Journal of Experimental Botany, Vol. 51, No. 345, pp. 747-754,
April 2000
© 2000 Oxford University Press
Alterations in the plasma membrane polypeptide pattern of tomato roots (Lycopersicon esculentum) during the development of arbuscular mycorrhiza
Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín (CSIC), Profesor Albareda 1, 18008, Granada, Spain
Received 20 August 1999; Accepted 12 November 1999
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Abstract |
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Changes induced by arbuscular mycorrhizal (AM) formation in the plasma membrane polypeptide pattern of tomato roots have been assessed by 2D-PAGE analysis. Plasma membrane fractions were isolated by aqueous two-phase partitioning from control and mycorrhizal tomato root microsomes. Analysis of 2D-PAGE gels revealed that AM colonization induces at the plasma membrane level two major changes in protein synthesis: down-regulation of some constitutive polypeptides and synthesis of new polypeptides or endomycorrhizins. A comparison of changes induced by two different levels of AM colonization showed that 16 polypeptides were differentially displayed at both AM colonization stages, while some others were transiently regulated. Five of the differentially displayed plasma membrane polypeptides at both AM colonization stages were selected for N-terminal amino acid sequencing. Reliable sequences were obtained for two of the selected spots. Sequence alignment search indicated that one of the sequenced polypeptides showed 75% identity to the N-terminal sequence of the 69 kDa catalytic subunit of the vacuolar type H+-ATPase of several plants. The possible significance of these findings is discussed in relation to the functioning of the AM symbiosis.
Key words: Arbuscular mycorrhizas, plasma membrane, 2D-PAGE, endomycorrhizins, V-type H+-ATPase.
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Introduction |
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Arbuscular mycorrhizal (AM) fungi belonging to the order Glomales (Zygomycota) are obligate biotrophs that form mutualistic symbioses with most of the agriculturally important plant species (Barea and Jeffries, 1995
). Plant colonization by the AM fungus and the development of an active symbiosis induces considerable morphological and physiological changes in both symbionts (Bonfante and Perotto, 1995). These changes are presumably the result of a complex sequence of interactions between the fungus and the plant root. As in other plant–microbe systems, it is likely that regulation of these interactions requires a continuous exchange of signals between both partners, which, when perceived through the corresponding receptors, induce a cascade of events leading to changes in the expression of certain genes (Koide and Schreiner, 1992; Boller, 1995). Although the nature of those signals and receptors in AM symbiosis is yet unknown, there is increasing evidence that the AM association is controlled by the differential expression of certain genes (Harrison, 1997).
The existence of plant mutants unable to form AM symbiosis confirms the hypothesis that specific plant genes are involved in the establishment of the symbiosis (Duc et al., 1989; Gianinazzi-Pearson et al., 1995; Barker et al., 1998; Wegel et al., 1998). This has been corraborated recently (Samra et al., 1997), when it was shown that the 2D-PAGE pattern of root proteins isolated from the AM-compatible (wild-type) and the AM-resistant (myc-nod-) pea genotypes were different and that the changes induced by AM colonization in both genotypes were also very different.
It is well known that in AM symbiosis the plant benefits from a better nutritional status in exchange for photosynthate to the fungus. This bidirectional nutrient transport is crucial for the functional integration of the symbionts (Smith and Smith, 1996). The symbiotic interfaces where the plasma membranes of both symbionts are associated, are considered to play a central role in regulating nutritional and signal exchanges between both partners (Smith and Smith, 1990). Therefore, it is expected that the activity, composition and structure of plasma membranes of both organisms are altered at the symbiotic interfaces. In this sense, it has been shown that one of the most dramatic alterations in the colonized host cell occurs at the interface level, where the plasma membrane invaginates and proliferates around the hyphal branches of the arbuscule, increasing 4–10-fold its surface area (Alexander et al., 1989). This peri-arbuscular plasma membrane has a high H+-ATPase activity, contrasting with the low activity of plasma membranes of uncolonized cells (Gianinazzi-Pearson et al., 1991). Furthermore, 2D-PAGE analysis of tomato root microsomal fractions has shown that AM colonization induces up-regulation and down-regulation of some constitutive membrane-bound polypeptides as well as induction of some new membrane polypeptides or endomycorrhizins (Benabdellah et al., 1998). Interestingly, immunological studies using monoclonal antibodies raised against membrane components of pea nodules revealed that some of the antigenic components were also expressed in the peri-arbuscular membrane (Perotto et al., 1994). Moreover, cloning and expression studies of genes encoding a plant plasma membrane H+-ATPase isozyme (Murphy et al., 1997), two phosphate transporters (Liu et al., 1998) and a sugar transporter (Harrison, 1996) have evidenced that AM symbiosis induces regulation of genes involved in membrane transport processes.
Although the above-mentioned studies indicate that the host plasma membrane undergoes multiple changes as a consequence of AM formation, further studies using isolated plasma membrane vesicles need to be done. The aim of the present work was to study by 2D-PAGE, a useful technique for the global analysis of protein synthesis, changes induced by AM colonization in the polypeptide pattern of tomato root plasma membrane vesicles isolated by aqueous two-phase partitioning of microsomal fractions. Additionally, some of the differentially displayed plasma membrane proteins were N-terminal sequenced.
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Materials and methods |
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Plant material and growth conditions
Tomato (Lycopersicon esculentum Mill cv. Earlymech) seeds were surface-sterilized and pregerminated in sterile vermiculite. Seedlings were grown in containers (1.0 l) containing a sterile mixture of sand/vermiculite (1/1, v/v). Half of the plants were inoculated with a soil–sand-based inoculum containing fungal propagules and chopped mycorrhizal roots of the AM fungus Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe (BEG 12). Control plants received a filtrate (<20 µm) of the AM inoculum to provide the microbial populations accompanying the mycorrhizal fungus but free from AM propagules.
Plants were grown in a growth chamber (25/18 °C day/night temperature, 70% relative humidity and 16 h photoperiod at 400 µmol m-2s-1), and watered three times per week with a low-phosphorus-content (25%) Long Ashton nutrient solution (Hewitt, 1952). Plants were harvested 4 weeks and 6 weeks after transplanting. At harvest, shoot and root weights were determined and AM colonization was estimated on stained (Phillips and Hayman, 1970) root samples (about 1 g per plant), using the gridline intersect method (Giovannetti and Mosse, 1980). Each experiment consisted of 40 replicate plants per treatment, and was repeated three times.
Plasma membrane isolation and purification
The root systems of 40 plants were used in each treatment to obtain significant amounts of purified plasma membrane. Root apices were discarded to minimize dilution of the plasma membrane of AM colonized cells.
Plasma membranes were purified by differential centrifugation and aqueous two-phase partitioning (Larsson et al., 1987). Roots were homogenized at 4 °C in a cold grinding medium (1/3, w/v), as previously described (Benabdellah et al., 1998). The homogenate was filtered through four layers of cheesecloth, centrifuged at 13 000 g for 15 min and the supernatant centrifuged again at 80 000 g for 35 min. The microsomal pellet was resuspended in 3 ml of 330 mM sucrose, 5 mM phenylmethylsulphonyl fluoride (PMSF), 5 mM K-phosphate buffer pH 7.8 and added to 9 g of a fresh phase mixture to yield a 12 g phase system containing 6.3% (w/w) dextran (T-500; Pharmacia, Uppsala, Sweden), 6.3% (w/w) polyethylene glycol (3350, Sigma, St Louis, USA), 330 mM sucrose, 5 mM K-phosphate buffer pH 7.8, and 4.5 mM KCl. After mixing, separation of the phases was achieved by centrifugation in a swinging rotor at 2000 g for 5 min. The upper phase (U1), containing mainly plasma membrane vesicles, was further purified by performing a second partition. For this purpose, about 90% of the U1 fraction was carefully removed with a Pasteur pipette and placed on a fresh bottom dextran phase that has had 90% of the top polyethylene glycol phase removed. After mixing and separation of the phases by centrifugation, the new upper phase (U2) was diluted with a washing buffer containing 2 mM BTP-TRIS pH 7.6, 250 mM sucrose, 1 mM DTT, and 0.1 mM PMSF. Following centrifugation at 105 000 g for 30 min, the plasma membrane pellet was resuspended in 2 mM TRIS-HCl pH 8.0, 250 mM sucrose, 10% glycerol, 1 mM DTT, l mM PMSF, and 20 mg ml-1 chymostatin and stored at -80 °C. Plasma membranes were obtained from three independent experiments, and two 2D-PAGE gels were run for each plasma membrane preparation.
Marker enzymes
To assess the purity of the plasma membrane preparations obtained from control and mycorrhizal tomato roots, the microsomal and plasma membrane fractions were characterized using marker enzymes associated with the different subcellular membranes (Ferrol and Bennett, 1996). Vanadate-sensitive ATPase, nitrate-sensitive ATPase and azide-sensitive ATPase were selected as plasma membrane, tonoplast and mitochondrial membrane markers, respectively. Antimycin-A insensitive NADH-cytochrome c reductase was used as endoplasmic reticulum marker, and latent UDPase for Golgi membranes.
Two-dimensional gel electrophoresis
For 2D-PAGE, plasma membrane proteins were solubilized (according to the method of Hurkman and Tanaka, 1986) with minor modifications as previously described (Benabdellah et al., 1998). Briefly, plasma membrane fractions containing 100 µg of proteins were extracted with water-saturated phenol and precipitated with 0.1 M ammonium acetate in methanol and the resulting pellet was solubilized in a urea buffer containing 9.5 M urea, 4% (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulphonate), 0.5% Nonidet P-40, 2% 2-mercaptoethanol, 2 mM PMSF, and 2% ampholytes (pH range 3.5–10, Bio-Rad). 2D-PAGE was performed as described by O'Farrell (O'Farrell, 1975). Isoelectric focusing was performed for 15 min at 200 V, 15 h at 300 V and 1 h at 800 V. Following isoelectric focusing, gels were calibrated in 2% SDS, 15 mM DTT, 62 mM TRIS-HCl pH 6.8, and 10% glycerol. Separation in the second dimension was performed in 11% polyacrylamide (SDS-PAGE) gels.
Image processing
Proteins on analytical 2D-PAGE gels were visualized by silver staining (Blum et al., 1987) and digitized with a HP desk scan run under Photoshop 3.0 (Adobe Mountain View, CA). The resulting images were transferred into a Macintosh Performa 5200 computer and analysed using the NIH image program developed at the US National Institute of Health. Results obtained by the computer-aided evaluation were rigorously compared by visual analysis of the original gels. The apparent molecular weight of proteins was determined by co-electrophoresis with broad range (Bio-Rad 161–0371) SDS-PAGE molecular weight standards, and the apparent isoelectric points (pI) by measuring the pH range in each set of IEF electrophoresis. Changes in specific polypeptides were recorded only when they occurred in all the replicated gels.
Electroblotting, N-terminal sequencing and amino acid analysis
Preparative gels for the electroblotting were made as analytical gels, except that 2 mg of plasma membrane proteins were loaded onto the gels. After 2D-PAGE, proteins were electrotransferred to polyvinylidene difluoride membranes (Bio-Rad, Sequi-Blot, PVDF membrane) using 50 mM TRIS- boric acid pH 8.2 as transfer buffer (Matsudaira, 1987). Electrotransfer was made at 0.4 A for 80 min. The membrane was stained with 0.1% Coomassie Blue R-250 in methanol/water/acetic acid (50/40/10, by vol) for 30 s. The membrane was air-dried and selected spots were excised from the membranes. N-terminal amino acid sequencing was performed on a PROCISE sequencer (Applied Biosystem Procise) using a standard sequencing procedure. Amino acid sequence homology searches against GenBank and Swiss-Prot databases were made with the FASTA program (GCG package). Multiple alignment of amino acid sequences was carried out with the program CLUSTAL W (version 1.5; Thompson et al., 1994).
Immunoblotting analysis
One-dimensional electrophoresis was performed according to the method of Laemmli (Laemmli, 1970). Plasma membrane proteins (100 µg) were precipitated with trichloroacetic acid, and pellets were resuspended in SDS-PAGE buffer containing 50 mM TRIS-HCl pH 6.8, 2% SDS, 10% glycerol, 5% mercaptoethanol, 2 mM PMSF, and 100 µg ml-1 chymostatin and incubated for 30 min at room temperature before electrophoresis (Ferrol and Bennett, 1996). Proteins were separated on 10% SDS-polyacrylamide gels on a Bio-Rad (Richmond, CA, USA) Protean II, and electrotransferred to nitrocellulose membranes at 100 V for 1.5 h in a Bio-Rad transfer cell with a buffer consisting of 10 mM 3-cyclohexylamino-1-propane sulphonic acid-NaOH, pH 11.0 and 10% (v/v) methanol. Blots were immunodetected with polyclonal antibodies raised against the 69 kDa subunit of the red beet vacuolar ATPase (kindly supplied by Dr Alan B Bennett, University of California, Davis), at dilutions 1:1000 using goat-anti-rabbit IgG-alkaline phosphatase conjugate as the second antibody.
Protein assays
Protein content of the microsomal and purified plasma membrane fractions (U2) was determined by the Bradford assay (Bradford, 1976). Protein concentration of the solubilized plasma membrane proteins in the loading urea buffer was determined using a method described previously (Ramagli and Rodriguez, 1985). In both assays, bovine serum albumin (BSA) was used as standard.
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Results |
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Plant growth and mycorrhizal colonization
Four weeks after transplanting, both shoot and root fresh weight of mycorrhizal plants were greater than their non-mycorrhizal counterparts. Two weeks later mycorrhizal plants still had a greater root fresh weight than non-mycorrhizal plants, but shoot fresh weight was similar in both sets of plants (Table 1
). Root colonization by the AM fungus reached 40% and 57% of total root length 4 and 6 weeks after inoculation, respectively. At the first harvest time (4 weeks), AM colonization consisted mostly of intercellular hyphae and arbuscules, and 2 weeks later some vesicles were also present.
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Plasma membrane isolation and marker enzymes
The marker enzyme analyses of the microsomal and plasma membrane (U2) fractions are shown in Table 2
. In the U2 fraction isolated from both treatments, the specific activity of the plasma membrane marker (vanadate- sensitive ATPase) was increased by more than 100% over the activity in the microsomal fraction. Golgi and endoplasmic reticulum marker enzyme activities were reduced by approximately 83% in the U2 fraction of both treatments and the activity of the marker enzymes for the tonoplast and mitochondria were not detected. These data indicate a similar purification index for the plasma membrane marker and comparable removal of contaminant membranes from both control and mycorrhizal tomato roots, indicating that plasma membranes from both treatments can be isolated with the same procedure. The plasma membrane marker activity in both U2 fractions was about 95% of the total marker enzyme activities, while it was about 45% in the microsomal fractions. These data also show that the two phase partitioning technique is quite effective for plasma membrane purification, although protein recovery was very low, approximately 3% of the microsomal proteins. The purity of the plasma membrane fraction was similar at both harvest times (data not shown).
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Plasma membrane protein profile
Using 2D-PAGE, about 200 and 150 reproducible plasma membrane spots were resolved from 4- and 6-week-old plants, respectively. The pIs of these polypeptides ranged from 4.4 to 7.1 and the molecular weights from 29 to 150 kDa, approximately (Figs 1, 2). Analysis of the polypeptide patterns revealed that synthesis of some polypeptides (numbered from 1 to 16 in Figs 1 and 2) was affected in the same way at 4 and 6 weeks after transplanting, while other polypeptides were differentially displayed only at one of the two harvest times. The polypeptides differentially displayed only at the first harvest time were numbered from 17 to 26 in Fig. 1 and those differentially displayed at the second harvest time from 27 to 32 in Fig. 2.
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); up-regulated (
), and new polypeptides (
) are indicated in both gels.
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From the 16 differentially regulated polypeptides at both harvest times, ten of them were newly synthesized and six down-regulated (Table 3
). It is interesting to note that the intensity of the newly-induced polypeptides 4, 5, 12, and 13 increased with the level of colonization, while the intensity of polypeptide 16 decreased (Figs 1
, 2). From the 10 polypeptides that were only differentially displayed by AM colonization 4 weeks after inoculation, six of them were new, three down-regulated and one up-regulated (Table 4). Polypeptides that were newly-synthetized only 6 weeks after inoculation form a cluster of acidic polypeptides with molecular weights ranging from 50 to 38 kDa (Fig. 2; Table 4). Polypeptide 27, which appeared in control roots six week after transplanting, was down-regulated by AM colonization.
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N-terminal sequencing
To characterize some of the differentially regulated plasma membrane polypeptides, an attempt was made to obtain the N-terminal sequences of spots 1, 2, 11, 14, 15, and 16. Unfortunately, no sequence information was obtained for polypeptides 11, 14, 15, and 16 because they were N-terminally blocked. Reliable sequence data to eight residues was obtained for the newly synthesized spot 1 (MAEETKKQ) and to 24 residues for the down-regulated polypeptide 2 (Fig. 3). Sequence alignment searches indicated that spot 1 did not show significant homology to previously described proteins. N-terminal sequence of spot 2 showed 75% identity to the N-terminal sequence of the 69 kDa catalytic subunit of the vacuolar H+-ATPase of several plants (Fig. 3). Down-regulation of this polypeptide in mycorrhizal roots was confirmed by Western blot analysis of plasma membrane proteins using antibodies raised against the red beet V-ATPase catalytic subunit (Fig. 4).
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Discussion |
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Proteome analysis, a technique that allows for a reverse-genetic approach to identify genes of interest, has been used to evaluate changes in the plasma membrane polypeptide pattern of tomato roots in response to AM colonization. The results presented in this paper show that AM colonization induces extensive changes in the synthesis of tomato root plasma membrane proteins and that these changes depend on the physiological status of the symbiosis.
Previous studies of changes induced by AM colonization in total, soluble and microsomal protein patterns have indicated three major changes in protein synthesis: up- or down-regulation of some constitutive polypeptides and synthesis of new ones (Dumas-Gaudot et al., 1994; Benabdellah et al., 1998). However, results presented in this paper show that at the plasma membrane level, AM colonization induces mainly two types of changes: synthesis of new polypeptides and down-regulation of some constitutive ones. In fact, among the 26 differentially displayed polypeptides 4 weeks after inoculation only one was up-regulated and 2 weeks later none were up-regulated. In a previous work (Benabdellah et al., 1998) it was observed that AM colonization increased the expression of 13 microsomal constitutive polypeptides in tomato roots. Comparison of the changes induced by AM colonization in the microsomal and plasma membrane protein profiles of tomato roots allows to conclude that the microsomal up-regulated polypeptides are located in the endomembranes, and this could be due to the increase in the number of organelles observed in the colonized host cells (Bonfante and Perotto, 1992).
The additional plasma membrane polypeptides detected in AM roots could be endomycorrhizins (mycorrhizal-specific polypeptides) or polypeptides of the fungal partner. Discrimination between these two possibilities requires comparison of the plasma membrane polypeptide pattern of mycorrhizal roots with that of the AM fungus. However, the character of obligate symbiont of AM fungi has hampered collection of enough AM fungus amount to obtain an enriched plasma membrane fraction to be analysed by 2D-PAGE. Taking into account that transport processes across membranes in the symbiotic interfaces are of capital importance for the functioning of the mycorrhizal association (Harrison, 1999), it is likely that the newly-synthesized plasma membrane polypeptides are proteins involved in the bidirectional transfer of nutrients and signals between both symbionts. However, the role of the differentially displayed polypeptides remains to be elucidated.
N-terminal microsequencing was performed to assign a biological function to some of the differentially displayed polypeptides; however, this approach allowed only one of them to be identified. The down-regulated polypeptide 2 has been identified as the catalytic subunit of the vacuolar H+-ATPase. This H+-ATPase is a member of the V-type ATPases that acidify organelles of all eukaryotic cells (Forgac, 1989). V-ATPases can be found in a wide variety of plant endomembranes, such as the tonoplast (Sze et al., 1992), the Golgi apparatus (Chanson and Taiz, 1985), coated vesicles (Oberbeck et al., 1994), and endoplasmic reticulum (Herman et al., 1994; Rouquié et al., 1998), as well as in plant plasma membranes (Robinson et al., 1996). Detection of a V-ATPase in plasma membranes isolated by phase partitioning could be due to tonoplast contamination. However, the recent immunocytochemical detection of V-ATPases in plasma membranes of pea cotyledons demonstrates the presence of this protein in the plasma membrane of plant cells (Robinson et al., 1996). Except for the tonoplast where V-ATPases are involved in the regulation of vacuolar pH and play a crucial role in the maintenance and regulation of cell turgor and in the transport and storage of various ions and metabolites (Sze et al., 1992), very little is known about their physiological role in other membrane systems. These results show that although the V-ATPase is present in the plasma membrane of tomato roots, the corresponding enzymatic activity was not detected in this fraction. Our data agree with the results previously obtained (Robinson et al., 1996) in plasma membrane fractions isolated from pea cotyledons. Based on the detection of the V-ATPase in the plasma membrane and in the trans-Golgi elements, structures known to be involved in the endocytotic pathway in plants, a temporary presence of the V-ATPase on the plasma membrane en route towards the vacuole was suggested (Robinson et al., 1996).
At this stage of research it is not known if the decreased levels of the V-ATPase in the plasma membranes isolated from mycorrhizal roots reflects a decreased level in the vacuolar membrane, which could be linked to the role of the vacuole in maintaining the ionic homeostasis in the cytosol. Further studies using enriched-tonoplast vesicles isolated from control and mycorrhizal tomato roots are necessary to understand the physiological significance of vacuolar ATPases in AM symbiosis. Moreover, methods for internal microsequencing of the differentially displayed polypeptides have to be developed to get some insights about the biological significance of the changes induced by AM colonization in the synthesis of plasma membrane proteins.
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Acknowledgments |
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We wish to thank Dr José Miguel Barea for helpful discussions and critical review of the manuscript and Mrs Custodia Cano for excellent technical assistance. This research was supported by the EU, AIR 3-CT 94–0809 project and TMR Network, ERB FMR XCT 960039. NF was supported by a post-doctoral contract from the Ministerio de Educación y Ciencia, Spain.
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Notes |
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1 To whom correspondence should be addressed. Fax: +34 958 129600. E-mail:nferrol{at}eez.csic.es
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