Leghaemoglobin biosynthesis in a new single cell system from soybean root nodules

Single cells were effectively released from 35–45-day-old soybean ( Glycine max L. cv. Yaefusanari) nodules by treatment with an enzymic solution containing 1 mg/ml maceration enzyme (Pectolyase Y-23), 0.5 M mannitol, 2% (w/v) sucrose and 0.5% (w/v) potassium dextran sulfate. Bacteroid-containing cells were purified by Percoll density gradient centrifugation. Electron microscopic observation showed that these cells were protoplasts enclosed by a thin wall and with well preserved internal structures including bacteroids. The single cells obtained were stable against centrifugation and vigorous pipetting. The cells retained the ability to synthesize proteins including leghaemoglobin. The ratio of leghaemoglobin components synthesized in the single cells was similar to that of components synthesized in the nodules. The bacteroidal cell fraction was further separated into three fractions by a Percoll density gradient centrifugation. Comparison of the absolute and relative leghaemoglobin content, the activity of glutamine synthetase in the cytoplasm and the activity of 3-hydroxybutyrate dehydrogenase in the bacteroid suggests that these fractions contained cells in different stages of symbiosis. This new single cell system should provide a useful experimental system for analyzing events in the root nodule.