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Ethanol-soluble organic acid, carbohydrate, and amino acid constituents of alfalfa (Medlcago sativa) roots and nodules (cytosol and bacteroids) have been identified by gas-liquid chromatography and high performance liquid chromatography. Among organic acids, citrate was the predominant compound in roots and cytosol, with malonate present in the highest concentration in bacteroids. These two organic acids together with malate and succinate accounted for more than 85% of the organic acid pool in nodules and for 97% in roots. The major carbohydrates in roots, nodule cytosol, and bacteroids were (descending order of concentration): sucrose, pinitol, glucose, and ononitol. Maltose and trehalose appeared to be present in very low concentrations. Asparagine, glutamate, alanine, y-aminobutyrate, and proline were the major amino acids in cytosol and bacteroids. In addition to these solutes, shrine and glutamine were well represented in roots. When alfalfa plants were subjected to 0.15 M sodium chloride stress for 2 weeks, total organic acid concentration in nodules and roots were depressed by more than 40%, whereas lactate concentration increased by 11, 27, and 94% in cytosol, roots, and bacteroids, respectively. In bacteroids, lactate became the most abundant organic acid and might contribute partly to the osmotic adjustment. On the other hand, salt stress induced a large increase in the amino acid and carbohydrate pools. Within the amino acids, praline showed the largest increase, 11.3-, 12.8-, and 8.0-fold in roots, cytosol, and bacteroids, respectively. Its accumulation reflected an osmoregulatory mechanism not only in roots but also in nodule tissue. In parallel, asparagine concentration was greatly enhanced; this amide remained the major nitrogen solute and, in bacteroids, played a significant role in osmoregulation. On the contrary, the salt treatment had a very limited effect on the concentration of other amino acids. Among carbohydrates, pinitol concentration was increased significantly, especially in cytosol and bacteroids (5.4-and 3.4-fold, respectively), in which this cyclitol accounted for more than 35% of the total carbohydrate pool; pinitol might contribute to the tolerance to salt stress. However, trehalose concentration remained low in both nodules and roots; its role in osmoregulation appeared unlikely in alfalfa.

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...

Previous studies indicate that methylated cyclitols are potentially important osmolytes in plants. In a search for genetic diversity for pinitol (D‐3‐O‐methyl‐chiro‐inositol) accumulation in soybean (Glycine max (L.) Merr.), two‐ to three‐fold differences in pinitol accumulation in leaf blades were found among Chinese plant introductions. Furthermore, it was found that genotypes that accumulated high concentrations of pinitol, when grown under well‐watered conditions, had been selected for performance in regions of China having low rainfall. Among the carbohydrates analysed, pinitol accumulation was uniquely associated with adaptation to dry areas of China. A detailed study of pinitol accumulation in the soybean plant showed two‐ to three‐fold gradients in pinitol concentration from the bottom to the top of the plant. The gradient shifted during plant development, with consistently higher concentrations of pinitol in the uppermost leaves. Pinitol accumulation was not correlated with activity of the key biosynthetic enzyme, inositol methyl transferase. This result and other lines of evidence indicated that shifting patterns of pinitol accumulation were due to translocation of the cyclitol from lower to upper nodes. Pinitol, proline, and sugars accumulated in leaf blades on soybean plants subjected to drought, but the molar concentration of pinitol in stressed plants was greater than the concentrations of proline or sugars. Although the mechanism by which pinitol participates in drought tolerance is not fully known, our results provide additional correlative evidence linking pinitol and drought tolerance in soybean.

In leaves, the anaerobic accumulation of alanine was accompanied by a loss of aspartate, and these changes preceded -yaminobutyrate accumulation and glutamate loss. Changes in keto acid content did not appear to be the cause of amino acid changes. Accumulation of y-aminobutyrate was due to acceleration of glutamate decarboxylation and arrest of -y-aminobutyrate transamination. Changes in enzyme content did not explain the changes in reaction rates in vivo. Most of the aspartate may be converted anaerobically to alanine via oxalacetate and pyruvate.Several reports on the anaerobic accumulation of -y-aminobutyrate in higher plant tissues have appeared in the last 10 years (6,7,10,11,25). GAB2 becomes markedly more radioactive under anaerobic than aerobic conditions when labeled glutamate (18) or CO2 (8) is administered to plants. Uncom-bined alanine accumulated anaerobically in quantities greater than those of GAB (6,7,11,25). Anaerobiosis also induces accumulations of GAB in Chlorella (14,29) and in mammalian brain tissue (31).Most of the previous studies on this subject have involved relatively long incubation periods (12 to 72 hr), whereas, in our studies, short incubations have been employed to explore the mechanisms underlying anaerobic accumulation of GAB and alanine.MATERIALS AND METHODS Plant Material and Leaf Incubations. Mature leaves of the radish plant Raphanus sativus L., var. Champion, were used in all experiments. One anaerobic incubation system (applying to Figs. 3,4,7,and 8 and Table I) involved placing each leaf (in a small test tube with solution of radioactive compound) in an inverted 3-liter flask through which oxygen-free N, was circulated at 300 to 500 ml/min prior to and after insertion of the leaf. This system resulted in a slower development of complete anaerobiosis than the second system. With the second system (applying to Figs. 1 The effluent from the column was warmed to room temperature, and pH was adjusted to 7.0 + 0.4. Most (90%) of the material absorbing at 340 nm was removed without any loss of keto acids by mixing the extract with activated charcoal (1.0 g for extract from 15 g of leaves), allowing the mixture to stand at room temperature for 15 min, and filtering. The filtered extract was assayed immediately so that total elapsed time was less than 2 hr. most of which time the extract was at 2 to 4 C. a-ketoglutarate-"C, extraction was the same as described above except that 0.13 N tungstic acid (2) was used. Aliquots of the combined supernatants were counted for determination of total acid-soluble radioactivty.For the analysis of a-ketoglutarate-'4C, two 5.0-ml aliquots of the tungstic acid extract were each mixed with 0.5 ml of a saturated solution of 2,4-dinitrophenylhydrazine in 3 N HCI and allowed to stand for 0.5 hr at room temperature. Dinitrophenylhydrazones were purified (20) and separated by paper chromatography in tert-amyl alcohol-ethanol-water, 50:17:40 (v/v) on paper buffered with 0.2 M phosphate, pH 6.3. An ascending run over 10 to 12 hr separated a-ketog...