Proteomic and physiological responses of the halophyte Cakile maritima to moderate salinity at the germinative and vegetative stages

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Proteomic and physiological responses of the halophyte Cakile maritima to moderate salinity at the germinative and vegetative stages☆ Ahmed Debeza, b,⁎, Hans-Peter Braunc , Andreas Pichd , Wael Taamallia , Hans-Werner Koyroe , Chedly Abdellya , Bernhard Huchzermeyerb a

Laboratoire des Plantes Extrêmophiles (LPE), Centre de Biotechnologie à la Technopole de Borj-Cedria (CBBC), BP 901, Hammam-Lif 2050, Tunisia Institut für Botanik, Leibniz Universität Hannover, Herrenhäuser-Str. 2, D-30419 Hannover, Germany c Institut für Pflanzengenetik, Dept V Plant Proteomics, Leibniz Universität Hannover, Herrenhäuser-Str. 2, D-30419 Hannover, Germany d Institute of Toxicologie, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany e Institut für Pflanzenökologie, Justus-Liebig Universität Gieβen, Heinrich-Buff-Ring 26‐32, D-35392 Gieβen, Germany b

AR TIC LE I N FO

ABS TR ACT

Article history:

Responses of the halophyte Cakile maritima to moderate salinity were addressed at germination

Received 6 June 2012

and vegetative stages by bringing together proteomics and eco-physiological approaches.

Accepted 14 August 2012

75 mM NaCl-salinity delayed significantly the germination process and decreased slightly the

Available online 29 August 2012

seed germination percentage compared to salt-free conditions. Monitoring the proteome profile between 0 h and 120 h after seed sowing revealed a delay in the degradation of seed storage

Keywords:

proteins when germination took place under salinity, which may explain the slower

Germination

germination rate observed. Of the sixty-seven proteins identified by mass spectrometry,

Halophytes

several proteins involved in glycolysis, amino acid metabolism, photosynthesis, and protein

Leaves

folding showed significantly increased abundance during germination. This pattern was less

Proteome

pronounced under salinity. At the vegetative stage, 100 mM NaCl-salinity stimulated

Salt-tolerance

significantly the plant growth, which was sustained by enhanced leaf expansion, water content, and photosynthetic activity. Comparative proteome analyses of leaf tissue revealed 44 proteins with different abundance changes, most of which being involved in energy metabolism. A specific set of proteins predominantly involved in photosynthesis and respiration showed significantly higher abundance in salt-treated plants. Altogether, combining proteomics with eco-physiological tools provides valuable information, which contributes to improve our understanding in the salt-response of this halophyte during its life cycle. © 2012 Elsevier B.V. All rights reserved.

1.

Introduction

Saline regions are often regarded as marginal and unproductive areas, although they are rich in naturally salt-tolerant plants,

i.e. halophytes. These species have evolved several and complex salt-tolerance strategies, which makes them particularly interesting models for understanding the physiological impact of such mechanisms [1]. Recent reviews have also

☆ This paper is a modest tribute dedicated to the memory of Professor Claude Grignon (1942–2010), from B&PMP (“Biologie et Physiologie Moléculaire des Plantes” (CNRS-INRA-SupAgro-University Montpellier2, France) for his valuable contribution in forming generations of plant eco-physiologists in Tunisia. Professor Claude Grignon was also closely involved in the conception of this study. ⁎ Corresponding author. Tel.: +216 79 325 848; fax: + 216 79 325 638. E-mail address: [email protected] (A. Debez).

1874-3919/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jprot.2012.08.012

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pointed out the strong domestication potential of halophytes based on their economical (fodder, seed oil, and food production), medicinal (source of natural antioxidant compounds), and environmental (landscaping, heavy metal phytoremediation) potentials [2–4]. Cakile maritima (Brassicaceae) is a C3 halophyte closely related to the model plants for salt tolerance studies, Arabidopsis thaliana and Thellungiella salsuginea. It is frequently found on the Tunisian seashore, where it contributes to sandy dune fixation due to its deep root system, and shows high seed oil content (up to 39% on dry weight basis), mainly in the form of triacylglycerols (TAG) (97% of the total fatty acids) with erucic acid as major fatty acid [5]. These seed traits typical of conventional oleaginous species confer to this species significant economical potential since erucic acid is widely used, notably in plastic and painting industries [6]. Generally C. maritima behaves as a salt-sensitive species at the early developmental stages, whereas it displays a typical halophytic response at the vegetative and reproductive stages. Indeed, moderate salinities significantly improved both biomass and seed production without altering seed viability or seed oil content [7,8]. Yet, a significant variability in the salt-tolerance aptitude of C. maritima depending on the geographical origin and the plant developmental stage was highlighted [9,10]. Adaptation to salt stress is a multigenic complex response, from the whole plant to the cellular level, notably characterized by changes in gene expression that lead to changes in the protein profile. In addition, proteins undergo significant post-translational modification such as phosphorylation under salinity, which makes quantitative analysis of gene expression at the protein level necessary when addressing plant responses to this issue [11]. As a consequence of recent technical developments in the postgenomics era, emerging molecular ‘omic’ approaches including proteomics are gaining increasing interest and proved to be advantageous for addressing the proteome responses to environmental issues such as salinity [12,13]. While salt impact on the proteome of crops is relatively well documented, leading to the identification of about 2200 proteins [14], reports on halophytes are rather scarce. Only a dozen of species including Aeluropus lagopoides [15], Aster tripolium [16], the mangrove Bruguiera gymnorhiza [17], Porteresia coarctata [18], Puccinellia tenuiflora [19], Salicornia europaea [20], Suaeda aegyptiaca [21], and Thellungiella halophila [22]—despite the latter is rather a facultative halophyte—have been considered so far, but curiously without investigating the germination process. The present study addresses the salt-induced changes in the proteome profile of C. maritima germinating seeds sown with 0 or 0 75 mM NaCl. This crucial developmental stage was emphasized since it greatly determines the successful establishment and development of plants native to saline regions. Furthermore since leaves tightly govern the whole plant biomass production and the seed yield under salinity [23], the leaf proteomic responses upon one month exposure to moderate salinity (100 mM NaCl) were also addressed along with determining physiological data including the plant relative growth rate, leaf expansion, salt accumulation, water status, and the photosynthetic capacity, assessed by pigment content, gas exchange and chlorophyll fluorescence.

Our objectives were to get a global overview of the proteome at two developmental stages of C. maritima when salt-treated. Information gained by the proteomic approach were also integrated with physiological data so that the behavior of this promising oilseed halophyte in its natural habitat can be better understood.

2.

Material and methods

2.1.

Plant material and salt treatments

Mature seeds of C. maritima were harvested from Sousse, a region located on the Mediterranean coast 140 km south of Tunis, and characterized by semi-arid Mediterranean climate. Previous studies reported that germination of C. maritima is maximal in the range of 0–100 mM NaCl. [7,9,10]. Therefore, in the present study, seeds were sown on two layers of filter paper imbibed with 10 ml of either 0 or 75 mM NaCl solution. Seeds were kept in covered Petri dishes to avoid evaporation. Moisture level was monitored regularly. Four replicates of 25 seeds each were used for each treatment. Germination was carried out in the dark at 20 °C (seeds were considered germinated when emerging radicle became visible). Germinated seeds were counted daily for 5 days. In a second experiment conducted under greenhouse conditions (22 ± 2 °C temperature, 60 ± 10% relative humidity, 700 μmol m− 2 s− 1 PAR, 16 h/8 h light/dark regime), seeds were germinated in pots filled with 3 kg inert and humid sand. Ten-day-old seedlings were then watered for one week with a half-strength Hoagland solution and for a further week with the full-strength nutrient solution. After the initial harvest, plants were irrigated at alternate days with 200 ml of the nutrient solution at 0 or 100 mM NaCl (10 replicates per treatment). Salinity level was stepwise increased by 50 mM until reaching the final concentration. Plants were harvested after 1 month of salt treatment. The photosynthetic activity and leaf pigment content of young fully expanded leaves (5 replicates per treatment) were measured prior to the final harvest.

2.2.

Modelling of germination kinetics parameters

In addition to the final germination percentage, the germination kinetics was assessed using a mathematical model, based on the assumption that the germination process has two steps: latency of duration (t0) during which the seeds acquire the aptitude to germinate, followed by the germination itself [7]. After the latency, the probability (k) of germination per unit of time is the same for all the seeds and constant with time. The formal translation of this model is as follows: Y(t) = Ymax (1 + e−k (t − t0)). Y(t) represents the number of germinated seeds at the time t, Ymax is the plateau reached by y(t) and corresponds to the number of viable seeds. k is related to the time for germination of 50% of the viable seeds by T50% = t0 + ln(2)/k. The values of Ymax, k, and t0 were determined by fitting the observed values to the above equation using the nonlinear regression module of the StatisticaTM (StatSoft, Inc.) program.

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2.3.

Growth, leaf water status, ion, and pigment contents

At the final harvest, leaf number, leaf surface area and whole plant dry weight (DW) were determined. The plant relative growth rate (RGR) was calculated as [24]: RGR; d

−1

¼ ΔM=MΔt:

Δ is the difference between the DW values at the final and initial harvests, t the salt treatment duration (days), and M the logarithmic mean of M, the whole plant dry weight (g) as: M ¼ ΔM=Δ lnðMÞ: Leaf succulence ratio (ml cm−2) was used as indicator of leaf water status. It was calculated by ratioing leaf water content to leaf surface area [7]. After extraction in 0.5% HNO3, sodium and chloride were assayed by emission photometry (Corning, UK) and coulometry (Büchler chloridometer), respectively. Chlorophyll (Chl) and carotenoids were extracted from two 10 mm diameter leaf discs in 5 ml 100% acetone. After centrifugation for 5 min at 500 × g, absorbance of extracts was measured at 470, 645 and 662 nm [25]. For anthocyanins, two 10 mm diameter discs were homogenized in 2 ml MeOH: H2O:HCl (3 M) (6:3:1) and kept in the dark for 24 h at 4 °C. Total anthocyanin concentration was estimated as: A530– 0.24 A653 [26].

2.4.

Gas exchange and chlorophyll fluorescence analysis

Gas exchange parameters were measured with an IRGA (LCi, Analytical Development Company Ltd, Hoddesdon, UK). Net photosynthetic rate (A) and stomatal conductance (gs) were determined under the following conditions: 2000 μmol m−2 s−1 PAR, 65± 5% relative humidity, 350 mmol mol−1 ambient CO2 concentration and 29± 2 °C leaf temperature. Chl fluorescence was measured using a PAM-210 Chl fluorimeter (Heinz Walz GmbH, Effeltrich, Germany). Leaves were 30 min dark-adapted before minimal fluorescence (Fo) value was registered by applying a low-intensity red light source of 650 nm. Maximal fluorescence (Fm) was recorded by imposing a saturating light pulse of 3500 mmol m−2 s−1. After leaves were illuminated with actinic light, the steady-state fluorescence (Fs), minimal (F′o) and maximal (F′m) fluorescence of light-adapted leaves were recorded. Based on parameters determined in both dark and light conditions, the maximum quantum efficiency (Fv/ Fm =(Fm −Fo)/Fm) and the quantum yield of PSII (ΦPSII =(F′m −Fs)/F′m) were calculated [27]. Gas exchange and Chl fluorescence data were recorded between 10:00 and 12:00 AM.

2.5.

Protein extraction

Plant material to be used in 2D-PAGE experiments was either seeds at 0, 18, 36, 72, and 120 h of imbibition with 0 or 75 mM NaCl solution, or leaves of plants exposed for one month to 0 or 100 mM NaCl. Samples were weighed and finely ground in a bead mill using liquid nitrogen. About 0.5 g of the obtained powder was then mixed with 750 μl of the extraction buffer pH 8.0 (700 mM sucrose, 500 mM Tris, 50 mM EDTA, 100 mM KCl, 2% (v/v) β-mercapto-ethanol, and 2 mM PMSF). After 10 min incubation in ice, 750 μl of water-saturated phenol

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(Amresco Biotech Chemicals) was added and the mixture was vortexed, shaken at 300 rpm at room temperature for 30 min (Mixer 5432, Eppendorf), and centrifuged for 10 min at 12,000 ×g and 4 °C. The centrifugation step was repeated after the upper phenolic phase containing soluble proteins had been removed carefully and the initial sample volume had been restored by addition of extraction buffer. The proteins extracted in the resulting phenolic phase were precipitated at −20 °C over night by adding 100 mM ammonium acetate in methanol (precipitating solution). This mixture was then centrifuged for 3 min at 15,000 ×g and 4 °C and the pellet was resuspended in 1 ml of the precipitating solution before re-centrifugation. This latter step was repeated three times. The pellet was then rinsed with 80% (v/v) ice-cold acetone, re-centrifuged, and air-dried at room temperature for 10 min.

2.6.

Two-dimensional gel electrophoresis (2D-PAGE)

The extracted proteins (three replicates per treatment) were separated in the first dimension by isoelectric focusing (IEF). In the second dimension separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used. Before IEF was performed, 300 μg of the protein pellet were re-suspended in 350 μl of the rehydration buffer containing 8 M urea, 2% (w/v) CHAPS, 0.5% (v/v) IPG buffer (3–11 NL, GE Healthcare, Munich, Germany), 30 mM DTT, and 2–4 mg Bromophenol Blue. The suspension was vortexed and centrifuged for 5 min at 17,000 ×g and 4 °C. IEF was carried out for 12 h with 18 cm dry gel strips (IPG strips, pH 3–11 non-linear, GE Healthcare, Munich, Germany) using the IPGphor system (GE Healthcare, Munich, Germany) with a current limit of 50 μA/strip at 20 °C. Sample rehydration was performed for 12 h at 30 V, followed by focusing in four steps 500 V (1 h), 1000 V (1 h), 8000 V (1 h), and 8000 V (6 h). Gel strips were then equilibrated for 15 min with a solution containing 50 mM Tris–HCl (pH 8.8), 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 1% (w/v) DTT, and 2 mg Bromophenol Blue. A second equilibration was carried out using the same solution in which DTT was replaced by 2.5% (w/v) iodoacetamide to alkylate free thiol groups of the proteins. Equilibrated IPG strips were then horizontally placed on a 12% Tricine SDS-PAGE gel [28] and sealed with a solution containing 0.5% agarose and 2–4 mg Bromophenol Blue in 100 ml tricine gel buffer pH 8.45 (3 M Tris and 0.3% SDS). SDS-PAGE was carried out at 35 mA for 18–20 h at 20 °C using Protean II xi electrophoresis unit (BioRad, Hercules, CA, USA).

2.7.

Protein staining, gel scanning and image analysis

Two-dimensional (2D) gels were fixed using a fixing solution containing 40% (v/v) methanol and 10% (v/v) acetic acid) for 2 h and stained over night with colloidal Coomassie Blue (0.1% (w/v) CBB-G250, 10% (w/v) ammonium sulphate, and 2% ortho-phosphoric acid in 20% methanol). Gels were then carefully washed with bi-distilled water to remove the background due to staining. Stained gels were scanned at 300 dpi resolution and stored as .tif files. For spot detection and volume quantification, .tif files were transformed into .mel files and analyzed using ImagemasterTM 2D PLATINUM software 6.0 (GE Healthcare, USA). Three images representing

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2.8. Mass spectrometry, protein identification, and hierarchical clustering Spots were excised manually, destained two times with 20 μl of 50% acetonitrile (ACN), 50 mM NH4HCO3 at 37 °C for 30 min, dehydrated by adding 10 μl ACN, and dried [30]. Twenty microliters of 4 ng ml−1 sequencing grade trypsin (Promega) was added and after 30 min incubation on ice remaining trypsin solution was discarded. Digestion was continued at 37 °C for 4 h or overnight and stopped by adding 0.1% TFA, and 50% ACN. Tryptic peptides were extracted with two times 20 μl 50% ACN, 0.1% formic acid (FA) for 30 min at 37 °C, and 10 μl ACN for 30 min at room temperature. All extracts were dried in a vacuum centrifuge. MALDI-ToF/ToF analysis was started by re-solubilizing dried samples in 2 μl of 2 mg/ml alpha cyano hydroxy cinnamic acid (CHCA), 50% ACN, and 0.1% TFA. One microliter of the solution was spotted onto a stainless steel target (AB Sciex) and analyzed with an AB Sciex TOF/TOF 5800 mass spectrometer. Internal calibration based on autolytic porcine trypsin peptides was applied for precursor MS spectra. External calibration with Glu-Fib fragments was used for MS/MS spectra. Mass spectrometrical data were searched against the SwissProt and NCBI Database with carbamidomethylation of cysteins as static and oxidation of methionine as variable modification. At least two peptides with a Mascot peptide ion score higher than 20 each or one peptide with a score higher than 55 were used as a threshold for protein identification for the reference map. Because of the incomplete data publicly available for C. maritima genome, for several protein spots no C. maritima protein was identified but a orthologous protein from a different plant. These orthologous proteins should however represent the similar protein from C. maritima. Protein classification was achieved using the database available on the “Proteomics of oilseeds” platform: http:// www.oilseedproteomics.missouri.edu and Expasy. Hierarchical clustering of identified proteins was performed using Gene Cluster software 3.0 with centered correlation and average linkage of log-transformed and standardized spot volumes (3 spots per treatment and per organ). Phylogenic trees were then established using TreeView software (http://rana.lbl. gov/downloads/TreeView/TreeView_vers_1_60.exe) [31].

3.

Results

3.1. Salt impact on the germination process: seed germination capacity and proteome profile Exposure to moderate salinity (75 mM NaCl) impaired slightly but significantly the germination of C. maritima seeds. The germination process was delayed especially in the 0 h-72 h time range, as directly reflected by the higher values of both the latency time and the time to reach 50% germination (t0 and T50%, respectively) (Fig. 1 and Table 1). Seed final germination percentage (observed or calculated) and seedling length were also slightly but significantly reduced in salt treated plants compared to control plants (Fig. 1 and Table 1). As a next step, protein fractions from salt-treated and control seedlings were extracted at defined time-points (0, 18, 36, 72,120 h after imbibition) and resolved by 2D IEF/SDS PAGE. Inspection of the resulting 2D gels allowed identification of six zones which include especially many proteins of changed abundance (Fig. 2). These zones are shown in detail in Fig. 3. Overall, the 67 proteins of changed abundance during germination in salt-free conditions were identified by mass spectrometry, most of which (37 proteins) representing seed storage proteins (SSPs), followed by proteins involved in energy metabolism and photosynthesis (12 proteins), other pathways of primary metabolism (9 proteins), stress-related proteins (5 proteins), proteins involved in folding and stability (2 proteins), and proteins with unknown function (2 proteins) (Table 2). Several distinct protein spots on the 2D gels were found to represent identical proteins, most likely reflecting posttranslational modifications. Seeds germinating in salt-free medium showed 26 storage proteins SSPs of significantly reduced and 11 SSPs of significantly increased abundance (Figs. 3 and 4A, Table 1 Supplementary material). SSP degradation was asynchronous

a

a

a

a

a

a

a

b

b

b

b

b

100

Germination percentage

three independent biological replicates of either germinating seeds or leaves exposed to salinity were grouped to calculate the mean volume of all the individual protein spots. The spot abundance was normalized as relative volume according to the normalization method provided by the software to obtain the individual relative spot volume (%), i.e. the spot volume of one spot in relation to the sum of all detected spots on the gel. This method eliminates eventual protein loading differences [29].

75

bc c

50

d

25

f g

0

75 mM NaCl

h

0 0

g

h

i

12

24

36

48

60

72

84

96

108

120

Time ( hours after sowing)

2.9.

Statistical analysis

Mean values were compared using the Student's t-test at P < 0.05. 2D germination data were analyzed with a two-way ANOVA at the same significance level with time and salinity as factors. In case of significant differences, Duncan post hoc tests were performed. The SPSS 13.0 statistical program (IBM) was used.

Fig. 1 – Time-course changes of C. maritima seed germination expressed as % of sown seeds with 0 or 75 mM NaCl solution. The observed values were subsequently used for calculating germination kinetics parameters presented in Table 1. Means of 4 replicates ± standard error. For each parameter, means with at least one same letter were not significantly different at P < 0.05.

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Table 1 – Salt effect on the final germination percentage, the parameters of germination kinetics, and the seedling length of C. maritima. Means of 4 replicates ± SE. For each parameter, means followed by at least one same letter were not significantly different at P < 0.05. NaCl (mM)

Final germination percentage a

Calculated final germination percentage: Ymax

0 75

100.00 ± 3.11a 91.66 ± 4.02b

100.00 ± 2.22a 94.00 ± 2.62b

a

t0

k

t50%

0.52 ± 0.06b 0.69 ± 0.05a 1.00 ± 0.12b 0.71 ± 0.07a 0.47 ± 0.06b 1.47 ± 0.21a

Seedling length (cm) 4.8 ± 0.28a 4.12 ± 0.22b

As determined by visual inspection.

and likely reflects the mobilization of seed energy and nitrogen reserves, a crucial step of the germination process (Figs. 3 and 4A). With respect to SSP degradation four kinetics classes were identified as follows: strong degradation after 18 h (s12 and s19), 36 h (e.g. s1, s2, and s3), 72 h (e.g. s5 and s2), and 120 h (e.g. s46, s47, and s48) of seed imbibition. Most of the SSPs showed marked reduced abundance at 72 h and four spots were no more detectable after 72 h (s20) and 120 h (s1, s15, and s25) of seed imbibition. Exposure to 75 mM NaCl salinity triggered a significant delay in the degradation of 16 SSP (e.g. s67, s9, s13, s14, s15, s16, s25, s26, s46, and s64), mainly in the 72 h–120 h time range (Figs. 3 and 4A, Table 1 Supplementary material). S1 was still detectable at 120 h after seed sowing whereas it was absent from the 2D-map of control seedlings. As found for the control, 11 SSPs displayed significantly increased abundance in saline conditions, despite to a lesser extent for s31, s33, s34, s35 and s55. In the control, seven (s17, s18, s39, s42, s44, s57, and s63) out of the 12 spots representing energy metabolism- and photosynthesis-related proteins were constitutively present, the remaining being detectable at 72 h of seed imbibition (Figs. 3 and 4B). Induced proteins were fructose bisphosphate aldolase (s21), glyceraldehyde-3-phosphate dehydrogenase (s23 and s24) and RuBisCO small and large subunit (s40 and s52, respectively). Some proteins involved in energy metabolism showed significant increased abundance during seed germination, except for putative aldolase (s63) which was constant. This pattern was significantly less pronounced under salt treatment for four proteins: phosphoglycerate kinase (s44), enolase (s42), and RuBisCO small subunit and large chain (s40 and s52, respectively) (Figs. 3 and 4B, Table 1 supplementary material). Yet, it is noteworthy that salt-impact occurred relatively late, mainly at 72 h, i.e. after radicle protrusion which corresponds to the seedling establishment stage. According to their change in abundance, four sub-groups of primary metabolism-related proteins could be distinguished in control experiments (Figs. 3 and 4C, Table 1 Supplementary material): the first one was composed by a single protein with unchanged abundance (s45, S-adenosylmethionine synthetase), the second one included constitutively present proteins with significantly increased abundance (s56, s65, and s66, respectively, identified as cobalamin-independent methionin synthase, serine hydroxymethyltransferase, and 3-keto-acyl-CoA thiolase 2), the third one comprised proteins of significantly increased abundance which first were detectable 18–36 h after seed sowing (s43, s54, and s62, respectively, identified as S-adenosylmethionine synthetase, nucleoside diphosphate

kinase 3, and glutamine synthetase), and the last group included proteins also characterized by significantly increased abundance, but which were later detectable, i.e. at 72 h (s22 and s58, respectively, identified as glutamine synthetase, and beta-glucosidase). Compared to the control, time-dependent changes in the abundance of primary metabolism-related proteins were similar although to a lesser extent when germinating seeds were exposed to 75 mM NaCl, salinity notably delaying the induction of the proteins s43, s54, and s62 (Figs. 3 and 4C, Table 1 Supplementary material). Stress-related proteins identified were putative small HSP (s51) and catalase (s59, s60, s61, and s67), s60 and s61 being induced at 18 h and 36 h, respectively (Figs. 3 and 4D). In salt-free conditions, the first protein was of significantly lower abundance during germination, whereas catalase showed significantly increased abundance. This protein abundance pattern was significantly less pronounced under saline conditions, especially for s61 and s67 which were induced later as compared to the control (Figs. 3 and 4C, Table 1 Supplementary material). Folding proteins identified were GroEL protein chaperonin (s38) and protein disulfide isomerase (s37). In the control, the latter protein showed significantly decreased abundance until 72 h after seed sowing, before significantly increasing. Both proteins were unaffected by salinity (Figs. 3 and 4E, Table 1 Supplementary material). Finally, two proteins (s11 and s41), which correspond to accessions AC069144 NID and ATH271468 NID, represent proteins with unknown function. The first protein was of significantly lower abundance during the germination in salt-free conditions, whereas the second remained constant. Abundance of both proteins was unaffected by mild salinity (Figs. 3 and 4E, Table 1 Supplementary material).

3.2. Growth, salt accumulation, water relations, and photosynthetic capacity The whole plant relative growth activity (RGR) was significantly stimulated at 100 mM NaCl (+14% as compared to the control). This was concomitant with a significant increase of the leaf surface area and the leaf number per plant (+18% and +22% as compared to the control, respectively) (Table 3). Leaf water status, assessed, by leaf succulence ratio, was significantly improved by moderate salinity, although sodium, and to a lesser extent chloride, were strongly accumulated in leaves (Table 3). Leaf pigment (total chlorophyll, carotenoids, and anthocyanins) contents increased significantly upon exposure to mild salinity (+19%, +22%, and +25% as compared to the control,

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Salinity ( mM NaCl ) 0

75

pI 3

pI 11

100 KDa

VI

VI III

III

I 0

I

IV

IV

V

II

V

II

10 KDa

VI

VI III

III

I 18

I IV

IV

Germination duration (h)

V

II

VI

V

II

VI III

III

I

I

36

IV

IV

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V

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VI III

III

I

I

72

IV

IV

V

II

VI

V

II

VI III

III

I

120

I IV

V

IV II

V

II

Fig. 2 – Kinetics of the proteome profile of C. maritima germinating seeds as affected by salinity. Proteins were extracted at 0 h, 18 h, 36 h, 72 h and 120 h of seed imbibition in the presence of either 0 or 75 mM NaCl solution. Proteins were separated according to their IEPs by isoelectric focusing (pH 3–11) in the first dimension and by Tricine SDS-PAGE in the second dimension. Gels were CBB-stained. Within six framed zones (I to VI), 67 differentially expressed proteins, marked by their corresponding numbers, were identified by mass spectrometry. The six framed zones are enlarged in Fig. 3.

respectively) (Table 4). CO2 net assimilation rate (A), stomatal conductance (gs), and PSII quantum yield (ΦPSII) were also significantly enhanced at 100 mM NaCl (+21%, +30%, and +21%

as compared to the control, respectively) whereas maximum quantum efficiency (Fv/Fm) was unaffected by 100 mM NaCl salinity (Table 4).

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3.3.

Leaf proteomic responses

Finally, comparative proteome analyses were carried out for leaf tissue of 100 mM NaCl-treated and control plants. On the corresponding 2D gels (Fig. 5), five zones including especially many proteins constitutively present and of differential abundance were identified. Of the 44 spots which were detected in these zones, the majority (35) corresponded to proteins involved in photosynthesis and energy metabolism.

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Twenty-seven spots included the large or the small subunit of RuBisCO (Table 5). The remaining proteins within this class were F1-type ATP synthase alpha and beta subunits (s7 and s8, respectively), enolase (s11), glyceraldehyde-3-phosphate dehydrogenase (s14), RuBisCO activase (s17), a subunit of PSII (s30), oxygen evolving-enhancer protein (s33), and PSII oxygen evolving complex protein 2 (s36). The other gel zones included proteins involved in stress-response, like catalase, oxalic acid oxidase, and dehydroascorbate reductase fragment (S10, S34,

Fig. 3 – Enlargement of the six zones (I–VI) within the 2D-PAGE gels showing the time-course changes in the abundance of the 67 protein spots identified upon salt-exposure of C. maritima germinating seeds. For each zone, white arrows indicate the spot position on the gel at a given time and salt concentration.

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Fig. 3 (continued).

and s35, respectively), primary metabolism, like glutamine synthetase precursor and putative 3-beta hydroxysteroid dehydrogenase/isomerase) (s16 and s29, respectively), proteins involved in folding and stability, like peptidyl-prolyl cis-trans isomerase CYP20-3, chloroplastic and heat shock protein HSP70(s37 and s44, respectively), cell growth factor (s13, putative chloroplast Elongation factor, EF-Tu), and a protein with unknown function (s32, putative uncharacterized protein) (Table 5). Salt treatment had no impact on the abundance of 27 proteins. In contrast, 17 proteins showed significantly higher abundance. Most of these proteins are involved in energy metabolism and photosynthesis (RuBisCO activase 1, RuBisCO small and large subunits as well as alpha and beta subunits of F1-type ATP synthase), stress-response (s10 and s34, respectively, identified as catalase and oxalic acid oxidase), and cell growth (s13, putative chloroplastic elongation factor) (Figs. 5B, C, 6, and 7).

4.

Discussion

The present study documents the salt-induced changes in the proteome profile of the halophyte C. maritima at two developmental stages. It was also designed to bring together data from molecular (using the proteomic tool) and ecophysiological (notably using non-destructive methods)

approaches so that the salt-responses of this halophyte can be more accurately deciphered. We focused on the germinative and autotrophic stages since the aptitude of halophytes to cope with salinity differs during its life cycle. Our previous results highlighted this phenomenon in tunisian accessions of C. maritima which showed to be salt-sensitive to relatively salt-tolerant at the early developmental stages whereas behaving either as salt-requiring or facultative halophyte, respectively, at the vegetative and reproductive stages [7,32,9] and [10].

4.1. Moderate salinity impacts C. maritima germination by delaying the mobilization of seed reserves: proteomic evidence Germination is a critical step in the halophyte life cycle, especially for annuals, which at the early developmental stage are generally as salt-sensitive as glycophytes. Salt-free conditions were optimal for C. maritima seed germination, since moderate salinity delayed the germination process and led to a slight but significant reduction of the final seed germination percentage and 5 day-old seedling length. The adverse effect of salinity has also been reported for germinating seeds of several other halophytes including Limonium emarginatum, Crithmum maritimum, and Polypogon monspeliensis [33–35]. To accomplish germination and seedling establishment, seeds deprived from a functional photosynthetic apparatus

J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 56 6 7 –5 69 4

5675

Fig. 3 (continued).

and the ability to take up nutrients, rely on utilization of storage products (mainly lipids, proteins, and starch) that were gradually accumulated during seed maturation [36]. Monitoring the proteome of germinating seeds revealed that besides fatty acids, C. maritima seed storage compounds include 11S globulins and cruciferins. These proteins were characterized by an asynchronous degradation, starting early in the germination phase and extending up to the seedling stage. Hence, in C. maritima, seed storage protein (SSP) mobilization starts early during the germination process, resulting in proteolytic fragments (as reflected by the significantly increased abundance of 11 SSP spots on 2D gels shown in Fig. 2). This phenomenon was documented in A. thaliana [37], rice [38], subclover [39], and more recently in Medicago sativa [40]. Mobilization of SSPs may facilitate their further proteolytic degradation during the

last stages of seed germination and seedling establishment [41]. Moderate salinity caused a significant delay in degradation of 16 of the 26 SSPs characterized by significantly lower abundance during germination, which suggests that the impairment of C. maritima germination under salinity has to be ascribed to a certain extent to slower mobilization of SSPs. It therefore provides a reasonable evidence for the physiological findings showing significantly slower germination rate (reflected by the increasing latency time and the time for 50% germination) at 75 mM NaCl. A salt-induced delay in reserve mobilization during germination was also shown for wheat [42], flax [43] and cashew [44] seeds. In the latter, storage protein hydrolysis was delayed by NaCl salinity since seed germination and the salt-induced inhibition of seedling growth was closely related to the delay of reserve

5676

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 56 6 7 –56 9 4

Fig. 3 (continued).

mobilization and the accumulation of products of reserve hydrolysis in the cotyledons. With respect to protein degradation, a water-requiring process (hydrolysis), which was significantly slowed-down under saline conditions, one may hypothesize that: (i) salinity restricted imbibition of C. maritima seeds through its osmotic effect [7], so that less water would be available for SSP degradation. This finding is of environmental significance since it may partly explain the necessity of salt leaching by rain so that halophyte seeds can germinate. (ii) Despite no data could be inferred from the present study, one may also assume that salinity likely impaired activity of proteases (e.g. cysteineproteinases, aspartic-proteinases) involved in SSP degradation, due to Na + binding to the functional groups of the proteins [45] or to post-translational modifications leading to misfolding protein and/or altered gene expression [46–48], which may explain the delay in SSP mobilization at 75 mM NaCl. (iii) Regulatory effects of protein phosphorylation, for instance, may be crucial for the observed effects. Several identified proteins in this present investigation were found to be represented by more than one of the salt responsive spots. Similar observations have been made when analyzing salt stress effects on the proteome of rice roots [11]. This finding is calling for a more detailed investigation of the phosphoproteome of C. maritima when exposed to salinity.

4.2. Moderate salinity causes only less pronounced changes in abundance of primary metabolism- and stress-related proteins Primary metabolism proteins identified (12 spots) were involved in four major pathways: photosynthesis (RuBisCO), glycolysis (Glyceraldehyde-3-phosphate dehydrogenase, fructose bisphosphate aldolase, putative aldolase, and enolase), gluconeogenesis (phosphoglycerate kinase), and the glyoxylate cycle (malate synthase). These proteins were mostly constitutively expressed, indicating that C. maritima seeds are well equipped to set off metabolic pathways like gluconeogenesis and glyoxylate cycle during germination to provide energy and carbon skeletons from stored lipids. In oilseed plants like C. maritima, the conversion of lipids to sucrose is triggered by germination. The reaction sequence begins with hydrolysis of TAGs stored in oil bodies to form free fatty acids. In a subsequent reaction fatty acids are oxidized to produce acetyl-CoA. This process requires the coordinated activation of a number of metabolic reactions located in different cellular compartments (oleosomes, glyoxysomes, mitochondria, and cytosol) [49]. Briefly, lipases hydrolyze stored triacylglycerol to its glycerol and fatty acid components. Fatty acids are then transported to glyoxysomes, where they are activated to acyl CoA derivatives, which will be turned over via ß-oxidation to form acetyl CoA. Subsequently acetyl CoA may feed into the

J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 56 6 7 –5 69 4

5677

Fig. 3 (continued).

glyoxylate cycle to compensate for consumption of four-carbon compounds by anabolic pathways [50]. Reserve mobilization is a major event during the postgerminative stages, but it can also occur during seed imbibition, as reported from experiments using A. thaliana seeds. It was found that partial mobilization of seed reserves increased carbon availability or provided signalling molecules, contributing to dormancy release and germination induction [51]. Consistent with our data, a significant augmentation in abundance of proteins related to photosynthesis and energy metabolism has been also reported during germination of rice [38], Beta vulgaris [52], the oilseed species Jatropha curcas [36] and M. sativa [40]. Interestingly, time-course changes in protein abundance for enzymes involved in energy metabolism were similar under salinity, despite being less pronounced for 4 proteins (phosphoglycerate kinase, enolase, and the RuBisCO small and large chain). It should be also pointed out that salt impact on enzymes involved in energy metabolism was not

exerted at early stages of the germination process, but rather at the seedling stage (at 72 h of seed sowing). This could be related to enhanced sensitivity to salt stress at later germination phases (due to their energy demand). Our findings on enolase, the key enzyme of glycoloysis, were also documented in Brassica campestris [53] and rice [54]. With respect to RuBisCO, two of the three spots were induced after the radicle emergence (72 h after seed sowing), i.e. at the seedling stage. The RuBisCO large subunit binding protein has been suggested to function as chaperone showing significantly higher abundance under water deficit and heat stresses [55,56]. Proteins involved in primary metabolism (9 spots) were either constitutively expressed or asynchronously induced. The identified proteins were involved in amino acid metabolism (glutamine synthetase, S-adenosylmethionine synthetase, and cobalamin-independent methionine synthase), ATP metabolism (nucleoside diphosphate kinase 3), polysaccharide catabolism (beta-glucosidase), one-C metabolism (serine hydroxymethyl

5678

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 56 6 7 –56 9 4

Fig. 3 (continued).

transferase), and beta-oxidation (3-ketoacyl-CoA thiolase 2, peroxisomal). Most of the proteins were of significantly higher abundance whether seed germination took place in salt-free or in saline conditions, although this tendency was less marked under salinity. For instance, the induction of 3 spots (s43, s54, and s62, respectively S-adenosylmethionine synthetase, nucleoside diphosphate kinase 3, and glutamine synthetase) appeared to be delayed by salt exposure. 3-keto-acyl-CoA thiolase 2, involved in β-oxidation cycle, showed significantly increased abundance during seed germination, which supports the

abovementioned assumption concerning the mobilization of fatty acid reserves during the germination of C. maritima seeds. As the content of amino acids increases upon seed imbibition, due to the hydrolysis of reserve proteins, several proteins involved in amino acid (especially methionine and glutamine) metabolism show higher activity. This was highlighted by proteomic data in pea [57], B. vulgaris [52], and M. sativa [40]. Methionine, which is implicated in protein synthesis, is the direct precursor of adenosylmethionine, the universal donor of

5679

J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 56 6 7 –5 69 4

Table 2 – Classification of proteins identified during seed germination. Spot a

Accession number b

Protein c

Organism d

Score e MW f

I. Storage Q2TLW0_SINAL

11S globulin precursor

Sinapis alba

155

56.5

MS

MS/MS

4

2

Abundance pattern h

4

Relative abundance

1

N° MP g

3

2

1

0 0

Q2TLW0_SINAL

11S globulin precursor

Sinapis alba

130

56.5

2

2

18

36

72

120

Time, h

4 Relative abundance

2

3

2

1

0

Q2TLW0_SINAL

11S globulin precursor

Sinapis alba

131

56.5

4

3

Relative abundance

0

3

18

36

72

120

Time, h

0

18

36

0

18

36

72

120 Time, h

0

18

36

72

120 Time, h

0

18

36

72

120 Time, h

0

18

0

18

36

72

120 Time, h

0

18

36

72

120

0

18

36

72

120 Time, h

0

18

36

72

120 Time, h

0

18

36

72

120 Time, h

0

18

36

72

120 Time, h

0

18

36

72

120 Time, h

0

18

36

72

4

3

2

1

0

Q7XB53_BRANA

Cruciferin (fragment)

Brassica napus

140

51.3

8

2

72

120

Time, h

4

Relative abundance

4

3

2

1

0

S08510

Cruciferin precursor

Arabidopsis thaliana

176

50.6

5

2

4

Relative abundance

5

3

2

1

0

S08510

Cruciferin precursor

Arabidopsis thaliana

170

50.6

6

2

4

Relative abundance

6

3

2

1

0

S08509

Cruciferin precursor

Arabidopsis thaliana

135

32.6

4

3

4

Relative abundance

7

3

2

1

0

Q7XB52_BRANA

Cruciferin (fragment)

Brassica napus

164

51.1

6

2

36

72

120

Time, h

4

Relative abundance

8

3

2

1

0

Q7XB52_BRANA

Cruciferin (fragment)

Brassica napus

136

51.1

6

3

4

Relative abundance

9

3

2

1

0

S08510

Cruciferin precursor

Arabidopsis thaliana

164

50.6

7

2

Time, h

4

Relative abundance

10

3

2

1

0

S08509

Cruciferin precursor

Arabidopsis thaliana

122

52.6

4

2

4

Relative abundance

12

3

2

1

0

Q2TLV9_SINAL

11S Globulin precursor

Sinapis alba

99

57.9

6

3

3

Relative abundance

13

2

1

0

Q2TLV0_SINAL

11S Globulin precursor

Sinapis alba

64

56.5

4

2

3

Relative abundance

14

2

1

0

Q7XB53_BRANA

Cruciferin (fragment)

Brassica napus

196

51.3

8

3

3

Relative abundance

15

2

1

0

Q7XB53_BRANA

Cruciferin (fragment)

Brassica napus

77

51.3

10

1 Relative abundance

16

3

2

1

0 120

Time, h

(continued on next page)

5680

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 56 6 7 –56 9 4

Table 2 (continued) Accession number b

Protein c

Organism d

19

Q7XB53_BRANA

Cruciferin (fragment)

Brassica napus

Score e MW f 225

51.3

N° MP g 11

Abundance pattern h

3

3

Relative abundance

Spot a

2

1

0

Q2TLW0_SINAL

11S Globulin precursor

Sinapis alba

118

56.5

6

2

0

18

36

72

120

Time, h

0

18

36

72

120

Time, h

0

18

36

72

120

Time, h

0

18

36

72

120

Time, h

0

18

36

72

120

Time, h

0

18

36

72

120

Time, h

0

18

3

Relative abundance

20

2

1

0

S08509

Cruciferin precursor

Arabidopsis thaliana

81

52.6

6

2

3

Relative abundance

25

2

1

0

Q7XB53_BRANA

Cruciferin (fragment)

Brassica napus

192

51.3

10

3

3

Relative abundance

26

2

1

0

S08509

Cruciferin precursor

Arabidopsis thaliana

122

52.6

2

2

3

Relative abundance

27

2

1

0

Q2TLW0_SINAL

11S Globulin precursor

Sinapis alba

77

56.5

6

2

6

Relative abundance

28

5 4 3 2 1 0

Q2TLW0_SINAL

11S Globulin precursor

Sinapis alba

75

56.5

5

5

12

Relative abundance c

29

10 8 6 4 2 0

S14762

Cruciferin CRU4

Brassica napus

130

51.4

8

3

36

72

Time, h

120

12

Relative abundance

30

10 8 6 4 2 0 0

S08509

Cruciferin precursor

Arabidopsis thaliana

77

52.6

6

1

18

36

72

Time, h

120

14 12

Relative abundance

31

10 8 6 4 2 0

32

S14762

Cruciferin CRU4

Brassica napus

107

51.4

8

2

36

72

Time, h

0

18

120

0

18

36

72

0

18

36

72

0

18

36

72

120

Time, h

0

18

36

72

120

Time, h

0

18

36

72

120

Time, h

0

18

12

Relative abundance

10 8 6 4 2 0

S08510

Cruciferin precursor

Arabidopsis thaliana

72

50.6

4

2

120

Time, h

10

Relative abundance

33

12

8 6 4 2 0

S14762

Cruciferin CRU4

Brassica napus

52

51.4

4

1

120

Time, h

8

Relative abundance

34

6

4

2

0

Q7XB52_BRANA

Cruciferin

Brassica napus

171

51.1

10

4

12

Relative abundance

35

10 8 6 4 2 0

A35540

Cruciferin precursor

Brassica napus

95

56.4

7

3

10

Relative abundance

36

8 6 4 2 0

S26217

Cruciferin fragment pAF7

Raphanus sativus

144

26.2

11

5

8 7

Relative abundance

46

6 5 4 3 2 1 0 36

72

120

Time, h

5681

J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 56 6 7 –5 69 4

Table 2 (continued) Accession number b

Protein c

Organism d

47

Q41164_RAPSA

Cruciferin fragment pAF7

Raphanus sativus

Score e MW f 133

26.1

N° MP g 11

Abundance pattern h

2

8

Relative abundance

Spot a

7 6 5 4 3 2 1 0

CAA40979

Cruciferin precursor

Brassica napus

132

12

3

1

0

18

36

72

120

Time, h

0

18

36

72

120

Time, h

7

Relative abundance

48

6 5 4 3 2 1 0

S26221

Cruciferin fragment pBB6

Raphanus sativus

91

12.3

3

1

8 7

Relative abundance

49

6 5 4 3 2 1 0 0

CAA40979

Cruciferin precursor

Brassica napus

242

12

4

2

18

36

120 Time, h

72

8

Relative abundance

50

9

7 6 5 4 3 2 1 0 0

53

S25091

Cruciferin fragment pAF7

Raphanus sativus

85

26.1

14

2

Cruciferin BnC2

Brassica napus

91

54.3

7

3

18

36

72

120

Time, h

120

Time, h

5

Relative abundance

55

Q41164_RAPSA

4

3

2

1

0 0

Q2TLW0_SINAL

11S Globulin precursor

Sinapis alba

134

56.5

5

2

18

36

72

10

Relative abundance

64

8

6

4

2

0 0

G3PC_SINAL

Glyceraldehyde-3-phosphate DH, cytosolic

Sinapis alba

185

37

8

3

36

120 Time, h

72

3

Relative abundance

II. Energy 17

18

2

1

0

G3PC_SINAL

Glyceraldehyde-3-phosphate DH, cytosolic

Sinapis alba

270

37

15

3

0

18

36

72

120

Time, h

0

18

36

72

120

Time, h

0

18

36

72

120

Time, h

0

18

36

72

120

Time, h

0

18

5

Relative abundance

18

4 3 2 1 0

T47550

Fructose bisphosphate aldolase

Arabidopsis thaliana

127

38.5

14

2

7 6

Relative abundance

21

5 4 3 2 1 0

Q2KMD9_QMYRT

Glyceraldehyde-3-phosphate DH

Pternandra multiflora

139

13.1

12

1

3 Relative abundance

23

2

1

0

24

Q4FAA7_BRANA

Glyceraldehyde-3-phosphate DH

Brassica napus

268

17.6

12

2 Relative abundance

3

2

1

0

Q7YNV2_CAKMA

RuBisCO large chain

Cakile maritima

557

47.6

39

11

36

72

120

Time, h

6

Relative abundance

39

7

5 4 3 2 1 0

Q7YNV2_CAKMA

RuBisCO large chain

Cakile maritima

188

47.6

24

2

0

18

36

0

18

36

72

120 Time, h

3

Relative abundance

40

2

1

0 72

120

Time, h

(continued on next page)

5682

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 56 6 7 –56 9 4

Table 2 (continued) Accession number b

Protein c

Organism d

42

Q6W7E8_BRACM

Enolase

Brassica campestris

Score e MW f 239

47.3

N° MP g 19

Abundance pattern h

2

6 5

Relative abundance

Spot a

4 3 2 1 0

H96826

Phosphoglycerate kinase

Arabidopsis thaliana

117

42.1

16

1

0

18

36

0

18

36

72

120

Time, h

3

Relative abundance

44

2

1

0

52

S37292

RuBisCO small subunit, chloroplastic

Brassica napus

326

20.2

21

3

72

120

Time, h

18 16

Relative abundance

14 12 10 8 6 4 2 0 0

SYRPMA

Malate synthase, glyoxysomal

Brassica napus

189

63.6

29

3

72

Time, h

18

36

120

0

18

36

72

120

Time, h

0

18

36

72

120

Time, h

4

Relative abundance

57

3

2

1

0

Q39414_BRACM

Putative aldolase

Brassica campestris

121

38.5

11

1

3

Relative abundance

63

2

1

0

III. Primary metabolism 22 S52040

Glutamine synthetase

Raphanus sativus

133

38.5

12

2 Relative abundance

4

3

2

1

0 0

JQ0410

S-adenosylmethionine synthetase 2

Arabidopsis thaliana

179

43.2

15

3

72

36

120

Time, h

3

Relative abundance

43

18

2

1

0 0

Q9LUT2_ARATH

S-adenosylmethionine synthetase

Arabidopsis thaliana

117

42.8

12

3

18

36

72

120

Time, h

5

4 Relative abundance

45

3

2

1

0 0

Q9LRJ2_BRACM

Nucleoside diphosphate kinase 3

Brassica campestris

65

21.5

6

1

18

36

72

120

Time, h

7 6

Relative abundance

54

5 4 3 2 1 0 0

Q6KCR2_ARATH

Cobalamin-independent methionine synthase

Arabidopsis thaliana

65

84.3

15

1

18

36

72

120

Time, h

3

Relative abundance

56

4

2

1

0 0

58

O24434_BRANI

Beta-Glucosidase

Brassica nigra

108

50.7

17

18

36

72

120

1 Relative abundance

5

4

3

2

1

0

62

S52040

Glutamine synthase

Raphanus sativus

215

38.5

13

3

0

18

36

0

18

36

0

18

0

18

72

120

Time, h

6

Relative abundance

5

4

3

2

1

0

Q9FPJ3_ARATH

Serine hydroxymethyltransferase

Arabidopsis thaliana

140

51.8

17

2

72

120

Time, h

3

Relative abundance

65

2

1

0

T52110

3-ketoacyl-CoA thiolase 2, peroxisomal

Arabidopsis thaliana

251

48.5

16

6

36

72

120

Time, h

6

5

Relative abundance

66

4

3

2

1

0 36

72

120

Time, h

Time, h

5683

J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 56 6 7 –5 69 4

Table 2 (continued) Accession number b

IV. Stress-related 51

B84697

Protein c

Organism d

Putative small HSP (At2g29500)

Arabidopsis thaliana

Score e MW f

88

17.6

N° MP g

14

Abundance pattern h

1

5

4 Relative abundance

Spot a

3

2

1

0 0

Q95YT7_BRAJU

Catalase

Brassica juncea

187

56.9

23

2

18

36

72

120

Time, h

4

Relative abundance

59

3

2

1

0

Q95YT7_BRAJU

Catalase

Brassica juncea

206

56.9

23

3

0

18

36

72

120 Time, h

0

18

36

72

120 Time, h

0

18

3

Relative abundance

60

2

1

0

61

Q9M4X3_RAPSA

Catalase

Raphanus sativus

161

56.8

24

1

5

Relative abundance

4

3

2

1

0

Q9ZPK6_BRAJU

Catalase

Brassica juncea

250

56.8

45

2

36

72

120 Time, h

4

Relative abundance

67

3

2

1

0 0

V. Folding and stability 37 Q38HW3_BRACI

Protein disulfide isomerase

Brassica carinata

62

55.7

11

1

18

36

72

120 Time, h

5

Relative abundance

4

3

2

1

0 0

Q9LJE4_ARATH

GloEL protein; chaperonin

Arabidopsis thaliana

186

63.8

26

4 Relative abundance

38

18

36

72

120

Time, h

36

72

120

Time, h

120 Time, h

3

2

1

0 0

AAG51119

AC069144 NID

Arabidopis thaliana

109

31.2

11

2

4

Relative abundance

VI. Unkown 11

18

3

2

1

0

CAL81058

ATH271468 NID, mitochondrial

Arabidopsis thaliana

244

63.3

41

4

0

18

36

72

0

18

36

72

4

Relative abundance

41

3

2

1

0 120

Time, h

a

Spot number as indicated on the 2D-PAGE master gels (see Fig. 3) and functional classification of the matching protein identified during germination of C. maritima seeds in the presence of salt. Protein classification was achieved using the database available on the “Proteomics of oilseeds” platform: http://www.oilseedproteomics.missouri.edu and Expasy. b Accession number according to the best hit of MASCOT search against SwissProt and NCBI databases. c The identified protein according to the best hit of MASCOT search against SwissProt and NCBI databases. d The corresponding species according to the best hit of MASCOT search against SwissProt and NCBI databases. e Probability score for protein identification based on MS analysis and MASCOT search. f Theoretical molecular mass of the identified protein. g Number of matching peptides. h Time-course changes in the protein abundance during seed germination of C. maritima as affected by salinity. Data on the Y axis represent the relative volume of each spot (Means of 3 replicates ± standard error).

methyl groups, and is also the precursor of polyamines, ethylene and biotin [58]. In A. thaliana, germination and seedling establishment were impaired in presence of the specific inhibitor of methionine biosynthesis, d,l-propargylglycine [59]. S-adenosylmethionine (SAM) synthetase catalyzes the formation of SAM from its substrates methionine and ATP. SAM is required for maintenance and recycling of methylation in

plants, for instance [60]. SAM synthetase also was shown to play a potent role in metabolism transition from a quiescent to a highly active state during A. thaliana seed germination [59]. Using proteomic data, the relative salt tolerance of the wheat cultivar Jing-411 was partly ascribed to a higher amount of SAM synthetase in salt-treated leaves [61]. Unlike our findings, significantly higher abundance of glutamine synthetase has

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been documented in Setaria italica salt-exposed germinating seeds, in concomitance with a significant increase of proline content [62]. More generally, Met cycle constitutes a “hub” that controls metabolic activity during germination process concomitantly with the action of the principal hormones and

signalling molecules involved in regulation of seed germination and seedling establishment [63]. Two stress-related proteins, either of significantly lower or higher abundance (putative small HSP s51 and catalase, respectively) during seed germination, were identified. Salt

Fig. 4 – Hierarchical clustering of the proteins identified during the germination of C. maritima under salinity based on the mean values of protein relative volume (three replicates per sample) inferred by using ImagemasterTM 2D PLATINUM software 6.0. The color gradient from bright green to bright red indicates increasing protein abundance. Grey cases represent undetected proteins. Log-transformed and standardized means of spot relative abundance were subjected to centered correlation and average linkage for clustering using TreeView software (Shi et al., 2010). Clustering was performed on the following protein categories: Storage (A), energy (B), primary metabolism (C), stress (D), Folding and stability, and proteins with unknown function (E).

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Fig. 4 (continued).

treatment had a significant effect on catalase, notably by delaying the induction of two spots among the four representing this enzyme. Oxidated or misfolded proteins or peptides may accumulate in plant cells subjected to abiotic stress [64], but plants may employ chaperone proteins to prevent and reverse incorrect interactions of proteins and to facilitate correct protein folding [65]. Interestingly, folding proteins (GroEL protein chaperonin and protein disulfide isomerase) were found to be unaffected by salinity which is of high significance for the establishment of this halophyte. The involvement of stress and chaperonins in the germination process could be related to the massive production of H2O2 in the peroxisomes as a by-product of fatty acid ß-oxidation [52,39]. With respect to stress perception and regulation of plant stress response, it has to be mentioned that oxidation of acy-CoA is a ROS generating reaction, i.e. ß-oxidation of fatty acids requires sufficient catalase activity to prevent building up of eventually toxic H2O2 concentrations. Therefore an inhibition of lipid mobilization will safe seedlings from ROS peroxidation when scavenging capacity (catalase activity, for instance) is impaired by salt stress. During germination the ROS scavenging system of C. maritima apparently was significantly impaired by salt stress. We measured for instance a significant salt-induced reduction of catalase activity (data not shown). But in preliminary experiments we did not find severe peroxidation effects (by monitoring malondialdehyde production) (data not shown). Moreover, salt stress did not interfere with the seeds viability: Indeed, when transferred into distilled water, seeds showed full germination recovery capacity subsequent to salt stress (data not shown). We therefore argue that inhibition of hydrolysis of storage proteins

as well as that of lipid degradation prevents seedlings from ROS toxicity. (As mentioned above, H2O2 is produced during oxidation of acyl-CoA, for instance). Peroxidative damage, due to reduced ROS scavenging capacity, could be expected if salt stressed seeds would germinate at similar rates as observed in control experiments. Hence, during germination phase, C. maritima seeds reduce their exposure stress by remaining dormant but viable. In absence of rain fall diluting the salts accumulated in the soil, this survival strategy may allow the plant to preserve seeds in saline environments by delaying their germination until favorable conditions occur.

4.3. Leaf proteomic data support the halophytic character of C. maritima at the vegetative stage The halophytic character of C. maritima seems to be acquired at the vegetative stage. This is indicated by a slight but significantly enhanced growth activity (expressed as RGR) and leaf morphological traits (leaf number and leaf surface area) of plants treated with moderate salinity (100 mM NaCl). Suboptimal growth in salt free conditions has been also reported in salt-requiring halophytes like S. aegyptiaca, S. europaea, Batis maritima, and A. lagopoides [21,20,66,15]. Interestingly, a leaf proteome map of the control C. maritima plants allowed to identify a set of proteins constitutively expressed and involved in several metabolic paths. Among the proteins related to energy metabolism and carbon fixation that were predominant among proteins identified from the leaf proteome (35 of 44 spots), 33 spots concerned photosynthesis (27 spots for RuBisCO small and large subunit, two spots for the ATP synthase alpha and beta subunits, and one spot each for RuBisCO activase, a subunit of

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Table 3 – Plant relative growth rate (RGR), leaf morphological traits, water status, and ion contents of plants cultivated for one month with 0 or 100 mM NaCl. Means of 10 replicates ± standard error. NaCl (mM)

RGR (d−1)

0 100

0.091 ± 0.007 0.104 ± 0.01*

Leaf parameters Surface area per plant (cm2)

Number per plant

Sodium content (meq g−1 DW)

Chloride content (meq g−1 DW)

Succulence ratio (ml cm−2)

400.11 ± 29 474.21 ± 33*

83 ± 8 101 ± 9*

0.491 ± 0.08 2.552 ± 0.30*

0.481 ± 0.08 1.488 ± 0.20*

0.052 ± 0.003 0.058 ± 0.003*

Asterisk indicates significant effect at P < 0.05.

the PSII protein complex, oxygen evolving-enhancer protein, and PSII oxygen evolving complex) whereas two proteins were involved in glycolysis (glyceraldehyde-3-phosphate dehydrogenase and enolase). Other proteins were related to stressresponse, folding and stability, cell growth, and an unknown protein. Leaf proteome profile of salt-treated plants appeared to be mostly stable. Seventeen spots representing proteins involved in photosynthesis (RuBisCO small and large subunits, RuBisCO activase 1, and as ATP synthase alpha and beta subunits), stress-response (catalase and oxalic acid oxidase), and cell growth (putative chloroplastic elongation factor) significantly increased in abundance as compared to the control (Figs. 5B, C, 6, and 7). According to a recent review [14], the majority of salt-responsive proteins in halophytes are related to photosynthesis, energy metabolism, ROS scavenging, and ion homeostasis, reflecting the evolution of salt tolerance mechanisms in these plants. Similar salt-induced changes in the whole plant growth activity, leaf number, and leaf surface area strongly suggest that salinity stimulated growth through its positive effects on initiation and expansion of new leaves. This assumption is further strengthened by a significantly higher abundance of cell growth-related protein putative chloroplastic elongation factor. Despite sodium and to lesser extent chloride were accumulated at high extents in leaves, leaf water status was improved as shown by a significantly higher succulence ratio. This observation confirms our previous findings on the efficient salt compartmentalization in leaf vacuoles in conjunction with an increased activity of the vacuolar (v-type) H+-ATPase in the 100–300 mM NaCl range [67]. Removing sodium and chloride away from the cytoplasm is considered as a sine qua none requirement for salt-tolerance and succulence is a trait of vital importance for halophytes, since it

enables the reduction of the cytosolic sodium content in leaves, and allows utilization of this ion for osmotic adjustment [68]. In C. maritima, this process is also achieved by synthesis of organic osmolytes (glycine-betaine, proline, and polyols) compatible with cytosolic metabolism [14,69]. Leaf pigment (chlorophyll, carotenoids, and anthocyanins) contents were also significantly enhanced by salinity confirming previous findings on C. maritima [70,71]. While carotenoids prevent photoinhibition, anthocyanins are powerful antioxidants [72] and contribute to light attenuation (shading effect), which is of high significance for plants native to sunny biotopes [73]. With respect to the antioxidative response, these findings could be linked to (i) proteomic data showing a significantly higher abundance of stress-response (catalase and oxalic acid oxidase) enzymes, (ii) potent leaf antioxidative activity (both enzymatic and non-enzymatic) characterizing C. maritima when salt-challenged [32,70,71], and (iii) the plant ability to release excess light energy as heat (non-photochemical quenching) [23]. Taken together, these data provide strong evidence for the plant ability to cope with the oxidative stress following salt-exposure. As documented in experiments using several salt-treated halophytes [74,33,75], plant growth was strictly correlated to photosynthetic performance. Values of photosynthetic gas exchange parameters were maximal at 100 mM NaCl. At this concentration level PSII functional integrity was not impacted by salinity. PSII quantum yield (ФPSII) was even significantly higher as compared to control plants when plants were grown at 100 mM NaCl. On the other hand, photosynthetic CO2 fixation is one of the most salt-sensitive metabolic processes, impaired by stomatal and/or non-stomatal effects [76]. It is well established in the literature that the redox potential of light activated chlorophyll allows direct electron transfer to

Table 4 – Leaf pigment content and photosynthetic activity, assessed by gas exchanges and chlorophyll fluorescence parameters. Means of 5 replicates ± standard error. For each parameter, means with at least one same letter were not significantly different at P < 0.05. NaCl (mM)

0 100

Leaf pigment content

Photosynthetic activity-related parameters

Total chlorophyll (mg cm−2)

Anthocyanins (mg cm−2)

Carotenoids (mg cm−2)

A (μmol m–2 s–1)

gs (μmol m–2 s–1)

0.058 ± 0.003 0.069 ± 0.004*

0.009 ± 0.001 0.011 ± 0.002*

0.057 ± 0.005 0.071 ± .002*

13.88 ± 1.1 16.85 ± 1.3*

0.50b ± 0.06 0.65 ± 0.07*

Asterisk indicates significant effect at P < 0.05.

Fv/Fm

ΦPSII

0.78 ± 0.04 0.52 ± 0.03 0.78 ± 0.03 0.63 ± 0.04*

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molecular oxygen producing an oxygen radical (Mehler reaction) [77–79]. Thus, photosynthetic electron transport and the Mehler reaction are competing for electrons from activated chlorophyll a of the reaction centers. Under physiological conditions, ROS production at reaction centers is limited due to fast electron transfer from activated chlorophyll to phaeophytin

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and subsequent redox partners possessing less negative redox potentials, not allowing direct ROS production. Permanent recycling of NADP+, the final acceptor of photosynthetic electron transport, is a pre-requisite for such fast electron transfer. This means that there will be an increasing risk of over-reduction of the redox chain (i.e. an increasing risk of ROS production) if

Fig. 5 – Salt effect on the leaf proteome profile of C. maritima (A). Plants exposed for one month to 0 or 100 mM NaCl salinity were harvested at the flowering stage. Extracted proteins were separated according to their IEPs by isoelectric focussing (pH 3–11) in the first dimension, and by Tricine SDS-PAGE in the second dimension. Gels were CBB-stained. Within five framed zones (A to E), 44 differentially expressed proteins, marked by their corresponding number, were identified by mass spectrometry. The protein location on the gel master is indicated by a black arrow (B and C).

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Fig. 5 (continued).

photosynthesis rate does not come up with energy capture of the reaction centers [80,81]. Therefore there are three strategies for plants to prevent ROS toxicity: (i) reduction of energy transfer to chlorophyll a of the reaction centers; (ii) increase off-flow rate of electrons from the reaction centers by enhancing consumption of redox energy; (iii) build detoxification capacity (stimulate scavenging of ROS by the ascorbate–glutathione cycle, for instance). Recent data on salt-requiring halophytes indicated a significant down-regulation of light harvesting complexes [82,15,19]. This resembles the first mentioned strategy: Reduction of the light harvesting antenna will reduce the rate of chlorophyll a activation. Less electrons will be available per time interval and photosynthesis rate, though affected by salt stress, can more easy compete for electrons with ROS production. The positive effect of mild salinity (100 mM NaCl) on photosynthetic performance of C. maritima is strengthened by the significant increase of RuBisCO activity in leaves [67]. This indicates that C. maritima is using the second strategy and compensates inhibitory effects of salt stress on turnover rate of the Calvin cycle by increased expression of genes encoding relevant proteins to improve its photosynthetic capacity. Thus off-flow of electrons from reaction centers remains high and low availability of activated chlorophyll molecules is reducing the risk of ROS

production. Similar observations have been reported from a salt tolerant variety of canola, for which the gene encoding RuBisCO activase was up-regulated upon salt-treatment [83] whereas it was significantly down-regulated in soybean [84]. RuBisCO activase mediates the release of inhibitory sugar phosphates from the active sites of RuBisCO so that CO2 can activate the enzyme by carbamylation. This enzyme contributes to energy dissipation in two ways: (i) it reactivates RuBisCO for CO2 fixation and (ii) it plays a role as a chaperon [85]. The increased abundance of ATP synthase fits well with increased photosynthetic activity and the up-regulation of the Calvin cycle enzymes. Moreover, enhanced ATP synthesis in salt-challenged plants may reflect as well the requirements of processes such as active transport mechanisms across the plasmamembrane and the tonoplast membrane. As activity of the chloroplast adenylate transporter can be neglected in the light [86], energy supply of the cytosol can be achieved by shuttle systems. Here we have documented a salt stress increased the abundance of proteins representing enzymes of carbohydrate metabolism and glycolysis. This may be interpreted as a hint that energy transfer from chloroplasts to the cytosol is mediated by the DHAP/GAP– 3-P-glycerate shuttle [86] and [87]. Up-regulation of this energy valve can meet extra energy demand of increased activity of H+ATPases required for increased antiporter activity at the plasma and tonoplast membranes during salt stress [67] and [88].

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Table 5 – Classification of leaf proteins identified at the vegetative stage. Spot a

Accession b

Protein c

I. Energy 1 Q7YNV2_CAKMA 2 Q7YNV2_CAKMA 3 Q7YNV2_CAKMA 4 Q7YNV2_CAKMA 5 Q7YNV2_CAKMA 6 Q7YNV2_CAKMA 7 Q95FW9_BATMA 8 BAA84370 9 Q6Q517_CUSPE 11 Q6W7E8_BRACM 12 Q1WFN1_ARALY 14 C96497

Organism d

Score e MW f

N° MP g MS MS/ MS

15 17 18

RuBisCO large chain RuBisCO large chain RuBisCO large chain RuBisCO large chain RuBisCO large chain RuBisCO large chain ATP synthase beta subunit ATP synthase subunit alpha, chloroplastic RuBisCO large subunit Enolase RuBisCO large subunit fragment Glyceraldehyde-3-phosphate dehydrogenase B, chloroplastic Q7YNV2_CAKMA RuBisCO large chain fragment Q9AXG1_GOSHI RuBisCO activase 1 Q9MT27_9APSA RuBisCO large chain fragment

19 20 21 22 23 24

Q7YNV2_CAKMA Q4PZZ9_9MAGN Q8MDI2_BRARP Q7YNV2_CAKMA Q9MSW3_FROFL Q8WHAO_9CARY

RuBisCO large RuBisCO large RuBisCO large RuBisCO large RuBisCO large RuBisCO large

25 26 27 28 30 31 33 36 38 39 40 41 42 43

Q8MDI2_BRARP Q7YJD9_FILI Q8M9K6_9AQUA Q8MDI2_BRARP Q6DV58_BRAOL Q8MDI2_BRARP B85065 PA0013 Q8MDI2_BRARP S06772 S06772 S06772 S06772 RKRPS

RuBisCO large chain fragment RuBisCO large chain fragment RuBisCO large chain fragment RuBisCO large chain fragment Photosystem II protein RuBisCO large chain fragment Oxygen-evolving enhancer protein 3–2, chloroplastic Photosystem II oxygen evolving complex protein 2 RuBisCO large chain RuBisCO small chain RuBisCO small chain RuBisCO small chain RuBisCO small chain RuBisCO small chain, chloroplastic

Cakile maritima Gossypium hirsutum Apodolirion lanceolatum Cakile maritima Penthorum sedoides Brassica rapa Cakile maritima Froelichia floridana Dyerophytum africanum Brassica rapa Ophioglossum costatum Ilex laurina Brassica rapa Brassica oleracea Brassica rapa Arabidopsis thaliana Arabidopsis thaliana Brassica rapa Sinapis alba Sinapis alba Sinapis alba Sinapis alba Brassica rapa

II. Primary metabolism 16 O04851_BRANA 29 Q65XW4_ORYSA

glutamine synthetase precursor Putative 3-beta hydroxysteroid dehydrogenase/isomerase

Brassica napus Oryza sativa

86 60

47.4 31.3

11 5

2 1

III. Stress-related 10 Q9ZPK6_BRAJU 34 Q58QP9_BRANA 35 Q1G149_SOLTU

Catalase Oxalic acid oxidase Dehydroascorbate reductase fragment

Brassica juncea Brassica napus Solanum tuberosum

60 53 63

56.8 21.5 15.7

15 2 2

0 1 1

IV. Folding and stability 37 B53422 44 Q9LTX9_ARATH

Peptidyl-prolyl cis-trans isomerase CYP20-3, chloroplastic Heat shock protein 70

Arabidopsis thaliana Arabidopsis thaliana

128 158

28.2 76.9

10 13

2 3

V. Cell growth 13 Q8GTE7_ARATH

putative chloroplast Elongation factor, EF-Tu

Arabidopsis thaliana

84

51.6

11

1

chain chain chain chain chain chain

fragment fragment fragment fragment fragment fragment

Cakile maritima Cakile maritima Cakile maritima Cakile maritima Cakile maritima Cakile maritima Batis maritima Arabidopsis thaliana Cuscuta pentagona Brassica campestris Arabidopsis lyrata Arabidopsis thaliana

289 198 314 400 330 313 214 120 84.5 58 121 76

49.4 49.4 49.4 49.4 49.4 49.4 52.5 55.3 48 47.3 38.9 47.6

23 19 19 36 31 31 15 11 8 9 11 11

4 2 3 3 2 2 2 2 1 2 2 1

149 183 153

49.4 48 26.3

17 22 9

2 2 2

275 225 106 249 119 96

49.4 25.3 15.9 49.4 49.9 48.4

19 3 3 16 12 11

3 3 1 3 1 2

150 103 163 168 154 128 182 94 77 285 131 95 299 439

15.9 45.6 17.5 15.9 36.8 15.9 24.6 1.43 15.9 9.6 9.6 9.6 9.6 20.3

9 12 7 10 2 8 9 1 3 5 7 2 6 17

3 1 1 2 2 1 3 1 1 5 1 2 6 6

(continued on next page)

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Table 5 (continued) Spot a

Accession b

VI. Unknown 32 Q84PB4_ORYSA

Protein c

Putative uncharacterized protein

Organism d

Oryza sativa

Score e MW f

115

22.02

N° MP g MS MS/ MS 7

3

a

Spot number as indicated on the 2D-PAGE master gels (see Fig. 5B and C) and functional classification of the corresponding protein identified in leaves of C. maritima plants exposed for one month to 0 or 100 mM NaCl. Protein classification was achieved using the database available on the “Proteomics of oilseeds” platform: http://www.oilseedproteomics.missouri.edu and Expasy. b Accession number according to the best hit of MASCOT search against SwissProt and NCBI databases. c The identified protein according to the best hit of MASCOT search against SwissProt and NCBI databases. d The corresponding species according to the best hit of MASCOT search against SwissProt and NCBI databases. e Probability score for protein identification based on MS analysis and MASCOT search. f Theoretical molecular mass of the identified protein. g Number of matching peptides.

Fig. 6 – Salt-related changes in the relative abundance of C. maritima leaf proteins identified by mass spectrometry. Means of 3 replicates ± standard error. Asterisk indicates significant effect of salinity at P < 0.05.

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Fig. 7 – Hierarchical clustering of the proteins identified upon one month-long exposure of C. maritima plants to 0 or 100 mM NaCl based on the mean values of protein relative volume (three replicates per sample) inferred by using ImagemasterTM 2D PLATINUM software 6.0. Proteins were classified according to their functional category. The color gradient from bright green to bright red indicates increasing level of protein abundance. For clustering procedure see Fig. 5. Clustering of proteins involved in energy metabolism proteins is shown in (A), clustering of remaining proteins in (B).

5.

Conclusion and perspectives

Despite germination is a crucial step for halophytes, their proteomic responses at this stage have not been emphasized so far. The combination of proteomics and physiological approaches allowed to further progress in understanding salt adaptation strategy evolved by C. maritima at the early developmental (germination and seedling) and vegetative stages. First, seeds of this halophyte appeared to be equipped with proteins encoded by constitutively expressed genes, a “survival kit” [57] of high significance for this halophyte in its saline environment, since it allows to (i) deliver various

nutritional protein and non-protein compounds both early during the germination process and for the establishing seedling, (ii) readily accomplish germination by a machinery already present in the seed, and (iii) cope relatively successfully with salinity. Interestingly, physiological data showed that moderate salinity delayed germination process and inhibited only slightly germination capacity, which correlated well with the concomitant slowing down in degradation of storage compounds (lipids and proteins). As pointed out, we interpret this finding as indicating a ROS avoidance strategy: As catalase activity is impaired by salinity, fatty acid metabolism and respiration might result in eventually toxic ROS concentrations and inhibit germination capacity.

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At the autotrophic stage, plants clearly showed a response of typical salt-requiring halophytes as indicated by improved growth activity at 100 mM NaCl which was associated with leaf expansion and a higher photosynthetic activity. Forthcoming experiments will aim at a more comprehensive investigation of the proteome map in different organs of this promising halophyte upon salt treatment, enabling identification of to date unidentified proteins and should also address post-translational modifications like protein phosphorylation and other post-translational modifications. Thus, proteome analysis will lead to a better understanding of signaling and regulation of metabolic pathways on cellular level. These findings will correlate with relevant salt stress responses to further elucidate salt tolerance strategies of C. maritima. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jprot.2012.08.012.

Acknowledgments This work was financially supported by the Alexander von Humboldt Foundation. The excellent technical assistance of Dagmar Lewejohann and the help of Malte Regelin with respect to the proteomic experiments and the 2D-PAGE gel analysis respectively are gratefully acknowledged. The critical reading of Dr. Wahbi Djebali was highly appreciated.

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