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.

Bacteroids isolated from alfalfa nodules induced by Rhizobium melifoti 102F34 transported glycine betaine at a constant rate for up to 30 min. Addition of sodium salts greatly increased the uptake activity, whereas other salts or non-electrolytes had less effect. The apparent K,,, for glycine betaine uptake was 8.3 pM and V was about 0.84nmol min-' (mg protein)-' in the presence of 200mM-NaCI which gave maximum stimulation of the transport. Supplementing bacteroid suspensions with various energy-yielding substrates, or ATP, did not increase glycine betaine uptake rates. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), and the respiratory inhibitor potassium cyanide strongly inhibited glycine betaine uptake, but arsenate was totally inactive. Glycine betaine transport showed considerable structural specificity: choline, proline betaine, y-butyrobetaine and trigonelline did not competitively inhibit the system, although choline and proline betaine were transported by bacteroids. Both a high-affinity activity and a low-affinity activity were found for choline uptake. These osmoprotective compounds might have a significant role in the maintenance of nitrogenase activity in bacteroids subjected to salt stress.

In medium of low or high osmolarity, bacteroids isolated from Medicago sativa nodules induced by Rhizobiurn rnefifoti 102F34 rapidly catabolized [ 4C]choline. Trimethylamine was never detected and glycine betaine was the predominant product within the ethanol-soluble fraction of bacteroids subjected to salt stress (0.4 M-NaCI). Both choline oxidase and glycine betaine aldehyde dehydrogenase activities were characterized; the apparent K,,, values for choline and glycine betaine aldehyde were 2.0 and 0.4 mM, respectively. A 3 h incubation of the bacteroids in medium of high osmolarity, supplemented or not with choline, did not significantly modify the specific activity of the two enzymes. Similarly, salinization of the host plants for 2 weeks with 0.15 M-NaCl had only a slight effect on both enzymic activities. Thus, the cholineglycine betaine biosynthetic pathway was not modulated by the external osmotic pressure. Glycine betaine itself was actively degraded by bacteroids suspended in low-osmolarity medium, but the demethylation process producing sarcosine and glycine was extremely slow in bacteroids subjected to salt stress. Thus, high concentration of glycine betaine can be maintained in salt-stressed bacteroids.