Analysis of grape berry cell wall proteome: a comparative evaluation of extraction methods

Different methods were tested for the extraction of proteins from the cell wall-enriched fraction (CWEf) obtained from a sample formed by skin and seeds of ripe berries of Vitis vinifera L. cv. Cabernet Sauvignon. The CWEf was isolated using a disruptive approach that involves tissue homogenization and precipitation by centrifugation. To extract proteins, the CWEf was treated with CaCl(2) and LiCl in two successive steps or, alternatively, with phenol. The efficiency of the protocols was evaluated by measuring protein yield and by analyzing two-dimensional gel electrophoresis (2-DE) gels for the highest detectable spot number and the greatest spot resolution. The phenol method was also adopted for the extraction of proteins from the cytosolic fraction (CYf). The comparison of 2-DE reference maps of protein extracts from CWEf and CYf indicated the presence of both common traits and unique characteristics. To survey this aspect some spots detected in both fractions or present in only one fraction were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Of the 47 spots identified, some were found to be cell wall proteins, while others were proteins not traditionally considered as localized in the apoplastic space. The data presented here provide initial information regarding the apoplastic proteome of grape berry tissues, but also raise the issue of the technical problems that characterize the isolation of cell wall proteins from these very hardy tissues.     

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.     

Possible role of soluble invertase in the gibberellic acid berry-sizing effect in Sultana grape

It is well known that post-bloom applications ofgibberellic acid (GA₃) increase seedless grapeberry size by enhancing cell division, or cellenlargement, or both. As a consequence, total waterand sugar per berry are increased. Soluble invertaseis considered to be one of the key enzymes in theaccumulation of sugar in grape berries. To study apossible role of invertase in the GA₃berry-sizing effect, different rates of post-bloomGA₃ were applied to seedless grape cv. Sultanaand hexose concentration and invertase activity weremeasured. GA₃ stimulated both parameters as earlyas 24 and 32 h after applications, respectively.Moreover, the increment in sugar content and enzymeactivity remained throughout the growing of the berries period and, at ripening, increases in hexosescontent (102%) and invertase activity (60%) weredetected when GA₃ was applied at a rate of 45 ppm.At the same GA₃ rate the pericarp cellsdoubled in size. Furthermore, positive correlationswere found between berry-size, invertase activity andhexose content, suggesting that GA₃ stimulationof invertase could be one of the factors involved in theberry sizing-effect of GA₃.     

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

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

Development and histochemistry of the cells,cell walls,and cuticle of the dermal system of fruit of the grape,Vitis vinifera L.

Summary The dermal system comprises the outer epidermis of the pericarp, its covering of wax and cuticle and the collenchymatous hypodermal cells. During the first of the two post-anthesis phases of fruit growth, differentiation occurred with respect to cell and nuclear volume, content of polyphenolic substances, and wall thickening. Walls of the presumptive dermal system cells developed massive primary thickenings which stained intensely with fluorescent brightener dyes. In the second phase of fruit growth these cells were redifferentiated, their walls becoming thinner as they enlarged to accommodate fruit expansion. Binding of the fluorescent brightener dye was reduced and confined to the outer edges of the walls. At maturity, the walls of the cortical cells adjacent to the dermal system underwent autolysis.The cuticle was evident during the first 16 days after anthesis as a thin layer which reacted positively with neutral lipid dyes and which contained periodate sensitive vinyl groups. Differentiation of a secondary cuticle followed, and a number of distinct layers were detected by autofluorescence, and staining with auramine 0, Nile blue, and PAS. Cuticle thickness and complexity was maintained throughout the second growth phase.     

A cDNA from grapevine (Vitis vinifera L.), which shows homology to AGAMOUS and SHATTERPROOF,is not only expressed in flowers but also throughout berry development

An AGAMOUS/SHATTERPROOF homologue (Vvmads1) was isolated from grapevine by differential display between berry and leaf mRNA. The predicted protein sequence of the full-length clone shows a high degree of homology to PLENA (77% identity) and to SHP1 and SHP2 (75% and 74% identity respectively), and is grouped with AGAMOUS/PLENA homologues when the conserved MADS and K domains are compared. Vvmads1 is expressed only in the later stages of flower development and throughout berry development, although expression is reduced after ripening commenced. When Vvmads1 was over-expressed in tobacco, the resulting plants display altered morphologies in the outer two floral whorls. In the most extreme cases, the inner whorls were surrounded by a carpelloid structure created by the modified sepals. Within these sepals were petals which had been split into sections and which were attached at the base of the flower by structures with the appearance of filaments. The results of this study suggest that Vvmads1 has a regulatory role in flower development before fertilisation and a role in fruit development after fertilisation.     

In situ fixation of grape berries

Summary Usual immersion protocols in aldehyde solutions fail to fully preserve the fine structure of both exocarp and mesocarp cells of grape berries, especially for theveraison (onset of ripening) and post-veraison stages. In exocarp cells, fixative diffusion is hampered by the thick polysaccharide cell walls. In mesocarp cells, plasma membrane and tonoplast are disrupted before aldehyde crosslinking occurs, owing to the high osmotic pressure and cell wall texture. The fixative was therefore injected under pressure as small droplets in the outer and inner parts of the fruit, with limited changes in the steady-state organization of fruit tissues. Compared to a selective range of immersion protocols, a striking improvement in cell preservation was observed for all berry tissues, allowing new information on various compartments of grape berry cells. The preservation of organ integrity and local concentration of aldehyde molecules are the most critical parameters of improved fixation. This technique may be applicable to a large array of fleshy fruits containing mainly cells comprising a high volumetric proportion of vacuoles accumulating large amounts of organic acids and sugars and bounded by thick-walled exocarp cells.     

Somatic embryogenesis from grapevine cells. I-Improvement of embryo development by changes in culture conditions

In conventional culture conditions without auxin, somatic embryos arising from suspension cultures of grapevine rootstock 41B (Vitis vinifera cv. Chasselas x Vitis berlandieri) are arrested at the heart stage of development. Starting from indications that inhibitors excreted in the culture medium could be responsible for this arrest, new culture conditions based on daily subculturing embryos in fresh medium have been successfully used to obtain full embryo development. From this technique, a microassay was devised for screening small amounts of extracellular molecules as potential inhibitors of embryonic development. Our results show that extracellular macromolecules of molecular weight higher than 10 kDa are likely involved in the inhibition of caulinary meristem initiation. However, other factors obviously cooperate to inhibit embryo development in conventional culture conditionsAbbreviations CH76 cv. Chardonnay clone 76 (Vitis vinifera) - NOA 2-naphthoxyacetic acid     

Accumulation of peonidin 3-glucoside enhanced by osmotic stress in grape (Vitis vinifera L.) cell suspension

Cell cultures of grapes, Vitis vinifera L. cv Gamay Fréaux were grown under different conditions of external osmotic potential induced by an increase of sucrose concentration or by the addition of mannitol to the culture medium. Addition of 82 mM mannitol or increasing sucrose concentration to 132 mM had similar effects on repressing growth. Cyanidin 3-glucoside, peonidin 3-glucoside and peonidin 3-p-coumaroylglucoside are three main anthocyanins of Vitis cells. Increasing osmotic potential from –0.43 MPa to –0.8 MPa in the medium resulted in a significant intracellular accumulation of anthocyanin especially peonidin 3-glucoside in the pigmented cells. High osmotic potential appears to stimulate the methylation of anthocyanins. Osmotic potential is an important culture factor and may be useful in the controlling of anthocyanin production and composition.     

Direct shoot organogenesis and plant regeneration from leaves of grape (Vitis spp.)

Adventitious shoots developed from in vitro-grown leaves of Vitis vinifera cultivars Cabernet Sauvignon, French Colombard, Grenache, Thompson Seedless (syn. Sultana) and White Riesling, V. rupestris cv. St. George (syn. du Lot) and V. vinifera × rupestris cv. Ganzin 1. Leaf explants less than 15 mm long were excised from nodal cultures and cultured on Murashige and Skoog or Nitsch and Nitsch-based regeneration media with 0, 1, 2 or 4 mgl^-1 6-benzylaminopurine (BAP). Adventitious shoots developed within 4 weeks at the petiolar stub and occasionally from wounded lamina tissues. Shoot organogenesis occurred only on media containing BAP and at a higher frequency with 2 mgl^-1 than with 1 or 4 mgl^-1. On media containing 2 mgl^-1 BAP, 47, 67, 60, and 42%, respectively, of leaf explants of Cabernet Sauvignon, French Colombard, Thompson Seedless, and White Riesling produced adventitious shoots compared to 14, 14, and 29%, respectively, for Grenache, St. George, and Ganzin 1. Solid culture medium was superior to liquid medium and transfer frequency on solid medium did not affect the regeneration frequency. Further shoot growth was promoted by the transfer of regenerating tissues to fresh regeneration medium. More than 80% of explants initially producing adventitious buds exhibited further shoot growth, developing an average of more than 6 shoots each. Shoots rooted easily and the resulting plants appeared morphologically identical to parent vines.     

Effects of high ammonium concentrations on growth and anthocyanin formation in grape (Vitis vinifera L.) cell suspension cultured in a production medium

Cultivating Vitis vinifera cell suspensions in a production medium which is characterized by high sucrose and low nitrate concentrations (132 mM and 6.25 mM respectively) repressed growth but enhanced the intracellular accumulation of anthocyanins, especially peonidin 3-glucoside. Increasing the ammonium concentration of the production medium from 2 to 8–16 mM increased growth and decreased the accumulation of anthocyanins and peonidin 3-glucoside specifically. Instead, peonidin 3-p-coumaroylglucoside accumulated. At 24 mM ammonium concentration, growth was inhibited and accumulation of peonidin 3-p-coumaroylglucoside was significant (p<0.05) and represented 42% of total anthocyanins after 12 days of culture compared with 19% in the production medium with 2 mM ammonium.Contribution Number 217.     

Water deficits accelerate ripening and induce changes in gene expression regulating flavonoid biosynthesis in grape berries

Water deficits consistently promote higher concentrations of anthocyanins in red winegrapes and their wines. However, controversy remains as to whether there is any direct effect on berry metabolism other than inhibition of growth. Early (ED) and late (LD) season water deficits, applied before or after the onset of ripening (veraison), were imposed on field grown Vitis vinifera “Cabernet Sauvignon”, and the responses of gene expression in the flavonoid pathway and their corresponding metabolites were determined. ED accelerated sugar accumulation and the onset of anthocyanin synthesis. Both ED and LD increased anthocyanin accumulation after veraison. Expression profiling revealed that the increased anthocyanin accumulation resulted from earlier and greater expression of the genes controlling flux through the anthocyanin biosynthetic pathway, including F3H, DFR, UFGT and GST. Increases in total anthocyanins resulted predominantly from an increase of 3′4′5′-hydroxylated forms through the differential regulation of F3′H and F3′5′H. There were limited effects on proanthocyanidin, other flavonols, and on expression of genes committed to their synthesis. These results demonstrate that manipulation of abiotic stress through applied water deficits not only modulates compositional changes during berry ripening, but also alters the timing of particular aspects of the ripening process.     

A PCR-based diagnostic tool for distinguishing grape skin color mutants

Berry skin color mutants are phenotypically different from their original cultivars, but they show identical molecular profile if analysed by using microsatellite markers. This work gives an easy, inexpensive and quick diagnostic tool to discriminate these somatic variants. We distinguished some grape (Vitis vinifera L.) skin color mutants from white to red or pink and from black to grey, pink or white and we investigated their molecular bases by single-strand conformational polymorphism (SSCP), single base primer extension and coding sequence analysis of anthocyanin biosynthetic enzyme genes and by polymerase chain reaction (PCR) analysis of VvmybA1 regulatory gene. Analyses of structural genes did not reveal polymorphisms between wild type and mutant cultivars but only among different varieties, whereas the study of VvmybA1 regulatory gene has given important outcomes for color mutants characterisation. The discrimination between white wild type and its derived colored mutant and between black wild type and white mutant has been obtained through a simple test of amplification for presence/absence. The discrimination between black wild type and less colored mutant has occurred through a quantitative result on agarose gel confirmed by real-time PCR analysis: the amount of functional allele in less colored somatic variants genome was about one-fourth of the correspondent quantity in original black cultivars genome.     

Microcutting leaf area,weight and position on the stock shoot influence root vigour,shoot growth and incidence of shoot tip necrosis in grape plantlets in vitro

The physiological parameters of microcuttings, namely leaf area, weight (wt) and position on the stock shoot, had significant effects on root vigour, microshoot growth and incidence of shoot tip necrosis (STN) in cultures of grape (Vitis vinifera L.) ‘Arka Neelamani’. Single-node leafy cuttings cultured in MS medium containing 1 μM IAA and 0.1 μM GA₃ generally rooted first and subsequently sprouted into a single shoot. Small leafed cuttings exhibited slow root emergence, poor quality roots, early sprouting and weak shoot growth. Large leafed cuttings on the other hand, showed early rooting, vigorous roots, delayed sprouting and healthier shoots. Significant correlations were observed between fresh root wt per plantlet at 1 month in culture and wt of lamina, total wt and leaf area of the cuttings in that order. A significant correlation also existed between wt of roots and height or wt of the sprout that developed. The study suggests that the weight or area of the leaf governed the root growth in a microcutting. STN was observed in some plantlets particularly those derived from large leafed cuttings. Such cuttings showed vigorous roots and delayed but fast sprout growth that ended in STN. Plantlets showing STN had less Ca⁺⁺ and Mg⁺⁺ in the shoot tissue than in the shoots of normal plantlets while the roots showed similar Ca⁺⁺ or higher Mg⁺⁺ contents. Both had comparable amounts of cytokinins in shoot and root tissues. This revised version was published online in June 2006 with corrections to the Cover Date.     

Using urea phosphate to enhance the effect of gibberellin A3 on grape size

Gibberellic acid (GA₃) is widely used to enlarge the berries of seedless grapes (Vitis vinifera L). In cv. Sultana (Thompson Seedless) the addition of 1000 mg/L urea phosphate (UP) to GA₃ solutions after fruit set reduced the pH of the solutions to a stable pH 2.9 and enhanced the effect of GA₃ on berry size and delayed maturation. Addition of citrate buffer, pH 2.9, to GA₃ sprays did not affect berry size or maturation. The possibility of improved GA penetration due to the low pH is considered. The nutritional effect of UP and direct enhanced penetration by the urea ion are also discussed.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 1735-E, 1986 series.     

Implication of signaling pathways involving calcium, phosphorylation and active oxygen species in methyl jasmonate-induced defense responses in grapevine cell cultures

Perception of elicitors triggers plant defense responses via various early signal transduction pathways. Methyl jasmonate (MeJA) stimulates defense responses in grapevine (Vitis vinifera). We investigated the involvement of various partners (calcium, ROS, reversible phosphorylation) in MeJA-induced responses by using a pharmacological approach. We used specific calcium channel effectors and inhibitors of serine/threonine phosphatases, superoxide dismutase and NAD(P)H oxidase and investigated production of stilbenes (resveratrol and its glucoside, piceid, the major form), which are the grapevine phytoalexins. RNA accumulation of two genes encoding enzymes involved in stilbene synthesis (PAL and STS), three genes encoding pathogenesis-related proteins (CHIT4C, PIN and GLU) and one gene encoding an enzyme producing jasmonates (LOX) were also assessed. Calcium and its origin seemed to play a major role in MeJA-induced grapevine defense responses. Phytoalexin production was strongly affected if calcium from the influx plasma membrane was inhibited, whereas calcium from the intracellular compartments did not seem to be involved. ROS production seemed to interfere with MeJA-stimulated defense responses, and protein phosphorylation/dephosphorylation events also played a direct role.     

Effects of low nitrate and high sugar concentrations on anthocyanin content and composition of grape (Vitis vinifera L.) cell suspension

In pigmented cells of Vitis vinifera suspension cultures, best accumulation of anthocyanins was obtained when nitrate concentration was reduced from 25 mM to 6.25 mM and when sucrose concentration was increased from 88 mM to 132 mM. Under such conditions growth was greatly decreased. However, cell viability was maintained. The increases in anthocyanins in pigmented cells were due largely to increases in peonidin — glucoside. The high sucrose and the low nitrate concentrations can be one of the important culture factors in controlling of anthocyanin production by cell cultures.     

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

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