Invertase activity, grape berry development and cell compartmentation

The effect of gibberellic acid on grape (Vitis vinifera L., ev. Sultanina) growth, β-fructofuranosidase (EC 3.2.1.26) activity and carbohydrate levels was investigated throughout berry development and ripening. Although the fruits responded to hormone application with the expected increase in size, growth was not correlated with enzymic activity and hexose accumulation. This suggests that there is no direct regulatory relationship between invertase and the rate of assimilate import. However, fructose:glucose ratios changed from 0.1 in green berries to 1.0 in mature samples. The latter situation can be reconciled with the 1:1 stoichiometry of sucrolysis by invertase. It is suggested that this is attributable to a spatial separation of substrate and enzyme in green tissue. Compartmentation studies indicate that mesocarp cell integrity gradually deteriorates during ripening, which allows invertase to leak out of the vacuole into the surrounding tissue. In fact, the protein fraction retrieved from a buffered medium after incubation of ripening berry slices contained a soluble invertase of presumably vacuolar origin with an acid pH-activity profile and a pI of about 4.     

Mycobacterial proteome extraction: comparison of disruption methods

Mycobacterium avium subsp. paratuberculosis has long been recognized as the causative agent of Johne's disease, a chronic inflammatory intestinal disease of sheep, cattle and other ruminants. Mycobacterial cells are extremely hardy, and proteomic analyses require the use of harsh conditions to effect their disruption. We compared the effectiveness of bead beating and sonication as cell lysis methods for the extraction of the proteomes of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis. Broad and narrow range two-dimensional gel electrophoresis was used to compare the numbers of silver stained protein spots that were observed in mycobacterial lysates. Despite differences in the yield of total protein from either species, and at different ages, the two methods appeared to give similar representations of the mycobacterial proteomes analyzed. Bead beating therefore represents a rapid and effective method of extracting the proteomes of mycobacterial species without the risks associated with an open tube sonication procedure.     

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.     

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.     

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.     

Proteomic analysis of somatic embryogenesis in <Emphasis Type="Italic">Vitis vinifera</Emphasis>

Two dimensional gel electrophoresis coupled to mass spectrometry has been used to study the somatic embryogenesis in Vitis vinifera, by comparing embryogenic and non embryogenic calluses of the Thompson seedless cv. More than 1,000 spots were reproducibly resolved in colloidal Coomassie brilliant blue stained gels over a pI nonlinear range of 3–10 in the first dimension and using homogeneous 12.5% polyacrylamide gels in the second dimension. The expression pattern of 35 spots differed significantly between the two samples. These spots were processed by mass spectrometry analysis and the protein identity was assigned by using both the non-redundant protein and EST databases. Several responsive proteins, some already known to be involved in the somatic embryogenesis process while others, for the first time put into relation with this process, have been described. Moreover, they have been subdivided in functional categories, and their putative role is discussed in terms of their relevance in the somatic embryogenesis process.     

Proteomics of Medicago sativa cell walls

A method for the sequential extraction and profiling by two-dimensional gel electrophoresis (2-DE) of Medicago sativa (alfalfa) stem cell wall proteins is described. Protein extraction included freezing, grinding in a sodium acetate buffer, separation by filtration of cell walls from cytosolic contents, and extensive washing. Cell wall proteins were then extracted sequentially with a solution containing 200 mM CaCl2 and 50 mM sodium acetate, followed by extraction with 3.0 M LiCl and 50 mM sodium acetate. Cell wall proteins from both the CaCl2 and LiCl fractions were profiled by 2-DE. Approximately 150 protein spots were extracted from these two gels, digested with trypsin, and analyzed using nanoscale HPLC coupled to a hybrid quadrupole time-of-flight (Q-tof) tandem mass spectrometer (LC/MS/MS). More than 100 proteins were identified and used in conjunction with the 2-DE profiles to generate proteomic reference maps for cell walls of this important legume. Identified proteins include classical cell wall proteins as well as proteins traditionally considered as non-secreted. Two unique extracellular proteins were also identified.     

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.     

A comparative study on cell wall antigens and cell surface hydrophobicity in clinical isolates ofCandida albicans

Characterization of common cell surface-bound antigens inCandida albicans strains, particularly those expressed in the walls of mycelial cells might be useful in the diagnosis of systemic candidiasis. Hence, antigenic similarities among wall proteins and mannoproteins fromC. albicans clinical serotype A and B isolates, were studied using polyclonal (mPAbs) and monoclonal (MAb 4C12) antibodies raised against wall antigens from the mycelial form of a commonC. albicans serotype A laboratory strain (ATCC 26555). Zymolyase digestion of walls isolated from cells of the different strains studied grown at 37°C (germination conditions), released, in all cases, numerous protein and mannoprotein components larger than 100 kDa, along with a 33–34 kDa species. The pattern of major antigens exhibiting reactivity towards the mPAbs preparation was basically similar for all the serotype A and B isolates, though minor strain-specific bands were also observed. The immunodeterminant recognized by MAb 4C12 was found to be absent or present in very low amounts inC. albicans isolates other than the ATCC 26555 strain, yet high molecular weight species similar in size (e.g., 260 kDa) to the wall antigen against which MAb 4C12 was raised, were observed, particularly in wall digests from serotype A strains. Cell surface hydrophobicity, an apparently important virulence factor inC. albicans, of the cell population of each serotype B strain was lower than that of the corresponding serotype A counterparts, which is possibly due to the fact that the former strains exhibited a reduced ability to form mycelial filaments under the experimental conditions used.Abbreviations CSH cell surface hydrophobicity - IIF indirect immunofluorescence     

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.     

Separation and identification of soybean leaf proteins by two-dimensional gel electrophoresis and mass spectrometry

To establish a proteomic reference map for soybean leaves, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 260 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. Fifty-three of these protein spots were identified by searching NCBInr and SwissProt databases using the Mascot search engine. Sixty-seven spots that were not identified by MALDI-TOF-MS analysis were analyzed with liquid chromatography tandem mass spectrometry (LC-MS/MS), and 66 of these spots were identified by searching against the NCBInr, SwissProt and expressed sequence tag (EST) databases. We have identified a total of 71 unique proteins. The majority of the identified leaf proteins are involved in energy metabolism. The results indicate that 2D-PAGE, combined with MALDI-TOF-MS and LC-MS/MS, is a sensitive and powerful technique for separation and identification of soybean leaf proteins. A summary of the identified proteins and their putative functions is discussed.     

Composition of the cell wall of Chlorella fusca

Isolated cell walls of mature Chlorella fusca consisted of about 80% carbohydrate, 7% protein, and 13% unidentified material. Mannose and glucose were present in a ratio of about 2.7:1 and accounted for most of the carbohydrate. Minor components were glucuronic acid, rhamnose, and traces of other sugars; galactose was absent. After treatment with 2 M trifluoroacetic acid or with 80% acetic acid/HNO₃ (10/1, v/v), a residue with a mannose/glucose ratio of 0.3:1 was obtained, probably representing a structural polysaccharide. An X-ray diffraction diagram of the walls showed one diffuse reflection at 0.44 nm and no reflections characteristic of cellulose. Walls from young cells contained about 51% carbohydrate, 12% protein, and 37% unidentified material. Mannose and glucose were also the main sugars; their absolute amounts per wall increased 6–7 fold during cell growth. Walls isolated with omission of a dodecylsulphate/mercaptoethanol/urea extraction step had a higher protein content and, with young walls, a significantly higher glucose and fucose content. These data and other published cell wall analyses show a wide variability in cell wall composition of the members of the genus Chlorella.Abbreviations GLC gas liquid chromatography - TFA trifluoroacetic acid     

Hydroxyproline: its asymmetric distribution in a cell wall glycoprotein

The main structural glycoprotein of the cell wall of Chlamydomonas reinhardii has been cleaved by thermolysin into glycopeptides which have been separated into three fractions, T1, T2 and T3. These correspond to three distinct domains within the glycoprotein, characterized by the asymmetric distribution of both sugars and amino acids, in particular hydroxyproline. T2 is very rich in hydroxyproline (43 mol %) and is highly glycosylated, while T3 is poor in hydroxyproline and contains very little carbohydrate. The results are discussed in terms of cell wall glycoproteins and their function.Abbreviations PAGE polyacrylamide gel electrophoresis - Tris Tris(hydroxymethyl)-methylamine - SDS Sodium dodecyl sulphate - PAS periodic acid-Schiff This is the seventh paper in a series entitled Structure, composition and morphogenesis of the cell wall of Chlamydomonas reinhardii. The last paper in this series was Catt et al. (1978)     

N-Glycyl-glucosamine: A novel constituent in the cell wall of Halococcus morrhuae

The dinitrophenyl-derivative of N-glycylglucosamine was isolated from partially hydrolyzed dinitrophenylated cell walls of Halococcus morrhuae CCM 859. To increase the yield of amino-terminal glycine residues, halococcal cell walls were treated with alkali or acid prior to dinitrophenylation. Authentic N-glycyl-glucosamine was used as a reference substance. A substitution of the amino group of glucosamine by an amino acid has so far not been found in any other wall of a pro- or eucaryotic cell. Since only 5% of the glycine residues reveal an unsubstituted carboxyl group within intact cell walls, glycine may play a role in connecting glycan strands through peptidic linkages between the amino group of glucosamine and the carboxyl group of an uronic acid or gulosaminuronic acid.Abbreviations DNP dinitrophenyl     

Local database and the search program for proteomic analysis of sperm proteins in the ascidian Ciona intestinalis

Separation of proteins by two-dimensional electrophoresis and following mass spectrometry (MS) is now a conventional technique for proteomic analysis. For proteomic analysis of a certain tissue with a limited information of primary structures of proteins, we have developed an analytical system for peptide mass fingerprinting in gene products in the testis of the ascidian Ciona intestinalis. Ciona sperm proteins were separated by two-dimensional gel electrophoresis and the tryptic fragments were subjected to MALDI-TOF/MS. The mass pattern was searched against on-line databases but resulted in less identification of these proteins. We have constructed a MS database from Ciona testis ESTs and the genome draft sequence, along with a newly devised, perl-based search program PerMS for peptide mass fingerprinting. This system could identify more than 80% of Ciona sperm proteins, suggesting that it could be widely applied for proteomic analysis for a limited tissue with less genomic information.     

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.     

The cell wall and secretory proteome of a tobacco cell line synthesising secondary wall

The utility of plant secondary cell wall biomass for industrial and biofuel purposes depends upon improving cellulose amount, availability and extractability. The possibility of engineering such biomass requires much more knowledge of the genes and proteins involved in the synthesis, modification and assembly of cellulose, lignin and xylans. Proteomic data are essential to aid gene annotation and understanding of polymer biosynthesis. Comparative proteomes were determined for secondary walls of stem xylem and transgenic xylogenic cells of tobacco and detected peroxidase, cellulase, chitinase, pectinesterase and a number of defence/cell death related proteins, but not marker proteins of primary walls such as xyloglucan endotransglycosidase and expansins. Only the corresponding detergent soluble proteome of secretory microsomes from the xylogenic cultured cells, subjected to ion‐exchange chromatography, could be determined accurately since, xylem‐specific membrane yields were of poor quality from stem tissue. Among the 109 proteins analysed, many of the protein markers of the ER such as BiP, HSP70, calreticulin and calnexin were identified, together with some of the biosynthetic enzymes and associated polypeptides involved in polymer synthesis. However 53% of these endomembrane proteins failed identification despite the use of two different MS methods, leaving considerable possibilities for future identification of novel proteins involved in secondary wall polymer synthesis once full genomic data are available.     

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.     

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.