars

Food (energy) consumption rates ofMeloidogyne incognita were calculated on Vitis vinifera cv. French Colombard (highly susceptible) and cv. Thompson Seedless (moderately resistant). One-month-old grape seedlings in styrofoam cups were inoculated with 2,000 or 8,000 M. incognita second-stage juveniles (J2) and maintained at 17.5 degree days (DD - base 10 C)/day until maximum adult female growth and (or) the end of oviposition. At 70 DD intervals, nematode fresh biomass was calculated on the basis of volumes of 15-20 nematodes per plant obtained with a digitizer and computer algorithm. Egg production was measured at 50-80 DD intervals by weighing 7-10 egg masses and counting the number of eggs. Nematode growth and food (energy) consumption rates were calculated up to 1,000 DD based on biomass increase, respiratory requirements, and an assumption of 60 % assimilation efficiency. The growth rate of a single root-knot nematode, excluding egg production, was similar in both cultivars and had a logistic form. The maximum fresh weight of a mature female nematode was ca. 29-32 μg. The total biomass increase, including egg production, also had a logistic form. Maximum biomass (mature adult female and egg mass) was 211 μg on French Colombard and 127 μg on Thompson Seedless. The calculated total cost to the host for the development of a single J2 from root penetration to the end of oviposition for body growth and total biomass was 0.535 and 0.486 calories with a total energy demand of 1.176 and 0.834 calories in French Colombard and Thompson Seedless, respectively.     

无核葡萄品种的无核性来源分析

系谱分析表明,无核白是世界范围内无核葡萄育种的主要无核性来源,以其为最早的亲本材料,经过简单杂交或多亲多代杂交,培育了一系列的无核葡萄品种;无核紫和俄罗斯无核是部分无核品种的原始亲本材料之一;有核葡萄的无核芽变也是无核品种选育的重要途径。     

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.     

Agroecological management of a soil‐dwelling orthopteran pest in vineyards

The efficacy of different combinations of undervine and inter‐row treatments for managing a soil‐dwelling orthopteran pest, weta (Hemiandrus sp.), in vineyards was investigated over 2 seasons. This insect damages vine buds, thus reducing subsequent grape yield. The undervine treatments comprised pea straw mulch, mussel shells, tick beans [Vicia faba Linn. var minor (Fab)], plastic sleeves on vine trunks (treated control) and control (no intervention), while inter‐rows contained either the existing vegetation or tick beans. Treatments were arranged in a randomized complete block design with 10 replicates. Data were collected on weta densities, damage to beans and components of yield. The latter were numbers of bud laid down per vine, shoots per bud, clusters per shoot, grape bunches per vine, bunch weight and yield. The undervine treatments significantly affected all variables except the number of shoots per bud. In contrast, none of the variables was significantly affected by the inter‐row treatments or their interaction with undervine treatments, apart from weta density. At the end of the experiment, weta density in the shell treatment was about 58% lower than in the control. As a result, there was about 39% significant yield increase in that treatment compared to the control. Although the undervine beans and sleeves treatments increased yield, there were no reductions in weta density. With undervine beans, the insect fed on the bean plants instead of vine buds. Thus, yield in that treatment was approximately 28% higher than in the control. These results demonstrate that simple agroecological management approaches can reduce above‐ground damage by soil‐dwelling insects.     

Transgenic parthenocarpic eggplants: superior germplasm for increased winter production

Winter production of three eggplant hybrids transgenic for the parthenocarpic gene DefH9-iaaM was compared, in an unheated greenhouse, to the performance of two untransformed control hybrids and the commercial parthenocarpic cultivar Talina. Each hybrid was either treated or untreated with a commercial formulation of phytohormones to induce fruit set and growth. The productivity of the transgenic parthenocarpic hybrids was not influenced by the hormonal treatment. On the contrary, the productivity of untransformed hybrids was significantly improved by hormonal treatment of the flower buds. The yield of the transgenic hybrids was significantly higher than that obtained in the corresponding untransformed hybrids, even when the latter were treated with phytohormones. The yield increment due to the parthenocarpic trait was particularly evident when compared to the yield of the two corresponding hybrid combinations, which are identical except for the presence of the DefH9-iaaM gene. The transgenic hybrids allowed an increase in productivity of ca. 25%. This increment coincided with a 10% reduction in cultivation cost, mainly due to the labour needed for the hormonal sprays, and to the production of fruits of better quality. Thus, the DefH9-iaaM gene is a biotechnological tool superior to both agronomic and traditional genetic parthenocarpic mutants.     

<Emphasis Type="Italic">Muscadinia rotundifolia</Emphasis> ‘Noble’ defense response to <Emphasis Type="Italic">Plasmopara viticola</Emphasis> inoculation by inducing phytohormone-mediated stilbene accumulation

Downy mildew (DM), one of the most devastating grape diseases worldwide, is caused by the biotrophic oomycete Plasmopara viticola (Pv). In general, grapevine responds to Pv infection with the accumulation of phytoalexins as part of the innate immune system, and diverse phytoalexins are induced on grapevines with different DM-resistance levels in response to Pv invasion. However, the regulation of phytoalexin biosynthesis during grapevine against Pv is still unclear. Herein, we detected stilbenes by UPLC-ESI-MS/MS and found that resveratrol was accumulated to higher level and earlier in the DM-immune Muscadinia rotundifolia ‘Noble’ than that in the DM-susceptible Vitis vinifera ‘Thompson Seedless’ after Pv inoculation. Additionally, a considerable amount of pterostilbene and ε-viniferin was found in ‘Noble’, while a little was detected in ‘Thompson Seedless’. Resveratrol was glycosylated into piceid both in ‘Noble’ and ‘Thompson Seedless’ after Pv inoculation. The qPCR analysis of gene expression indicated that the resveratrol-synthesis gene (STS) was induced by Pv inoculation earlier in ‘Noble’ than that in ‘Thompson Seedless’, while the pterostilbene-synthesis gene (ROMT) was induced in ‘Noble’ but not in ‘Thompson Seedless’ at all. The piceid-synthesis gene (GT) was generally up-regulated in both cultivars. Sequence analysis of STS, ROMT, and GT promoters revealed that they contained cis-regulatory elements responsive to phytohormones and pathogens. Following Pv inoculation, the level of SA, MeJA, and ABA was found to be consistently higher in ‘Noble’ than those in ‘Thompson Seedless’. The results of exogenous hormone elicitation further demonstrated that the accumulation of stilbenes was regulated by phytohormones. The earlier and higher accumulation of phytohormones and consequent induction of stilbene synthesis may play an important role in grapevine defense against downy mildew disease.     

Ripening grape berries remain hydraulically connected to the shoot

Berry diameter was monitored during dry-down and rewatering cycles and pressurization of the root system of Vitis vinifera (cv. Merlot) and Vitis labruscana (cv. Concord) to test changes in xylem functionality during grape ripening. Prior to veraison (onset of ripening), berries maintained their size under declining soil moisture until the plants had used 80% of the transpirable soil water, began to shrink thereafter, and recovered rapidly after rewatering. By contrast, berry diameter declined slowly but steadily during post-veraison water stress and did not recover after rewatering; irrigation merely prevented further shrinking. Preconditioning vines with a period of water stress after flowering did not influence the berries' reaction to subsequent changes in transpirable soil water. Pressurizing the root system led to concomitant changes in berry diameter only prior to veraison, although some post-veraison Concord, but not Merlot, berries cracked under root pressurization. The xylem-mobile dye basic fuchsin, infused via the shoot base, moved throughout the berry vasculature before veraison, but became gradually confined to the brush area during ripening. When the dye was infused through the stylar end of attached berries, it readily moved back to the plant both before and after veraison. Our work demonstrated that berry-xylem conduits retain their capacity for water and solute transport during ripening. It is proposed here that apoplastic phloem unloading coupled with solute accumulation in the berry apoplast may be responsible for the decline in xylem water influx into ripening grape berries. Instead, the xylem may serve to recycle excess phloem water back to the shoot.     

Endophytic colonization of Vitis vinifera L. by Burkholderia phytofirmans strain PsJN: from the rhizosphere to inflorescence tissues

The colonization pattern of Vitis vinifera L. by Burkholderia phytofirmans strain PsJN was determined using grapevine fruiting cuttings with emphasis on putative inflorescence colonization under nonsterile conditions. Two-week-old rooted plants harbouring flower bud initials, grown in nonsterile soil, were inoculated with PsJN:gfp2x. Plant colonization was subsequently monitored at various times after inoculation with plate counts and epifluorescence and/or confocal microscopy. Strain PsJN was chronologically detected on the root surfaces, in the endorhiza, inside grape inflorescence stalks, not inside preflower buds and flowers but rather as an endophyte inside young berries. Data demonstrated low endophytic populations of strain PsJN in inflorescence organs, i.e. grape stalks and immature berries with inconsistency among plants for bacterial colonization of inflorescences. Nevertheless, endophytic colonization of inflorescences by strain PsJN was substantial for some plants. Microscopic analysis revealed PsJN as a thriving endophyte in inflorescence organs after the colonization process. Strain PsJN was visualized colonizing the root surface, entering the endorhiza and spreading to grape inflorescence stalks, pedicels and then to immature berries through xylem vessels. In parallel to these observations, a natural microbial communities was also detected on and inside plants, demonstrating the colonization of grapevine by strain PsJN in the presence of other microorganisms.     

Pre-anthesis CPPU Treatment Modifies Quality and Susceptibility to Post-harvest Berry Cracking of <Emphasis Type="Italic">Vitis vinifera</Emphasis> cv. ‘Thompson Seedless’

Plant growth regulators (PGRs), especially gibberellic acid (GA), are often used in table grape (Vitis vinifera) production to increase berry size. However, overuse of this PGR (for example, more than 200 g·ha^?1·season^?1 of GA) may result in problems with berry condition at the post-harvest stage. A synthetic cytokinin, florchlorfenuron (CPPU), could be considered an alternative to GA. Thus, our objective was to study the effects of pre-anthesis CPPU treatment on berry quality and susceptibility to post-harvest cracking of table grapes. CPPU applied to ‘Thompson Seedless’ inflorescences 48 or 36 days prior to anthesis increased ovary size, number of cells in the ovary pericarp at bloom and berry size at harvest. It also increased the amount of soluble solids and decreased titratable acidity of the fruit, thus allowing an earlier harvest. Finally, if GA was applied during berry growth, CPPU at pre-anthesis improved berry potential tolerance to post-harvest cracking and spoilage. This work demonstrates that application of CPPU at the pre-anthesis stage modifies grape development, improving berry size and potential post-harvest fruit performance facilitating also an early harvest.