The halophyte Mesembryanthemum crystallinum adapts to salt stress by salt uptake and switching from C3 photosynthesis to CAM (crassulacean acid metabolism). An important role in this process is played by transport proteins in the tonoplast of the central vacuole. In the present study we examine dynamic changes in the protein composition during salt-stress adaptation in microsomes from M. crystallinum leaves. Plants challenged with 400 mM NaCl accumulate salt by day 4 of treatment and malic acid only at day 12; a switching to CAM hence follows any initial steps of salt adaptation with a delay. Using a label-free and semiquantitative approach, we identified the most dramatic changes between the proteome of control plants and plants harvested after 12 days of the treatment; the abundance of 14 proteins was significantly affected. The proteomic data revealed that the majority of the subunits of V-ATPase (vacuolar H+-ATPase) holoenzyme. The salt treatment somewhat decreased the abundance of all subunits in the short term (4 days). Long-term adaptation, including the switching to CAM, goes together with a strong increase in the representation of all detectable subunits. Because this increase is subunit-specific, with the highest rise occurring for subunits E and c, the data suggest that long-term adaptation to salt stress correlates with a change in V-ATPase subunit stoichiometry and highlight the structural plasticity of this holoenzyme.

INTRODUCTION

Mesembryanthemum crystallinum is a model plant, which is widely used to examine the molecular strategy of salt-stress adaptation. It is clear from many studies that the main strategy of adaptation in this plant is centred on mechanisms that minimize the loss of water. For this purpose M. crystallinum accumulates NaCl in the leaf and root cells in order to generate a negative water potential [1]. A further strategy to reduce the loss of water is based on the metabolic shift from C3 to CAM (crassulacean acid metabolism) photosynthesis [2,3]. In this way the plants can open their stomata during the night to acquire CO2 with a minimal loss of water via transpiration [4]. An important role in this scenario is played by the central vacuole. This cellular compartment serves as deposit into which the cells sequester the accumulated Na+ and Cl [1,5]. The vacuole is therefore essential for keeping the cytoplasmic Na+ concentration low; it also provides the main storage compartment for osmotically active solutes. As in other salt-tolerant plants, in M. crystallinum this stress induces the expression of transport proteins in the tonoplast. Important representatives of such salt-induced transport tonoplast proteins are Na+/H+ exchangers and different subunits of V-ATPase (vacuolar H+-ATPase) [57].

A number of studies have already shown that a stress-induced modulation of V-ATPase activity is a key player in stress adaptation. In this context it was reported that in M. crystallinum salt stress causes a differential transcription of individual subunits of the V-ATPase [712]. During short-term adaptation it was found that the V-ATPase subunits are regulated in a non-stoichiometric fashion [10]. The data suggest that a non-co-ordinated increase in these subunits is a marker for the rapid osmotic adaptation to salt stress. For long-term adaptation to salt stress, M. crystallinum has been found to react via a profound metabolic rearrangement with a final switch to CAM. Transcriptional analyses suggest that this goes together with a concerted and overall increase in transcripts of all V-ATPase subunits. This should result in an overall increase in the number of active V-ATPase complexes in the tonoplast [9,10,13,14]. A more detailed scrutiny of the transcripts of individual subunits, however, implies that they increase with different rates. The consequence should be a variation in the architecture of V-ATPase with a different subunit stoichiometry. It has been proposed that such a modification in the stoichiometry composition of the V-ATPase could be a mechanism that contributes to a stress-related and organ-specific modulation of V-ATPase activity [8].

In the present study we used a proteomic approach to analyse the way in which the salt-induced modification of the transcripts of different V-ATPase subunits is reflected in the differential translation and abundance of the individual subunits at the protein level. Advances in LC (liquid chromatography)-MS have in recent years facilitated the investigation into the qualitative and quantitative proteomics of large numbers of proteins [15,16]. Indeed, MS-based quantification has rapidly made an impact on the analysis of differential protein expression and during the last few years several quantification methods have been proposed [1720]. For M. crystallinum only a few proteomic studies have so far been conducted [21]. None of the work has up to now has reported a systematic investigation by means of high-throughput proteomic technologies. In the present study we investigated the dynamics of V-ATPase subunits in the halophyte M. crystallinum in response to salt stress and during the subsequent shift from C3 to CAM. By using a MudPIT (multidimensional protein identification technology) proteomic approach [22,23] we performed a semiquantitative analysis based on label-free quantification. In this way we can identify statistically significant changes in salt stress-induced abundance and stoichiometry of V-ATPase subunits in M. crystallinum plants harvested before (C3 metabolic condition) and after (CAM condition) 12 days of NaCl treatment.

EXPERIMENTAL

Plant materials and growth conditions

M. crystallinum plants were from the collection of the Botanical Garden in Darmstadt (Germany). At 4–5 weeks old the plants were stressed by irrigating with 400 mM NaCl in a climate-regulated growth chamber with a 12 h day/night light cycle. QTI-AGRO 400 W daylight lamps (Philips) provided 300–350 mE·m−2·s−1 at the plant level. Temperature and relative humidity were 27°C and 60% during the day and 20°C and 80% during the night respectively. Control plants were irrigated with water during the same timeframe.

Malic acid and osmolality determination

The differences in leaf malic acid concentration at the beginning and the end of the light period were determined as a basic indicator of CAM. Disks of leaf samples from three different plants were collected and frozen in liquid nitrogen in order to break the cell walls, thawed and centrifuged for 5 min at 12000 g. The supernatant was used for the enzymatic determination of malic acid concentration using a spectrometer (λ=340 nm) as described by Möllering [24]. Leaf osmolality was detected at the end of the light period by direct measurement at the osmometer (Osmomat 030, Gonotec) of the mesophyll sap of a mixture of leaf samples of three plants harvested at the same time.

Na+ determination in leaves

In order to determine the amount of Na+ accumulated into the leaves, M. crystallinum plants were harvested at the late light period, before salt stress was induced, after 4 and 12 days of 400 mM NaCl treatment. Each sample, generated as a mixture of leaves from three different plants, was burned to ashes at 540°C for 24 h, resuspended in 1 M HCl and then incubated overnight at 37°C. Finally, the ashes were pelleted and the supernatant was subjected to CE (capillary electrophoresis) with a Beckman P/ACE system 5510 in a 100 μm capillary. Samples were hydrodynamically pressure-injected for 30 s from microvials and separated for 4 min at 256 V/cm. Na+ ions were detected indirectly by UV irradiation at 214 nm. Waters IonSelect™ lowmobility cation electrolyte buffer was used in order to separate ions within the capillary.

Enrichment of the membrane fraction

M. crystallinum plants were harvested at the beginning of the light period at days 0, 4 and 12 of NaCl treatment. Fresh leaves were collected from three different plants harvested at the same treatment time and then homogenized with an electronic blender in HM buffer [100 mM Tricine, 3 mM MgSO4, 3 mM EGTA and 3% (w/v) PVP (polyvinylpyrrolidone) (pH 7.5) with a Tris base]. The HM buffer osmolality was adjusted with mannitol as described previously [25,26] with some adaptations. The homogenate was filtered through 50-μm diameter nylon net and the supernatant was centrifuged twice at 16000 g at 4°C for 30 min. The pellet-containing membranes was collected by a final centrifugation at 200000 g at 4°C for 30 min and resuspended in 100 mM Tris/HCl (pH 7.5). The resuspension was then frozen and thawed twice in liquid nitrogen in order to break every vesicle. The membranes were finally centrifuged three times at 200000 g for 4°C for 15 min and resuspended in 100 mM Tris/HCl (pH 7.5). The membrane-enriched fractions obtained were stored as dried pellets at −80°C. For Western blot analysis, tonoplast-enriched fractions were isolated according to a standard protocol [26].

SDS/PAGE and Western blot analysis

SDS/PAGE was performed on polyacrylamide slab gels containing 12.5% (w/v) acrylamide and electrophoretically transferred on to teflon membranes (Immobilon P, Millipore) as described previously [26]. The free protein-binding sites of the teflon membrane were blocked by incubation in PBS containing 1% (w/v) fat-free milk powder for 1 h at room temperature (20–25°C) prior to incubation for 1 h at room temperature in an antiserum diluted 1:1000 with PBS and directed against the V-ATPase holoenzyme of Kalanchoe daigremontiana [27,28]. Immunodetection was performed using the Western-Light chemiluminescence detection kit (Serva Tropix); secondary antibody was diluted 1:15000 with PBS and the chemiluminescence substrate CSPD (disodium 3-{4-methoxyspiro[1,2-dioxetane-3,2′-(5′-chloro)tricyclo(3.3.1.13,4)decan]-4-yl}phenyl phosphate) was diluted 1:1000. V-ATPase subunit bands on X-ray films were scanned (Scan Jet IIP, Hewlett Packard) and their densitometry was analysed. The intensity of the mayor bands for the ATPase subunits in the gel increases linearly as a function of the tested protein concentration from 5 to 10 μg (results not shown).

Protein digestion and peptide purification

The membrane-enriched fractions were dissolved in 0.5% Rapigest (Waters) according to the manufacturer's protocol. The protein concentration was adjusted with 100 mM ammonium carbonate (pH 7.9). Total protein was digested with sequence-grade modified trypsin (Promega) at an enzyme/protein ratio of 1:50, and incubated overnight at 37°C. The reaction was stopped by adding TFA (trifluoroacetic acid) until a pH value of 2 was reached. The mixture was treated according to Norrgran et al. [29]; the collected supernatant was desalted using PepClean C-18 spin columns (Pierce Biotechnology), concentrated (Concentrator 5301, Eppendorf) and resuspended in 0.1% formic acid.

LC-MS/MS (tandem MS)

Four sets of plants were grown independently under the same conditions over a timeframe of several weeks and harvested at days 0, 4 and 12 of NaCl treatment. For each treatment condition, the leaves from three different plants were collected and homogenized together generating three samples per set of preparation. Thus the whole experimental plan produced 12 samples per four independent sets of plant preparations (see Supplementary Figure S1 at http://www.biochemj.org/bj/450/bj4500407add.htm). With the purpose of limiting the experimental and instrumental noise, samples belonging to the same set of plant preparation were trypsin-digested and analysed by MudPIT at the same time. Two technical replicates per sample were performed. Runs were carried out using a ProteomeX-2 system (Thermo Fisher Scientific) implemented on a LTQ (linear trap quadrupole) mass spectrometer.

The digested peptide mixtures were loaded on to a cation exchange column [0.32 i.d. (internal diameter)×100 mm, 5 μm; BioBasic-SCX column, Thermo Fisher Scientific] and eluted stepwise with ammonium chloride salt injections of increasing molarity (0, 20, 40, 80, 120, 200, 400, 600 and 700 mM). Fractions were captured in peptide traps (0.3 i.d.×5 mm, 5 μm, Zorbax 300 SB-C18, Agilent Technologies) for concentration and desalting prior to final separation by a reversed-phase C-18 column (0.180 i.d.×100 mm, 5 μm, Biobasic-18, Thermo Electron) with an acetonitrile gradient (eluent A, 0.1% formic acid in water, and eluent B, 0.1% formic acid in acetonitrile). The gradient profile was 5% eluent B for 5 min, 5–65% B for 45 min, 65% B for 3 min and 65–95% B for 10 min; the flow-rate was 1 μl/min. The eluted peptides were directly electrosprayed into a LTQ linear ion-trap mass spectrometer equipped with a nano-LC electrospray ionization source (Thermo Fisher Scientific). Nanospray was achieved using a coated fused silica emitter [360 μm o.d. (outer diameter)/50 μm i.d. and 730 μm tip i.d.; New Objective) held to 1.5 kV, whereas the heated capillary was held at 185°C. Full mass spectra were acquired in positive mode over a range 400–2000 m/z, followed by five MS/MS events sequentially generated in a data-dependent manner on the first, second, third, fourth and fifth most intense ions selected from the full MS spectrum scans using dynamic exclusion for MS/MS analysis (collision energy 35%).

Data processing of MS results

Experimental mass spectra were correlated with the theoretical spectra, calculated using 416 M. crystallinum protein entries in the NCBI database, by using Bioworks 3.3.1 based on the SEQUEST algorithm [30]. Matching between spectra was retained at a minimum cross-correlation of 1.5 for the +1 charge state, 2.0 for the +2 charge state and 2.5 for the +3 charge state. In addition, the threshold of peptide/protein probability was determined to 10−3 and the score value ≥10. Identification of the FDR (false discovery rate) was performed by processing the raw mass spectra using a decoy database [31], which comprised reversed amino acid sequences of M. crystallinum. The rate calculation considering the total number of peptides identified by target and decoy database revealed an FDR lower than 5% (results not shown).

In order to identify new peptides, which are not reported in the M. crystallinum protein database, experimental mass spectra were further processed using the ARAMEMNON database [32]. It contains a wide collection of putative membrane proteins of Arabidopsis thaliana, Oryza sativa and other 300 seed plants. To ensure a correct peptide identification on the basis of sequence homology, we used more stringent thresholds for peptide probability (≤10−4) and Xcorr against Charge (2 for single charge, 2.5 for double charge and 3 for triple charge).

WoLF PSORT was used to predict the subcellular localization of proteins on the basis of their amino acid sequences [33]. The predicted subcellular localization of the identified proteins was compared with the subcellular localization of the whole dataset of M. crystallinum protein sequences; from these data we estimated the successful enrichment in membrane proteins. MAProMa was further used to achieve the semiquantitative comparison of the protein lists [34]; R-package (http://www.r-project.org/) was used to perform PCA (principal component analysis) and hierarchical clustering coupled to Ward's minimum variance method, where the distance between two clusters is the ANOVA sum of squares between the two clusters added up over all of the variables [35].

Label-free quantification

On the basis of the direct correlation between the SEQUEST-based SCORE values and the relative abundance of the identified proteins [36,37], proteins differentially represented in the three different physiological conditions were semiquantitatively evaluated by the DAVE (differential average) and DCI (differential confidence index) algorithms of the MAProMa software [34,38]. DAVE, which evaluates the variation of protein expression, is defined as (X−Y)/(X+Y)×0.5, whereas DCI, which indicates the confidence of the differential expression, is defined as (X+Y)×(X−Y)/2, where X and Y represent the SEQUEST-based SCORE values in two samples.

To better assess the accuracy of the differentially represented proteins, the evaluation of SpC (spectral count) values were also taken into account [20]. In this context, statistically significant differences were evaluated using the G-test, according to eqn 1 [39]:where f1 and f2 are the normalized SpC for the protein in sample 1 and sample 2 respectively, and and are the expected SpC for the protein in sample 1 and sample 2 respectively. If we assume that the protein is equally expressed (eqn 2):

 
formula
(2)

then G-test values >3.8, corresponding to statistically significant protein differences (P<0.05), were retained. Finally, independent biological replicates (n=4) were statistically investigated by two-way ANOVA (P<0.05) coupled to Tukey's honestly significant difference test (Supplementary Table S6 at http://www.biochemj.org/bj/450/bj4500407add.htm). SpC and SEQUEST-based SCORE values were normalized applying the total signal normalization procedure [40], before any semiquantitative evaluation.

RESULTS AND DISCUSSION

Validation of the plant material

Salt stress evokes in M. crystallinum an adaptation mechanism, which includes Na+ detoxification of the cytoplasm, regulation of the osmotic potential and diurnal vacuolar accumulation of malic acid [1,2,4,5]. After treatment with 400 mM NaCl, M. crystallinum plants were tested for these parameters. After 4 days of salt stress, Na+ accumulation into the leaves was already 1.9±0.15-fold higher than in the control plants (Figure 1A). The osmolality of leaf cells was at the same time in the stressed plants 1.6±0.1-fold higher than at the onset of treatment (Figure 1B). In contrast, at day 4 of stress the plants did not yet show any nocturnal net accumulation of malic acid in the vacuoles of mesophyll cells. After 12 days of NaCl treatment, Na+ and osmolality were up to 5.3±0.6- and 2.8±0.1-fold respectively higher than the control. At this same time point malic acid was up to 28.5±0.7-fold higher than at day 0 of the treatment (Figure 1C). These data are in agreement with the findings of Cosentino et al. [5], who demonstrated that M. crystallinum plants were already adapting their osmotic conditions to salt stress at day 4 of NaCl treatment; only thereafter they switch their photosynthetic metabolism to CAM for a long-term adaptation to stress.

M. crystallinum physiological conditions upon salt treatment

Figure 1
M. crystallinum physiological conditions upon salt treatment

Na+ concentration (A), osmolality (B) and the difference in malic acid concentration between the onset and end of light period (C) in leaves of plants treated with 400 mM NaCl and harvested at the reported time of treatment. Differences are significant according to Student's t test (P<0.05). Error bars are S.D. (n=3). FW, fresh mass.

Figure 1
M. crystallinum physiological conditions upon salt treatment

Na+ concentration (A), osmolality (B) and the difference in malic acid concentration between the onset and end of light period (C) in leaves of plants treated with 400 mM NaCl and harvested at the reported time of treatment. Differences are significant according to Student's t test (P<0.05). Error bars are S.D. (n=3). FW, fresh mass.

Proteomic analysis of the leaf microsomal fractions

Under salt-stress conditions M. crystallinum plants sequester Na+ into the central vacuole and for long-term adaptation they shift from C3 photosynthesis to CAM [1,4]. Salt stress regulates, in this context, the expression of transport proteins in the tonoplast [57]. To detect stress-induced changes in protein levels we enriched membranes according to Valot et al. [25]; they used this procedure for a proteomic study of membrane-associated proteins in Medicago trunculata. In the present study we analysed microsomal preparations from the leaves of salt-stressed plants of M. crystallinum by MudPIT.

The subcellular localization of the identified proteins was predicted using WoLF PSORT [33]. Even when the accuracy of the bioinformatic prediction for tonoplast localization was poor, the analysis showed that the enrichment procedure increased the relative amount of plasma membrane and tonoplast proteins by 65% and 45% respectively; at the same time it decreases the fraction of plastidial proteins by 25%, compared with the protein from the of the M. crystallinum dataset.

The advantage of this enrichment strategy in the present context is that it has no methodological bias. It is known in M. crystallinum that the physicochemical properties of the tonoplast change during development [41] and that the salt-induced C3 to CAM shift leads to a higher stability of the V-ATPase holoenzyme in the presence of detergents [42]. This change in stability may alter the protein yield when some tonoplast isolation methods are used.

Moreover a high degree of tonoplast purification is also not essential in this strategy because the MudPIT proteomic approach allows the simultaneous identification of hundreds of proteins, without limits related to pI, molecular mass or hydrophobicity. It is important to note that the presence of unavoidable contaminants does not affect the analysis because each protein is detected independently. The combination of the MudPIT technique with the enrichment of a microsomal fraction is therefore sufficient to identify proteins from the tonoplast with good statistical support. The combined approach is particularly suitable for the detection of membrane proteins, which are key players in salt-stress adaptation and which remain frequently undetected by conventional proteomic methods [43]. The present analysis has been conducted on plants harvested at days 0, 4 and 12 of NaCl treatment and allowed the overall identification of 114 entries, corresponding to approximately 20% of the M. crystallinum entries available in the NCBI protein database. Among these, 69 proteins were non-redundant and about 72% of them were identified with high significance on the basis of at least a total SpC equal to 10 (see Supplementary Tables S1 and S2 at http://www.biochemj.org/bj/450/bj4500407add.htm).

At the three physiologically interesting time points, i.e. 0, 4 and 12 days of NaCl treatment, we have identified 64, 56 and 55 proteins respectively. A total of 44 proteins were present under all three conditions, whereas seven proteins were exclusively identified in one of the three biological states. In particular, six proteins were only found in the controls (0 days of NaCl treatment), whereas 12 days of salt stress evoked the exclusive presence of one protein (see Supplementary Table S3 at http://www.biochemj.org/bj/450/bj4500407add.htm).

Replicate analyses have been processed by PCA and their protein profiles grouped into distinct clusters, which reflect the investigated biological conditions (Figure 2). The highest similarity was observed between profiles of plants harvested at day 0 and day 4 of NaCl treatment. Conversely, the profiles of plants harvested at day 12 of the treatment grouped separately. A similar result was obtained by unsupervised hierarchical clustering (see Supplementary Figure S2 at http://www.biochemj.org/bj/450/bj4500407add.htm).

PCA of M. crystallinum membrane-enriched protein profiles obtained by MudPIT

Figure 2
PCA of M. crystallinum membrane-enriched protein profiles obtained by MudPIT

PCA has been performed using variables such as the normalized SpC values of the proteins identified with high confidence in multiple replicate analyses. PC1, PC2 and PC3 account for 71% of the variance.

Figure 2
PCA of M. crystallinum membrane-enriched protein profiles obtained by MudPIT

PCA has been performed using variables such as the normalized SpC values of the proteins identified with high confidence in multiple replicate analyses. PC1, PC2 and PC3 account for 71% of the variance.

The PCA results validate the reproducibility of the MudPIT analysis. Moreover they also suggest that after 4 days of NaCl treatment, M. crystallinum is still in a C3 metabolic state even though the plant is already adapting to high salinity by Na+ accumulation in the vacuoles. This partially contrasts with previous transcriptomic studies, which indicate that M. crystallinum starts adaptation in a short timeframe after salt stress [3,12]. However, although the number of proteins which are considered in our analysis is lower than the entries evaluated in a transcriptomic study, our proteomic data indicate a mostly constant level of protein expression over the first days of M. crystallinum stress induction; major changes only occur over a long period after stress and they go together with the physiological active shift to CAM. In this respect and in support of our results, Cosentino et al. [5] observed that in M. crystallinum the vacuolar Na+/H+ antiporter transcripts slightly increase in leaves up to 3 days after the salt-stress induction. Only later do they undergo a burst of expression, which reaches a maximum level between days 8 and 10 of the treatment. Moreover a previous proteomics analysis conducted in Thellungiella halophyla showed that this salt-tolerant plant is already inclined to cope with the stress before the stress actually occurs [44]. This strategy allows the plant to save energy, which otherwise would be expended in activating pathways to lower the stress effects [44].

Semiquantitative evaluation of differentially represented proteins

Protein lists of replicated analyses were aligned through a specific MAProMa tool and normalized SEQUEST-based SCORE values were compared by the DAVE and DCI algorithms. Because the proteomic profiles between day 0 and day 12 of NaCl treatment have shown the highest differences (Figure 2), we focused the analysis on the differences between data from these days. On the basis of this approach we compared, in the present study, in a pair-wise manner, samples belonging to the same set of plant preparation (see Supplementary Figure S1). In order to increase the differential trend reliability of the proteins, a threshold of acceptability for each DCI (DCI>200 and DCI<−200) and DAVE (DAVE>0.2 and DAVE<0.2) value was imposed [45,46]. The statistical significance of the protein differences was evaluated by the G-test with a P<0.05. In addition, only proteins which were differentially represented between the two pairs and which were at least detected in three out of the four pair-wise preparations were considered for further analysis. From these analyses it was found that 14 proteins (equivalent to 2.6% of the protein M. crystallinum database) were differentially represented with a high significance in the two treatments: eight of which were mainly detected in plants after 12 days of NaCl treatment, i.e. plants that had fully switched to CAM with the other six proteins having increased representation in untreated plants (day 0), i.e. plants that perform C3 photosynthesis. The results of this analysis are listed in Table 1 and confirm previous transcriptional studies on responses of M. crystallinum to salt stress, for example the stress-related increases in transcript levels of enolase [12,21], glucose 6-phosphate translocator [12,47], cysteine proteinase [12] and phosphoenolpyruvate carboxylase 1 [12],. The decrease in the transcript levels of triose phosphate/phosphate translocator [47] results, in the present study, in a corresponding change in the respective protein representation. Malate dehydrogenase is responsive to salt stress after 12 days NaCl treatment and this finding is similar to data from Ndimba et al. [48] on A. thaliana. Moreover, MipE also decreased upon treatment, a result which is expected from transcript profiling of salt-stressed M. crystallinum plants [3]. Finally, the results of the present study uncover for the first time a decrease in the protein content of Ras-related Rab7A, glutamine synthetase, pyruvate phosphate dikinase and of a putative adenylate transporter upon salt stress.

Table 1
Protein representation changes among M. crystallinum plants harvested at days 0, 4 and 12 of NaCl treatment

Only proteins which exhibit significant changes at least in three out of the four pair-wise preparations are reported in the Table. Negative and positive DAVE values indicate proteins which are less or more represented at day 12 of NaCl treatment respectively. Differences are significant according to G-test (P<0.05).

   SpC value Comparison of days 0–12 
NBCI GI Uniprot ID Protein name 0 days 4 days 12 days DAVE DCI Differential 
4884970 Q9XFJ1 Glutamine synthetase 71 32 −1.7 −5809 More represented at day 0 
559471 Q42910 Pyruvate, phosphate dikinase 11 −1.5 −290  
1657950 O24050 MipE 64 25 31 −0.8 −2638  
61651616 Q59IV1 Putative adenylate transporter 53 24 39 −0.9 −2942  
1842069 P93267 Ras-related Rab7A 11 −0.7 −576  
9295273 Q9MSB6 Triose phosphate/phosphate translocator 60 38 36 −0.6 −649  
13235340 Q9AVU8 V-ATPase subunit A 343 219 1108 0.2 21807 More represented at day 12 
27125515 Q8GUB1 V-ATPase subunit a 96 65 283 0.3 4241  
1143509 Q40272 V-ATPase subunit E 70 47 340 0.5 2522  
9295277 Q9MSB4 Glucose 6-phosphate/phosphate translocator 51 1.5 846  
1524121 O24047 Malate dehydrogenase 39 1.6 753  
1087071 Q43130 Enolase 65 1.6 2348  
19536 P10490 Phosphoenolpyruvate carboxylase 1 (PPC1) 166 1.9 5165  
944916 Q40261 Cysteine proteinase 75 2.0 1257  
   SpC value Comparison of days 0–12 
NBCI GI Uniprot ID Protein name 0 days 4 days 12 days DAVE DCI Differential 
4884970 Q9XFJ1 Glutamine synthetase 71 32 −1.7 −5809 More represented at day 0 
559471 Q42910 Pyruvate, phosphate dikinase 11 −1.5 −290  
1657950 O24050 MipE 64 25 31 −0.8 −2638  
61651616 Q59IV1 Putative adenylate transporter 53 24 39 −0.9 −2942  
1842069 P93267 Ras-related Rab7A 11 −0.7 −576  
9295273 Q9MSB6 Triose phosphate/phosphate translocator 60 38 36 −0.6 −649  
13235340 Q9AVU8 V-ATPase subunit A 343 219 1108 0.2 21807 More represented at day 12 
27125515 Q8GUB1 V-ATPase subunit a 96 65 283 0.3 4241  
1143509 Q40272 V-ATPase subunit E 70 47 340 0.5 2522  
9295277 Q9MSB4 Glucose 6-phosphate/phosphate translocator 51 1.5 846  
1524121 O24047 Malate dehydrogenase 39 1.6 753  
1087071 Q43130 Enolase 65 1.6 2348  
19536 P10490 Phosphoenolpyruvate carboxylase 1 (PPC1) 166 1.9 5165  
944916 Q40261 Cysteine proteinase 75 2.0 1257  

Long-term adaptation to salt stress and CAM is correlated with an increase in V-ATPase subunit abundance

Several reports have already suggested, on the basis of transcriptional analysis, that plants react to salt stress with a differential expression of individual subunits of the V-ATPase [712]. In the present study we examined the effect of stress on the protein level of the V-ATPase subunits. Figure 3 shows a histogram with the average SpC values for each detected V-ATPase subunit; the data are normalized to the values obtained at day 0. The mean values were statistically analysed using two-way ANOVA and Tukey's test (P<0.05). In our proteomic analysis we can detect all of the V-ATPase subunits which are so far known in plants, with the exception of subunits D, F, C and e.

Histogram of the average SpC values for the identified V-ATPase subunits

Figure 3
Histogram of the average SpC values for the identified V-ATPase subunits

SpC values of the V-ATPase subunits identified in M. crystallinum plants harvested at days 0, 4 and 12 of NaCl treatment. The SpC values were normalized for each subunit by protein length [50] and on the value at day 0. Differences between average SpC values are statistically significant, as determined by two-way ANOVA and Tukey's test (P<0.05), for all of the represented V-ATPase subunits. Error bars show S.D. (n=4).

Figure 3
Histogram of the average SpC values for the identified V-ATPase subunits

SpC values of the V-ATPase subunits identified in M. crystallinum plants harvested at days 0, 4 and 12 of NaCl treatment. The SpC values were normalized for each subunit by protein length [50] and on the value at day 0. Differences between average SpC values are statistically significant, as determined by two-way ANOVA and Tukey's test (P<0.05), for all of the represented V-ATPase subunits. Error bars show S.D. (n=4).

Subunits D and F so far have been only identified by partial EST (expressed sequence tag) sequences (CA835172 and CA833068 respectively). Because the wrong association of peptide sequences with the parent protein of not yet fully sequenced subunits could generate a false non-stoichiometric expression of subunits during homology search, M. crystallinum ESTs CA835172 and CA833068 have been further investigated. These sequences are homologous with V-ATPase subunits D and F respectively of A. thaliana and they do not share any similarity neither with other sequences present in the M. crystallinum protein database, nor with other V-ATPase subunits. For these reasons we can conclude that the lack of subunit D and F of M. crystallinum V-ATPase is not expected to affect the peptide identification; however, as there are no data in the M. crystallinum protein database for identifying these subunits unequivocally they were not considered in the analysis.

Subunits C and e were not found at a significant level either in the control or in salt-treated plants. One possibility for the low abundance of these proteins in the mass spectra is that their level of expression is very low. However, because the defined stoichiometry of the V-ATPase holoenzyme requires at least one subunit C and e per enzyme complex, it is more probable that these proteins are underrepresented in the analysis and evade detection for technical reasons. Subunit C is reported in the M. crystallinum protein database as a partial peptide of 76 amino acids. It is possible to assume that this short sequence lowers the probability of detections; however, subunit e is also located in the tonoplast and is nevertheless detected in our analysis [49]. It is also probable that the detection of subunit c could be negatively affected by the Rapigest detergent, which removes them from the membrane. However subunit e is even shorter than subunit c (70 and 165 amino acids respectively) and subunit c should hence have a higher probability for being detected than subunit e.

Independent of the fact that the some subunits remain undetected we can analyse the relative abundance of the remaining subunits because they all generate a clear-cut signal. A semiquantitative analysis of these positively identified subunits shows that long-term stress adaptation goes together with a significant rise in V-ATPase subunits A, B, E, G, H, a, c and d (see Supplementary Table S4 at http://www.biochemj.org/bj/450/bj4500407add.htm). Figure 3 shows that all detected subunits slightly decrease in the short-term. At day 4 of NaCl treatment they reach an average of 0.6±0.1 with respect to the normalized reference value at day 0. It is important to note that the ratio of the individual subunits remains constant between the 2 days of data collection.

A dramatic effect occurs in the long-term response to salt stress. After 12 days of treatment, all subunits increase substantially. The normalized data [50] further show that the representation of all of the subunits is modulated in a more or less concerted manner (Figure 3). Hence the switch from C3 to CAM goes together with a major up-regulation of V-ATPase. For a short-term adaptation to the accumulation of salt it appears that a major change in V-ATPase protein abundance is not required. This implies that the stimulation of the V-ATPase activity, which is induced rapidly after salt stress [26], can generate sufficient protonmotive force for salt accumulation in the vacuole via a Na+/H+ antiporters even without any de novo synthesis of V-ATPase [5,51]. This post-translational adaptation seems to be a specific response mechanism in halophytes, which is missing in glycophytes such as A. thaliana [52].

For all subunits analysed in the present study the ratio of detection at day 12 divided by the starting detection level at day 0 ranged between 2 and 4.8. A more detailed analysis showed that it is possible to distinguish among three classes of increments. In particular subunits E and c are those that increased with the highest significance, roughly 4-fold (4.8- and 4.2-fold respectively). Subunits A, B, a and d increase around 3-fold after 12 days of NaCl treatment (3.2-, 3.0-, 3.0- and 2.8-fold respectively). In contrast subunits G and H are those that increased the lowest, they roughly double (2.0- and 2.3-fold respectively; see Supplementary Table S5 at http://www.biochemj.org/bj/450/bj4500407add.htm). The differences in ratios are all statistically significant. They indicate that the stoichiometry of subunits in the V-ATPase of M. crystallinum indeed changes when salt stress induces the switch from C3 to CAM.

Thus subunits E and c are, after salt stress, more abundant by a factor of 2 than subunits G and H. Also the increments of subunits A, B, a and d exceed those of subunits G and H by a factor of 1.5. The analysis indicates that the stoichiometry of subunits in the V-ATPase of M. crystallinum is sensitive to environmental changes, i.e. salt stress. The proteomic data are in general agreement with results of Löw et al. [8], who found that in M. crystallinum among three tested subunits (A, B and c), subunit c is the most sensitive to stress at the transcript level. In the present study we find that this is reflected also at the protein level and that subunit E is even more responsive than c. A more recent proteomic analysis [21] indicated B and d as the only salt-responsive V-ATPase subunits in M. crystallinum, whereas subunit E appeared salt-insensitive. These data are in contrast with previous findings, which reported an increase in V-ATPase subunit c under salt stress [53]. Furthermore the authors do not report any evidence for a full switch of the treated plants to CAM. In the present study we report that an imbalance in the V-ATPase stoichiometry occurs after a complete salt-induced switch from C3 to CAM in M. crystallinum plants (i.e. 0 and 12 days of NaCl treatment respectively). Thus an incomplete metabolic switch to CAM in the work of Pantoja and co-workers [21] could explain the apparent difference in salt effect on subunit E with the results of the present study.

Finally, when the MS spectra were analysed using the ARAMEMNON database [32], a plant membrane protein repository with A. thaliana as a reference model plant, the results were comparable with the ones obtained with the M. crystallinum protein database. The V-ATPase subunits A, B, E, H, G, d and D, which are missing from the M. crystallinum protein repository, were identified in homologous A. thaliana proteins; a homologous subunit c was identified in Vitis vinifera. These results confirm further the strength of the M. crystallinum MS data.

Western blot analysis confirms the non-stoichiometric appearance of the V-ATPase subunits under salt stress

To test the non-stoichiometric increase in the V-ATPase subunits with an independent method, we performed a Western blot analysis on the enriched tonoplast fractions from the control and NaCl-stressed plants (Figure 4). An antiserum directed against the V-ATPase holoenzyme of K. daigremontiana has been used previously to detect the V-ATPase subunits in M. crystallinum [27,28]. The blots displayed in Figure 4(A) show the typical pattern of V-ATPase subunits observed on the same gel for control and NaCl-treated plant. In the control the antibody detected mostly subunits A (67 kDa), B (55 kDa), D (34 kDa) and c (16 kDa). In response to salt treatment the intensity of subunits A, B and D clearly increased and subunit C (41 kDa) became more evident. Subunit c, which generally gives a low signal in the control, appeared at a higher intensity in the NaCl-treated sample. The Western blot analysis furthermore shows that the switch to CAM also leads to the occurrence of the polypeptide Di (32 kDa) as the result of an in vivo proteolytic processing of subunit B [13,28]. It has been suggested that this post-translational modification could be involved in regulatory function [13]. These data confirm that different V-ATPase subunits are represented with a different stoichiometry after salt treatment. To quantitatively compare the Western blotting observations with the proteomics data, we estimated the relative abundance of subunits A, B, D and c in salt-treated plants compared with those in the control (Figure 4B). The data from pair-wise comparisons on the same gel of the respective V-ATPase subunits from the NaCl-treated and control plants shows that subunits A, B and D roughly increase in parallel. Only subunit c shows a nearly 2-fold higher increase over the aforementioned three subunits in the transition to CAM. The results of these experiments are fully consistent with the quantification obtained with the proteomic approach (Figure 3).

Western blot analysis of V-ATPase subunits in tonoplast-enriched membrane fractions

Figure 4
Western blot analysis of V-ATPase subunits in tonoplast-enriched membrane fractions

(A) Immunostaining was performed on tonoplast-enriched fractions of M. crystallinum using an antiserum directed against the K. daigremontiana V-ATPase holoenzyme [30,31]. The characters below gels indicate V-ATPase subunits (for the apparent molecular masses see the Results section). The grey value intensity of the gel bands for the two treatments (grey, untreated plants and black, plants treated with NaCl) is shown at the top of the gel. The arrow indicates the appearance of polypeptide C in salt-treated plants. (B) Mean relative abundance of subunits A, B, D and c in the NaCl-treated compared with control plants. Data are from a pair-wise comparison of Western blot data as in (A) with material from the NaCl-treated and control plants analysed on the same gel. To compare the data from different experiments the relative increase in the subunit intensity between the control and NaCl-treated samples was normalized to the relative increase in subunit A, which occurred between NaCl-treated and control tissue (ANaCl/Acontrol). Error bars show S.D. (n=4).

Figure 4
Western blot analysis of V-ATPase subunits in tonoplast-enriched membrane fractions

(A) Immunostaining was performed on tonoplast-enriched fractions of M. crystallinum using an antiserum directed against the K. daigremontiana V-ATPase holoenzyme [30,31]. The characters below gels indicate V-ATPase subunits (for the apparent molecular masses see the Results section). The grey value intensity of the gel bands for the two treatments (grey, untreated plants and black, plants treated with NaCl) is shown at the top of the gel. The arrow indicates the appearance of polypeptide C in salt-treated plants. (B) Mean relative abundance of subunits A, B, D and c in the NaCl-treated compared with control plants. Data are from a pair-wise comparison of Western blot data as in (A) with material from the NaCl-treated and control plants analysed on the same gel. To compare the data from different experiments the relative increase in the subunit intensity between the control and NaCl-treated samples was normalized to the relative increase in subunit A, which occurred between NaCl-treated and control tissue (ANaCl/Acontrol). Error bars show S.D. (n=4).

V-ATPase subunit E identification

Because subunit E is often detected in higher plants in two or more isoforms, a BLAST search has been performed in order to identify all subunits of the E isoforms in the EST repository of M. crystallinum. Subunit E isoforms from A. thaliana (NCBI accession numbers Q39258, Q9C9Z8 and P0CAN7), Thellungiella halophila (NCBI accession number Q8S2S1), O. sativa japonica (NCBI accession numbers Q8SA35, Q5KQI7 and Q5NB63) and M. crystallinum (NCBI accession number Q40272) [54] were used for a TBLASTN search within the EST M. crystallinum repository [3]. This analysis identified 52 different ESTs matching with a V-ATPase subunit E consensus sequence (see Supplementary Table S5). Despite the expected three isogenes encoding subunit E in M. crystallinum [14], the results of the present study indicate that 51 ESTs group in a single contig alignment together with the known M. crystallinum subunit E (results not shown). The latter EST BE035527 (GenBank® gi 8330536) perfectly accounts for nearly half of its sequence with the known M. crystallinum subunit E; however, the remaining nucleotides show several mismatches with the reference subunit E, even if it is still belonging to a subunit E-type isogene. In particular since one of the fragments detected in the MS analysis is also matching with EST BE035527, we strongly believe that our analysis comprises all the putative V-ATPase subunit E isoforms represented in the M. crystallinum EST database.

Salt stress alters the stoichiometric balance of V-ATPase subunits in M. crystallinum

Subunits E and c are involved in the proton translocation and the regulation of the V-ATPase in M. crystallinum [13,14]. The change in stoichiometry under salt stress, which is detected in the present study at the proteome level, confirms that NaCl directly affects the V-ATPase activity by altering the amount of its catalytic subunits. This has been anticipated already earlier on the basis of indirect evidence [10,11,26,51]. Previous findings already observed structural changes in the size of the V-ATPase Vo domain of M. crystallinum under salt stress. It had been speculated that they are due to a variation of the subunit composition of Vo and in particular to an increase in the copy number of subunit c [13,53]. The modification of the Vo domain might be correlated with changes in the coupling ratio of the V-ATPase holoenzyme [13].

With such an altered coupling the V-ATPase can pump protons into the vacuole with a lower coupling ratio of protons/ATP. Furthermore, under salt-induced CAM nocturnal malic acid transport into the vacuole has to be performed against a high concentration gradient of solutes. Under these conditions a lower coupling ratio could be beneficial because the energy available from hydrolysis of ATP is sufficient for a steep uphill transport of protons. The current data indeed indicate that more copies of subunit c are available for incorporation in the enzyme complex at day 12 of NaCl treatment. In other words the CAM state has a direct functional impact on the coupling ratio of the V-ATPase [13].

The fact that all identified subunits remain roughly constant over the first 4 days of salt stress implies that any change in activity in this time window must result from a modulation in enzyme activity of the V-ATPases that are already present. The proteomics data, which show that the V-ATPase remains more or less constant over a short period after salt stress, are therefore not in agreement with extrapolations on the basis of transcript studies. The latter have anticipated an unco-ordinated rise in ATPase subunits over a short period after salt stress [12]. The present proteomic data however confirm the extrapolations from several other transcriptional analyses. In these studies it was reported that transcription of subunits A, B, E, F, G and c was elevated upon salt stress in M. crystallinum leaves [10,11]. It was also speculated that the transcript levels of subunit E increased in the leaves of older plants, but were unaffected in the leaves of juvenile plants, although these leaves accumulated considerable amounts of Na+ [10]. The present proteomic data show that the detection of subunit E is significantly increased after 12 days of NaCl treatment, confirming the hypothesis that the availability of this subunit is causally linked to a long-term adaptation to salt stress. Moreover the degree of increase in subunit E in leaves upon salt stress correlates favourably with the 4–6-fold increase in transcript levels of the leaves of plants after 2 weeks of treatment [10,12]. Also our findings on subunit c reflect their changes in transcript levels; it was shown that the transcript of subunit c increased upon salt stress in the leaves of M. crystallinum [9]. Any partial discrepancy between the level of transcript and the corresponding level of protein is not completely unexpected because of the low correlation between proteomic and transcriptomic data identified previously [55].

In summary the results of the present study show that the bulk abundance and stoichiometry of V-ATPase subunits is kept constant in M. crystallinum leaves over the first 4 days of salt stress. Hence the requirement for a higher protonmotive force, requested by the Na+/H+ antiporters for the vacuolar Na+ accumulation, must be accomplished by a post-translational up-regulation of V-ATPase activity [27]. Then in the course of the long-term adaptation, i.e. the time in which the plants converted their metabolism from C3 into CAM photosynthesis, the protein representation of the detectable V-ATPase subunits increases in a concerted, but subunit-distinct, fashion, demonstrating that the V-ATPase of M. crystallinum can undergo structural changes which make this holoenzyme of considerable plasticity.

Proteotypic peptides

When used in proteomic analysis PTPs (proteotypic peptides) are expected to represent an important source of information [56]. Using the most common definition of PTPs [57], 128 peptides, corresponding to 39 proteins, have been selected (see Supplementary Table S7 at http://www.biochemj.org/bj/450/bj4500407add.htm). These data were used to evaluate the peptide peak area intensity [AUC (area under the curve)] of the proteins reported in Table 1. By linear regression analysis, the AUC highly correlated with the SCORE (or SpC) values confirming the semiquantitative nature of these parameters [37] (see Supplementary Figures S3 and S4 at http://www.biochemj.org/bj/450/bj4500407add.htm). The present study demonstrates that the MudPIT method is a valid system for detecting membrane proteins in plants. The data on the response of V-ATPase subunits to salt stress in M. crystallinum are in overall agreement with previous expectations on the basis of transcript analysis; this validates the system of analysis.

Abbreviations

     
  • AUC

    area under the curve

  •  
  • CAM

    crassulacean acid metabolism

  •  
  • DAVE

    differential average

  •  
  • DCI

    differential confidence index

  •  
  • EST

    expressed sequence tag

  •  
  • FDR

    false discovery rate

  •  
  • i.d.

    internal diameter

  •  
  • LC

    liquid chromatography

  •  
  • LTQ

    linear trap quadrupole

  •  
  • MS/MS

    tandem MS

  •  
  • MudPIT

    multidimensional protein identification technology

  •  
  • PCA

    principal component analysis

  •  
  • PTP

    proteotypic peptide

  •  
  • SpC

    spectral count

  •  
  • V-ATPase

    vacuolar H+-ATPase

AUTHOR CONTRIBUTION

Cristian Cosentino designed the project, managed the plants, prepared the biological material, analysed data and wrote the paper. Dario Di Silvestre performed MS experiments, analysed data and wrote the paper. Elke Fischer-Schliebs performed the Western blots and revised the paper; Ulrike Homann revised the paper. Antonella De Palma and Claudio Comunian performed MS experiments. Pier Luigi Mauri supervised the MS analysis and revised the paper. Gerhard Thiel supervised the project and wrote the paper.

AKNOWLEDGEMENTS

We thank Rossana Rossi (Istituto di Tecnologie Biomediche–CNR) and Sylvia Haase for technical assistance and we particularly acknowledge the contribution of the late Dr R. Ratajczak who laid the ground for this work.

FUNDING

This work was supported by the Deutsche Forschungsgemeinschaft the framework of Graduiertenkolleg 340 ‘Communication in biological systems: from the molecule to the organism in its environment’.

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Author notes

1

These authors have contributed equally to the work.