JXB Advance Access published online on June 11, 2008
Journal of Experimental Botany, doi:10.1093/jxb/ern156
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RESEARCH PAPER |
Grape berry plasma membrane proteome analysis and its differential expression during ripening
1College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China
2College of Agriculture and Biotechnology, China Agricultural University, Beijing, China
3College of Biological Sciences, China Agricultural University, Beijing, China
To whom correspondence should be addressed. E-mail: swchen{at}cau.edu.cn
Received 14 March 2008; Revised 4 May 2008 Accepted 6 May 2008
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Abstract |
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High purity berry plasma membranes (PMs) of Vitis vinifera L. cv. Cabernet Sauvignon were isolated by two-phase partitioning of microsome fractions at different stages of berry ripening. PM proteins resolvable by the detergent cocktail of CHAPS and ASB-14 were separated by two-dimensional electrophoresis. A total of 119 protein spots from pre-véraison berry PMs on 2-D gels detected with silver staining were subjected to MALDI-TOF mass spectrometry analysis. Sixty-two spots were identified as putative PM proteins, with 1–6 predicted transmembrane helices, including true PM proteins such as ATP synthase, ABC transporters, and GTP-binding proteins reported in plants. They were then grouped into eight functional categories, mainly involved in transport, metabolism, signal transduction, and protein synthesis. Another 11 spots were identified as proteins of unknown function. The véraison and post-véraison samples stained 98 and 86 spots on the gels, respectively. During the berry ripening process, total PM protein content gradually decreased. Among all identified proteins, 12 showed significant differences in terms of their relative abundance. Increasing ubiquitin proteolysis and cytoskeleton proteins were observed from pre-véraison to post-véraison. Zeatin O-glucosyltransferase peaked at véraison, while ubiquitin-conjugating enzyme E2-21 was down-regulated at this stage. This proteome research provides the first information on PM protein characterization during the grape berry ripening process.
Key words: Grape berry, MALDI-TOF-MS, plasma membrane, proteomics, two-dimensional electrophoresis
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The grapevine (Vitis vinifera L.) is a major economic crop for its use in wines, as raisins, and as the fruit itself. Grapes are classified as a non-climacteric fruit, and berry development can be divided into three phases on the basis of berry growth. Ripening is characterized by a number of changes, including an increase in grape volume, the development of skin colour (in red grapes), softening, the catabolism of organic acids, the formation of compounds for flavour and aroma, and the intense accumulation of soluble sugars (Coombe and McCarthy, 2000; Terrier et al., 2001). Dramatic metabolic changes take place in the cells during the different berry development stages, especially before and after véraison. As a boundary of cells, the plasma membrane (PM) is thought to play a critical role in terms of its barriers, channels, exchanges, and communication in the cell processes. Many essential functions of the PMs are carried out by their proteinaceous complexes, including molecular transport, cell–cell interactions, ligand binding, signal transduction, and environmental sensing (Ephritikhine et al., 2004; Komatsu et al., 2007).
To understand the nature of the fundamental cell process, there is a need to investigate the protein profiles of cells, tissues, and organs at the specific development stages. In recent years, proteomics-based technology has been successfully applied to grapevine in different cell processes and pathways, such as herbicide reaction (Castro et al., 2005), water deficit and salt stress responses (Vincent et al., 2007; Jellouli et al., 2008), single gene transformation-induced protein changes (Sauvage et al., 2007), and somatic embryogenesis-induced protein changes (Marsoni et al., 2008). The first two-dimensional electrophoresis (2-DE) analysis of the total polypeptides in ripe red grapevine berries (Tesnières and Robin, 1992) has launched proteomic studies on grape berries. Sarry et al. (2004) identified 67 mesocarp proteins of ripening berry of different grape genotypes, Vincent et al. (2006) optimized the method of protein extraction of grape berry for proteomic analysis. More recently, Giribaldi et al. (2007) investigated the protein expression during different stages of grape berry development, and Deytieux et al. (2007) analysed the proteins changed in berry skins during ripening. These studies improved our understanding of proteomic changes of grapevine. However, all the above publications up to now were limited to total proteins in grape; proteomic analysis of grape berry at the subcellular compartment level has not been reported.
The PM is particularly important for all biological processes. However, proteomic studies on PM proteins have so far been rare. The limited information on PM proteomics, including those of grape berry, is mainly due to difficulties in PM protein analysis, such as physicochemical heterogeneity of these proteins, the fact that many hydrophobic proteins cannot be solubilized in isoelectric focusing (IEF) sample buffers, and low abundance proteins are beyond the detection limits of standard proteomics techniques (Ephritikhine et al., 2004). Despite all of these difficulties, in the past few years, several PM protein proteomics studies have been applied to different plants and algae, such as Arabidopsis (Santoni et al., 2000, 2003; Alexandersson et al., 2004; Marmagne et al., 2004), rice (Tanaka et al., 2004; Chen et al., 2007), Spinacia (Kjell et al., 2004), tobacco (Mongrand et al., 2004), and Synechocystis (Huang et al., 2002, 2006). The PM is usually separated by two-phase partitioning from the microsome fraction of tissues homogenates. With this method, Alexandersson et al. (2004) isolated the Arabidopsis PM; 238 putative PM proteins involved in transport, signal transduction, membrane trafficking, and stress responses were identified. Grape berry contains very high concentration of polyphenols, proanthocyanidins, and condensed tannins, which add extra difficulties in the study of PM proteins by interfering with the PM separation and protein extraction steps. To date, there is no report on the proteomics of grape berry PMs.
In this study, the PM fractions were prepared by modified two-phase partitioning of the microsome fractions from berries of three development stages. 2-DE together with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis was used to construct a reference proteome map of the berry PM and to reveal changes during the ripening process.
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Materials and methods |
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Plant materials
Berries of V. vinifera L. cv. Cabernet Sauvignon were collected from Sino-French Demonstration Farm, Huailai, China, in 2007. Fifty berries from different bunches were measured every 5 days after flowering (DAF) to record the growth rate and soluble sugar content. Berries were harvested at stage II before véraison, that is 50 DAF (Sa, sample a), at véraison (75 DAF, Sb, sample b), and at stage III, after véraison (95 DAF, Sc, sample c) for PM isolation and proteome analysis. Random samples of at least 30 grape clusters were selected for each sample. The berries were deseeded, immediately frozen in liquid nitrogen, and stored at –80 °C for further use.
Preparation of PM fractions
The PMs of grape berry were isolated by differential centrifugation to obtain the PM-rich microsomal membrane fractions, followed by two-phase partitioning to purify the PM fractions, as described by Gallagher and Leonard (1982) and Soudain et al. (1992) with modification. In brief, the frozen deseeded berries were pulverized with a steel roll-on mechanical grinder half-filled with liquid nitrogen. The frozen powder (50 g) was resuspended in 250 ml of buffer containing 0.25 M sucrose, 3 mM EDTA, 10% (v/v) glycerol, 0.4% (w/v) bovine serum albumin (BSA), 0.2% (w/v) polyvinylpolypyrrolidone (PVPP), 1 mM phenylmethylsuphonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), 5 mM vitamin C, 80 mM TRIS, and 5 mM BTP-MES, pH 9.0. After filtering through two layers of nylon, the combined homogenate was centrifuged at 10 000 g for 15 min, and the supernatant was centrifuged at 100 000 g for 1 h. The pellet was resuspended in a buffer containing 0.25 M sucrose, 10% (v/v) glycerol, 5 mM TRIS-MES pH 7.8. The crude PM fraction was further purified with the two-phase partition system containing 6.2% (w/v) polyethylene glycol (PEG)-3350, 6.2% (w/v) dextran T-500, 0.25 M sucrose, 5 mM KCl, and 5 mM phosphorus buffer pH 7.8. After being partitioned by centrifugation at 1500 g for 5 min, repeated three times, the upper phases from three centrifugations were recovered and centrifuged at 100 000 g for 1 h. The pellet was washed with ddH2O twice. Marker enzyme activity assays were conducted as previously described (Gallagher and Leonard, 1982). The resulting pellet was resuspended in IEF solubilization buffer [7 M urea, 2 M thiourea, 1% (w/v) CHAPS, 1% (w/v) ASB-14, 1% (w/v) DTT, and 1% (v/v) IPG buffer]. The suspension was incubated at room temperature for 2 h with shaking every 10 min, followed by centrifugation at 20 000 g for 1 h to remove the insoluble polymers. The membrane protein concentration was assessed using a 2-D Quant Kit (GE Healthcare; Amersham Biosciences), according to the manufacturer's instructions, using BSA as the standard.
2-DE and image analysis
Total membrane protein (100 µg) was loaded onto GE Healthcare 18 cm IPG strips (pH 4–7 NL) (GE Healthcare; Amersham Biosciences) by active overnight rehydration. The IEF by the IPGphor system (GE Healthcare; Amersham Biosciences) was programmed as follows: 200 V for 2 h, 500 V for 1 h, 1000 V for 1 h, ramping until 8000 V for 2 h, 8000 V for 7 h, finally to achieve a total of 66 kVh. Strips were then consecutively equilibrated twice by shaking in 50 mM TRIS-HCl, 6 M urea, 10% (v/v) glycerol, 2% (w/v) SDS, and trace amounts of bromophenol blue. The first equilibration was with addition of 2% (w/v) DTT (15 min) and the second was with 2.5% (w/v) iodoacetamide (15 min). The second-dimension SDS–PAGE was performed in 12.5% acrylamide gels using Ettan Dalt twelve (GE Healthcare; Amersham Biosciences), 0.5 h at 1 W per gel, then at 6 W per gel until the dye front reached the gel bottom. Proteins were detected by silver nitrate stain, and scanned at 300 dpi (Perfection 4990 scanner, Epson). Image elaboration and analysis were carried out with the ImageMaster 2-D Platinum version 5 software (GE Healthcare; Amersham Biosciences). The gels corresponding to the three berry samples (Sa, Sb, and Sc) were compared two by two and matched in order to attribute a common spot identify to the same spots derived from different images. The volume of each spot from three replicate gels was normalized against the total spot volume, quantified, and subjected to analysis of variance (ANOVA) (P <0.05). Spots of varying intensities were excised manually.
In-gel digestion
Protein spots were excised and transferred into a 0.6 ml tube, and digested following the method of Shevchenko et al. (1996). For proteins of lower abundance, the coordinated spots from all the replicate gels were collected, pooled, and digested in a single tube. The protein spots were washed twice with MilliQ water, destained twice with 100 mM sodium thiosulphate and 30 mM potassium ferricyanide, and then rinsed with 25 mM ammonium bicarbonate in 50% (v/v) acetonitrile (ACN). Gel pieces were dehydrated with 100% ACN, dried under vacuum on a centrifugal evaporator, rehydrated in digestion solution composed of 25 mM ammonium bicarbonate, 1 mM CaCl2, and 0.015 mg ml–1 trypsin for 45 min at 4 °C, then incubated overnight at 37 °C. The resulting tryptic fragments were eluted by diffusion into 50% (v/v) ACN and 0.1% (v/v) trifluoroacetic acid (TFA) for mass spectrometric analysis.
Protein identification by MALDI-TOF MS
MS analysis was performed using an AUTOFLEX II TOF-TOF (Bruker Daltonics, Germany). Digested protein samples were spotted on a 384-MPT AnchorChip plate (1 µl) twice and then 0.5 µl of recrystallized 
-cyano-4-hydroxycinnamic acid (CHCA) matrix (Bruker Daltonics) dissolved in 0.1% (v/v) TFA and 70% (v/v) ACN was spotted once on top. Each sample spot was desalted twice with 0.1% (v/v) TFA. The mass spectrometer was operated under 19 kV accelerating voltage in the reflection mode and an m/z range of 600–4000. The peptide ions generated by autolysis of trypsin (with m/z 2163.333 and 2273.434) were used as internal standards for calibration. The list of peptide masses from each peptide map fingerprinting (PMF) was saved for database analysis.
Data analysis
The peptide lists generated by MASCOT software were searched in the non-redundant (nr) protein database of NCBI or the SWISS-PROT database. The search parameters were as follows: Viridiplantae (green plant) for taxon consideration, oxidation of methionine, and carbamidomethylation of cysteine were specified as variable and fixed modifications, one miscleavage of trypsin, and 0.2 Da mass tolerances for peptide. The identification of the proteins was repeated at least once using spots from different gels. The identified proteins were assigned to MIPS funcats (http://www.mips.gsf.de/projects/funcat), according to the annotation described in the literature. The prediction of transmembrane helices of identified proteins was performed using the TMpred program (http://www.ch.embnet.org/software/TMPRED_form.html).
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Results and discussion |
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Isolation of PMs from microsome of grape berry homogenate extract
Three grape berry samples were characterized by progressively increased single berry weight and sugar content (Fig. 1). The isolation of PM fractions from mature grape berries is much more difficult than from other fruits such as apple (Zhang et al., 2001), muskmelon (Lester et al., 1993), and tomato (Soudain et al., 1992). Besides the remarkably higher concentration of sugars, a large amount of specific polyphenols and very high acidity increased the interference, limiting the efficiency of PM extraction and separation from berry samples. To overcome these difficulties, a suitable grounding buffer was modified from previous work (Zhang et al., 2001) to improve the protection of the membrane proteins. For this, 80 mM TRIS and 5 mM BTP-MES buffer were selected, with addition of BSA to reduce the adsorptive activity of proanthocyanidins and condensed tannins, and extra PVPP to prevent the polyphenol oxidase, with differential centrifugation successfully providing a high quality microsomal membrane preparation. The PMs were purified from the grape berry microsome fractions using a two-phase partitioning consisting of PEG and dextran. The purity of the PM was estimated by enzymatic assays; the major part (
90%) of the total ATPase activity associated with the PM fraction was vanadate-sensitive and the enrichment in PM was estimated at 5–6 times compared with the microsome membrane. The results indicated that a high quality PM fraction was obtained from the grape berries (Table 1).
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Identification of protein spots
The low solubility and low abundance of proteins associated with the grape berry PM is the major challenge for 2-DE. Different chaotropes and detergents were tested to enhance the solubility of PM proteins. Santoni et al. (2000) and Huang et al. (2006) introduced ASB-14 as a powerful surfactant to improve the solubilization of hydrophobic proteins. The solubilization efficiency depends not only on the nature of membrane proteins, but also on the lipid content and the sample preparation prior to final solubilization (Santoni et al., 2000). Many of the plasma membrane proteins have several membrane-spanning domains, deeply embedded in lipid membranes, which increases the difficulty of resolving them in the sample preparation (Van Wijk, 2001). It was found that ASB-14 was also effective for grape berry. When a urea–thiourea chaotropic mixture was used, 1% ASB-14 and 1% CHAPS became more efficient. Most of the grape berry PM proteins in the IEF on the immobilized linear pH gradient gel strip were distributed in the range pH 4–7. For the second dimension, 12.5% SDS–PAGE was used. A total of 119 protein spots were detected across the three replicate gels of the Sa sample, as shown in Fig. 2. Along with the fruit ripening, the three PM samples showed a consecutive decrease in the number of protein spots, from 119 (Sa) to 98 (Sb) and then 86 (Sc) (Fig. 3). A total of 200 spots were collected from the three stage samples and submitted to MALDI-TOF analysis; 62 proteins were successfully identified by PMFs via MALDI-TOF MS and MASCOT database searching (Table 2).
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For all the MALDI-TOF analysis samples, data were collected only when the spot parameters corresponded to the following criteria: (i) the deviation between the experimental and theoretical peptide masses was <50 ppm; (ii) at least four predicted peptide masses matched the observed mass for an identification to be considered valid; and (iii) the coverage of protein sequences by matching peptides reached a minimum of 10%. In addition, a MOWSE score was obtain from MASCOT, which rates scores as significant if they are above the 95% significance threshold (P <0.05) (Marmagne et al., 2004; Komatsu et al., 2007). The 62 identified spots were classified into eight groups by their function according to the annotation in the MIPS Arabidopsis thaliana database (Fig. 4). Most of the identified proteins were involved in transport (28%), metabolism (18%), signal transduction (11%), and protein synthesis or fate (13%). About 18% (11 of 62) of the proteins identified in the study were considered as unknown proteins, which have no match with proteins of already known function in other plants. The number of predicted transmembrane helices of the identified proteins is listed in Table 2. As predicted by the TMpred program, 21 of the identified proteins have one transmembrane helix, 14 have two transmembrane helices, 12 have three transmembrane helices, and the rest have four or six transmembrane helices (Table 2). This indicates that the majority of the resolved and identified proteins are peripheral PM proteins.
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Transport proteins
As expected for the PM of the Sa sample, 28% (18 of 62) of the identified protein spots were involved in transport, such as ATP synthase, ATP-binding cassette (ABC) transporter, and sugar transporter. The ATP synthase belongs to the ATPase family, produces ATP from ADP in the presence of a proton gradient across the membrane, and the
chain is regulatory. In the present research, ATP synthase was identified in spots 2, 15, 50, 51, 52, and 53. The presence of ATP synthase, a respiratory chain component, on the PM was also reported in cyanobacteria (Norling et al., 1997; Huang et al., 2002, 2006). In grapes, Giribaldi et al. (2007) identified two ATP synthases from total protein extracts of grape berry by 2-DE and MALDI-TOF, and found that the ATP synthases chain decreased after véraison when berry chloroplasts disappear. In the present study, the ATP synthases were not differentially expressed at three development stages during ripening, which could be important to provide a stable energy resource for the function of the PM. Vacuolar ATP synthase catalytic subunit A (spots 54–57) was found in high abundance in the gels. There are at least three publications reporting the presence of V-ATPase in plant PM studies, that is Arabidopsis (Alexandersson et al., 2004; Marmagne et al., 2004) and tobacco (Mongrand et al., 2004). V-ATPase was also found in the tonoplast of Arabidopsis (Shimaoka et al., 2004). Sarry et al. (2004) reported that an -subunit of V-ATP synthase was identified in the soluble proteins of grape berry. As there was a low level of contamination by other membranes according to the present tests, V-ATPase could be an overlap protein which has more than one cell membrane localization. Two protein spots (spots 9 and 10) were identified as ribose import ATP-binding protein (Table 2 and Figure 2), a type of ABC transporter. This transporter is involved in ribose import and is responsible for energy coupling with the transport system. To our knowledge, this is the first report of an ABC transporter in the PM of grape berry. ABC transporters are the largest family of membrane transport proteins, they are ubiquitous and powerful for the translocation of multisubstrates across the membrane, often against a concentration gradient by hydrolysing ATP. Besides functioning as primary pumps, some also modulate the activity of heterologous channels (Theodoulou, 2000). Although most ABC transporters characterized in plants have been localized to the tonoplast, Alexandersson et al. (2004) reported that two ABC transporters (PDR8 and MDR11) were in the Arabidopsis PM, and indicated that members of both PDR8 and MDR11 subfamilies of ABC transporter may be located in the PM. Giribaldi et al. (2007) also found that ABC transporters were increased after véraison in ripening berries, but could not indicate their locations. There were 2202 entries of ABC transporters in the GenBank dbEST; only five entries came from grapevine, with two of them found in the berry (dbEST Id: 7948072 and 6120368).
The mechanism of sugar accumulation in ripening berries is still not very clear. According to the present results, no sucrose or hexose transporter was identified in the berry PM. A putative UDP-galactose transporter MSS4 was identified (spot 48). However, the localization of UDP-galactose transporters was only reported in the endoplasmic reticulum and Golgi apparatus in plants; its presence could be due to contamination by other membrane systems. The observed pI was also lower than the expected value, and the difference may reflect the modification of this protein.
Some less well characterized transport proteins were also found. Patellin-3 (spot 8) is a carrier protein that may be involved in membrane trafficking events associated with cell plate formation during cytokinesis, binds to some hydrophobic molecules such as phosphoinositides, and promotes their transfer between different cellular sites. Peterman et al. (2004) reported that patellin was recruited from the cytoplasm to the expanding and maturing cell plate. Ras-related protein Rab7 (spot 38) belongs to the small GTPase superfamily, involved in protein transport, localized to the cell membrane, probably in vesicular trafficking (Drew et al., 1993). The presence of these proteins in the berries at all three stages suggested that active cellular processes were carried out during the rapid berry mesocarp cell expansion and dramatic sugar accumulation.
Proteins associated with signal transduction
Three different types of GTP-binding protein have been found in the Sa sample: SAR1A (spots 19 and 20), Rac-like GTP-binding protein 1 (spots 21–24), and yptV5 (spot 49) (Fig. 2, Table 2). The classification of small GTP-binding proteins is based on the identification in the sequences of conserved domains such as RAS, RAB, RAF, and SAR. Ras-like GTPases are involved in upstream signalling for mitogen-activated protein kinase cascade activation, and also involved in sphingolipid elicitor (SE)-dependent defence signalling (Lieberherr et al., 2005). Tthe Rac-like GTP-binding protein 1 was identified in four protein spots. The small GTP-binding proteins play key roles in the signal pathway, vesicular trafficking, and targeting to the PM. Marmagne et al. (2004) identified several GTP-binding proteins in the PM of Arabidopsis, and reported a putative site of prenylation in their C-terminus; the two ADP-ribosylation factors are predicted to be myristoylated. SAR1A and Rac-like GTP-binding protein were abundant in pre-véraison berries and decreased to a very low level in véraison and post-véraison berries. SAR1A is involved in transport from the endoplasmic reticulum to the Golgi apparatus. Kim et al. (1997) reported the presence of a Sar1 gene family in Brassica campestris that suppresses a yeast vesicular transport mutation Sec12-1. More recently, GTP-binding proteins were also identified in ripe grape berries (Sarry et al., 2004; Giribaldi et al., 2007). YptV5 (spot 49) was found as a major protein component and the most abundant protein of PM in all the three berry stages (Fig. 3). YptV5 is membrane-associated protein which is localized to the cell periphery (PM or PM-associated vesicles). It may be a long half-life protein in grape berry, due to its high spot density in the PM 2-D gel versus very low frequency in dbEST records. YptV5 was first identified in the Golgi complex of the green alga Volvox carter (Fabry et al., 1993).
Proteins associated with cellular biogenesis and protein synthesis or fate
Different heat shock proteins (HSPs; spots 27, 39, and 60), well known as intracellular chaperonins, have been found in the PM of the grape berries at all three stage. HSPs are of highly conserved families which play essential roles in protein folding, unfolding, and transport within both prokaryotic and eukaryotic cells. Cicconi et al. (2004) have shown that a fraction of the 60 kDa HSPs, which is typically a mitochondrial protein, is located on the cell membrane in Daudi cells. HSPs were also identified in the PM of Arabidopsis (Alexandersson et al., 2004). Several actin (spots 14 and 26) and tubulin (spot 7) proteins were identified in this research. The tubulin and actin were identified as a group of major membrane proteins in the ripening berry occurring together with rapid enlargement of the cells, in which extensive cytoskeleton rearrangement took place. Actins play an important role in cytoplasmic streaming, cell shape determination, cell division, organelle movement, and extension growth. Tubulin is the major constituent of microtubules; it binds 2 mol of GTP, one at an exchangeable site on the β chain and one at a non-exchangeable site on the chain. Giribaldi et al. (2007) reported that the expression of most tubulins decreased during berry development and was very low after véraison, whereas the actins increased throughout berry development. The actins and tubulins have also been reported in grape berry (Sarry et al., 2004). However, the subcellular location of those proteins in grape berries was not discussed. From the present research, they were found to be located in the PM and with relatively stable densities in all the three development stages (Fig. 3). Two studies reported that actins and tubulins were located in the PM in Arabidopsis (Alexandersson et al., 2004; Marmagne et al., 2004). Two spots (5 and 6) have been identified as elongation factor 1-
, which probably plays a role in anchoring the complex to other cellular components; elongation factors were also identified in PM of Arabidopsis (Marmagne et al., 2004) and Synechocystis (Huang et al., 2002). Mongrand et al. (2004) reported that elongation factor 1- was identified in the tobacco PM by 2-DE and HPLC-MS/MS. Other proteins were found in the present research, such as the seed-type storage proteins provicilin (spots 28 and 29) and the 40S ribosomal protein SA (spot 62), whose location was reported to be cytoplasmic.
Proteins associated with metabolism, transcription, and energy
Nine identified spots (1, 4, 17, 25, 32, 45–47, and 61) were proteins associated with metabolism, three spots (36, 37 and 58) were related to energy production, and one (spot 13) was a transcription factor. Some of the metabolism-, transcription-, and energy-related proteins have previously been reported to be associated with the PM, such as alcohol dehydrogenase 1 (spot 47). It might be a cytoplasmic protein, which could be co-isolated with the PM; its identification was reported in the PM of rice (Chen et al., 2007) and Arabidopsis (Alexandersson et al., 2004), and it was also found in grape berries (Sarry et al., 2004; Giribaldi et al., 2007). 14-3-3-like protein A (spot 32) was detected in the present study; it was also reported in tobacco PM (Mongrand et al., 2004). Spot 1 was identified as a probable xyloglucan endotransglucosylase, which catalyses xyloglucan endohydrolysis or endotransglycosylation, cleaves and religates xyloglucan polymers, is an essential constituent of the primary cell wall, participates in cell wall construction of growing tissues, and exists in diverse vascular plants (Vissenberg et al., 2003). 4-Coumarate-CoA ligase (spot 4) is a key enzyme in the phenylpropanoid pathway which produces CoA thioesters of a variety of hydroxy- and methoxy-substituted cinnamic acids, as precursors of several phenylpropanoid-derived compounds, including anthocyanin and flavonoids (Hamberger and Hahlbrock, 2004). Spots 45 and 16 were identified as photosystem I assembly protein ycf3, a peripheral membrane protein of chloroplast loosely associated with the thylakoid membrane. One spot (36), apocytochrome f precursor, which belongs to the cytochrome f family, a chloroplast-encoded protein, may mediate electron transfer between photosystem II and photosystem I, cyclic electron flow around PSI, and state transitions (Tichy and Vermaas, 1999). These groups of proteins might be contaminants due to the fact that grape skin was included in the extracts, and may suggest that berries undergo important changes in energy and metabolism during véraison.
Differential proteome expression in PM during ripening
The detergent cocktail-resolvable proteins of the PM fraction of grape berries from three development stages were analysed to illustrate the changes in ripening fruits. The three samples showed 119 (Sa), 98 (Sb), and 86 (Sc) spots (Fig. 3) on the 2-DE gel, respectively. The PM protein content in grape berries decreased during ripening; this result is in agreement with the patterns of total berry cellular proteins reported by other investigators (Ghisi et al., 1984; Giribaldi et al., 2007). Twelve out of a total of 62 proteins showed a significant difference in their abundance (P >0.05%) during berry ripening (Table 3).
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Three proteins (Fig. 3B), iron-binding protein (spot 11), hypothetical protein (spot 12), and TGACG-motif-binding protein (spot 13), were found only in sample Sa. Two proteins (Fig. 3D, F), apocytochrome f precursor (spot 36) and hypothetical protein NeolCp091 (spot 31), were found only in samples Sa and Sb. Two unnamed proteins (spots 34 and 35) (Fig. 3E) were differentially regulated in three samples. A probable xyloglucan endotransglucosylase (spot 64) (Fig. 3C), an essential constituent of the primary cell wall which participates in cell wall construction of growing tissues, was absent in the Sa sample, and high in the Sb and Sc samples. NPL4-like protein 2 (spot 65) (Fig. 3C), which was not found in Sa and Sc, is expressed during véraison; it may be part of a complex that binds ubiquitinated proteins and is necessary for the export of misfolded proteins from the endoplasmic reticulum to the cytoplasm. ATP-dependent Clp proteolytic complex subunit CLPP (spot 66), a component of the ClpAP chaperone–protease complex, was up-regulated from pre-véraison to post-véraison (Fig. 3E), indicating that the degradation of misfolded proteins was strengthened during berry ripening. Spot 63 (Fig. 3A), which was identified as zeatin O-glucosyltransferase, was first up-regulated from pre-véraison to véraison, and then down-regulated after véraison. Zeatin is the most active and ubiquitous cytokinin, involved in bud formation, leaf expansion, retarding senescence, seed germination, and chloroplast formation. It can be rapidly induced in response to mycotoxin deoxynivalenol exposure, and weakly induced by salicylic acid, jasmonic acid, and 1-aminocyclopropylcarbonic acid treatments (Poppenberger et al., 2003). The down-regulation of zeatin O-glucosyltransferase after véraison was in agreement with the senescence of the berry. Ubiquitin-conjugating enzyme E2-21 (spot 43) (Fig. 3G) was down-regulated at this stage.
Concluding remarks
This is the first proteomic study on PM proteins of grape berry with differential expression during the ripening process. Sixty-two out of 200 protein spots were identified; most of them were involved in transmembrane transportation, signal transduction, and other functions, together with 18% (11 of 62) with unknown functions. Although more proteins are probably associated with grape berry PM, the present 2-DE technology could not provide complete information on all of them. The proteins found in the PM (i.e. ATP synthase, ABC transporter, and GTP-binding protein) which have been reported as true PM proteins in different plant species provided sound evidence of the quality of the sample preparation. However, there could be contaminant components from other subcellular fractions. Functional classification of the identified PM proteins found that most of them were involved in metabolic and cellular processes, and demonstrated that during berry ripening the PM is a vigorous system in signal transduction and transportation of metabolites; dynamic for energy metabolism, protein trafficking, and proteolysis. Further investigation will address confirmation of the localization and function of the unknown proteins, and will develop more specific methods for purification of PM fractions in proteomic analysis.
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Acknowledgements |
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This work was financially supported by National Natural Science Foundation of China (30471212 and 30500347).
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Footnotes |
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* These authors contributed equally to this work.
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Abbreviations |
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ACN, acetonitrile; ASB-14, tetra-decyanoylamido-propyl-dimethylammoniopropane-sulphonate; CHAPS, 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate; CHCA,
-cyano-4-hydroxycinnamic acid; DAF, day after flowering; MIPS, Munich Information Center for Protein Sequences; MALDI-TOF-MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; MATDB, MIPS Arabidopsis thaliana database; PM, plasma membrane; TFA, trifluoroacetic acid; 2-DE, two-dimensional electrophoresis. |
References |
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