COMPOSITION AND DISTRIBUTION OF ADENYLATES IN SOYBEAN (GLYCINE-MAX L)NODULE TISSUE

Adenylates (ATP, ADP, and AMP) may play a central role in the regulati on of the O-2-limited C and N metabolism of soybean nodules. To be abl e to interpret measurements of adenylate levels in whole nodules and t o appreciate the significance of observed changes in adenylates associ ated with changes in O-2-limited metabolims, methods were developed fo r measuring in vivo levels of adenylated pools in the cortex, plant ce ntral zone, and bacteroid fractions of soybean (Glycine max L. Merr cv Maple Arrow x Bradyhrizobium japonicum strain USDA 16) nodules. Intac t nodulated roots were either frozen in situ by flushing with prechill ed Freon-113(-156 degrees C) or by rapidly (<1 s) uprooting plants and plunging them into liquid N-2. The adenylate energy charge (AEC = [AT P + 0.5 x ADP]/[ATP + ADP + AMP]) of whole-nodule tissue (0.65 +/- 0.0 1, n = 4) was low compared to that of subtending roots (0.80 +/- 0.03, n + 4), a finding indicative of hypoxic metabolism in nodules. The co rtex and central zone tissues were dissected apart in lyophilized nodu les, and AEC values were 0.84 +/- 0.04 and 0.61 +/- 0.03, respectively . Although the total adenylated pool in the lyophilized nodules was on ly 41% of that measured in hydrated tissues, the AEC values were simil ar, and the lyophilized nodules were assumed to provide useful materia l for assessing adenylate distribution. The nodule cortex contained 4. 4% of whole-nodules and the use of marker enzymes or compounds to corr ect for recovery of bacteroids and cross-contamination of the bacteroi d and plant fractions resulted in estimates that 36.2% of the total ad enylated pool was in bacteroids, and 59.4% was in the plant fraction o f the central zone. These are the first quantitative assessments of ad enylated distribution in the plant and bacteroid fractions of legume n odules. These estimates were combined with theoretical calculations of rates of ATP consumption in the cortex (9.5 nmol g(-1) fresh weight o f nodule s(-1)), plant central zone (38 nmol g(-1) fresh weight of nod ule s(-1)), and bacteroids (62 nmol g(-1) fresh weight of nodule s(-1) ) of soybean nodules to estimate the time within each nodule fraction. The low values for time constant (1.6-5.8 s for total adenylate, 0.9- 2.5 s for ATP only) in each fraction reflect the high metabolic activi ty of soybean nodules and provide a background for further studies of the role of adenylates in O-2-limited nodule metabolism.