JXB Advance Access originally published online on March 14, 2003
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Journal of Experimental Botany, Vol. 54, No. 386, pp. 1335-1341,
May 1, 2003
© 2003 Oxford University Press
A germin-like protein of wheat leaf apoplast inhibits serine proteases
Received 15 October 2002; Accepted 24 January 2003
1 Instituto de Investigaciones Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, CC 1245, B7600GTQ, Mar del Plata, Argentina
2 Departamento de Biología, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, CC 1245, B7600GTQ, Mar del Plata, Argentina
3 To whom correspondence should be addressed. E-mail: segarra{at}mdp.edu.ar
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Abstract |
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A protein resistant to heat and proteolysis that inhibits serine proteases was isolated from wheat leaf apoplasts. Based on trypsin inhibition, its more active form was a 66–69 kDa oligomer. It was dissociated in an 18–21 kDa monomer having an amino terminal sequence identical to the Box A of germins and germin-like proteins. Like these proteins, it was glycosylated and showed manganese superoxide dismutase activity. The monomer displayed three forms when examined by 2D western blot: two of 19 kDa, pI 5.8 and 6.2; and one of 21 kDa, pI 5.8. It was found that the protein controls serine protease activity in the apoplast of plants challenged with the fungus Septoria tritici.
Key words: Apoplast, germin, serine protease inhibitor, wheat leaf.
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Introduction |
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Proteolysis is required for protein turnover and processing in the cellular and extracellular events of both healthy and diseased plants (Koiwa et al., 1997). Inhibitors habitually control the activity of proteolytic enzymes. Thus, several non-homologous families of protease inhibitors are recognized among plants, animals and micro-organisms (Laskowski et al., 1988). Similarly, proteins that establish complexes with proteases and inhibit their activities are widespread in nature. Important amounts of protease inhibitors are often found in fluids and tissues exposed to foreign proteases (Neurath, 1984), such as in blood serum (Travis and Salvensen, 1983), pancreatic acinar cells, and storage tissues of plants (Richardson, 1980; Ryan, 1988). Protease inhibitors are mainly proteins (Bode and Huber, 1992) classified in about ten families (Ryan, 1990). The most abundant and extensively studied group is that acting on serine proteases (Mosolov, 1998). Proteolysis participates in plant development and plant–pathogen interaction (Ryan and Walker-Simmons, 1981). Thus, leaf damage produced by herbivores induces a fast activation of defence genes in several plant species (Ryan and Pearce, 1998). an accumulation of protease inhibitors occurs as part of this response (Green and Ryan, 1972; Brown and Ryan, 1984; Hilder et al., 1987; Pautout et al., 1991; Rickauer et al., 1992; Yamagishi et al., 1993; Nielsen et al., 1995; Stevens et al., 1996).
The intercellular washing fluid is part of the apoplast, where pathogens often start plant colonization (Bowles, 1990). However, the properties of apoplastic protease inhibitors and their genes are scarcely known. Extracellular inhibitors have been found in Carica papaya, potato tubers challenged with Phytophtora infestans, and tomato roots (Odani et al., 1996; Valueva et al., 1998; Narváez-Vásquez et al., 1993, respectively). In spite of these data, little is known about the inhibitor-mediated control of proteolytic activities in the leaf apoplast. Recently, the behaviour of leaf extracellular serine protease activity in wheat plants challenged with the fungus Septoria tritici was described (Segarra et al., 2002). These data suggest that this activity could be controlled by an inhibitor(s).
In this study, the aim was to investigate the presence of serine protease inhibitor(s) in wheat leaf apoplasts. The results obtained indicate that a germin-like protein acts as serine protease inhibitor in leaf apoplast.
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Materials and methods |
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Plant material
Wheat (Triticum aestivum L.) seeds from cv. Pigüé and cv. Isla Verde were generously supplied by Ing. A Bariffi from the Estación Experimental Balcarce, Instituto Nacional de Tecnología Agropecuaria, Argentina. Seedlings were grown in chambers at 24 °C and illuminated by Grolux lamps (Sylvania) at an average photosynthetically active radiation of 250 µmol m–2 s–1 for 14 h d–1. The substrate was vermiculite soaked in Hoagland’s nutrient solution. Most of the experiments described here were used 10-d-old seedlings of cv. Pigüé.
Inoculation with Septoria tritici
Seedlings from wheat cultivars Pigüé and Isla Verde were sprayed with a Septoria tritici conidial suspension when 7-d-old as described by Segarra et al. (2002). Afterwards, they were stored for several days in humidified chambers at 18 °C under a 14 h photoperiod.
Preparation of the intercellular washing fluid
The intercellular washing fluid (IWF) was obtained from leaves as described by Pinedo et al. (1993), except that TRIS-HCl was replaced by Na-phosphate as buffer. This change allows a quick estimation of proteolytic activity using the fluorescamine reaction (Pinedo et al., 1993). In short, 80–100 leaves (nearly 5 g fresh weight) were dipped in 30 ml of a solution containing 50 mM Na-phosphate buffer, pH 7.5, 0.1% (v/v) 2-mercaptoethanol, 0.6 M NaCl, and 0.13% (v/v) Tween 20 at 4 °C, and exposed to vacuum for three 10 s periods separated by 30 s. Afterwards, they were dried on filter paper, placed in a fritted glass filter mounted on a tube and centrifuged at 400 g for 20 min. Each gram of leaf yielded nearly 0.25 ml of IWF. Preparations were routinely inspected to ensure that the extraction of IWF proteins was not contaminated with intracellular proteins (Pinedo et al., 1993). As indicated, IWF was heated at 70 °C for 30 min and cleared by centrifugation at 16 000 g for 15 min. This fraction was named IWF-70.
Protein determinations
The protein content of samples was measured by the bicinchoninic method (Smith et al., 1985), using bovine serum albumin as standard.
Protease inhibition measurements
Casein substrate solution was prepared as follows: 100 mg of casein were dissolved in 1 ml of 100 mM NaOH, heated for 30 min at 40 °C, cooled, neutralized with 100 mM HCl, and diluted with water to a 20 mg ml–1 concentration. Protease activity in either the absence or the presence of inhibitors was determined as described by Pinedo et al. (1993). A mixture containing either 0.25 µg of trypsin or 25–200 µg of IWF protein, distinct amounts of inhibitor, and 50 mM Na-phosphate buffer, pH 7.5 in 0.4 ml, was preincubated for 15 min at 37 °C. The reaction was triggered by the addition of 0.1 ml casein solution. Incubation was carried out for 1 h at 37 °C. Reactions were stopped by the addition of 50 µl of 50% (w/v) trichloroacetic acid (TCA) and stored 30 min at 4 °C. The TCA-soluble material was collected by centrifugation at 2000 g for 20 min. Aliquots of 200 µl were neutralized with 200 µl of 0.3 M KOH. Then, they were mixed with 400 µl of 0.4 M potassium borate, pH 9.7, and 400 µl of a solution of 0.3 mg ml–1 fluorescamine in acetone (Udenfriend et al., 1972). The fluorescence of fluorescamine-adducts was measured at 390/470 nm (excitation/emission) respectively, using 100–200 nmol of leucine as standard. All measurements were made in triplicate. The proteolytic activity of samples was estimated in units. One unit was defined as the activity necessary to release 30 nmol of leucine h–1.
Gel electrophoresis and in-gel trypsin inhibition assays
Proteins present in IWF-70 were precipitated with acetone 90% (v/v) at –20 °C and dried. Pellets were dissolved in sample buffer to contain 1 µg protein µl–1. Twenty µg were analysed by SDS-PAGE according to Laemmli (1970) in a Mini Protean II cell (Bio-Rad). Slab gels containing 12% acrylamide were 0.75 mm thick. When it was indicated, samples were heated at 100 °C for 3 min. Analysis in SDS-PAGE slabs containing 0.1% gelatine was performed as described by Heussen and Dowdle (1980). Samples containing 10–20 µg protein were run on slab gels 1 mm thick. After washing with 25 mM TRIS-HCl pH 8.0 containing 1% Triton X-100 and subsequently 10 mM CaCl2 to remove SDS, gels were incubated in 40 µg ml–1 trypsin solution for 3 h at 37 °C. Then they were washed with distilled water and stained with Coomassie Brilliant Blue. Blue stained bands revealed a protease inhibitor activity, while transparent background indicated a complete proteolytic digestion of gelatine. For analysis under non-denaturing conditions, 20 µg of IWF-70 protein were dissolved in 30 µl of 20% (v/v) glycerol and run on 0.75 mm thick slab gels containing 10% acrylamide (Pinedo et al., 1993). The TNIMAGE computer program was used to obtain spot densitograms of scanned gels.
Electroelution of SDS-PAGE bands
Polypeptide bands separated by SDS-PAGE were excised for electroelution. This was performed in a 422 Bio-Rad electroeluter following the manufacturer’s protocol. The eluted samples were exhaustively dialysed against 25 mM Na-phosphate buffer, pH 7.4, prior to testing their capacity for the inhibition of proteases.
Amino terminal sequencing
For SDS-PAGE separation, IWF-70 (20 µg protein) was dissolved in sample buffer (Laemmli, 1970) and heated at 100 °C for 3 min. After the run, proteins were transferred to a polyvinylidene difluoride membrane (PVDF) using an Applied Biosystems ProBlott equipment as prescribed by the manufacturer. Membrane was stained with a solution of Coomassie Blue R-250 0.1% (w/v), methanol 40% (v/v) and acetic acid 1% (v/v). A polypeptide of 18–21 kDa with the activity of a protease inhibitor was excised for sequencing. Sequence analysis was performed using a Beckman PI 2090E microsequencer in the Peptide Analysis and Synthesis Shared Facility at the University of Alabama, Birmingham, USA. Searches for homology to the N-terminal sequences were performed in the Swiss Prot Database with the BLAST FAQs program (Altschul et al., 1997).
Glycoprotein assays
Protein samples (20 µg) were subjected to 12% SDS-PAGE and stained with the periodic acid–Schiff reagent for the detection of glycoprotein (Segrest and Jackson, 1972).
Superoxide dismutase activity assays
Identification of superoxide dismutase (SOD) activity was carried out on samples subjected to non-denaturing polyacrylamide gel electrophoresis as described by Beauchamp and Fridovich (1971). After the run, gels were incubated for 30 min at 30 °C in the dark in 50 mM Na-phosphate buffer pH 7.5 containing 2.5 mM methyl thiazole tetrazolium. Then gels were transferred to a solution containing 0.1 mM EDTA, 0.2% (v/v) TEMED and 3 mM riboflavin and exposed to a 400 W lamp for 10–15 min. When clear bands over a dark background appeared, gels were washed with distilled water. Superoxide dismutase was also assessed by the inhibition of nitro blue tetrazolium (NBT) reduction (Beauchamp and Fridovich, 1971; Scebba et al., 1998). In short, the reaction medium comprised 200 µg of IWF-70 protein, 0.6 ml of 50 mM HEPES, pH 7.6, 0.1 mM EDTA, 50 mM Na HCO3, 13 mM methionine, 0.025% Triton-X-100, 75 µM NBT, and 2 µM riboflavin. Reactions were carried out for 5 min at 30 °C in a water-bath fitted with a 22 W fluorescent lamp (Phillips), and the absorbance of samples at 560 nm was measured. When indicated, samples were pre-incubated with 200 mM H2O2 (Yamahara et al., 1999; Carter and Thornburg, 2000).
Immunization and antiserum preparation
Dried IWF-70 (80 µg protein) was dissolved in SDS sample buffer at 100 °C for 3 min. Then it was run on SDS-PAGE and stained with Coomassie Blue R-250 as described above. The main 18–21 kDa band (85x2x1 mm; 500 µg protein) was cut and blended with 2 ml of 25 mM Na-phosphate buffer pH 7.4. The suspension was mixed with 2 ml of Freund’s adjuvant. Four 1 ml injections of the antigen mixed with complete adjuvant were given subcutaneously in an adult white rabbit. Eight weeks later, the rabbit received 500 µg of the antigen mixed with incomplete adjuvant. Four weeks later, blood was collected at weekly intervals from the ear vein and allowed to clot. The serum was separated by centrifugation at 2000 g for 15 min and stored frozen at –20 °C. Title was tested by dot immunobinding according to Hawkes (1986).
Two-dimensional gel electrophoresis
The IWF fraction was obtained as described above, except that 1 mM DFP was added to the extraction solution. The first dimension gel solution contained 6.25% (v/v) ampholytes, pH range 3–10 (Pharmacia). Six µl of IWF (85 µg protein) were mixed at 37 °C with 6 µl of a solution containing 4% (w/v) CHAPS, 0.2 M DTT, 18 M urea, and deposited under 10 µl of ampholytes, pH range 3–10. Gels were run at 10 °C for 10 min at 500 V and then for 3.5 h at 700 V. Gel was 1 mm thick and 60 mm long, with an acrylamide concentration of 5.4%. The second dimension was performed as in the conventional 2D-PAGE described by Garrels (1983). Briefly, the first-dimensional tube gels were extruded from the basic end and treated for 4 min with equilibration buffer C (SDS 3%, DTT 50 mM, TRIS-HCl 0.5 M, pH 6.8) followed by treatment with equilibration buffer D (SDS 3%, iodoacetamide 0.2 M, TRIS-HCl 0.5 M, pH 6.8). The gels were then layered onto the surface of a 12.5% SDS-polyacrylamide slab gel (1 mm thick, 90 mm wide, 75 mm long), and electrophoresed at 10 °C. The protein spots were revealed using the silver staining technique developed by Blum et al. (1987).
Immunoblot assays
After 2D-PAGE, proteins were transferred onto nitrocellulose paper. Carbohydrate epitopes were removed from proteins by periodate oxidation according to Heimgartner et al. (1990) as follows: the paper was incubated in a solution containing 10 mM periodic acid and 100 mM Na-acetate, pH 5, for 30 min at 20 °C in the dark. Then it was washed with water and blocked with TRIS-buffered saline solution, pH 8.0, containing 0.2% Tween-20 and 4% dried milk for 2 h. Then it was incubated for 16 h with a 1:2000 dilution of rabbit antibody against the 18–21 kDa inhibitor, washed with blocking solution, and incubated for 2 h with horseradish peroxidase-conjugated secondary antibody diluted 1:10 000. Immunoreactive bands were detected with the SuperSignal West Pico Chemiluminescent Substrate for Western Blotting (Pierce). Controls with preimmune serum (dilution 1:50) were processed.
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Protease inhibitory activity of IWF
The leaf IWF heated at 70 °C and cleared of precipitated proteins represented IWF-70. This fraction, earlier found lacking in protease activity (Segarra et al., 2002), inhibited both trypsin and protease activity present in whole IWF (Fig. 1). The extent of inhibition depended on the amount of IWF-70 in the assay and was greater for trypsin than for IWF protease activity.
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The fungus Septoria tritici stimulates leaf IWF protease activity in wheat cv. Pigüé while it reduces it in cv. Isla Verde (Segarra et al., 2002). These cultivars are resistant and susceptible to the disease caused by the fungus, respectively. Whether these responses depend on the inhibitory activity of IWF-70 was examined at 4, 8, and 12 d after fungal inoculation. On average, the percentage inhibition related to healthy plants decreased by 22% in cv. Pigüé while it increased by 65% in cv. Isla Verde (Fig. 2).
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SDS-PAGE characterization of IWF-70
The SDS-PAGE pattern of IWF-70 depended on its pretreatment (Fig. 3A). Dissolved in sample buffer at room temperature, it displayed a main band of size range 66–69 kDa (100%). However, mixed with sample buffer and heated at 100 °C for 3 min, it showed 33–36 kDa and 18–21 kDa bands representing 22% and 76% of the total protein, respectively. After SDS-PAGE separation in slabs containing co-polymerized gelatine, high and low size forms inhibited trypsin activity (Fig. 3B). However, the 33–36 kDa and 18–21 kDa forms inhibited trypsin less efficiently than the 66–69 kDa form. Furthermore, as shown in Fig. 1, 20 µg of the electroeluted 18–21 kDa band inhibited trypsin with a low potency compared with 20 µg of the 66–69 kDa form (0.12±0.02 and 0.68±0.16 units, respectively). Thus, the inhibitor displayed greater activity in the oligomeric form.
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N-terminal amino acid sequence
The N-terminal amino acid sequence of the 18–21 kDa polypeptide showed ten invariant residues as follows: LTQDFCVADL. This sequence was identical to that of Box A, one of the three highly conserved oligopeptides of germins (Bernier and Berna, 2001). Further searches in data banks proved that this region is 100% identical to the N-terminal sequence of the following germin-like proteins: GLP 1-barley (G:I 7447326); GLP 5-rice (GI: 7447328); GLP rice (GI: 7447327); GLP Oryza sativa (GI: 4239821); and adenosine diphosphate glucose pyrophosphatase Hordeum vulgare (GI: 13160411).
Glycoprotein property
After SDS-PAGE, IWF-70 displayed a 66–69 kDa band stained with the Schiff’s reagent for glycoproteins (Fig. 4A).
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Superoxide dismutase activity
After separation by non-denaturing gel electrophoresis, IWF-70 displayed a SOD activity band (Fig. 4B). Measured by inhibition of NBT reduction, this activity was not affected by H2O2.
Two-dimensional western blot characterization
Complete IWF run on 2D-PAGE, transferred to a membrane and tested for reaction with antibodies against the 18–21 kDa polypeptide revealed three spots (Fig. 5B). Two of them were of 19 kDa, pI 5.8 and 6.2; one was of 21 kDa, pI 5.8 (average values).
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Discussion |
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The leaf IWF is part of the apoplast where plants express, among others, proteins involved in the defence against pathogens (Bowles, 1990). Some of these proteins are hydrolytic enzymes, like ß-1,3 glucanases (Legrand et al., 1987), chitinases (Kombrink et al., 1988) and proteases (Segarra et al., 2002). After inoculation with the fungus S. tritici, this activity increases in leaves of the wheat cv. Pigüé and decreases in those of cv. Isla Verde (Segarra et al., 2002). The cultivars Pigüé and Isla Verde are tolerant and susceptible to septoriosis, respectively. Furthermore, extracts containing this protease activity restrains the growth of S. tritici (Segarra et al., 2002). Thus, the serine protease activity of the leaf apoplast can participate in the defence of wheat plants against S. tritici.
This work reports the isolation of a protein from wheat leaf apoplasts, which is resistant to heat and inhibits trypsin. It also inhibits apoplastic serine-like protease activity, the control of which could be exerted on plants infected with S. tritici in a cultivar-dependent way. Based on its capacity for trypsin inhibition, it could act in vivo as a 66–69 kDa tetramer (or larger) constituted by an 18–21 kDa subunit. The N-terminal sequence of this subunit is similar to that of Box A, one of the three highly conserved oligopeptides of germins and germin-like proteins (GLPs) (Bernier and Berna, 2001). As with germin and GLPs, it is glycosylated and displays SOD activity resistant to H2O2 like Mn-SOD (Yamahara et al., 1999; Carter and Thornburg, 2000). With one exception, all GLP family members contain at least one N-glycosylation site (Carter et al., 1998). N-glycosylation is a major modification of protein in plant cells. This process starts in the endoplasmic reticulum by the co-translational transfer of a precursor oligosaccharide to specific asparagine residues of the nascent polypeptide chain. Processing of this oligosaccharide occurs in the secretory pathway as the glycoprotein moves from the endoplasmic reticulum to its final destination (Lerouge et al., 1998). Such a mechanism could lead to the extracellular location of this GLP. In general, the earliest reactions detectable in plant pathogen recognition are the opening of specific ion channels and the formation of reactive oxygen intermediates (Somssich and Hahlbrock, 1998). It is proposed that GLPs function primarily as SODs in order to protect plants from the effects of oxidative stress (Khuri et al., 2001). In 2D western blots the momomer has two forms of 19 kDa (pI 5.8 and 6.2) and one of 21 kDa (pI 5.8). These features are reasonably comparable with the 26 kDa forms of pI 6.5 and 6.3 reported for germins (Hurkman et al., 1991).
Germin was first described in sprouting wheat grain as an apoplastic, multimeric (130 kDa), glycosylated enzyme with resistance to heat, H2O2 action and proteolysis (Thomson and Lane, 1980; Dunwell et al., 2000). Indeed, germins and GLPs are present in all plant organs playing several processes (Bernier and Berna, 2001). They belong to the cupin superfamily, which is composed of functionally diverse proteins present in archaea, eubacteria and eukaryota (Dunwell et al., 2000). Mature cupins are made of three highly conserved oligopeptides (boxes A, B, C). The benefit of the tertiary structure of cupin domains is the flexibility of the ‘active site’ within the centre of a ß-barrel (Dunwell et al., 2000). The functions of certain germins and GLPs have been discussed (Khuri et al., 2001). However, the majority of GLPs have been detected by genome sequencing and are still of unknown function. Many GLPs are induced by a range of either biotic (Thordahl-Christensen et al., 1997; Schweizer et al., 1999) or abiotic stresses (Hurkman and Tanaka, 1996; Vallelian-Bindschedler et al., 1998).
Although this is the first report about GLPs bearing protease inhibitor activity, some germins and GLPs are connected with plant defence against stresses (Vallelian-Bindschedler et al., 1998; Hurkman et al., 1991; Berna and Bernier, 1999; Michalowski and Bohnert, 1992; Dumas et al., 1995; Carter and Thornburg, 2000). The present findings agree with the theory that several protein families described as protease inhibitors hold various other functions. It is then possible that they derive from ancestral storage proteins which evolved into defence proteins capable of inhibiting insect and microbial proteases and amylases (Mosolov, 1998). Indeed, the inhibitor from Ragi (Eleusine coracana Gaertneri) inhibits both trypsin and 
-amylase throughout independent active sites (Maskos et al., 1996).
In conclusion, the presence in wheat leaf apoplasts of a GLP-serine protease inhibitor could be connected to the control of apoplastic serine proteases. This protein has additional functions, which could be connected with metabolic events of the apoplast under either physiological or stress conditions.
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Acknowledgements |
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Grants of the Universidad Nacional de Mar del Plata, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Fundación Antorchas and IFS-Sweden supported this work. We gratefully acknowledge the criticism of our colleagues at the IIB, Dr SM Goicoechea for advice on protein sequencing, ME Aued-Rau and MC Luciano for technical assistance. RD Conde is a career researcher from CONICET.
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