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placed in sealed Plexiglas boxes as described and incubated under constant light at >300 µE·m−2·s−1 for 24 h at 28°C (12).

Suspension-Cultured Cells. Suspension-cultured cells of L. peruvianum (cells kindly provided by A. Schaller; ref.9) were cultivated under constant light as described (6). Cells were subcultured weekly and used for experiments 4–8 days after subculturing. For the experiments, 1.5 ml of cell suspension cultures were transferred to multiwell plates that were vigorously shaken on an orbital shaker under ambient room light and temperature conditions. After an ≈1-h equilibration period, the pH of the medium had reached a starting pH of 4.8 ± 0.2.

Alkalinization Assay. Test compounds were supplied to 1.5 ml of cell suspension cultures at the times indicated in the text. The pH of the medium was measured with a pH meter (Orion, Model EA940; Beverly, MA) with a semimicro combination electrode (Orion 8103BN, Beverly, MA). Addition of systemin or oligosaccharide elicitors to the cultured cells causes an alkalinization of the medium within 1–4 min. Over the next 6–15 min, the pH increases by about 0.6–1.2 pH units, remains at the high pH for 5 –10 min, and thereafter decreases. The pH in untreated controls consistently increased less than 0.3 pH units over the course of an experiment. Changes in the pH of the medium are indicated as “∆ pH” above the controls. Aqueous solutions of systemin and chitosan were prepared as described (12). The β-glucan elicitor from Phytophtora megasperma f. subsp. glycinea (pmg elicitor; partially purified according to Ayers et al., ref.33) was a generous gift of E. Farmer (University of Lausanne, Lausanne, Switzerland).

Myelin Basic Protein (MBP) Kinase Assay. At the times indicated, cells were mixed with extraction buffer (12) and frozen in liquid nitrogen. Extracts were obtained by sonicating the cells twice for 20 s (Model 300 Sonic Dismembrator, microtip; Fisher) and subsequent centrifugation at 30,000 × g. Supernatants were analyzed for MBP kinase activity by an in-gel kinase assay with MBP (Sigma) as an artificial substrate as described (12).

Immunocomplex Kinase Assay. Phosphotyrosine-containing proteins in cell extracts were immunocomplexed with the monoclonal phosphotyrosine-specific antibody PY20 coupled to Sepharose 4B (Stratagene) and assayed for MBP kinase activity as described (34).

Radioreceptor Assay. The binding of the systemin derivative 125I-Tyr-2,Ala-15-systemin to cell-surface binding sites was performed as described (6). Suramin was applied 1–10 min before addition of 125I-Tyr-2,Ala-15-systemin.

Photoaffinity Labeling. Photoaffinity labeling of the systemin receptor was performed in the presence or absence of competing systemin (200-fold excess; 150 pmol) or suramin (1 mM). Preparation of the analog 125I-N-(4-[p-azidosalicylamido]butyl)-3′(2′-Cys-3, Ala-15-systemindithiol)propionamide and photoaffinity labeling were performed as described (6). After photocrosslinking, microsomal membranes were prepared by adding the cells to 250 µl of extraction buffer containing 0.5 M D-sorbitol, 50 mM Mops, 10 mM Tris·HCl, 10 mM EGTA, 2.5 mM potassium metabisulfite, 2.0 mM salicylhydroxamic acid, 1 mM PMSF, plant protease inhibitor mixture (diluted 1/100; Sigma), and 1% β-mercaptoethanol, pH 7.6 (KOH). The membranes were extracted by sonication (Model 300 Sonic Dismembrator) for 1 min on ice. After sonication, 1 ml of extraction buffer was added, and the extract was vortexed and centrifuged at 12,000 × g for 5 min at room temperature. A 1-ml volume of the supernatant was removed and added to 9 ml of extraction buffer. The 10-ml extracts were ultracentrifuged for 1 h at 100,000 × g, after which the supernatant was removed and the pellets were gently rinsed with 2 ml of 10 mM Tris/1% sodium-dodecylsulfate, pH 8.0. The pellets were resuspended in 100 µl of the same buffer, and 30 µl was analyzed by SDS/7.5% PAGE and subsequent phosphorimaging of the dried gel.

Table 1. EC50s for medium alkalinization in response to elicitors

 

EC50

Elicitor

−SUR

+SUR

Systemin, pg/ml

360

9,600

Chitosan, ng/ml

2

200

pmg elicitor, µg/ml

22

250

EC50, elicitor concentration for half-maximal medium alkalinization ofsuspension-cultured tomato cells in the absence (−SUR) and presence(+SUR) of suramin. EC50s were calculated based on Fig. 1.

Results

Proteinase Inhibitor Synthesis in Response to Wounding and Systemin Is Inhibited by Suramin. Suramin had been shown to interfere with binding of several animal polypeptide cytokines and growth factors to their plasma-membrane receptors (see the introduction). Because the 18-amino acid wound signal systemin is perceived by a membrane-bound receptor, we investigated whether suramin would inhibit the systemin-induced defense response in 14-day-old tomato plants. Supplying young excised plants simultaneously with 2.5 nM systemin and 700 µM suramin inhibited systemin-induced synthesis of serine proteinase inhibitors I and II in leaves by 95 ± 5% and 75 ± 18%, respectively. Synthesis of the two inhibitors in leaves of excised plants in response to wounding was 100% inhibited when the plants had been pretreated for 1 h with 700 µM suramin, a level consistent with its inhibition of animal growth factor–cell-surface receptor interactions.

Alkalinization of L. peruvianum Cell Medium in Response to Systemin, Chitosan, and pmg Elicitor Is Inhibited by Suramin. To study suramin effects on early cellular responses related to the synthesis of defense-related proteins, suspension-cultured cells of L. peruvianum were used. Previous studies had shown that elicitors of plant defense responses cause a rapid potassium ion efflux and proton influx leading to alkalinization of the culture medium of suspension-cultured cells through inhibition of the plasma membrane H+-ATPase (9, 10, 35). The pH of the medium of cultured tomato cells had been shown to increase about 1 pH unit within 10 min in response to systemin and to remain at the higher pH for about 25 min (10). The EC50 for alkalinization of tomato suspension-cultured cells induced by systemin is shown in Table 1, compared with the alkalinization of the medium by the oligosaccharide elicitors chitosan (a glucosamine oligomer) and pmg elicitor (a β-glucan mixture derived from mycelial walls of P. megasperma f. subsp. glycinea; ref.33). The EC50 for the alkalinization response induced by pmg elicitor varied considerably from the EC50s of systemin and chitosan, but the pmg elicitor was purified only partially, whereas systemin and chitosan had been highly purified.

The effect of suramin on alkalinization of the medium in response to increasing concentrations of systemin, chitosan, and pmg elicitor was assayed by pretreating the cells with a constant suramin concentration of 700 µM for 10 min before the application of elicitors (Fig. 1). The alkalinization response to lower elicitor concentrations was inhibited by suramin, resulting in an increase in the EC50s (Table 1). In the presence of high concentrations of elicitor, the suramin inhibition was reversed (Fig. 1). The reversible inhibition of the medium alkalinization in response to all three elicitors suggested that suramin competes with elicitors for extracellular elicitor-binding sites.

With each elicitor, the alkalinization response was inhibited



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