JXB Advance Access originally published online on April 10, 2007
Journal of Experimental Botany 2007 58(7):1851-1862; doi:10.1093/jxb/erm049
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH PAPER |
Proteome analysis of grape skins during ripening
1UMR 1219, Faculté d' 
nologie-ISVV, Université V. Ségalen Bordeaux 2, 351 Cours de la Libération, F-33405 Talence, France
2Pole protéomique, Plateforme Génomique Fonctionnelle, Université V. Ségalen Bordeaux 2, Bordeaux, France
* To whom correspondence should be addressed. E-mail: oenobioc{at}oenologie.u-bordeaux2.fr
Received 7 November 2006; Revised 21 February 2007 Accepted 23 February 2007
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionConcluding remarksReferences |
|---|
The characterization of proteins isolated from skin tissue is apparently an essential parameter for understanding grape ripening as this tissue contains the key compounds for wine quality. It has been particularly difficult to extract proteins from skins for analysis by two-dimensional electrophoresis gels and, therefore, a protocol for this purpose has been adapted. The focus was on the evolution of the proteome profile of grape skin during maturation. Proteome maps obtained at three stages of ripening were compared to assess the extent to which protein distribution differs in grape skin during ripening. The comparative analysis shows that numerous soluble skin proteins evolve during ripening and reveal specific distributions at different stages. Proteins involved in photosynthesis, carbohydrate metabolisms, and stress response are identified as being over-expressed at the beginning of colour-change. The end of colour-change is characterized by the over-expression of proteins involved in anthocyanin synthesis and, at harvest, the dominant proteins are involved in defence mechanisms. In particular, increases in the abundance of different chitinase and ß-1,3-glucanase isoforms were found as the berry ripens. This observation can be correlated with the increase of the activities of both of these enzymes during skin ripening. The differences observed in proteome maps clearly show that significant metabolic changes occur in grape skin during this crucial phase of ripening. This comparative analysis provides more detailed characterization of the fruit ripening process.
Key words: Fruit ripening, proteome analysis, skin, Vitis vinifera L
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionConcluding remarksReferences |
|---|
Fruit ripening is accompanied by important changes in cell-wall composition, leading to fruit softening and greater susceptibility to pathogens (Brownleader et al., 1999; Robinson and Davies, 2000; Brummell et al., 2004). Grape ripening is a key growth phase for determining the quality of both wine and table grapes, as it immediately precedes harvesting. Grapes are classified as a non-climacteric fruit on the basis of respiration rates. It has been suggested that abscisic acid may play a role in the ripening process in grapes, as its concentration increases as berries ripen (Antonín et al., 2003; Geny et al., 2004; Okamoto et al., 2004). The onset of maturation begins at ‘veraison’, i.e. the onset of skin colour-change in black cultivars. Anthocyanin pigment accumulation starts in skin cells at veraison and continues through the ripening phase. Ripening is also characterized by an increase in grape size, softening, and cell expansion resulting in water and sugar accumulation in the mesocarp cell vacuoles (Kanellis and Roubelakis-Angelakis, 1993; Coombe and McCarthy, 2000). The characteristics used to judge grapes for commercial harvesting include combinations of changes in skin colour, titratable acidity, soluble solid concentrations, and volatile aroma compounds.
The characterization of skin tissue is apparently an essential parameter for understanding grape ripening, due to its key role in developing the main compounds responsible for wine quality. Furthermore, grape skins are of particular interest as these tissues are metabolically active during development and ripening, and may have an endocrinal function (Kanellis and Roubelakis-Angelakis, 1993). The skin also constitutes a physical barrier between the external environment and the inner tissues, and its integrity is a key factor in preventing pathogen infections. Surprisingly, there is little literature concerning skin as an isolated component during ripening.
Some proteins associated with fruit maturity have recently been detected on two-dimensional electrophoresis gels (Abdi et al., 2002; Barraclough et al., 2004). These proteome studies were generally conducted on whole fruit and no distinction was made between seeds, pulp, and skin. However, they present major differences in composition and structure. In recent years, proteome analysis has been successfully applied to a range of plant tissues: germinating tomato seeds (Sheoran et al., 2005), ripe grape mesocarp (Sarry et al., 2004), and leaves, shoots, and roots of grapevine plantlets (Castro et al., 2005). Plant cells contain many components that may interfere with protein extraction, separation, and purification and the skin has a particularly high content of compounds such as tannins, anthocyanins, terpenes, etc. (Saravanan and Rose, 2004). Considering this, efficient extraction of proteins from grape skins at the different stages of berry development is difficult and, to date, there are no specific protein extraction and separation methods for this isolated tissue.
Therefore, the objectives of this study were to adapt a method for monitoring skin proteome development using 2D-PAGE, and to apply this methodology to identify changes in protein expression during the ripening process.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionConcluding remarksReferences |
|---|
Plant materials
Grapes (Vitis vinifera L. cv. Cabernet sauvignon) were collected from a Bordeaux vineyard (France) during the 2004 growing season. Grape clusters were taken at different stages of ripening: 46 d after anthesis (DAA), corresponding to the onset of skin colour-change (veraison); 55 DAA, when all the grapes in the bunch were completely red; and 94 DAA, corresponding to full maturity. Random samples of seven grape clusters were selected for each stage, immediately frozen in liquid nitrogen and stored at –80 °C prior to protein extraction and anthocyanin determination, except for maturity characterization, requiring fresh grape clusters.
Physiological parameters studies
Fresh berries were wrapped in a double-layer of cheesecloth and crushed by hand. The juice obtained was used for immediate analyses of titrable acidity by titration with NaOH (Guymon and Ough, 1962) and soluble solids by refractometry (Amerine and Ough, 1980). Analytical data are the mean ±SD of four experiments, each experiment consists of 50 berries harvested and 100 berries for berry weight determination.
Phenolic compound extraction and assays
The skins of 10 berries were ground in liquid nitrogen and processed by carrying out two successive extractions in 40 ml of methanol containing 0.1% of 12 N HCl for 3 h, according to Gagne et al. (2006b). Tannin and anthocyanin contents were measured by colorimetric analysis according to Ribereau-Gayon and Stonestreet (1966). Data represent the means ±SD of three replicates.
Preparation of crude enzyme extracts
Protein was extracted by homogenizing frozen berry skin tissues (1 g fresh weight) at 4 °C, for 10 min, in 5 ml of 50 mM sodium acetate buffer, pH 5.0 containing 1 mM PMSF, 0.21% ascorbic acid and 0.1% ß-mercaptoethanol. The homogenate was centrifuged at 20 100 g for 10 min at 4 °C. The supernatant was recovered and clarified with 5% PVPP for 10 min and then centrifuged, as above. The resulting supernatant contained the crude enzyme extracts. Protein concentration was measured by Bradford (1976) method using a protein–dye reagent (Bio-Rad) and BSA as a standard.
Chitinase activity assay
Chitinase activity was assayed using a commercial enzyme substrate, CM-chitin-RBV solution (Loewe), based on the precipitability of a non-degraded, highly polymerized substrate when acid is added. Crude extract (100 µl) was diluted 1:10 and incubated at 37 °C with 150 µl of CM-chitin-RBV and 150 µl of 50 mM sodium acetate buffer pH 5.0 for 30 min. The reaction was stopped by adding 150 µl of 2 M HCl and cooling on ice for 10 min. The mixture was then centrifuged at 15 000 g for 5 min to precipitate the non-degraded substrate. The absorbance was measured at 550 nm against a blank reaction (incubation mixture without crude extract). Specific enzyme activity was defined as ‘absorbance at 550 nm min–1 mg–1 of protein’. Data represent the means ±SD (n=6) of two extractions and have been tested with Student's test.
Assay of ß-1,3-glucanase activity
Total of ß-1,3-glucanase activity was essayed with lamarin as substrate according to Humbert-Goffart et al. (2004). The reaction mixture contained 975 µl of crude extract (diluted 1:10) mixed with 25 µl lamarin (100 mg ml–1). Incubation was carried out at 23 °C for 1 h. Glucanase activity was measured by the release of glucose using the enzymatic test-combination D-glucose/D-fructose Boehringer-Mannheim (R-Biopharm, Darmstadt, Germany) after heat inactivation (100 °C, 1 min) followed by a 10-fold dilution with distilled water. The specific activities were expressed as mmol glucose min–1 mg–1 of protein. Data represent the means ±SD (n=4) of two extractions and have been tested with Student's test.
Preparation of total protein extract
The skin was isolated from 20 grapes after peeling frozen berries and all the proteins were extracted with phenol, followed by ammonium acetate precipitation, according to methods previously described for tomato (Saravanan and Rose, 2004). The corresponding tissue was ground to a fine powder in a mortar with liquid nitrogen, 3 vols of extraction buffer (0.1 M TRIS–HCl pH 7.5, 5 mM EDTA, 1 mM PMSF, 2% ß-mercaptoethanol, 0.1 M KCl, 0.7 M sucrose, 1% PVPP) were added, and the mixture was stirred at 4 °C for 1 h. Then, an equal volume of phenol-TRIS–HCl pH 7.5 was added and the mixture was agitated at 4 °C for 1 h. The homogenate was centrifuged (30 min, 9000 g, 4 °C) and the phenol phase was collected. The aqueous phase was re-extracted with 2 ml+2 ml of extraction medium and phenol, stirred for 30 min and centrifuged. The supernatant was combined with the first extract. This phenol phase was washed three times with an equal volume of extraction buffer, then the extracted proteins were precipitated by adding 5 vols 0.1 M ammonium acetate in methanol, and incubated overnight at –20 °C. The following day, the proteins were collected by centrifugation (30 min, 9000 g, 4 °C) and the pellets washed once with 0.1 M ammonium acetate in methanol, twice with cold methanol, and, finally, twice with ice-cold 80% acetone. The resulting pellet was dried under nitrogen, resuspended in 600 µl IEF solubilization buffer (7 M urea, 2 M thiourea, 25 mM DTT, 4% CHAPS, 1% IPG buffer) and sonicated for 5 min. The resulting mixture was centrifuged (10 000 g, 10 min) to remove insoluble polymers and the supernatant was transferred to a new tube. Protein concentration was determined over six replicated assays, according to the protocol previously described by Ramagli and Rodriguez (1985), using ovalbumine as standard.
Isoelectric focusing and second-dimensional gel
Proteins (600 µg) dissolved in IEF solubilization buffer were loaded by active overnight rehydration on immobiline gradient pH 3–10 drystrip. The IPGphor system (Amercham Biosciences, Uppsala, Sweden) was programmed for 12 h at 30 V, 1 h at 500 V, 1 h at 1000 V, and, finally, 8000 V h–1 to achieve a total of 64 000 V h–1. Strips were equilibrated in two steps with an equilibration solution (50 mM TRIS-HCl, 6 M urea, 2% SDS, 30% glycerol, bromophenol blue) and DTT (50 mM) in the first step and iodoacetamide (1235 mM) in the second. SDS-PAGE was carried out on batches of six gels per stage of development in a buffer (25 mM TRIS, 0.2 M glycine, 0.1% SDS) at 30 W for 30 min, then 90 W. The spots were stained with colloidal Coomassie Blue G-250 and the gels were scanned and analysed using Image Master 2D Platinum (GE Healthcare; Amersham Biosciences).
Image acquisition and analysis
The gels were scanned and the computerized 2-D gels analysed. The spots detected by the software were checked manually and added or removed if necessary. The volume of each spot corresponded to a gross value. The gels corresponding to the three stages in ripening were compared two by two and matched in order to attribute a common spot identity for the same spots derived from different images. Normalized spot volumes of the six replicated gels of the two ripening stages were compared, and were analysed according to Student's test to verify if they were significantly different (P <0.05). Only spots with volumes that varied significantly by at least a ratio of 2 were sequenced.
In-gel digestion
Spots were excised from the gel and washed in H2O/ACN (50:50, v/v). The solvent mixture was removed and replaced with ACN. When the gel pieces had shrunk, ACN was removed and the gel pieces were dried in a vacuum centrifuge. The gel pieces were rehydrated with the trypsin solution (10 ng µl–1 in 50 mM NH4HCO3), at 4 °C for 10 min and finally incubated overnight at 37 °C. The spots were incubated in 50 mM NH4HCO3 at room temperature with rotary shaking for 15 min. The supernatant was collected and a H2O/ACN/HCOOH (47.5:47:5, by vol.) extraction solution was added to the gel pieces for 15 min. This step was repeated and both supernatants were pooled and concentrated in a vacuum centrifuge to a final volume of 25 µl. Digests were finally acidified by adding 1.2 µl acetic acid (5% v/v) and stored at –20 °C.
On-line capillary HPLC nanospray ion trap MS/MS analyses
The peptide mixture was analysed by on-line capillary HPLC (LC Packings, Amsterdam, The Netherlands) coupled to a nanospray LCQ ion trap mass spectrometer. Ten microlitres of peptide digests were loaded onto a 300 µm inner diameter x5 mm C18 PepMapTM trap column (LC Packings, Amsterdam, The Netherlands) at a flow rate of 30 µl min–1. The peptides were eluted from the trap column onto an analytical 75 µm inner diameter x15 cm C18 PepMapTM column (LC Packings, Amsterdam, The Netherlands) with a 5–50% linear gradient of solvent B in 30 min (solvent A was 0.1% formic acid in 5% ACN, and solvent B was 0.1% formic acid in 80% ACN). The separation flow rate was set at 200 nl min–1. The mass spectrometer operated in positive ion mode at a 2 KV needle voltage and a 46 V capillary voltage. Data were acquired in a data-dependent mode alternating a MS scan survey over the range m/z 300-2000 and three MS/MS scans in an exclusion dynamic mode. MS/MS spectra were acquired using a 2 m/z units ion isolation window, a 35% relative collision energy, and a 0.5 min dynamic exclusion duration.
Identification by database research
Data were searched by SEQUEST through Bioworks 3.1 interface (ThermoFinnigan, San Jose, CA) against the NCBI database. DTA files were generated for MS/MS spectra that both reach a minimal intensity (5.104) and a sufficient number of ions (35). The DTA generation allowed the averaging of several MS/MS spectra corresponding to the same precursor ion with a tolerance of 1.4 Da. Spectra from the precursor ion higher than 3500 Da or lower than 500 Da were rejected. The search parameters were as follows: mass accuracy of the peptide precursor and peptide fragments was set to 1.5 and 0.5 Da, respectively. Only b- and y-ions were considered for mass calculation. Oxidation of methionine oxidation (+16) and carbamidomethylation of cysteine (+57) were considered as differential modifications. Two missed trypsin cleavages were allowed. Only peptides which Xcorr was over 1.5 (single charge), 2 (double charge), and 2.5 (triple charge) were retained. In all cases, 
Cn was above 0.1. All protein identifications were based on a minimum of two peptides assignments, except where indicated.
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionConcluding remarksReferences |
|---|
Characterization of the skin tissue during grape development
The grapes collected were characterized by an increase in berry weight, an increase in soluble contents, and a decrease in titratable acidity during ripening (Table 1). These modifications corresponded to a normal evolution profile during this growth phase (Coombe and McCarthy, 2000). In the skin, tannin contents decreased throughout berry ripening, although anthocyanins continuously increased, thereby increasing skin coloration. These findings are in accordance with the results of Gagne et al. (2006a). The ripening period in the Bordeaux region in 2004 can be described as a classic vintage with regard to temperatures and the intrinsic balance of the berry and wine components (http://www.oenologie.u-bordeaux2.fr). In consequence, the stages of ripening chosen in this study presented classic characteristics and the proteome analysis was related to a normal ripening process, without stress conditions.
|
Proteome changes in skin tissue during ripening: comparative analysis of 2-DE maps from ‘beginning colour-change’ and ‘100% colour-change’
Six replicated two-dimensional electrophoresis gels were prepared for each ripening stage. Each gel was stained with colloidal blue, then scanned and analysed. An average of about 700 spots was visible on the gels following this extraction protocol (Figs 1, 2). Comparative data were initially taken at two stages during the colour-change, 10% and 100%, to reflect the onsets of the ripening process and softening. Figure 1 shows examples of 2-DE gels obtained at these two stages in ripening, corresponding to 46 DAA and 55 DAA, respectively. Both samples were extracted rigorously in parallel to minimize variations due to replicate extractions. Spots, whose normalized volumes were in at least a 2:1 ratio between the two stages, were retained for identification. Ratios were calculated on the average volume of the spots on the six repeat gels. Thirty-seven proteins showed a modified expression pattern between the two stages.
|
|
Proteins over-expressed at the ‘beginning colour-change’ stage
The proteome map of the skin at the onset of colour transition indicated that 11 proteins were over-expressed at this stage of development, as compared with the end of colour-change (Fig. 1A). Table 2 lists the proteins identified. Of these, one has already been described in grapevines and three others in Arabidopsis thaliana. The proteins identified are involved in a specific functional distribution (defined here as the distribution of enzyme groups to produce a specific control) for carbohydrate and nitrogen metabolisms, carbon catabolism, stress response, and transcription.
|
Five over-expressed proteins are involved in photosynthesis and carbohydrate metabolisms. Four of them are different Rubisco isoforms. Senescent tissue often shows a decrease in Rubisco. Considering this phenomenon, the senescence of skin tissue may be initiated at the onset of ripening, starting at colour-change. From a cDNA microarray analysis of developing grape berry skin (Waters and Holton, 2005), a group of genes involved in photosynthesis and carbohydrate metabolism are down-regulated at veraison. This validates the results on proteome profile changes during this period. The presence of this group of proteins proves that the berry skin is a source of carbohydrate for the skin itself.
A glutamine synthetase, 3.5 times more abundant at 10% colour-change, was identified. This enzyme is known to catalyse glutamine synthesis using glutamate, which is an important reserve form in plants and a central intermediate in nitrogen incorporation. In the Cabernet sauvignon grape cv., the glutamate content is constant from flowering to colour-change, then disappears (Stines et al., 2000). Skin cells may transform large quantities of glutamate at the onset of ripening, via glutamine synthetase, in response to the increased nitrogen requirement during induction of the second growth phase.
Three small proteins related to stress response (HSP17.7) were identified as heat shock proteins. They were significantly over-expressed 2.5–3-fold compared with the end of colour-change. The corresponding mRNA has been found to accumulate in plant tissues in response to heat shock (>42 °C), abscisic acid or water stress (Coca et al., 1996). These factors regulate gene expression and may be considered transcription factors, but their real role in senescent tissue has not yet been clearly defined (Heller et al., 2000). Over-expression of these proteins may be a response to the onset of abscisic acid accumulation in the skin at this stage in development (Deytieux et al., 2004).
Proteins over-expressed at the end of colour-change
Comparative analysis of the proteome maps from the beginning and end of colour-change indicated that 20 proteins were over-expressed at the full red stage (Fig. 1B; Table 3). Among the excised spots, 18 were identified and 11 of them (61%) had previously been described in grapevines. These proteins apparently have a very different functional distribution from those that are over-expressed at the onset of ripening.
|
Most of the over-expressed proteins are involved in the anthocyanin biosynthesis pathway. Two isoforms of UDP-glucose:flavanoid 3-O-glucosyltransferase (UFGT) were identified; one was over-expressed 4-fold and the other 9-fold. In addition, a leucoanthocyanidin dioxygenase (LDOX), a flavonone 3-hydroxylase (F3H), and a chalcone synthase (CHS) were identified. The over-expression of numerous enzymes involved in anthocyanin synthesis reflects the gradual accumulation of these compounds in skin cells during this period, as shown in Table 1. Because enzymes involved in the anthocyanin biosynthetic pathway are difficult to assay, much of the information about this pathway comes from data from the expression of the genes encoding these enzymes. In Shiraz grape skins, the genes involved in anthocyanin synthesis are expressed both in early berry development and during ripening. Most of the genes in this pathway, such as CHS, F3H, and LDOX, are expressed in flowers and in berries for the first 2–4 weeks post-flowering, although no pigment synthesis is detected at that stage (Boss et al., 1996). Following veraison, there is a co-ordinated induction of all the genes in the pathway, including UFGT, one of the last enzymes of the pathway. In berry skin, Bogs et al. (2006) have recently observed a low expression of three flavonoid hydroxylase genes at the onset of ripening and an increase after veraison. This can cause the abundance of the corresponding proteins at the end of colour-change. This event coincides with the appearance of anthocyanin pigments in the skin. A similar observation has been made by Castellarin et al. (2006) about a correlation between F3H transcript profiles and the kinetic of accumulation of anthocyanins in the skin of ripening red berries. Moreover, abscisic acid has been described as the mediator of the expression of genes involved in the anthocyanin biosynthesis pathway (Hiratsuka et al., 2001). This hormone accumulates in skin tissue between the two stages of ripening that were compared (Gagne et al., 2006a). Therefore, the accumulation of proteins involved in the anthocyanin pathway is probably correlated with the accumulation of this hormone at veraison.
Proteins in the second major group are actively involved in carbon and organic acid metabolisms. Over-expression of these proteins facilitates skin cell energy production. The proteins identified are: (i) an aconitate hydratase (x7.8), which is involved in the glyoxylic acid cycle and plays an important role in lipid accumulation; (ii) a transketolase (x10.7), which provides a link between glycolysis and the pentose phosphate pathway, and also produces nucleotides, aromatic amino acids, and vitamin precursors; (iii) a phosphoenolpyruvate carboxylase; (iv) an oxalylCoA decarboxylase; and (v) an aldehyde dehydrogenase.
Three other proteins are considered protein chaperones, involved in molecular peptide stabilization: (i) cyclophilin, an ubiquitous protein catalysing linked peptide rotation that may constitute a signal during plant development, and also induced in response to abiotic stresses and pathogen infection (Kong et al., 2001); (ii) disulphide-isomerase; and (iii) methionine sulphoxide reductase, a chloroplastic protein involved in protein oxidation protection. Waters et al. (2005) found a cyclophilin cDNA up-regulated at week 12 of berry development, when compared with non-pigmented berry skin at veraison. This gene expression profile may reflect the maximum abundance of cyclophilin in proteome skin at the end of colour-change. As for cycliphilin protein, there are no specific bibliographic data for the real functions of this protein group during fruit ripening, nor in grapes. Their presence indicates that they may play significant roles in a range of signal transduction processes in relation with the time-course of grape-skin ripening. It is also possible that the metabolic disruption that occurred at the onset of ripening is treated by the plant as a stress condition, so that the abundance of this group of proteins increases.
An endo 1,3 ß-glucanase was more abundant at the end of skin coloration. Moreover, Fig. 3 shows that the corresponding enzyme activity also increases during skin pigmentation. These findings indicate a good correlation between the proteome profile and the activity of this enzyme. It is generally believed that endoglucanases fulfil different biological functions (Menu-Bouaouiche et al., 2003). Their role has been classically associated with pathogenesis-related defence. However, this protein has rarely been studied in grapevines. It has been found to be induced in grapevine leaves as a response to pathogen infection with Botrytis (Renault et al., 2000). Furthermore, it has also been suggested that ß-1,3-glucanases play a role in fruit ripening and/or softening (Cosgrove, 2000; Peumans et al., 2000). It may be the case in grape skins, as this study's samples were healthy.
|
A protein involved in cell-wall structuring was also identified: xyloglucan endo transglycosylase (XTH). Messenger RNAs for XTH were detected in grapes during ripening (Nunan et al., 2001). XTH gene expression was also detected before veraison, and increased markedly at veraison, so that its gene expression was closely related to berry softening (Ishimaru and Kobayashi, 2002). These findings, in addition to the increase in the abundance of the corresponding enzyme in skin proteome, suggest that XTH may be important in ripening-related cell-wall changes, leading to grape softening.
Grip61, a ripening-related protein without a clearly identified function, is also over-expressed. Grip61 is a thaumatin-like protein and shows homologies with proteins involved in water-stress or wounding response (Davies and Robinson, 2000). The corresponding transcripts have only been detected in ripe grapes and in the exocarp two weeks after veraison. This study's results indicate the presence of Grip61 earlier in the ripening process, as it is present in skin at 10% colour-change. This indicates that the corresponding transcripts must be present from the onset of ripening. This protein was also identified in a ripe flesh proteome study (Sarry et al., 2004). Further investigations are required to elucidate the function of Grip proteins during pericarp ripening. The presence of several protein groups with no clear functions in fruit ripening illustrates there is much to learn about the molecular and proteomic events that surround ripening in grape skins.
Comparative analysis of 2-DE maps from two stages in ripening: ‘end colour-change’ and ‘maturity’
Figure 2 shows examples of 2-DE gels of grape skins at end colour-change and at maturity, i.e. 55 DAA and 94 DAA, respectively. Proteins were extracted from both samples at the same time and comparative analysis of the normalized volumes of the spots made it possible to detect 25 spots with average volumes that met the 2:1 criterion described above.
Proteins over-expressed at the end of colour-change
The maps of Fig. 2A show that 12 proteins are over-expressed at the end of colour-change compared with full ripeness. These proteins become less abundant in skin tissue as the grapes ripen. Table 4 lists the proteins identified. These proteins are predominantly involved in anthocyanin synthesis and carbon and nitrogen metabolisms.
|
Several enzymes involved in flavonoid biosynthesis are over-expressed: LDOX, CHS, F3H, so that, during skin ripening, levels of these proteins peak at the end of colour-change. These results suggest a co-ordinated control of these proteins in the partitioning of the general phenylpropanoid pathway. A similar observation was made by Waters and Holton (2005) with the expression of numerous genes involved, i.e. CHS and F3H. These genes show similar expression profiles during berry skin development but these authors did not report a co-ordinated expression profile between LDOX cDNA and both CHS and F3H genes. According to Goto-Yamamoto et al. (2002), there are different CHS genes identified in grapes with independent transcriptions, suggesting that the products of CHS activity are utilized by a number of different pathways. Depending on the regime of transcriptional control, each member of the CHS gene family can lead to the production of different metabolites in addition to the anthocyanins and anthocyanidins. In parallel with these findings, these results show a concomitant evolution of three CHS isoforms.
Three SAMS isoforms were identified. This enzyme catalyses biosynthesis of SAM, the major methyl group donor in the transmethylation of proteins, nucleic acids, polysaccharides, and fatty acids. Furthermore, decarboxylated SAM serves as a propolyamine group donor in polyamine synthesis in plants. The presence of several sams genes (Peleman et al., 1989; Schröder et al., 1997) was justified by the metabolic importance of SAM.
ElF-5A, a translation initiation factor formerly known as elf-4D, was over-expressed at the end of colour-change. This protein is involved in initiating protein synthesis and is characterized by the presence of hypusine, a unique modified amino acid formed post-translationally via the transfer and hydroxylation of the butylamino group from the polyamine spermidine. Elf-5A is thought to be the intermediate for polyamine action (Chamot and Kuhlemeier, 1992). In tomato tissues, elF-5A induction has been found to facilitate the translation of the mRNA species required for programmed cell death (Wang et al., 2001), but there is no clear information on the characterization of this protein in grapevines.
Several proteins involved in photosynthesis are more abundant at the end of colour-change: a Rubisco and an oxygen-evolving enhancer protein 3. This finding reflects the fact that chlorophyll pigment levels decrease during skin ripening.
Proteins over-expressed at maturity
Comparative analysis indicates that 13 proteins are over-expressed at maturity (Fig. 2B; Table 5) and eight of them have previously been described in grapevines. The functional distribution of these proteins is specific to the mature stage: most of the over-expressed proteins are involved in defence mechanisms.
|
Numerous pathogenesis-related proteins are more abundant at maturity. Five chitinase isoforms (PR-4), a PR-10, and two ß-1,3-glucanase (PR-2) isoforms are identified. PR-4s are classified as endochitinases, although their specific activities on colloidal chitin vary over 100-fold (Brunner et al., 1998). The PR-10 family is structurally related to ribonucleases and it is hypothesized that these proteins provide protection from viruses (Van Loon and Van Strien, 1999). All these enzymes are present in skin tissue from the onset of ripening in the absence of any pathogen infection and are over-expressed at maturity. Figure 3a shows the changes in chitinase activity in the skins. Enzyme activity increases significantly between the end of colour-change and maturity, in correlation with the increase of the different spot volumes, identified as chitinase isoforms. Chitinases have also been identified on SDS gels of grape juices (Vincenzi and Curioni, 2005), suggesting that these proteins become predominant in the mesocarp, in the absence of pathogens. These results are in accordance with the observation that constitutive expression of a class IV chitinase gene coincides with grape ripening as chitinase activity increases (Robinson et al., 1997). Another important activity related to plant defence, ß-1,3-glucanase, has been assayed (Fig. 3b). This activity increases significantly during skin coloration, as described before, but no change in the activity is observed between the end of colour-change and maturity, although two spots increase in the levels between the two proteome maps compared. These observations suggest that the abundance in the proteome of these proteins, are not necessary well correlated with the enzymatic activity. These proteins are strongly present at the harvest, certainly in the prevention of pathogen attack. These observations mean that grape skin naturally accumulates several PR proteins and causes active chitinase and glucanase activities that increases during ripening, without any pathogen induction. This may be a form of protective mechanism induced during ripening so that the grapes have a pool of defensive enzymes present to respond rapidly in case of a pathogen attack. Terry and Joyce (2004) mentioned that during development of plant organs and in post-harvest, natural resistance generally declines leading to inevitable infection, disease, and ultimately death. In any case, the ripening process of the berries is characterized by an increase in pathogen susceptibility (McClellan and Hewitt, 1973; Commenil et al., 1997). Observations made on berries in the vineyards showed that berries become susceptible to Botrytis infection as they ripen (data not shown). Mature grapes remain prone to grey mould when conditions are favourable for B. cinerea development. This suggests that the natural presence of active PR proteins in skins is not sufficient to protect berries from pathogen infection.
The same XTH identified previously is also more abundant (by a factor of 2.7) at maturity. This protein, involved in cell-wall loosening, gradually increases in abundance in the skin from the onset of ripening to harvest. In addition, XTH gene expression was revealed to be closely related to berry softening (Ishimaru and Kobayaschi, 2002). These findings suggest that XTH plays a crucial role in changes in the skin cell-wall structure during ripening.
Surprisingly, an actin depolymerizing factor enzyme is 4.5-fold over-expressed at maturity. The corresponding gene has previously been found to be induced in grape cuttings during new root formation. This protein specifically acts by rapidly depolymerising and/or severing actin filaments. In addition, it may contribute to the root emergence by reorganizing the cells' actin cytoskeleton during grape harvest (Thomas and Schiefelbein, 2002). Cytoskeletal regulation of plant cell morphogenesis is associated with growth, so that the real contribution of this mechanism in ripe skin is still unclear, although it may be connected with intensive cell expansion during grape ripening.
One protein, identified as an aldo-keto reductase, is 72 times more abundant at full ripeness. This protein belongs to an enzyme superfamily of NAD(P)H-dependent oxidoreductases. The importance of the presence of such enzymes in mature skin calls for further research.
|
Concluding remarks |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionConcluding remarksReferences |
|---|
This first proteomic approach to grape skins provides a great deal of information likely to enhance our understanding of the effects of the ripening process on this specific tissue. A comparative analysis of proteome maps at three stages in ripening, yielding important information for characterizing V. vinifera black cultivar skin maturation, is reported here. From the onset of ripening to the end of colour-change, there is a decrease in proteins involved in energy and general metabolisms. There are also decreases in the levels of several heat shock proteins between the two stages, indicating that it will be useful to focus on their respective roles in the onset of ripening in further studies. At the end of colour-change, a number of enzymes involved in anthocyanin biosynthesis are over-expressed compared with the onset of colour-change or full ripeness. One protein associated with cell-wall loosening increases gradually during ripening, thus confirming the importance of XTH in skin cell-wall modifications. An in-depth study of this activity should enhance our understanding of grape softening. A ß-1,3-glucanase also increases gradually during ripening. The corresponding activity increases significantly during skin coloration. ß-1,3-glucanase is normally classified as a PR protein, but it may also play a role in the fruit ripening process. Towards the end of ripening, the skin also accumulates several chitinase isoforms, and the corresponding activity also increases, indicating that the tissue strengthens its defence mechanism to prevent pathogen attacks. Topics of further studies include a comparative micro-array analysis of developing grape skin. Another topic would be to determine whether the observed protein variations reflect changes in gene expression and changes in some enzyme activities. This is likely to be a powerful tool for characterizing the events during skin ripening, making it possible to identify a specific ripening marker for this tissue.
|
Acknowledgements |
|---|
The authors acknowledge the Conseil Interprofessionnel des Vins de Bordeaux (CIVB) for its financial support. We thank John Almy for the revision of the English.
|
Abbreviations |
|---|
DAA, days after anthesis; CHS, chalcone synthase; F3H, flavonone 3-hydroxylase; LDOX, leucoanthocyanidin dioxygenase; PR, pathogenesis-related; SAMS, S-adenosyl methionine synthase; UFGT, UDP-glucose:flavanoid 3-O-glucosyltransferase; XTH, xyloglucan endotransglycosylase.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionConcluding remarksReferences |
|---|
Abdi N, Holford P, McGlasson B. Application of two-dimensional gel electrophoresis to detect proteins associated with harvest maturity in stonefruit. Postharvest Biology and Technology (2002) 26:1–13.[CrossRef][Web of Science]
Amerine MA, Ough CS. Methods for analysis of musts and wines (1980) New York: John Wiley and Sons.
Antonin MC, Baigorri H, De Luis I, Aguirrezabal F, Geny L, Broquedis M, Sanchez-Diaz M. ABA during reproductive development in non-irrigated grapevines (Vitis vinifera L. cv. Tempranillo). Australian Journal of Grape and Wine Research (2003) 9:169–176.[Web of Science]
Barraclough D, Obenland D, Laing W, Carroll T. A general method for two-dimensional protein electrophoresis of fruit samples. Postharvest Biology and Technology (2004) 32:175–181.[CrossRef][Web of Science]
Bogs J, Ebadi A, McDavid D, Robinson SP. Identification of the flavonoid hydroxylases from grapevine and their regulation during fruit development. Plant Physiology (2006) 140:279–291.
Boss PK, Davies C, Robinson SP. Analysis of the expression of anthocyanin pathway genes in developing Vitis vinifera L. cv. Shiraz grape berries and the implication for pathway regulation. Plant Physiology (1996) 111:1059–1066.[Abstract]
Bradford M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry (1976) 72:248–254.[CrossRef][Web of Science][Medline]
Brownleader MD, Jackson P, Pantelides AT, Sumar S, Trevan M, Dey PM. Molecular aspects of cell wall modifications during fruit ripening. Critical Revue of Food Science and Nutrition (1999) 39:149–164.[CrossRef]
Brummell DA, Dal Cin V, Crisosto CH, Labavitch JM. Cell wall metabolism during maturation, ripening and senescence of peach fruit. Journal of Experimental Botany (2004) 55:2029–2039.
Brunner F, Stintzi A, Fritig B, Legrand M. Substrate specificities of tobacco chitinases. The Plant Journal (1998) 14:225–234.[Web of Science][Medline]
Castellarin SD, Di Gaspero G, Marconi R, Nonis A, Peterlunger E, Paillard S, Adam-Blondon F, Testolin R. Colour variation in red grapevines (Vitis vinifera L.): genomic organization, expression of flavonoid 3'-hydroxylase, flavonoid 3',5'-hydroxylase genes and related metabolite profiling of red cyaniding-/blue delphinidin-based anthocyanins in berry skin. BMC Genomics (2006) 7:1–12.[CrossRef][Web of Science][Medline]
Castro AJ, Carapito C, Zorn N, Magné C, Leize E, Van Dorsselaer A, Clément C. Proteomic analysis of grapevine (Vitis vinifera L.) tissues subjected to herbicide stress. Journal of Experimental Botany (2005) 56:2783–2795.
Chamot D, Kuhlemeier C. Differential expression of genes encoding the hypusine-containing translation initiation factor, elF-5A, in tobacco. Nucleic Acids Research (1992) 4:665–669.
Coca MA, Almoguera C, Thomas TL, Jordano J. Differential regulation of small heat-shock genes in plant: analysis of a water-stress-induced and developmentally activated sunflower promoter. Plant Molecular and Biology (1996) 3:863–876.
Commenil P, Brunet L, Audran JC. The development of the grape berry cuticle in relation to susceptibility to bunch rot disease. Journal of Experimental Botany (1997) 48:1599–1607.
Coombe BG, McCarthy MG. Dynamics of grape berry growth and physiology of ripening. Australian Journal of Grape and Wine Research (2000) 6:131–135.
Cosgrove DJ. Expansive growth of plant cell walls. Plant Physiology and Biochemistry (2000) 38:109–124.[CrossRef][Web of Science][Medline]
Davies C, Robinson SP. Differential screening indicates a dramatic change in mRNA profiles during grape berry ripening. Cloning and characterization of cDNAs encoding putative cell wall and stress response proteins. Plant Physiology (2000) 122:803–812.
Deytieux C, Geny L, Donèche B. Relation between hormonal balance and polygalacturonase activity in grape berry. Acta Horticulturae (2004) 682:163–170.
Gagne S, Esteve K, Deytieux C, Saucier C, Geny L. Influence of abscisic acid in triggering ‘veraison’ in grape berry skins of Vitis vinifera L. cabernet sauvignon. International Journal of Vine and Wine Science (2006a) 40:7–14.
Gagne S, Saucier C, Geny L. Composition and cellular localization of tannins in Cabernet sauvignon skins during growth. Journal Agricultural of Food and Chemistry (2006b) 54:9465–9471.[CrossRef]
Geny L, Deytieux C, Donèche B. Importance of hormonal profile on the onset of ripening in grape berries of Vitis vinifera L. Acta Horticulturae (2004) 682:99–105.
Goto-Yamamoto N, Wan GH, Masaki K, Kobayashi S. Structure and transcription of three chalcone synthase genes of grapevines (Vitis vinifera). Plant Science (2002) 162:867–872.
Guymon JF, Ough CS. A uniform method for total acid determination in wines. American Journal of Enology and Viticulture (1962) 13:40–45.
Heller R, Esnault R, Lance C. Perception et transduction des signaux hormonaux. In: Physiologie végétale; développement—Dunod, ed. (2000) Paris: Masson. 135–164.
Hiratsuka S, Onodera H, Kawai Y, Kubo T, Itoh H, Wada R. ABA and sugar effects on anthocyanin formation in grape berry cultured in vitro. Science Horticulturae (2001) 90:121–130.[CrossRef]
Humbert-Goffart A, Saucier C, Moine-Ledoux V, Canal-Llaubères RM, Dubourdieu D, Glories Y. An assay for glucanase activity in wine. Enzyme and Microbial Technology (2004) 34:537–543.[CrossRef][Web of Science]
Ishimaru M, Kobayaschi S. Expression of a xyloglucan endo-transglycosylase gene is closely related to grape berry softening. Plant Science (2002) 162:621–628.
Kanellis AK, Roubelakis-Angelakis KA. Grape. In: Biochemistry of fruit ripening—Seymour G, Taylor J, Tucker G, eds. (1993) London: Chapman and Hall. 189–234.
Kong HY, Lee SC, Hwang BK. Expression of pepper cyclophilin gene is differentially regulated during the pathogen infection and abiotic stress conditions. Physiological Molecular Plant Pathology (2001) 59:189–199.[CrossRef]
McClellan WD, Hewitt WD. Early Botrytis of grapes: time infection and latency of Botrytis cinerea Pers. in Vitis vinifera. Phytopathology (1973) 63:1151–1157.[Web of Science]
Menu-Bouaouiche L, Vriet C, Peumans WJ, Barre A, Van Damme EJM, Rougé P. A molecular basis for endo-ß-1,3-glucanase activity of the thaumatin-like proteins from edible fruits. Biochimie (2003) 85:123–131.[Medline]
Nunan KJ, Davies C, Robinson SP, Fincher GB. Expression of cell wall-modifying enzymes during grape berry development. Planta (2001) 214:257–264.[Web of Science][Medline]
Okamoto G, Kuwamura T, Hirano K. Effects of water deficit stress on leaf and berry ABA and berry ripening in Chardonnay grapevines (Vitis vinifera). Vitis (2004) 43:15–17.[Web of Science]
Peleman J, Saito K, Cottyn B, Engler G, Seurinck L, Van Montagu M, Inzé D. Structure and expression analyses of the S-adenosylmethionine synthetase gene family in Arabidopsis thaliana. Gene (1989) 84:359–369.[CrossRef][Web of Science][Medline]
Peumans WJ, Barre A, Derycke V, Rougé P, Zhang W, May GD, Delcour JA, van Leuven F, van Damme AJM. Purification, characterization and structural analysis of an abundant ß-1,3-glucanase from banana fruit. European Journal of Biochemistry (2000) 267:1188–1195.[Web of Science][Medline]
Ramagli LS, Rodriguez LV. Quantification of microgram amounts of protein in two-dimensional polyacrylamide gel electrophoresis sample buffer. Electrophoresis (1985) 6:559–563.[CrossRef][Web of Science]
Renault AS, Deloire A, Letinois I, Kraeva E, Tesniere C, Ageorges A, Redon C, Bierne J. ß-1,3-glucanase gene expression in grapevine leaves as a response to infection with Botrytis cinerea. American Journal of Enology and Viticulture (2000) 51:81–87.
Ribereau-Gayon P, Stonestreet E. Le dosage des anthocyanes dans le vin rouge. Chimie Analytique (1966) 48:188–195.[Web of Science]
Robinson SP, Davies C. Molecular biology of grape berry ripening. Australian Journal of Grape and Wine Research (2000) 6:175–188.
Robinson SP, Jacobs AK, Dry IB. IV chitinase is highly expressed in grape berries during ripening. Plant Physiology (1997) 114:771–118.[Abstract]
Saravanan RS, Rose JKC. A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues. Proteomics (2004) 4:2522–2532.[CrossRef][Web of Science][Medline]
Sarry JE, Sommerer N, Sauvage FX, Bergoin A, Albagnac G, Romieu C. Grape berry biochemistry revisited upon proteomic analysis of the mesocarp. Proteomics (2004) 4:201–215.[CrossRef][Web of Science][Medline]
Schröder G, Eichel J, Breini S, Schröder J. Three differentially expressed S-adenosylmethionine synthetases from Catharanthus roseus: molecular and functional characterization. Plant Molecular Biology (1997) 33:211–222.[CrossRef][Web of Science][Medline]
Sheoran IS, Olson DJH, Ross ARS, Sawhney VK. Proteome analysis of embryo and endosperm from germinating tomato seeds. Proteomics (2005) 5:3752–3764.[CrossRef][Web of Science][Medline]
Stines AP, Grubb J, Gockowiak H, Henschke PA, Hoj PB, Van Heeswijck. Proline and arginine accumulation in developing berries of Vitis vinifera L. in Australian vineyards: influence of vine cultivar, berry maturity and tissue type. Australian Journal of Grape and Wine Research (2000) 6:150–158.
Terry LA, Joyce DC. Elicitors of induced disease resistance in postharvest horticultural crops: a brief review. Postharvest Biology and Technology (2004) 32:1–13.[CrossRef][Web of Science]
Thomas P, Schiefelbein J. Cloning and characterization of an actin depolymerising factor gene from grape (Vitis vinifera L.) expressed during rooting in stem cuttings. Plant Science (2002) 162:283–288.
Van Loon LC, Van Strien EA. The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiological and Molecular Plant Pathology (1999) 55:85–97.[CrossRef][Web of Science]
Vincenzi S, Curioni A. Anomalous electrophoretic behaviour of a chitinase isoform from grape berries and wine in glycol chitin-containing sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels. Electrophoresis (2005) 26:60–63.[CrossRef][Web of Science][Medline]
Wang TW, Lu L, Wang D, Thompson JE. Isolation and characterization of senescence-induced cDNAs encoding deoxyhypusine synthase and eukaryotic translation initiation factor 5A from tomato. Journal of Biological Chemistry (2001) 276:17541–17549.
Waters LE, Holton TA. cDNA microarray analysis of developing grape (Vitis vinifera cv. Shiraz) berry skin. Functional Integrative Genomics (2005) 5:40–58.[CrossRef][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. Zhang, H. Ma, J. Feng, L. Zeng, Z. Wang, and S. Chen Grape berry plasma membrane proteome analysis and its differential expression during ripening J. Exp. Bot., August 1, 2008; 59(11): 2979 - 2990. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Troggio, S. Vezzulli, M. Pindo, G. Malacarne, P. Fontana, F. M. Moreira, L. Costantini, M. S. Grando, R. Viola, and R. Velasco ASEV Honorary Research Lecture 2007: Beyond the Genome, Opportunities for a Modern Viticulture: A Research Overview Am. J. Enol. Vitic., June 1, 2008; 59(2): 117 - 127. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||