Metabolites in Bradyrhizobium japonicum bacteroids and in Glycine max (L.) Merr. cytosol from root nodules were analyzed using an isolation technique which makes it possible to estimate and correct for changes in concentration which may occur during bacteroid isolation. Bacteroid and cytosol extracts were fractionated on ion-exchange columns and were analyzed for carbohydrate composition using ps-liquid chromatography and for organic acid and amino acid composition using high performance liquid chromatography. Analysis of organic acids in plant tissues as the phenacyl derivatives is reported for the first time and this approach revealed the presence of several unknown organic acids in nodules. The time required for separation of bacteroids and cytosol was varied, and significant change in concentration of individual compounds during the separation ofthe two fractions was estimated by calculating the regression ofconcentration on time. When a statistically significant slope was found, the true concentration was estimated by extrapolating the regression line to time zero. Of 78 concentration estimates made, there was a statistically significant (5% level) change in concentration during sample preparation for only five metabolites: glucose, sucrose, and succinate in the cytosol and D-pinitol and serine in bacteroids. On a mass basis, the major compounds in bacteroids were (descending order of concentration): myoinositol, D-chiro-inositol, a,a-trehalose, sucrose, aspartate, glutamate, Dpinitol, arginine, malonate, and glucose. On a proportional basis (concentration in bacteroid as percent of concentration in bacteroid + cytosol fractions), the major compounds were: a-aminoadipate (94) (18,19). In this report, the technique is extended to the analysis of individual carbohydrates, organic acids, and amino acids in the bacteroids and cytosol of soybean nodules. In addition, application to nodule extracts of a new method for the analysis of organic acids (10) About 50 g of nodules were pulled from roots at 64 d after planting. Nodules were chilled (1°C) as they were pulled, and after nodules were all pulled and chilled (about 30 mimi), the large sample was mixed. Subsamples of 3.0 g were used for analysis.Extraction and Preparation of Fractions. Chilled nodules samples were ground in a mortar in 10 ml of triple deionized water (1°C) and the mixture was filtered through four layers of cheesecloth into a 50 ml centrifuge tube. To provide different extraction times, mixtures were held at this stage-i.e. in centrifuge tubes in the cold room (1°C). Mixtures were centrifuged using a Beckman J2-21 centrifuge, a JA-20 angle-head rotor, and a speed of 20,000 rpm (48,400g) held for 2 min and followed by deceleration using maximum brake. The supernatant (cytosol) was immediately mixed with 20 ml of hot (75°C) 95% (v/v) ethanol. The surface of the bacteroid pellet was rinsed with 0.7 to 1 ml of ice-cold water and the rinse discarded. Twenty ml of hot ethanol was added to the tube and the pellet was suspended in the ethanol...
