Adenylate Gradients and Ar:O2 Effects on Legume Nodules. II. Changes in the Subcellular Adenylate Pools

Abstract

Central infected zone tissue of soybean (Glycine max L. Merr.) nodules was fractionated into separate subcellular compartments using density gradient centrifugation in nonaqueous solvents to better understand how exposure to Ar:O 2 (80:20%, v/v) atmosphere affects C and N metabolism, and to explore a potential role for adenylates in regulating O 2 diffusion. When nodules were switched from air to Ar:O 2 , adenylate energy charge (AEC) in the plant cytosol rose from 0.63 6 0.02 to 0.73 6 0.02 within 7 min and to 0.80 6 0.01 by 60 min. In contrast, AEC of the mitochondrial compartment of this central zone tissue remained high (0.80 6 0.02 to 0.81 6 0.02) following Ar treatment while that of the bacteroid compartment was unchanged, at 0.73 6 0.02, after 7 min, but declined to 0.57 6 0.03 after 60 min. These results were consistent with a simulation model that predicted Ar:O 2 exposure would first reduce ATP demand for ammonia assimilation and rapidly increase cytosolic AEC, before the Ar:O 2 -induced decline mediated by a decrease in nodule O 2 permeability reduces bacteroid AEC. The possibility that adenylates play a key, integrating role in regulating nodule permeability to oxygen diffusion is discussed.In active, N 2 fixing nodules, respiration and N 2 ase activity are O 2 limited and can be increased slightly, but significantly (2%-20%), by gradual increases in the external partial pressure of O 2 (pO 2 ; Hunt et al., 1989). However, following treatments such as defoliation, detopping, stem girdling, nitrate fertilization, or exposure to C 2 H 2 or Ar:O 2 that are known to inhibit nitrogenase activity and nodule respiration, nodules respond to increases in pO 2 with much larger enhancements of nitrogenase activity (Hartwig et al., 1987;Vessey et al., 1988;Denison et al., 1992;de Lima et al., 1994). These studies, together with measurements of infected cell O 2 concentration and nodule permeability (Denison and Layzell, 1991;King and Layzell, 1991), showed that the treatments apparently down-regulate metabolism by reducing the supply of O 2 through a decrease in the gaseous permeability of nodules.The mechanism that nodules use to regulate the diffusion barrier is unknown, but is correlated with the adenylate ratios (ATP-ADP; adenylate energy charge, AEC ¼ ð½ATP10:5½ADPÞ=ð½ATP1½ADP1½AMPÞ), in most, but not all treatments known to induce severe O 2 limitation of nodule metabolism (de Lima et al., 1994). For example, exposure of nodules to 10% O 2 (2-3 min), stem girdling (3 h), or nitrate fertilization (48 h) decreased nodule AEC values by approximately 0.12 units (de Lima et al., 1994). However, 60 min exposure of soybean (Glycine max) nodules to Ar:O 2 did not change the nodule AEC values (de Lima et al., 1994), even though the treatment dramatically lowered infected cell [O 2 ] (King and Layzell, 1991), and N 2 ase activity could be recovered at elevated pO 2 . Taken at face value, this result does not support the idea that adenylate pools or adenylate ratios in the infected cells are involved in the mechanism...

Adenylate-Coupled Ion Movement. A Mechanism for the Control of Nodule Permeability to O2 Diffusion

“…Within 5 to 10 min of Ar:O 2 exposure, nitrogenase declines as a result of an O 2 limitation imposed by a reduction in nodule permeability (King and Layzell, 1991). This observation is consistent with mathematical model and experimental results (Wei et al, 2004a(Wei et al, , 2004b linking the reduction in NH 3 production to reduced cytosolic ATP demand in support of Gln synthetase activity (Fig. 2), resulting in an increase in the cytosolic ATP/ADP ratio.…”

“…After each treatment, the nodulated roots were rapidly (,2 s) removed from the pots and plunged into liquid N 2 (2196°C) as described previously (Wei et al, 2004b). The nodules were picked from the frozen roots/sand mixture while being kept frozen by occasional flooding with liquid N 2 .…”

“…The nodules were picked from the frozen roots/sand mixture while being kept frozen by occasional flooding with liquid N 2 . They were then weighed and stored in liquid N 2 until they were lyophilized as described previously (Wei et al, 2004b). Each nodule was then carefully dissected to separate the cortex from the CZ tissue (Fig.…”

“…Soybean (Glycine max L. Merr cv Maple Arrow) seeds were inoculated at sowing with Bradyrhizobium japonicum USDA 16 and grown in silica sand in a growth chamber as described previously (Wei et al, 2004b). When plants were 4-to 5-weeks old, they were randomly selected to be placed in a control treatment, a stem-girdling treatment (the bark and phloem were removed from a 1-cm-wide ring around the hypocotyls and then left for 3 h), or one of three treatments in which a flow-through gas exchange system (Hunt et al, 1989) was used to provide either Ar:O 2 (80:20), 30% O 2 (balance N 2 ), or 10% O 2 (balance N 2 ) to the nodulated roots for 1 h.…”