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First published online April 2, 2004; 10.1104/pp.103.038547 Plant Physiology 134:1775-1783 (2004) © 2004 American Society of Plant Biologists Adenylate Gradients and Ar:O2 Effects on Legume Nodules. II. Changes in the Subcellular Adenylate Pools1Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6 (H.W., D.B.L.); and Botany, School of Plant Biology, The University of Western Australia, Nedlands, Western Australia 6907, Australia (C.A.A.)
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:O2 (80:20%, v/v) atmosphere affects C and N metabolism, and to explore a potential role for adenylates in regulating O2 diffusion. When nodules were switched from air to Ar:O2, adenylate energy charge (AEC) in the plant cytosol rose from 0.63 ± 0.02 to 0.73 ± 0.02 within 7 min and to 0.80 ± 0.01 by 60 min. In contrast, AEC of the mitochondrial compartment of this central zone tissue remained high (0.80 ± 0.02 to 0.81 ± 0.02) following Ar treatment while that of the bacteroid compartment was unchanged, at 0.73 ± 0.02, after 7 min, but declined to 0.57 ± 0.03 after 60 min. These results were consistent with a simulation model that predicted Ar:O2 exposure would first reduce ATP demand for ammonia assimilation and rapidly increase cytosolic AEC, before the Ar:O2-induced decline mediated by a decrease in nodule O2 permeability reduces bacteroid AEC. The possibility that adenylates play a key, integrating role in regulating nodule permeability to oxygen diffusion is discussed.
In active, N2 fixing nodules, respiration and N2ase activity are O2 limited and can be increased slightly, but significantly (2%–20%), by gradual increases in the external partial pressure of O2 (pO2; Hunt et al., 1989  ). However, following treatments such as defoliation, detopping, stem girdling, nitrate fertilization, or exposure to C2H2 or Ar:O2 that are known to inhibit nitrogenase activity and nodule respiration, nodules respond to increases in pO2 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 O2 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 O2 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,
Nevertheless, adenylates do seem to play a key role in the regulation of nodule metabolism. Kuzma et al. (1999)
The model also predicted that short-term exposure of nodules to an Ar:O2 atmosphere—a treatment that stops ATP demand for N assimilation by GS and asparagine synthetase activities, but not nitrogenase activity—would increase the infected cell's average cytosolic AEC from 0.70 to 0.77. If this prediction was correct, then the work of de Lima et al. (1994)
This study uses nonaqueous centrifugal density gradient fractionation of the central infected zone tissue of soybean nodules to permit reliable recovery of adenylate pools from subcellular compartments. The nonaqueous technique is particularly useful for legume nodules since their metabolism is highly compartmentalized, not only within cells, but also between cell and tissue types (Smith and Atkins, 2002
Ar:O2 Treatment and Nitrogenase Activities Out of three uniform populations of soybean plants, 18 were harvested after the N2:O2 (control) treatment, 18 after the 7-min Ar:O2 treatment, and 17 after the 60-min Ar:O2 treatment. Since no significant variations were observed in the measured apparent (ANA) and total (TNA) nitrogenase activities among plants destined for different treatments, the data were pooled for presentation in Figure 1A . The average ANA of nodules of plants selected for nonaqueous fractionation was 87 ± 9 µmol H2 g–1 DW(nod) h–1 (n = 18 + 18 + 17 = 53; Fig. 1A). After 7 min Ar:O2 exposure, H2 production increased to 240 ± 10 µmol H2 g–1 DW(nod) h–1 (TNA), resulting in an electron allocation coefficient (EAC = 1 – ANA/TNA) of 0.64 ± 0.03 (n = 18 + 17 = 35).
TNA declined sharply after 7 min in Ar:O2 and then recovered slightly and declined more gradually to 123 ± 8 µmol H2 g–1 DW(nod) h–1 (n = 17) after 60 min (Fig. 1A). This value was 51 ± 2% (n = 17) of the TNA value measured after 7 min Ar:O2 exposure.
To permit direct comparison of adenylate concentrations between different nodule tissues, all the measured values were expressed in µmol g–1 DW(nod) (Fig. 1, B–D). Consistent with an earlier study (Oresnik and Layzell, 1994 Ar:O2 exposure had no significant effect on total adenylate contents in either whole frozen nodules or dissected nodule CZ and cortex tissues (Fig. 1, B–D). The measured total adenylate pool in the frozen nodule averaged 1.8 ± 0.1 µmol adenylate g–1 DW(nod) (Fig. 1B). The AEC in frozen nodule tissues increased from 0.73 to 0.76 within 7 min Ar:O2 exposure but then declined to 0.72 after 60 min Ar:O2 exposure (Fig. 1B). The same trend was found for AEC of the CZ tissue under Ar:O2 treatment (Fig. 1C). In contrast, AEC in the cortex was 0.83 to 0.84 and was unchanged during Ar:O2 exposure (Fig. 1D). To assess adenylate losses following lyophilization and subsequent dissection, recovery was calculated as the sum of total adenylate in CZ and cortex [µmol g–1 DW(nod), Fig. 1, C–D], divided by the total adenylate in frozen whole nodules [µmol g–1 DW(nod), Fig. 1B]. Recovery of total adenylate was 85 ± 3%, 84 ± 2%, and 82 ± 2% (n = 3 in each case) for tissue samples from the N2:O2, 7-min Ar:O2, and 60-min Ar:O2 treatments, respectively. The protein content of frozen nodule tissues was 127 ± 6 mg g–1 DW(nod) (n = 9), whereas values for CZ and cortex tissues were 111 ± 5 and 11 ± 1 mg g–1 DW(nod) (n = 9), respectively. Therefore, recovery of protein following lyophilization and subsequent dissection was 96%.
There were no significant differences in the proportional distribution of protein or marker enzyme activities in the nonaqueous gradient fractions collected following centrifugation of sonicated tissues from nodules exposed to N2:O2, or after 7 or 60 min Ar:O2. Therefore, the data obtained from nine nonaqueous gradients (3 treatments x 3 replicates per treatment) were pooled to calculate the proportional distribution in each of the fractions (Fig. 2
). Hydroxybutyrate dehydrogenase (HBD), the marker enzyme for bacteroids, was mainly localized in the top five fractions of the density gradient, whereas glutamate dehydrogenase (GDH; mitochondrial marker) and phosphoenolpyruvate carboxylase (PEPC; plant cytosolic marker) were more widely distributed and showed additional peaks of activity in gradient fractions 7 to 9 (Fig. 2A). This pattern of fractionation was broadly similar to that in an earlier study (Kuzma et al., 1999
Adenylates in each of the 10 fractions were also measured, and the results for the three treatments are shown in Figure 3 . Only small differences in the relative ATP and ADP distributions were observed between the treatments, whereas the differences were more pronounced in the distribution of AMP across gradient fractions (Fig. 3).
To assess the effect of sonication on the recovery of soluble protein, total adenylate (ATP + ADP + AMP), and activities of marker enzymes, the sonicated homogenate before fractionation was assayed, and the measured values are expressed as a percentage of the amount in the lyophilized CZ tissue (Table I). Recovery of soluble protein, total adenylate, and activities of PEPC and GDH after sonication were close to 100%; however, HBD activity recovered after sonication was 149 ± 6% of that in the lyophilized CZ tissue (Table I), consistent with the observation of Kuzma et al. (1999) , who noted that sonication in nonaqueous solvents released more HBD activity than extraction in aqueous buffer. In addition, to assess the overall effect of sonication and the subsequent nonaqueous fractionation on the recovery of metabolites and activities of marker enzymes, the total of all values in the 10 fractions are expressed as a percentage of those in the lyophilized CZ tissue, and the results are presented in Table I.
Estimation of Subcellular Adenylate Pools under N2:O2 and Ar:O2 Treatments
Combining the results for adenylate assays (Fig. 3) with the marker enzyme distribution for each fraction (Fig. 2) and using the Bestfit program described by Riens et al. (1991)
In bacteroids, the ATP concentrations remained unchanged within 7 min Ar:O2 exposure, but declined to 78% of the initial level after 60 min in Ar:O2 (Fig. 4A). While the ADP levels in bacteroids were not significantly changed during Ar:O2 exposure, the AMP levels increased sharply by nearly 6-fold between 7 and 60 min Ar:O2 exposure (Fig. 4A). In plant cytosol, the ATP concentrations increased to 123% of the initial level following 7 min Ar:O2 exposure, and to 131% of the initial level after 60 min in Ar:O2, whereas the ADP and AMP levels declined gradually (Fig. 4B). In the mitochondrial compartment, there were no significant changes in the levels of ATP, ADP, or AMP after 7 and 60 min Ar:O2 exposure (Fig. 4C).
The total adenylate pools in bacteroids, plant cytosol, and mitochondria were 6.2 ± 0.4, 6.4 ± 0.4, and 1.4 ± 0.2 nmol mg–1 CZ protein, respectively. They accounted for 45 ± 2%, 45 ± 2%, and 10 ± 2%, respectively, of total adenylate in the infected cells. The summed percentage of adenylate pools in the plant compartment (plant cytosol + mitochondria) was 54 ± 2%, similar to that reported in an earlier study (Kuzma et al., 1999
The results of Figure 4 were used to determine the effect of Ar:O2 exposure on AEC and ATP-ADP ratio in the bacteroids, cytosol, and mitochondria of the CZ tissue (Fig. 5 ). Within 7 min Ar:O2 exposure, cytosolic AEC increased significantly from 0.63 ± 0.02 in N2:O2 to 0.73 ± 0.02 (0.05 > P > 0.02), and increased further to 0.80 ± 0.01 after 60 min Ar:O2 exposure (0.002 > P > 0.001; Fig. 5A). In contrast, AEC in the bacteroid compartment was unchanged (0.73 ± 0.02 in N2:O2) after 7 min Ar:O2 exposure, but then declined to 0.57 ± 0.03 after 60 min (0.01 > P > 0.002; Fig. 5A). In mitochondria, AEC was high under N2:O2 treatment (0.80 ± 0.02) and remained high following exposure to 7 and 60 min Ar:O2 (0.81 ± 0.02, 0.81 ± 0.02; Fig. 5A).
The AEC of the whole CZ tissue calculated as the sum of the AEC values for all three compartments was 0.70 ± 0.02, 0.74 ± 0.02, and 0.70 ± 0.02 for the N2:O2, 7-min, and 60-min Ar:O2 treatments, respectively (Fig. 5A). These values were similar to those measured in the lyophilized CZ tissue before fractionation (Fig. 1C). The calculated subcellular ATP-ADP ratios for the three compartments showed similar trends to those observed for AEC (Fig. 5B).
This study is, to our knowledge, only the second report (after Kuzma et al., 1999 ) that describes the use of a nonaqueous fractionation technique to study changes in metabolite pools in subcellular compartments of legume nodules.
Unlike the previous report with soybean nodules (Kuzma et al., 1999
The detection limit for nonaqueous fractionation of a metabolite in a particular compartment has been estimated at about 5% of the total amount in intact cells (Farre et al., 2001
Separation of subcellular compartments using this method was unable to resolve plastids, which would undoubtedly have contained some adenylates. The Bestfit program that was used (Riens et al., 1991
A mathematical model developed previously (Wei et al., 2004
Furthermore, the significant increase in the AEC of plant cytosol observed following Ar:O2 exposure supports the hypothesis being tested in this study and is consistent with a reduced demand for ATP in the cytosol where, in Ar:O2, NH4+ assimilation through GS activity would be negligible. Since nodule respiration (and presumably ATP synthesis) after 7 min Ar:O2 exposure is similar to that in air (King and Layzell, 1991
Within the first 7 min of Ar:O2 exposure, the bacteroid AEC was unchanged from the value in air (0.73 ± 0.02), but declined sharply (to 0.57 ± 0.03) after 60 min Ar:O2 exposure (Fig. 5A). Previous studies have shown that long-term exposure of legume nodules to an Ar:O2 atmosphere causes a major reduction in the permeability of the nodule to O2 diffusion, thereby reducing the infected cell O2 concentration (King and Layzell, 1991
Kuzma et al. (1999)
The average mitochondrial AEC in the CZ of soybean nodules was high before and after Ar:O2 treatment. This result was consistent with the bacteroid, rather than the plant compartment, being the site of O2 limitation in legume nodules (Kuzma et al., 1999
On a whole-nodule basis, there was little effect of extended Ar:O2 treatment on the summed or measured AEC, despite the fact that other treatments known to induce an O2 limitation of nodule metabolism (low O2, nitrate fertilization, stem girdling) have been shown to result in a significant decrease in the nodule AEC (de Lima et al., 1994
Curioni et al. (1999)
Previous studies (Larsson et al., 2000
The simulation model (Figure 5A in Wei et al., 2004
While there is abundant physiological evidence that a reversible mechanism controlling the permeability of nodules to gaseous diffusion has a central regulating role in determining rates of nitrogenase activity, the nature of the signals involved and the transduction pathway linking this mechanism to metabolic events in the nodule remains obscure (for review, see Minchin, 1997
The rapid and significant adjustment in plant cytosolic AEC and ATP-ADP ratio that accompanies the reduced flux of fixed N to the plant cell cytosol on exposure of nodules to 7 min Ar:O2 (80:20; v/v) raises the possibility that adenylates may, in fact, be signal molecules for the control of nodule O2 permeability. This might be mediated through activation of ion pumps (i.e. ATP-dependent K+- and/or Ca2+-pumps) or ATP-gated ion channels on the plasma membrane that results in a net efflux of K+ and Ca2+ from the cytoplasm of the infected cells to the intercellular spaces. The K+, Ca2+, and water in the intercellular spaces of the CZ region would be likely to move toward the inner cortex region because the intercellular spaces in the inner cortex are smaller and therefore have stronger hydrogen bonding between water molecules and the cell walls (i.e. capillary effect). Such a relocation of ions and water, from the intercellular space in CZ to that in the inner cortex, would decrease nodule permeability to O2 and reduce O2 availability to infected cells. That could account for the greater O2 limitation on nitrogenase activity and bacteroid metabolism that is known to be associated with Ar:O2 exposure (King and Layzell, 1991
Minchin et al. (1995)
A recent report has speculated that ATP might have a role in plants as an extracellular signal molecule, functioning in a manner similar to its role as a neurotransmitter in animal systems (Demidchik et al., 2003
Plant Culture
Seeds of soybean (Glycine max L. Merr. cv Maple Arrow) were inoculated at the time of planting with Bradyrhizobium japonicum USDA 16 at about 0.5 x 109 viable cells per plant. The seeds were planted 3 cm deep under the surface of silica sand (grade 16) in a gas-exchange pot and grown in a growth chamber (Kuzma and Layzell, 1994
Three uniform populations of 18 soybean plants were used. Within a population, six plants were selected for measurement of ANA (Hunt et al., 1989 The nodules were harvested by quickly uprooting the soybean plant and immediately immersing the roots with rooting medium attached in liquid N2 (–196°C). The nodules were picked from the frozen roots and solid matrix while being kept frozen by occasional treatment with liquid N2, weighed, and stored in liquid N2. Specific ANA and/or TNA values for each plant were calculated, taking care to include the weight of nodule tissue that was missed in the initial harvest (typically 10%–20% of total). The ANA values of the nodules from all plants except one for the 60-min Ar:O2 treatment were in the range of 70 to 100 µmol H2 g–1 DW(nod) h–1 (assuming a ratio of 4.8 for fresh to dry weight); therefore, one plant for the 60-min Ar:O2 treatment was discarded.
The soybean nodules from the same treatment within the same population were mixed. Therefore, the three plant populations now became nine samples of mixed nodules (i.e. 3 treatments x 3 replicates per treatment). To facilitate lyophilization, frozen large nodules (>2 mm diameter) were cut in half while immersed in liquid N2 using a small guillotine fabricated from razor blades. For each of the nine samples, two 100-mg aliquots of nodule halves were set aside for metabolite and marker enzyme assay. The remaining nodule material was lyophilized in Fast-Freeze Flasks (Labconco, Kansas City, MO) while being maintained at –15°C in a modified chest freezer for the first 2 d, and at room temperature for another 2 d. The cold trap temperature was set at –50°C and the vacuum at 6 to 8 microns (18 L Freeze Dry System, Labconco).
To provide homogenous CZ tissue for nonaqueous fractionation, lyophilized nodule halves were dissected into cortex and CZ tissues using a dissecting needle and microforceps. This was done in a partially closed chamber under a dissection microscope while the chamber was flushed continuously with a stream of compressed air to maintain a dry environment. The cortex and CZ tissues have different textures and water contents (Oresnik and Layzell, 1994
The dried CZ tissue was ground in a mortar and pestle in a gas bag filled with dry N2 gas and nonaqueous fractionation carried out using a modified method of Kuzma et al. (1999)
Three gradients were prepared simultaneously, one for each of the treatment groups from a plant population. The gradient was centrifuged at 25,000g for 2.5h (rotor SW28, Beckman l8-55M, Beckman Instruments, Fullerton, CA) and 10 fractions collected from each as described previously (Kuzma et al., 1999
Adenylates and marker enzymes were assayed in extracts of frozen nodules, dry cortex and CZ tissues, and in dry subfractions as described previously (Kuzma et al., 1999 To assess the stability of adenylates during extraction in 10% perchloric acid, a known amount of ATP, ADP, and AMP (10 or 20 nmol) was added to an aliquot of the fresh nodule tissues and also to the dried cortex and CZ tissue samples. After correction for endogenous adenylate content, recovery of ATP, ADP, and AMP was 91 ± 3%, 92 ± 5%, and 95 ± 6% (n = 9), respectively, and no differences were found between the three nodule tissues. Given the consistently high recovery rate, no adjustments were made to measured adenylate levels in subfractions. Recovery of adenylate pools, protein, and marker enzyme activities were also calculated by comparing the measured levels in dried CZ with those from all subfractions.
The proportion of each of the adenylates, soluble protein content, and marker enzyme levels in each gradient were entered into a computer program (Bestfit) developed by Riens et al. (1991) Total soluble protein was treated as a metabolite. Thus, the distribution of protein among the three compartments could also be calculated using the Bestfit program to analyze the measured contents of protein and the activities of marker enzymes in each fraction of the gradient.
We are grateful to Dr. Monika Kuzma for help with the nonaqueous fractionation technique and to Sara Porter and Li Sun for technical assistance. Received January 2, 2004; returned for revision February 3, 2004; accepted February 3, 2004.
1 This work was supported by the National Science and Engineering Research Council of Canada (grants to D.B.L.).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.038547. * Corresponding author; e-mail layzelld{at}biology.queensu.ca; fax 613–533–6617.
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ABSTRACT
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), in most, but not all treatments known to induce severe O2 limitation of nodule metabolism (de Lima et al., 1994
), PEPC (
), GDH (
), and protein (
).
) Ar:O2.
), ADP (
) in the bacteroid (A), plant cytosol (B), and mitochondria (C) compartments in CZ tissue from nodules of 4- to 5-week-old soybean plants. Values are presented as the mean (±SE; n = 3) for each of the three treatments (N2:O2, 7-min, and 60-min Ar:O2 exposure).
), plant cytosol (
