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in response to the arbuscular mycorrhizal (AM) symbiosis. Membrane proteins were extracted from. Medicago truncatula roots either inoculated or not with the ...
 Springer 2005

Plant Molecular Biology (2005) 59:565–580 DOI 10.1007/s11103-005-8269-2

Identification of membrane-associated proteins regulated by the arbuscular mycorrhizal symbiosis Benoıˆ t Valot1, Marc Dieu2, Ghislaine Recorbet1, Martine Raes2, Silvio Gianinazzi1 and Eliane Dumas-Gaudot1,* 1

UMR 1088 INRA/CNRS 5184/UB Plante-Microbe-Environnement, INRA/CMSE, BP 86510, 21065, Dijon cedex, France (*author for correspondence; e-mail [email protected]); 2Unite´ de Biochimie Cellulaire et Biologie, Spectrome´trie de Masse, Universite´ de Namur (FUNDP), 61 rue de Bruxelles, 5000 Namur, Belgium Received 9 March 2005; accepted in revised form 1 June 2005

Key words: comparative proteomics, Glomus intraradices, Medicago truncatula roots, microsomal proteins

Abstract A sub-cellular proteomic approach was carried out to monitor membrane-associated protein modifications in response to the arbuscular mycorrhizal (AM) symbiosis. Membrane proteins were extracted from Medicago truncatula roots either inoculated or not with the AM fungus Glomus intraradices. Comparative two-dimensional electrophoresis revealed that 36 spots were differentially displayed in response to the fungal colonization including 15 proteins induced, 3 up-regulated and 18 down-regulated. Among them, seven proteins were found to be commonly down-regulated in AM-colonized and phosphate-fertilized roots. Twenty-five spots out of the 36 of interest could be identified by matrix assisted laser desorption/ ionisation-time of flight and/or tandem mass spectrometry analyses. Excepting an acid phosphatase and a lectin, none of them was previously reported as being regulated during AM symbiosis. In addition, this proteomic approach allowed us for the first time to identify AM fungal proteins in planta. Abbreviations: 1-D, one-dimensional; 2-D, two-dimensional; AM, arbuscular mycorrhizal; BSA, bovine serum albumin; C/M, chloroform/methanol; EST, expressed sequence tag; IPG, immobilised pH gradient gel; MALDI-TOF, matrix assisted laser desorption/ionisation-time of flight; Mr, molecular weight; PAGE, polyacrylamide gel electrophoresis; pI, isoelectric point; PMF, peptide mass fingerprinting; TM, transmembrane

Introduction Root symbioses belong to the various strategies plants have evolved to cope with nutrient limitation in soils (Lum and Hirsch, 2002). Among them, the arbuscular mycorrhizal (AM) symbiosis, occurring in more than 80% of vascular plants, is the most widely observed (Harley and Smith, 1983). This association, involving plant roots and soilborne fungi belonging to the Glomeromycota, is characterized by a bilateral exchange between

the two symbionts: plants benefit from an improved mineral nutrient uptake from the soil (mainly phosphorus) while, in turn, AM fungi are supplied with the organic carbon forms essential for achieving their full life cycle (Harrison, 1999b; Ferrol et al., 2002). AM symbiosis development undergoes a well-defined series of morphogenetic events starting from the formation of appressoria at the root surface up to the fungal mycelium proliferation within the cortical parenchyma (Gianinazzi-Pearson, 1996). At this stage, the

566 fungus differentiates haustorial structures, called arbuscules, in which the fungal cell wall is completely surrounded by a modified root cortical cell plasma membrane defined as the periarbuscular membrane (Harrison, 1999a). Arbuscules are thought to be the major sites of solute transfer between the two symbionts. Phosphorus and nitrogen are among the major macronutrients acquired by mycorrhizal fungi and translocated to plant roots (Smith and Read, 1997; Subramanian and Charest, 1999). Phosphate transporters from various plant species were found specifically expressed in response to AM symbiosis (Rausch et al., 2001; Harrison et al., 2002; Paszkowski et al., 2002). Localization of phosphate transporter transcripts in Lycopersicon esculentum and Solanum tuberosum showed that the corresponding genes were highly expressed in arbusculecontaining cells (Rosewarne et al., 1999; Rausch et al., 2001). In Medicago truncatula, phosphate transporter proteins could be detected at the arbuscular level (Harrison et al., 2002). The Lycopersicon esculentum nitrate transporter gene NRT2;3 was also demonstrated to be overexpressed during AM symbiosis. Most interestingly, its expression extended to the inner cortical cells when the roots were colonized by AM fungal structures (Hildebrandt et al., 2002). Supporting the view of an active nutrient transport at the arbuscule level, a high H+-ATPase activity has been detected on both the periarbuscular and fungal plasma membranes (Gianinazzi-Pearson et al., 1991). Proton ATPase enzymes would generate the driving force necessary for the uptake and efflux of solutes across the plant-fungus interfaces (Portillo, 2000). Recently, H+-ATPase encoding genes from Nicotiana tabacum and Medicago truncatula were shown specifically expressed in arbuscule-containing cells (Gianinazzi-Pearson et al., 2000; Krajinski et al., 2002). High levels of expression of two plasma membrane H+-ATPase genes from the mycorrhizal fungus Glomus mosseae have also been detected during the intraradical development (Requena et al., 2003). Although a few intrinsic membrane proteins involved in nutrient transport have been investigated by molecular approaches, knowledge about other putative membrane proteins regulated during the functional AM symbiosis still remains very limited. Recently, proteomics-based approaches, combining 2-D electrophoresis, mass spectrometry

and bio-informatics, have been proved useful to monitor protein regulation in root-microbe interactions (Mathesius et al., 2003a; Rolfe et al., 2003; Bestel-Corre et al., 2004a; Ca´novas et al., 2004). Such studies have mainly been conducted with M. truncatula partly because genomic resources, including more than 190,000 expressed sequence tags (ESTs), are available for this model legume (Bestel-Corre et al., 2002; Mathesius et al., 2003b; Bestel-Corre et al., 2004b; Catalano et al., 2004; Colditz et al., 2004). To date, mycorrhiza-responsive proteins in M. truncatula have only been investigated within total soluble root proteins (Bestel-Corre et al., 2002, 2004b). In view of the AM symbiosis-highly specific membrane-related features, sub-cellular proteomics targeting the root microsomal fraction has been suggested to be more relevant to address core functional proteins in mycorrhiza. Alterations in the root membrane polypeptide patterns during the development of arbuscular mycorrhiza were first reported in tomato (Benabdellah et al., 1998, 2000). However, only one protein, corresponding to a down-regulated vacuolar plant H+-ATPase subunit, could be identified following N-terminal sequencing (Benabdellah et al., 2000). Therefore, in a previous study, we set up a fractionation process for characterizing microsomal root proteins of M. truncatula. Benefiting from the development of mass spectrometry and bio-informatics tools, a high percentage of successful protein identifications could be obtained when using M. truncatula clustered EST database for queries (Valot et al., 2004). In the present work, this procedure was carried out to investigate the membrane-associated proteins that were regulated in response to the fully established symbiosis between M. truncatula roots and the AM fungus Glomus intraradices. By using this sub-cellular comparative proteomic approach, we managed to identify 25 membrane proteins whose expression was significantly altered by the AM symbiosis. Most of them were not known previously as being regulated in mycorrhizal roots.

Materials and methods Biological material and growth conditions Seeds of Medicago truncatula Gaertn. cv Jemalong line J5, compatible with both AM and bacterial

567 nitrogen-fixing symbioses (Myc+Nod+), were surface-sterilized and germinated at 27 C in the dark on 0.7% sterile agar (Bestel-Corre et al., 2002). For non-inoculated plants, 2 day-old seedlings were transplanted into 400 ml pots containing a sterile mix (2:1 v/v) of terragreen (Agsorb, Oil Dry Corporation, IMC Imcore) and Epoisses soil (neutral clay loam). Mycorrhizal inoculation was performed by replacing the Epoisses soil with a soil-based inoculum (spores, roots and hyphae) of Glomus intraradices N. C. Schenck & G. S. Smith (DAOM 181602). Seedlings (3 per pot) were grown under controlled conditions (16 h photoperiod, 23C/18C day/night, 60% relative humidity, 220 lE m)2 s)1 photon flux density) for 5 weeks. Non-inoculated and inoculated plants were daily watered (25 ml) with demineralised water and once a week with a phosphate-starved but nitrogenenriched nutrient solution (Dumas-Gaudot et al., 1994). To investigate whether root microsomal proteins could be commonly regulated by the AM symbiosis and a phosphate supply, additional noninoculated plants were fertilized once a week with 25 ml of a phosphate-enriched nutrient solution (13 mM NaH2PO4, 2H2O). At harvest, pots were immersed in tap water to carefully remove the roots from the soil mix. Roots were then gently rinsed to eliminate any remaining soil particles. Mycorrhizal root fragments were randomly collected and stained with trypan blue (Phillips and Hayman, 1970) after clearing with potassium hydroxide. Mycorrhizal colonization parameters were estimated under light microscopy as described by Trouvelot et al. (1986). Following calculation with the MycoCalc program (http://www.dijon.inra.fr/ mychintec/Mycocalc-prg/download.html), the frequency of mycorhization (F%), the percentage of root cortex colonisation (M%) and the percentage of arbuscules (A%) reached F%=92%±4, M%=53%±17, A%=30%±5. For each treatment, the pooled root systems from three pots were collected. Independent experiments were performed four times for non-inoculated and G. intraradices-inoculated plants and twice for phosphate-fertilized plants. To investigate whether root microsomal protein modifications were related to the fully established AM symbiosis, the M. truncatula mutant TRV25 (dmi3/mtsym13), unable to develop functional AM and bacterial nitrogen-fixing symbioses (Myc) Nod)) (Sagan et al., 1995), was either

inoculated or not with G. intraradices as previously described for the J5 line. Five weeks later, randomly collected root fragments were stained as described above in order to verify the absence of root colonisation. For these complementary treatments, independent experiments were performed twice. Whatever the treatment, the collected roots were weighted, frozen into liquid nitrogen and stored at )80 C until protein extraction. Microsomal protein extractions For each experiment, 10 g of M. truncatula roots were ground in liquid nitrogen. Total membrane fractions (microsomes) were obtained by differential centrifugation as previously described (Valot et al., 2004). Then, membrane proteins were extracted by chloroform/methanol extraction according to the procedure of Ferro et al. (2000), with the following modifications. The microsomal fraction was slowly added to a cold mixture of CHCl3/MeOH (6:3 v/v). Following complete homogenization, the mix was stored on ice for 30 min. After centrifugation (15,000 · g, 30 min, 4 C), a small pellet corresponding to the Insoluble C/M fraction was observed between the two phases. These two phases corresponded to the Soluble C/M fraction. Both Insoluble and Soluble C/M fractions were carefully taken and dried under vacuum. The Insoluble fraction was then resuspended in 200 ll of 7 M urea, 2 M thiourea, 4% w/v CHAPS, 0.1% v/v Triton X-100, 2 mM TBP and 2% v/v IPG buffer, pH 3–10 (Amersham Biosciences, Uppsala, Sweden). Lipids were removed by a 30 min ultracentrifugation step at 170,000 · g (Airfuge, Beckman Coulter, Villepinte, France). The protein content was determined with the method of Schaffner and Weissmann (1973) using BSA as a standard. Samples were stored at )20 C before electrophoresis. The Soluble C/M fraction was resuspended in 50 ll of 0.5 M Tris–HCl, pH 7.5, 0.1 M KCl, 5 mM EDTA and 1 mM PMSF and the protein content was determined by the Bradford-derived method described by Bearden (1978) in presence of 0.01% v/v of Triton X-100 using BSA as a standard. After the addition of 4 volumes of cold acetone, proteins were precipitated overnight at )20 C and stored at )20 C before electrophoresis.

568 One-dimensional gel electrophoresis After centrifugation (150,00 · g, 30 min, 4 C) of the Soluble C/M fraction, the protein pellet was dried and suspended in the loading buffer (65 mM Tris–HCl, pH 6.8, 2% w/v SDS, 5% v/v b-mercaptoethanol, 10% v/v glycerol). The Insoluble C/M fraction was prepared by adding 1 v of a 2X loading buffer. The two fractions were then boiled 3 min at 95 C. Ten micrograms of proteins were then separated on linear 12%, pH 8.8, SDSpolyacrylamide gel (Protean II xi Cell, Bio-Rad, Hercules, CA). Separations were carried out at 40 mA for 5 h at 10 C. Two-dimensional gel electrophoresis Precast 18 cm nonlinear, pH 3–10, IPG strips (Amersham Biosciences) were rehydrated overnight with 350 ll of 7 M urea, 2 M thiourea, 4% w/v CHAPS, 2 mM TBP, 2% v/v IPG buffer, pH 3–10 and bromophenol blue. For analytical separations, 80 lg of proteins were loaded at the anodic end of the strips and allowed to focus at 20 C for 57 kVh (Multiphor, Amersham Biosciences). Standard proteins (2-D, Bio-Rad) were used to determine the Mr and the pI of polypeptides separated in the gels. For micropreparative analyses, 600 lg of proteins were loaded and the focusing was extended to 71 kVh. After being equilibrated according to Go¨rg et al. (1987), strips were then transferred onto 12%, pH 8.8, SDSpolyacrylamide gels (Hoefer DALT, Amersham Biosciences). Separations were carried out overnight at 10 C and 90 V. Staining procedures Mono and bidimensional analytical gels were silver-stained, while micropreparative gels were stained with Coomassie blue G-250 (Mathesius et al., 2001). Spots of interest were carefully excised from extensively water-rinsed micropreparative gels using sterile tips. Gel pieces were then dried and stored at room temperature before mass spectrometry analyses. Scanning and relative expression analysis Silver-stained gels were digitalized (300 dpi) using the Sharp JX-330 scanner (Amersham Biosciences)

and the Labscan 3.1 software. Two gels were run for each biological replicate. Gel pictures from non-inoculated and G. intraradices-inoculated J5 extracts were visually compared to detect induced, up- and down-regulated spots. Spot volumes were then quantified by using the Image Master 2D Elite software (Amersham Biosciences) and compared using the bilateral paired Student t-test (P £ 0.1). Only statistically significant differences occurring in all replicated experiments were taken into account. The modifications detected in response to the mycorrhizal colonisation were further searched between the protein profiles from non-inoculated phosphate-starved and phosphatefertilised J5 plants. Spot quantification and statistical analyses were performed as previously described. In gel trypsin digestion Gel pieces, containing proteins, were destained using successive washings with 50 mM ammonium bicarbonate and acetonitrile. After repeating twice to three times this step, gel pieces were dehydrated with acetonitrile and dried under vacuum. Polypeptide disulfide bonds were reduced with 10 mM dithiotreitol in 100 mM ammonium bicarbonate at 56 C for 45 min, and then alkylated for 30 min in the dark with 55 mM iodoacetamide in the same buffer at room temperature. After successive washing steps with acetonitrile/water (1:1 v/v), 100 mM ammonium bicarbonate and dehydration with acetonitrile, gels pieces were dried in a Speedvac. According to spot size, a solution containing 25–75 ng of Porcine modified-Trypsin (Promega, France) diluted in 50 mM ammonium carbonate was added and digestion was performed overnight at 37 C. MALDI-TOF mass spectrometry and peptide mass fingerprinting Peptide digests were extracted by 5% formic acid and desalted on C18 Geloader pipette Tips (Proxeon Biosystems, Denmark). They were directly eluted on the target with a mix (1:1 v/v) of a-cyano4-hydroxycinnamic acid (in 7:3 v/v acetonitrile/5% formic acid) and 2,5-dihydroxybenzoic acid (in 7:3 v/v acetonitrile/0.1% trifluoracetic acid). Peptide mass fingerprints were obtained using a TofSpec-2E MALDI-TOF mass spectrometer

569 (Waters, Milford, USA). The ionisation was performed with a nitrogen laser (337 nm, 3 Hz) with a coarse laser energy of 20%. Mass spectra were acquired in the positive reflectron mode with a 20 kV potential using the MassLynx 3.4 software (Waters). The trypsin autodigestion peak at 2211.104 Da was used for the internal calibration. Peptide masses, ranging from 800 to 3500 Da, were manually obtained after interpretation of each mass spectrum. The PMF search was performed on the clustered EST M. truncatula database available online (http://medicago.toulouse.inra.fr/Mt/EST/ DOC/MtB.html) or on the NCBI database (http:// www.ncbi.nlm.nih.gov/), using the protein prospector software (Clauser et al., 1999) (http://prospector.ucsf.edu/ucsfhtml4.0/msfit.htm). Parameters for peptide matching were a minimum of four matches with a tolerance of 50 ppm, a maximum of one missed cleavage and possible peptide modifications by carboxyamidomethylcysteine, methionine sulfoxide and pyro-glutamic acid or acetylated N-terminal residue. The confidence in the PMF was based on the MOWSE score level and was further confirmed by the accurate overlapping of the matched peptides with the major peaks of the mass spectrum. Protein identification by tandem mass spectrometry Peptide digests were extracted from the gel by the successive addition of acetonitrile and 5% formic acid. The pooled extracts were dried under vacuum and resuspended in 5% formic acid. They were desalted on Poros R-2 resin and loaded into nanospray capillary needles (Proxeon, Denmark). Mass spectra were acquired on a Q-Tof2 mass spectrometer (Waters) with the MassLinx 3.4 software and performed within the range of 400– 1400 m/z for MS. Peptide sequences were obtained using the MS/MS mode within the mass range of 50–2000 m/z and determined by de novo sequence interpretation with oxidised methionine, carbamidomethylation of cysteine and one possible missed cleavage. Homology searches were performed by BLAST using the PAM 30 matrix tool to enhance short nearly exact matches. The search computing process was carried out on clustered EST M. truncatula database (http://medicago.toulouse.inra.fr/Mt/EST/DOC/MtB.html), on the nr protein database and other EST database restricted to fungi (http://www.ncbi.nlm.nih.gov/BLAST).

The position of trypsin cleavage sites supported the search for peptides homology. In the case of EST, homologies were confirmed when occurring on accurate reading frames (http://www.expasy.org/tools/dna.html). Sequence analysis Putative transmembrane (TM) domains were searched in the TMpred server within a range of 19–25 residues and a minimum score of 1000 (http://www.ch.embnet.org/software/TMPRED_ form.html). The identification of signal peptides was performed using the SignalP server (Nielsen et al., 1997) (http://www.cbs.dtu.dk/services/SignalP/) and was reported in the corresponding table. Theoretical Mr and pI were determined using the Compute pI/Mw tool (http://www.expasy.org/ tools/pi_tool.html).

Results Separation of microsomal proteins To investigate mycorrhiza-responsive microsomal proteins in M. truncatula roots, we used a previously optimised method based on the differential solubilization of membrane proteins in chloroform/methanol mixtures (Seigneurin-Berny et al., 1999). With this procedure, both peripheral and partially hydrophobic proteins were precipitated resulting in the Insoluble C/M fraction. On the contrary, strictly hydrophobic proteins were solubilized in the solvent mix and formed the Soluble C/M fraction accounting for 5–10% of the total amount of membrane proteins (data not shown). Both membrane-protein fractions were visually compared by SDS-PAGE. After silver staining, around 30 and 70 bands, ranging from 10 to 150 kDa, were displayed for the Soluble and Insoluble C/M fractions, respectively (Figure 1). The lower complexity we observed for the Soluble fraction, as compared to the Insoluble one, has been previously reported (Seigneurin-Berny et al., 1999; Ferro et al., 2000). Because highly hydrophobic proteins still escape 2-D gels based-proteomics, only the latter fraction could be further analyzed by two-dimensional electrophoresis by using the protocol optimized to study membrane-associated proteins

570

Figure 1. SDS-PAGE of root membrane-associated proteins from non-inoculated J5 plants after chloroform/methanol extraction. Ten micrograms of proteins from the Soluble (Sol) and Insoluble (Ins) C/M fractions were loaded. Gels were silver-stained. Standard proteins (St) are indicated on the left.

from M. truncatula roots (Valot et al., 2004). In agreement with this previous work, 440 wellresolved spots were reproducibly displayed after silver-staining within a window of pI 3–10 and molecular mass 10–100 kDa (data not shown) Membrane-related proteins differentially displayed in response to the AM symbiosis Comparisons between non-inoculated and G. intraradices-inoculated J5 root microsomal protein patterns were performed after SDS-PAGE and 2-D electrophoresis for the Soluble and the Insoluble C/M fractions, respectively (data not shown). Neither quantitative nor qualitative modification in response to the AM symbiosis could be

reproducibly detected in the Soluble fraction polypeptide patterns after silver-staining. This could be explained by the fact that only 30 bands, probably accounting for the more abundant intrinsic proteins (Ferro et al., 2000), were observed in this fraction (Figure 1). The minor proteins, which could have been regulated during AM symbiosis, were not detected although whole amounts of hydrophobic proteins from the two treatments had been loaded. Concerning the Insoluble fraction, 40 spots were found differentially-displayed between the 2-D profiles from non-inoculated and G. intraradices-inoculated roots. Such modifications were reproducibly detected in the four independent experiments. Differences were found to be statistically significant for 37 of them (P £ 0.1). To assess whether these protein modifications were related to the fully established AM symbiosis, the 2-D profiles of the insoluble root microsomal fractions from the dmi3/mtsym13 mutant, inoculated or not with G. intraradices, were also compared. One spot was found commonly down-regulated in the J5 and TRV25 lines in response to the inoculation with G. intraradices (data not shown). Because the AM fungal development is restricted to appressoria at the root surface of the dmi3/ mtsym13 mutant, 36 membrane-associated protein modifications were finally considered as being strictly related to the functional AM symbiosis in M. truncatula, including 15 induced, 3 up-regulated and 18 down-regulated spots as illustrated in Figure 2. Their corresponding Mr, pI and relative expression level are indicated in Table 1. Membrane-associated proteins regulated in response to a phosphate supply One of the most documented AM symbiosisrelated features is the general improvement in plant phosphate acquisition (Schachtman et al., 1998; Ezawa et al., 2002; Ferrol et al., 2002; Smith, 2002). To assess whether root membraneassociated proteins may be or not commonly regulated by the AM symbiosis and a phosphate supply, differences in the accumulation of the previously detected mycorrhiza-responsive proteins were further quantified between the 2-D protein patterns from non-inoculated phosphatestarved and phosphate-fertilized J5 plants. A statistically significant decrease (P £ 0.1) in the

571

Figure 2. Areas from 2-D gels showing the membrane-associated proteins differentially displayed between non-inoculated (left) and G. intraradices-inoculated (right) J5 roots. Eighty micrograms of Insoluble C/M proteins were loaded and gels were silver-stained. Spot numbers on the left correspond to those indicated in Table 1. Protein modifications, indicated by arrows, were labelled as induced (A), up-regulated (B) and down-regulated (C).

abundance of seven proteins down-regulated in mycorrhizal J5 roots was also found to occur in the phosphate-supplemented plants as compared to the phosphate-starved ones. The decrease in the accumulation of two of them (21, 36) was significantly more pronounced (P £ 0.1) in the G. intraradices-inoculated plants than in the phosphate-fertilized ones (Figure 3). Identification of the differentially displayed proteins by mass spectrometry analysis All the spots whose accumulation was significantly modified in response to the fully established AM

symbiosis were first analyzed using MALDI-TOF mass spectrometry. Twenty-three out of the 36 spots of interest could be identified following PMF analyses (Table 2). Among the spots grouped together according to their closely related electrophoretic characteristics (Figure 2), some of them (1 and 16; 2 and 3; 4, 5 and 6; 8, 9 and 10; 26 and 27; 29 and 30) were found to display similar mass spectra (data not shown), indicating that they corresponded to the same protein with possible post-translational modifications (Gooley and Packer, 1997). Five of these six groups could be identified by MALDI-TOF mass spectrometry. Two of them (2 and 3; 4, 5 and 6) corresponded to

572 Table 1. Characteristics of the differentially displayed membrane-associated proteins in response to AM symbiosis in M. truncatula roots. Spot numbera

pIb

Mrb

Relative differencec

Induced proteins 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

5.85 8.45 8.85 4.75 4.80 4.85 5.60 6.95 7.10 7.25 5.75 6.90 7.20 5.00 5.75

48.0 46.0 46.0 44.5 44.5 44.5 44.0 39.0 39.0 39.0 31.0 22.0 22.0 17.5 16.0

a a a a a a a a a a a a a a a

Up-regulated proteins 16 17 18

6.00 6.00 8.10

46.0 26.0 16.0

134 (112) 121 (77) 262 (158)

Down-regulated proteins 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

6.00 6.20 5.65 5.15 5.25 5.35 4.90 5.45 5.45 5.40 5.50 5.60 5.30 5.45 6.00 7.70 6.15 5.40

44.0 40.5 38.0 34.0 34.0 34.0 29.0 28.0 27.0 25.0 24.0 24.0 21.0 21.5 19.0 18.0 17.0 16.0

)60 )64 )46 )70 )60 )42 )46 )27 )21 )41 )63 )40 )53 )67 )66 )71 )59 )74

(17) (22) (8) (16) (19) (24) (25) (7) (7) (9) (37) (16) (19) (10) (11) (14) (14) (7)

a

Spot numbers correspond to the numbers given in Figure 2. Molecular weight and isoelectric points were calculated with Image Master 2D Elite (Amersham Biosciences) according to the migration of coelectrophoresed standard proteins (Bio-Rad). c The difference between the spot volumes from mycorrhizal and control treatments was normalized to the spot volume from the control and expressed in percent. The mean value is indicated with the standard deviation into brackets. a refers to the spots only detected in mycorrhizal roots. b

ATP synthase subunits from yeast, while all the other identified spots matched with 15 proteins contained in the M. truncatula database (Table 2). Some of the most abundant spots that could not be identified by PMF search were then further analyzed by tandem mass spectrometry. In addi-

tion, the most abundant spots of each group (2 and 3; 4, 5 and 6), previously identified following crossspecies identification, were also included in this analysis. Putative amino-acid sequences, listed in Table 3, were obtained for four different proteins corresponding to eight spots (4, 5 and 6; 8, 9 and

573 Discussion Root membrane proteins differentially displayed in response to AM symbiosis

Figure 3. Comparison of the relative expression levels of the seven root membrane-associated proteins down-regulated by the AM symbiosis and the phosphate supply in J5 plants. The difference between the spot volumes from mycorrhizal (or phosphate-supplied) and control treatments was normalized to the spot volume from the control. Bars represent the standard deviation. Letters indicate significant differences (P £ 0.1).

10; 14; 17). The identification of spots 4, 5 and 6 as a b-ATP synthase, following cross-species identification by PMF search, was further confirmed by tandem mass spectrometry with two sequences fully matching to the ones previously determined from the G. intraradices fungal proteome (DumasGaudot et al., 2004). Four sequences were obtained for spots 8, 9 and 10, but without protein identification. The two sequences obtained for the spot 14 matched with a G. intraradices extra-radical cDNA. The four sequences determined for the spot 17 matched to an acid phosphatase protein retrieved from the M. truncatula database. In summary, among the 36 mycorrhiza-responsive microsomal root proteins from M. truncatula, 7 out of the 15 induced, the 3 up-regulated and 15 among the 18 down-regulated ones could be identified by one or the other mass spectrometry methods or by both. Among these 25 spots, 7 have a possible transmembrane domain, among which 6 (spots 17, 19, 26, 27, 34, 35) could correspond to a signal peptide (Nielsen et al., 1997) (Tables 2 and 3).

To gain knowledge about the root membrane protein regulation likely expected to occur in response to AM symbiosis, a comparative subcellular proteomic approach was performed in M. truncatula. Because this interaction is a welldocumented asynchronous process, we chose to collect mycorrhizal J5 plants 5 weeks after inoculation with G. intraradices, so that all the morphological events related to the symbiosis development could be contained within roots. From the 440 spots displayed when the Insoluble C/M fraction from non-inoculated J5 roots was resolved by 2-Delectrophoresis, 36 statistically significant spot modifications could be recorded as being related to the fully established symbiosis including 3 up-regulated and 18 down-regulated ones. In addition, 15 spots were recorded as induced in the mycorrhizal roots (Figure 2 and Table 1). The additional significant spot modification we detected was excluded as being related to the fully established symbiosis because also occurring when the M. truncatula mutant (TRV25), defective in the earliest stages of AM formation, was inoculated with G. intraradices. This low number of membrane proteins commonly regulated in the wild-type and mutant M. truncatula lines in response to G. intraradices is in agreement with a previous study revealing that only two genes showed similar expression patterns in the two genotypes when inoculated with the AM fungus G. mosseae (Gianinazzi-Pearson and Brechenmacher, 2004). Among the 36 membrane proteins differentially displayed in response to the symbiosis development, 25 could be identified. Such a high success rate in protein identification, particularly with regard to a similar study attempted in tomato (Benabdellah et al., 2000), likely accounts for the use of mass spectrometry analyses coupled with the genomic resources available for M. truncatula. Special care was taken in selecting criteria robust enough to ascribe confidence in our protein identification. A minimum of five matching peptides for the low Mr proteins (spots 34, 35 and 36) was obtained in PMF identification. This number reached up to a maximum of 20 for other spots. In addition, the matching peptides belonged to the most intense

574 Table 2. Membrane-associated proteins differentially displayed in mycorrhizal J5 roots identified following peptide mass fingerprinting analyses. Spot numbera

Database entry

Number of matching peptides

Coverage/score

Identification

TM domainb

1, 16 2, 3

MtC40073 P49375

12 9

24%/5.36e+6 16%/5350

– –

4, 5, 6

NP_012655

8

18 19 20

MtC10115 MtC30193 MtD00070

8 10 7

38%/8.51e+3 23%/1.94e+3 16%/431

21

MtC10110

10

34%/1.48e+5

25 26, 27 28 29, 30 31

MtC10091 MtC10055 MtC30069 MtC00127 MtC10318

7 7 12 15 20

41%/6.19e+3 31%7.4e+4 56%/2.65e+5 41%/3e+6 70%/4.25e+8

32 33 34 35

MtC10146.1 MtC10091 MtC00365.1 MtC10242

7 6 5 5

20%/670 38%/954 36%/418 23%/113

36

MtC00779

5

28%/462

Nodulin-like protein F(1)F(0)-ATPase complex, a subunit (Kluyveromyces lactis) F(1)F(0)-ATPase complex, b subunit (Saccharomyces cerevisiae) Hypothetical protein Aminoacylase-1 2-oxoglutarate dehydrogenase Cytosolic phosphoglycerate kinase DREPP Lectin Proteasome subunit a-2 Ferritin ATP synthase D chain, mitochondrial Lipoxygenase DREPP Hypothetical protein Nucleoside diphosphate kinase Thioredoxin H-type

20%/7530

–

68–87 22–41 (S) – – – 10–32 (S) – – – – – 12–31 (S) 54–74 (S) –

a

Spot numbers correspond to the numbers given in Table 1 and Figure 2. The number refers to the residues surrounding the predicted transmembrane domain. (S) indicates that the TM domain could correspond to a signal peptide. b

peaks of the mass spectra. Similarly, identification performed after de novo sequencing was validated when a minimum of two sequences longer than 8 amino-acid residues was found. Giving additional support to our identifications, a high congruence between theoretical and calculated pI and Mr was observed, with correlation coefficients reaching 0.938 and 0.985, respectively (Link et al., 1997). Success in protein identification happened to be quite different between induced spots (47%) and down- or up-regulated spots (86%). The higher success obtained for the latter ones is likely to account for the fact that down- and up-regulated spots correspond to M. truncatula root proteins. A similar percentage of successful identification was previously reported for M. truncatula root proteins using clustered EST database for PMF search (Valot et al., 2004). In contrast, spots reported as induced may correspond either to G. intraradices proteins expressed in planta or to M. truncatula proteins newly expressed in response to the AM symbiosis. The very low amount of EST data

available for the Glomeromycota still contributes to limit PMF identification (Natera et al., 2000). This situation will soon be improved as the G. intraradices genome sequence project is now into the pipeline (Martin et al., 2004). Anyway, the present study allowed to identify, using PMF search or/and de novo sequencing, 6 of the 15 induced spots as being of fungal origin, including two highly conserved proteins from yeast (five spots) and a G. intraradices extra-radical mycelium cDNA. To our knowledge, this is the first report of AM fungal protein identifications in planta by using a proteomic approach.

Induced root membrane proteins Among the induced proteins, spots 2 and 3 and spots 4, 5 and 6 were identified as a and b subunits of the fungal mitochondrial ATP synthase, respectively. These two subunits, forming part of the F(1)-ATPase complex, are localised

575 Table 3. Membrane-associated proteins differentially displayed in mycorrhizal J5 roots identified following tandem mass spectrometry analyses. Spot numbera

Observed masseb

Putative amino-acid sequencec

Mass differenced

Identification

TM domaine

4, 5, 6

1277.63 2747.4

TIAMDGTEGLVR SLQDIIAILGMDEL SEEDKQTVER

)0.01 )0.05

Nd

8, 9, 10

2034.24 2630.29 3041.74 3431.93

X-RTVALGDLNTLMVW-X PLSDAVDHD-X X-TPEYALLQVLR LTQLSSFNTG-X

– – – –

Glomus intraradices ATP synthase b-chain mitochondrial precursor (Dumas-Gaudot et al., 2004) Nd

14

2941.45 3361.63

X-INYLVPLGVLVAFIAYK X-INYLVPLGVLVAFIAYKYISG

– –

Nd

17

1085.5 1114.66 1350.68 2169.94

LPDPLYYI-X IAFLTGRPLK LPDPLYYI-X IIGNSGDQ-X-SDILGTNTGER

–

Glomus intraradices extra-radical mycelium cDNA (BI452104) Acid phosphatase (MtC00065.1)

1–19 (S)

0.02 – –

a

Spot numbers correspond to the numbers given in Table 1 and Figure 2. Masses were monoisotopic. c Leucine=Isoleucine; Methionines were oxidised; X- corresponds to peptide residues which were not determined with sufficient confidence. d The mass in Dalton corresponds to the difference between the observed peptide mass and the predicted peptide mass deduced from de novo sequencing. e The number refers to the residues surrounding the predicted transmembrane domain. (S) indicates that the TM domain could correspond to a signal peptide. b

in the inner mitochondrial membrane and play a key role in the final step of ATP synthesis (Capaldi and Aggeler, 2002). The b subunit ATP synthase was previously identified when the extra-radicular proteome of G. intraradices was analysed (DumasGaudot et al., 2004). Previous investigations concerning the presymbiotic responses of the AM fungus Gigaspora rosea to an active root factor have revealed the induction of several genes related to mitochondrial activity (Tamasloukht et al., 2003). In addition, a higher respiration rate was detected in the fungus and more mitochondria could be observed. As previously suggested by Becard et al. (2004) for the presymbiotic phase, the two fungal mitochondrial ATP synthase subunits we detected may provide the ATP required for sustaining energy-dependent functions in mycorrhizal roots including membrane ATPase activity (Ezawa et al., 2002; Ferrol et al., 2002). Up-regulated root membrane proteins Among the membrane proteins up-regulated in response to AM symbiosis, spots 1 and 16 show similarity with a 53-kDa nodulin first isolated

from the soybean symbiosome membrane (Winzer et al., 1999) and further identified into of the pea peribacteroid membrane proteome (Saalbach et al., 2002). Of unknown function, the 53-kDa nodulin was shown to be glycosylated and to display several spots after 2-D electrophoresis (Winzer et al., 1999). To our knowledge, this is the first report concerning the overexpression of a nodulin 53-like protein in mycorrhizal roots. The so-called nodulin genes concern plant genes that are induced during the formation and functioning of root nodules. Recently, a gene encoding a nodulin 26-like protein, a multifunctional aquaporin, was revealed up-regulated during root interactions between M. truncatula and G. mosseae or Sinorhizobium meliloti (Brechenmacher et al., 2004). From the existence of isogenic mutants defective for both mycorrhization and nodulation (Sagan et al., 1995; Marsh and Schultze, 2001) and of common gene expression during these symbioses (Fru¨hling et al., 1997; van Rhijn et al., 1997; Albrecht et al., 1998; Journet et al., 2001), it is widely admitted that mycorrhizal and nitrogen-fixing symbioses share common molecular pathways. However, compar-

576 ative root proteomics of M. truncatula interactions with G. mosseae or S. meliloti has failed so far to reveal polypeptides commonly regulated when attempted on total soluble proteins (BestelCorre et al., 2002). By focusing our study on root microsomes, we reported here that at least one protein is commonly regulated by the two symbioses. Because the 53-kDa nodulin was found located in the peribacteroid membrane of root nodules (Saalbach et al., 2002), it is tempting to speculate that this protein could also be associated with the periarbuscular membrane that develops in mycorrhiza. Spot 17 was identified as an acid phosphatase containing a possible peptide signal addressing the secretory pathway (Table 3). Its accumulation in root microsomes increased by 121% in response to AM colonisation. Using transcript profiling in M. truncatula roots, a putative defence-associated acid phosphatase was reported up-regulated in the initial stages of the symbiosis, in contrast to others that were down-regulated by a phosphate supply (Liu et al., 2003). An increase in the amount and activity of a similar acid phosphatase was also found to occur during nodule development in soybean (Penheiter et al., 1997). Although the role of this enzyme at the cellular level is not yet clear, it has been suggested to be important for efficient nodule metabolism. Its role in the AM symbiosis functioning remains to be investigated. Annotated as a hypothetical protein, the spot 18 seemed to be an intrinsic protein whose accumulation increased by 262% during AM colonisation. Down-regulated proteins Two spots (26 and 27) were identified as a lectin. Their relative accumulation decreased by 20% in response to G. intraradices colonisation unlike other lectin encoding genes previously reported induced in AM plants (Balestrini et al., 1999; Wulf et al., 2003; Kuster et al., 2004). Many roles have been proposed for plant lectins including their involvement in the bacterial nitrogen fixing symbiosis establishment (Hirsch, 1999; van Rhijn et al., 2001) and their functioning as plant defence mechanisms (Chrispeels and Raikhel, 1991). Whether the expression of some constitutive root lectins has to be lowered to allow AM fungal penetration of root tissues remains unknown. Spot 19 has been identified as

an aminoacylase-1 belonging to metallopeptidases located in the endoplasmic reticulum. This peptidase is involved in the maturation of secreted proteins by catalysing the release of a N-terminal amino acid from a polypeptide (Rawlings and Barrett, 1995). Its down-regulation (60%) may reflect the changes occurring in the membrane and cell wall structures of cortical arbuscule-containing cells (Harrison, 1999b). Two distinct mitochondrial proteins involved in primary metabolism were shown to decrease in G. intraradices-colonised roots. They corresponded to an ATP synthase D chain (spot 31) belonging to the F(0)-ATP synthase complex and to a 2-oxoglutarate dehydrogenase (spot 20), an enzyme from the tricarboxylic acid (TCA) cycle. The activity of TCA cycle enzymes was shown to increase in phosphate-deficient white lupin and tobacco (Toyota et al., 2003; Uhde-Stone et al., 2003), so that the down-regulation of the 2oxoglutarate dehydrogenase in mycorrhizal roots may reflect an improvement in phosphate availability. However, the down-regulation of this enzyme could not be recorded in our phosphatesupplied plants. Spot 32 was identified as the N-terminal sequence of a lipoxygenase. This protein of approximately 90 kDa could be cleaved to form a 30 kDa N-terminal chain that has been demonstrated to allow the linkage to biological membranes (Oliw, 2002). Lipoxygenases catalyze the dioxygenation of polyunsaturated fatty acids into reactive hydroperoxide derivatives that can be further transformed into biologically active compounds. In plants, linoleic acid and a-linoleic acid, the major fatty acids of membrane phospholipids, are the main substrates for lypoxygenases. The lipoxygenase pathway is induced during some plant-pathogen interactions leading to the production of signalling molecules, activation of defence genes and phytoalexins accumulation (Blee, 2002; Feussner and Wasternack, 2002; Porta and Rocha-Sosa, 2002). The present study revealed that the lipoxygenase shorter-fragment decreased by 67% in response to G. intraradices colonisation, suggesting that this enzyme might be down-regulated during AM symbiosis. Earlier analyses have shown that defence-response genes involved in pathogenesisrelated protein accumulation and phytoalexins biosynthesis were down-regulated as the AM symbiosis developed (Harrison and Dixon, 1993;

577 Gianinazzi-Pearson et al., 1996). Additional experiments are required to address whether this may also occur for the lipoxygenase pathway in mycorrhizal roots. Spot 34, whose accumulation decreased by 71% in response to AM colonisation, is annotated as a hypothetical protein containing a signal peptide. Two spots (25 and 33), exhibiting distinct pI/ Mr (Figure 2 and Table 1), matched with a developmentally regulated plasma membrane polypeptide (DREPP). DREPPs are putatively plant specific and are differentially regulated during plant development (Logan et al., 1997) As deduced from the MALDI-TOF mass spectrum examination (data not shown), spot 33 (Mr of 19 kDa) could be a fragment of the spot 25 (Mr of 29 kDa). These two spots (25 and 33) decreased in response to G. intraradices colonisation by 46 and 66%, respectively. The expression of spot 25 was also found down-regulated in response to a phosphate supply (Figure 3). The down-regulation of spots 25 and 33 could account for the root architecture modification observed in phosphate-starved plants complemented with phosphorus (Hammond et al., 2004) and in mycorrhizal plants (Cruz et al., 2004; Gamalero et al., 2004). Six additional spots were found down-regulated in AM-colonized and phosphate-fertilized roots (Figure 3). Among them, spots 21 and 35 were identified as a cytosolic phosphoglycerate kinase and a nucleoside diphosphate kinase, respectively. Catalyzing ATP synthesis, these two enzymes belong to an alternative metabolic pathway that is activated to enable plants to survive under phosphorus limiting conditions (Hammond et al., 2004). Both spots 29 and 30 corresponded to a ferritin. This protein allows the iron-storage into plastids and its amount is directly linked to the plant iron content (Briat et al., 1999). Because iron uptake is known to increase in phosphate-starved plant roots (Lakshmi and Narayanan, 1988; Shen et al., 2004), the decrease in the ferritin expression level, observed in both G. intraradices-colonized and phosphate-supplied plants, is likely to account for its down-regulation in phosphate-replenished plants. Spot 28 matched to a proteasome a-2 subunit, involved in ATP/ubiquitin-dependent proteolysis. Spot 36 corresponded to a thioredoxin h implicated in the cell redox regulation (Gelhaye et al., 2004). The down-regulation

of these two proteins in mycorrhizal and phosphate-supplied J5 lines may reflect less stressed plants due to their improved nutritional status.

Concluding remarks This sub-cellular proteomic approach, focussed on root microsomes, proved successful in revealing mycorrhiza-responsive proteins including induced (15), up-regulated (3), but also downregulated ones (18). Among them, 25 proteins could be further identified using mass spectrometry analyses. With regard to earlier studies, none was shown previously regulated in the AM symbiosis excepting a lectin and an acid phosphatase. Such a result likely accounts for the fact that most of the previous functional genomic approaches did not address down-regulated genes. Suppressive subtractive hybridisation together with ESTs sequencing have been performed to monitor gene regulation during functional AM symbiosis in Pisum sativum and M. truncatula (Wulf et al., 2003; Brechenmacher et al., 2004; Grunwald et al., 2004), but this approach only gave access to newly and/or upregulated genes. Recently, cDNA arrays have been used to examine transcript profiling in M. truncatula roots during colonisation by AM fungi (Liu et al., 2003; Kuster et al., 2004), but only the study from Liu et al. (2003) has taken into account genes which were down-regulated in response to AM symbiosis. The discrepancy we observed between transcript and protein regulations may also account for a low correlation between mRNA and protein amounts (Gygi et al., 1999). In addition, we focussed this study on membrane-associated proteins. Among the 25 mycorrhiza-responsive proteins we identified, 19 were not found regulated by a phosphate supply, suggesting they may be candidates as markers of the AM symbiosis. Further experiments would be required to determine at which step they might be relevant, including the time-course study of their expression and localization. The next challenge in identifying strictly mycorrhiza-related proteins will be to target the symbiosis-specific organ: the arbuscule. The sub-cellular analysis of the mycorrhizal plasma membrane looks very promising in this respect.

578 Acknowledgments The authors are grateful to Ge´rard Duc (INRA, Dijon, France) for providing TRV25 seeds and to Jonathan Negrel (INRA, Dijon, France) for critical reading of the manuscript. Benoıˆ t Valot was financially supported by a MNERT (Ministe`re de l’Education Nationale et de la Recherche Technique) grant. This work was funded through the ‘‘Conseil Re´gional de Bourgogne’’ (04 516 CP09 5327).

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