Inhibition of Ethylene Production by Rhizobitoxine

Abstract

Rhizobitoxine, an inhibitor of methionine biosynthesis in Salmonella typhimurium, inhibited ethylene production about 75% in light-grown sorghum seedlings and in senescent apple tissue. Ethylene production stimulated by indoleacetic acid and kinetin in sorgh-um was similarly inhibited. With both apple and sorghum, the inhibition could only be partially relieved by additions of methionine. A methionine analogue, a-keto-y-methylthiobutyric acid, which has been suggested as an intermediate between methionine and ethylene, had no effect on the inhibition. Incorporation of "4C from added methionine-'4C into ethylene was curtailed by rhizobitoxine to about the same extent as was ethylene production. These results suggest that rhizobitoxine interferes with ethylene biosynthesis by blocking the conversion of methionine to ethylene and not indirectly by inhibiting the biosynthesis of methionine. Ethylene production by Pemi-cillium digitatum, a fungus which produces ethylene via pathways not utilizing methionine as a precursor, was not affected by rhizobitoxine. Two model systems for the generation of ethylene in plant tissues have been described by Lieberman and co-workers, one utilizing methionine as a substrate (8), and the other utilizing linolenate (1 1). In addition, methionine can serve as a precursor of ethylene in plant tissues (2, 7). To help assess the physiological importance of methionine as an ethylene precursor, a specific inhibitor of methionine biosynthesis was sought. Rhizobitoxine appeared to offer that potential. Rhizobitoxine is a phytotoxin produced by certain strains of the soybean root nodule bacterium Rhizobium japonicum (15). It inhibits greening of new leaf tissue of many plants and causes the main visual symptom of the disease in soybean known as rihizobial-induced chlorosis (14). The precise structure of rhizobitoxine remains to be elucidated; however, it is known to be a basic sulfur-containing amino acid which yields an ether derivative of homoserine upon desulfurization (13). Rhizo-bitoxine inhibits the growth of Salmonella typhimurium by inhibiting /3-cystathionase, an enzyme in the methionine bio-synthetic pathway (12). It also irreversibly inactivates /B-cystathionase isolated from spinach leaves (4); however, the physiological effect of this lesion on the biosynthesis of methionine in spinach has yet to be assessed. We report here that rhizobitoxine inhibits ethylene bio-synthesis in sorghum seedlings and in senescent apple tissue by the unexpected mechanism of blocking the conversion of methionine to ethylene. MATERIALS AND METHODS Sorghum Experiments. Seeds of Sorghum vulgare var. Hegari were surface-sterilized by wetting with ethanol and then immersing in an aqueous solution of 0.2% HgCl2 + 1% HCl for 2 min. After rinsing well, the seeds were germinated on moist filter paper in a Petri dish at 27 C in the dark. Two days after imbibition, the seedlings were transplanted to 50-ml Erlenmeyer flasks constructed with a side arm to collect CO2. Six seedlings per flask (about 300 mg fresh wt) were supported on a nylon mesh screen held 1.0 cm above the flask bottom by three glass cylinders and 0.3 cm above the surface of the root solution (10 ml of water). The loosely capped flasks were placed in the dark at 27 C overnight, after which the treatments were added. One milliliter of 10% KOH was added to the side arm, and the flasks were closed with a rubber serum stopper and placed under a fluorescent lamp in the laboratory (about 250 ft-c) at about 25 C and 14-hr day length. At daily intervals, 02 was added to restore flasks to near atmospheric pressure with a gas-tight syringe. Three milliliters of gas were then withdrawn (replaced with air) for assaying ethylene. The total gas volume of the assembled flask system was 50 cc. Treatments were replicated three times and each experiment was performed at least twice except for the experiment with P. digitatum. Apple Tissue Experiments. Apple tissue samples were prepared from postclimacteric Stayman apples stored 6 to 9 months at 0 C or Summer Rambeau apples stored 2 weeks at 0 C and 1 week at 21 C. One apple was used for each experiment. Tissue plugs (1.0 cm in diameter, cut with a cork borer from 0.5-cm thick slices, and weighing approximately 1.5 g) were placed in 25-ml Erlenmeyer flasks containing 5 ml of solution and a small vial of KOH to absorb CO2. The incubation solution contained 0.4 M sucrose, 0.1 M NaHCO,, pH 8.5, and 0.05 mm EDTA. The flasks were sealed with serum stoppers and incubated in a shaker water bath at 30 C. Tracer Experiments with Apple Tissue. L-Methionine-"C was added to the experimental system described above. After incubation at 30 C for 5 hr, a 2-ml aliquot of the flask atmosphere was removed for ethylene analysis by gas chromatography. Then 86% of the remaining gas was removed with a gas-tight syringe, the ethylene was absorbed in cold mercuric perchlorate, and the radioactivity was determined by liquid scintillation counting as described (9). The radiochemical purity of L-methionine-`C(UL) (New England Nuclear Corp.) used in experiment 2 of Table II was determined by thin layer chromatography (cellulose) of a sample diluted with carrier L-methionine. Butanol-acetic acid-water (12:3:5 v/v) was used as the developing solvent, after which the cellulose was scraped off and the radioactivity was determined by placing directly in Bray's liquid scintillation solution. Only 10% of the radioactivity remained as methionine after storage in 50% ethanol for 6 months at-20 C.