METABOLITE CONCENTRATIONS IN SOYBEAN NODULES

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. Of78 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), trehalose (66), lysine (58), and arginine (46). The results indicate that metabolite concentrations in bacteroids can be reliably determined. As efforts to understand the functioning of legume nodules at the molecular level increase, knowledge of metabolite concentrations in nodules and especially in bacteroids becomes increasingly important. For example, the disaccharide a,a-trehalose is synthesized by bacteroids and, because of the potential toxicity of trehalose to the plant, it is of interest to know how much trehalose is released by bacteroids to the host (18). Recent evidence indicates the existence of a relatively large pool of glutamate in bacteroids (16), and an estimate of the actual concentration of glutamate in bacteroids would contribute to an understanding of the role of this amino acid. Estimates of metabolite concentrations in bacteroids and cy'Supported in part by the United States Department of Agriculture under agreement No. 84-CRCR-1-1426. Salaries and research support provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Journal article No. 83-87. tosol from legume nodules have been published recently (1, 8, 11, 22). However, a potential problem which has not been carefully considered is the exchange of compounds between bacteroids and cytosol or the metabolism of compounds during the isolation of bacteroids. In this report, the exchange of metabolites was estimated by allowing bacteroid/cytosol mixtures to stand (1°C) for times ranging from 0 to 44 min. Analysis of a series of 12 samples and extrapolation of a regression line to time zero provides an approximation of the original composition. Results for the application of this technique to the analysis of a few carbohydrate compounds have been presented previously (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) is described. MATERIALS AND METHODS Plant Growth. Soybeans (Glycine max [L.] Merr.) cv Beeson 80 were grown in silica sand in a greenhouse. Seeds were inoculated at planting time with Bradyrhizobium japonicum USDA 110, grown as previously described (18). Plant nutrient solution (18) contained 2 mm nitrate. 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. Tubes were heated at 75°C for 10 to 15 min and then kept at 1°C overnight. Solids were separated from the extract by centrifugation and were then reextracted twice with 80% (v/v) ethanol. The combined ethanol extracts were dried in vacuo and solids were dissolved in 5.0 ml water and stored at 1°C over approximately 0.5 ml ofchloroform. Extracts were divided into neutral, anionic, and cationic fractions using QAEand SP-Sephadex ion-exchange columns (15). Elution volume for QAE-Sephadex was increased by 50% over that recommended in order to provide essentially complete re-