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Adenylate-Coupled Ion Movement. A Mechanism for the Control of Nodule Permeability to O₂ Diffusion^1,[OA]

Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
In response to changes in phloem supply, adenylate demand, and oxygen status, legume nodules are known to exercise rapid (seconds to hours) physiological control over their permeability to oxygen diffusion. Diffusion models have attributed this permeability control to the reversible flow of water into or out of intercellular spaces. To test hypotheses on the mechanism of diffusion barrier control, nodulated soybean (Glycine max L. Merr.) plants were exposed to a range of treatments known to alter nodule O₂ permeability (i.e. 10% O₂, 30% O₂, Ar:O₂ exposure, and stem girdling) before the nodules were rapidly frozen, freeze dried, and dissected into cortex and central zone (CZ) fractions that were assayed for K, Mg, and Ca ion concentrations. Treatments known to decrease nodule permeability (30% O₂, Ar:O₂ exposure, and stem girdling) were consistently associated with an increase in the ratio of [K⁺] in cortex to [K⁺] in the CZ tissue, whereas the 10% O₂ treatment, known to increase nodule permeability, was associated with a decrease in the [K⁺]_cortex:[K⁺]_CZ. When these findings were considered in the light of previous results, a proposed mechanism was developed for the adenylate-coupled movement of ions and water into and out of infected cells as a possible mechanism for diffusion barrier control in legume nodules.

These treatments can be classified into three groups according to how they affect both the nodule's permeability to O₂ diffusion and the O₂-sufficient metabolic capacity of the nodules (Layzell, 1998). The first group includes the treatments that directly affect the O₂ status of the infected cells by either altering the supply or demand for O₂. For example, increases or decreases in the rhizosphere [O₂] (King et al., 1988), changes in nodule temperature (Kuzma and Layzell, 1994), or drought stress (Diaz del Castillo et al., 1994) result in alterations in nodule permeability, presumably to maintain control over the infected cell [O₂]. Typically, the nodule adjustment of diffusion barrier permeability compensates for the treatment to return the nodule to a similar state of O₂ limitation as that found in the nodules under the initial, control condition. The O₂ limitation coefficient is measured as the ratio of the total electron flow through nitrogenase (total nitrogenase activity [TNA]) in air divided by the peak electron flow through nitrogenase when the pO₂ is gradually increased (potential nitrogenase activity [PNA]).

A second group includes treatments that cause a large reduction in nodule O₂ permeability, infected cell [O₂], and TNA. In this case, the O₂ limitation coefficient also declines because there is little or no change in PNA. Example treatments include nodule exposure to Ar:O₂ (80:20) or 10% C₂H₂, both of which stop N₂ fixation, NH₄⁺ assimilation, and their associated ATP demand, and cause the diversion of electron flow through nitrogenase to the reduction of protons to H₂ (Ar:O₂ treatment) or C₂H₂ to C₂H₄ (C₂H₂ treatment). However, after a short period (5–10 min) of treatment, nodule O₂ permeability decreases, lowering the infected cell [O₂] (Kuzma et al., 1993) and inducing a decline in TNA that can be largely recovered, at least in the short term, by increasing external pO₂ (King and Layzell, 1991; Kuzma et al., 1993).

A third group includes treatments that inhibit nitrogenase activity by reducing both the O₂ permeability and the respiratory capacity (i.e. PNA) of nodules. For example, decreasing the carbohydrate supply to the nodules as in stem girdling (Vessey et al., 1988), defoliation (Hartwig et al., 1987), detopping (Denison et al., 1992), or application of fertilizer (Vessey et al., 1988), have all been shown to decrease nodule O₂ permeability, infected cell [O₂], and TNA, but increases in external pO₂ are not able to recover all of the initial activity, showing that the PNA is also reduced.

Various hypotheses have been proposed to explain how legume nodules adjust their permeability to O₂ diffusion (James et al., 1991; Streeter, 1992; Thomas and Minchin, 1992; Hunt and Layzell, 1993; Minchin et al., 1994; Minchin, 1997), most involving changes in the proportion of gas (high permeability) and water (low permeability) in the inner cortex of the nodule, a key region in the pathway of O₂ diffusion from the soil atmosphere to the central, infected zone of the nodule. In biological systems, movement of water is often linked with the movement of ions such as K⁺ or Mg²⁺ (Rao et al., 1987; Becker et al., 2003; Hosy et al., 2003). Therefore, ions may play a key role in the mechanism of nodule O₂ permeability control, potentially as a transducer of information on the status of the infected cells to the inner cortex where it is associated with the movement of water and the change in O₂ permeability.

This study tests this hypothesis by measuring changes in K⁺, Mg²⁺, and Ca²⁺ distribution between the central zone (CZ) and cortex of soybean nodules exposed to various treatments known to alter the O₂ diffusion barrier. The results are then used to propose a mechanistic model for the regulation of O₂ permeability in legume nodules.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Nodule Dissection and Ion Distribution

Lyophilized nodules, having a dry weight of 6 to 8 mg per nodule were selected for dissection and subsequent ion measurements. Preliminary experiments showed that tissue losses during the dissection of lyophilized nodules were less than 5% (data not shown). The CZ tissue accounted for 65% to 68% of the dry weight of the whole nodule.

The total K⁺ pool in whole nodules from the control treatment was 365 ± 15 µmol g^–1 DW(nod), with about 52% in the cortex and the balance in the CZ tissue. The total Ca²⁺ and Mg²⁺ pool in the whole nodules were 62 ± 3 and 120 ± 8 µmol g^–1 DW(nod), respectively, with about 44% and 16%, respectively, in the cortex and the balance in the CZ. The predominance of Mg²⁺ in the CZ region is consistent with the high metabolic activity and ATP concentration in this region (Gordon, 1991; Oresnik and Layzell, 1994).

Note that the K⁺ content in nodules was 6 to 9 times higher than Ca²⁺ and Mg²⁺, a difference consistent with the ion concentrations measured in nodule phloem and xylem saps (Jeschke et al., 1985).

Apparent Nitrogenase Activity in Plants Used in Various Treatments

Two complete sets of experiments were carried out, each with six replicate plants per treatment. The results presented here are for one of those experiments, although very similar results were obtained in the second experiment (data not shown). In each experimental treatment, the initial apparent nitrogenase activity (ANA) was measured before the plants were subjected to the treatment; no significant differences were observed in the initial ANA [92 ± 11 µmol H₂ g^–1 DW(nod) h^–1] among nodules for control plants (maintained in air, 80% N₂:20% O₂) and for those plants that were subsequently stem girdled or treated with 10% O₂, 30% O₂, or Ar:O₂, confirming that they were at a similar physiological state before being subjected to various treatments.

The 10% O₂ Treatment

The 10% O₂ treatment resulted in a rapid inhibition of nitrogenase activity, as measured by H₂ production, followed by a gradual recovery (Fig. 1A ) such that after 1 h, the ANA was approximately 75% of initial. After 1 h of treatment, no significant changes were observed in the concentration of K⁺ in either the nodule cortex () or CZ tissues (Fig. 1B). However, the ratio of declined by 14% (P < 0.05) from 2.0 in the control nodules to 1.70 after 1 h of the 10% O₂ treatment (Fig. 1B).

View larger version (39K): [in this window] [in a new window]   Figure 1. The effect of treatments of 10% O2 (A–D), 30% O2 (E–H), Ar:O2 (I–L), and stem girdling (M–P) on the rate of H2 production (A, E, I, K) and the concentration of K+ (B, F, J, N), Ca2+ (C, G, K, O), and Mg2+ (D, H, L, P) in N2-fixing nodules of soybean. Ion concentrations were measured in cortex (light shading) and CZ (dark shading) tissues dissected from lyophilized nodules. The ratios of ion concentration in the cortex to that in the CZ are provided with a scale on the right side. All values are presented as mean ± SE (n = 6). *, Significant difference at p 0.05; **, significant difference at P 0.01.

The 30% O₂ Treatment

The 30% O₂ treatment also resulted in a rapid inhibition of nitrogenase activity, as measured by H₂ production, followed by a gradual recovery (Fig. 1E ) such that after 1 h, the ANA was approximately 95% of initial.

View larger version (31K): [in this window] [in a new window]   Figure 2. A schematic describing the possible mechanism by which stem girdling, Ar:O2 treatment, or exposure to 10% or 30% O2 alters the nodule permeability to O2 diffusion; see text for details. CH2O, Carbohydrate; ETC, electron transport chain; e–, electrons/reducing power; G.S., Gln synthetase; Lb, leghemoglobin; N2ase, nitrogenase; T, transporter.

In contrast, no significant differences were observed in the cortex or CZ concentrations of Ca²⁺ or Mg²⁺ in response to 30% O₂. Neither was there an effect of 30% O₂ on the cortex to CZ ratio of ion concentrations (i.e. or ; Fig. 1, G and H).

The Ar:O₂ Treatment

The Ar:O₂ treatment caused an initial increase in nodule H₂ production of about 2.7-fold as the electron flow was diverted from N₂ reduction to H⁺ reduction. The peak rate of H₂ evolution in Ar:O₂ was taken as a measure of the TNA, resulting in an electron allocation coefficient of 0.63, a value typical in soybean nodules (Moloney et al., 1994). However, after a few minutes, the total electron flow through nitrogenase declined so that after 1 h, the rate of H₂ production in Ar:O₂ was only about 150% of the initial H₂ production in N₂:O₂ (Fig. 1I ).

View larger version (29K): [in this window] [in a new window]   Figure 3. Method to separate cortex and CZ fractions of soybean nodules. Rapidly frozen nodules at a known physiological state were lyophilized and then dissected into CZ and cortex tissues. These tissues were ground to a fine powder before being digested in nitric acid and the extracts assayed for K+, Ca2+, and Mg2+ concentration in the CZ and cortex fractions.

In contrast, no significant differences were observed in the cortex or CZ concentrations of Ca²⁺ or Mg²⁺ in response to Ar:O₂ treatment. Neither was there an effect of Ar:O₂ on the cortex to CZ ratio of ion concentrations (i.e. or ; Fig. 1, K and L).

The Stem-Girdling Treatment

Stem girdling the plants had no effect on the ANA for the first 20 to 30 min, but then caused a decline in H₂ production over the next 150 to 160 min such that after 3 h, the nitrogenase activity was only 60% ± 12% of the initial value (Fig. 1M).

Three hours after the start of the stem-girdling treatment, the cortex K⁺ concentration was decreased slightly by 6% (not statistically significant) compared to the control plants, whereas the CZ K⁺ concentration had decreased by 18% (P < 0.01) compared to control. Therefore, a significant 17% increase (P < 0.01) was observed in the ratio from 2.0 in the control nodules to 2.3 after 3 h of stem-girdling treatment (Fig. 1N).

In contrast, no significant differences were observed in the cortex or CZ concentrations of Ca²⁺ or Mg²⁺ in response to stem girdling. Neither was there an effect of stem girdling on the cortex to CZ ratio of ion concentrations (i.e. or ; Fig. 1, O and P).

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Intra-Nodule Movement of K⁺ Ion Correlated with Changes in Nodule Permeability

A number of environmental and physiological treatments are known to induce changes in nodule permeability to O₂ diffusion. This study clearly shows that these same treatments alter the distribution of K⁺ ion between the CZ and the nodule cortex. It is interesting to note that similar changes were not seen in the movement of Mg²⁺ or Ca²⁺ ions.

Three of the treatments employed here (30% O₂, Ar:O₂, and stem girdling; Fig. 1, E–P) were associated with an increase in the ratio, a change consistent with the treatment-induced movement of K⁺ from the infected cells in the CZ to the cortex, the region of the nodule implicated in regulating nodule permeability. Previous studies (Vessey et al., 1988; King and Layzell, 1991; de Lima et al., 1994) have shown that these three treatments are associated with a decrease in nodule permeability to O₂ diffusion.

In contrast, the 10% O₂ treatment showed a decrease in the ratio and this treatment is known (de Lima et al., 1994; Kuzma et al., 1999) to increase the permeability to O₂ diffusion. Consequently, there is an inverse correlation between the direction of the change in and the direction of the O₂ permeability change.

These results support the hypothesis that K⁺ movement in nodules plays a central role as a transducer in the regulation of nodule O₂ permeability in response to sensors of environmental or physiological change that threatens homeostasis within the nodule.

Treatment Effects on Total Nodule Ion Concentration

Over the time course of the treatments, the K⁺, Mg²⁺, and Ca²⁺ levels in whole nodules were not significantly different between the start and end of any treatment except the stem-girdling treatment. Over the 3 h stem-girdling treatment, there was no significant change in the Mg²⁺ or Ca²⁺ content, however the K⁺ content declined by 13% (P < 0.05). This was attributed to the fact that K⁺ supply to the nodules in the phloem would have been terminated by the stem-girdling treatment, whereas xylem export from the nodules would have not been affected, at least in the short term (Walsh, 1995). This conclusion is supported by the observation that although the stem-girdling treatment reduced K⁺ concentration in both the cortex and CZ, the declined much more than the .

Consequently, in all treatments, the observed changes in the ratio of are most readily explained as a redistribution of K⁺ within these nodules: a shift of K⁺ ions between cortex and CZ.

The results of this study support the suggestion that K⁺ movement between the CZ and cortex plays a key role in the regulation of nodule permeability to O₂ diffusion. However, a full explanation of the mechanism for controlling nodule permeability requires an account for (1) how the movement of K⁺ from the infected cells to the nodule cortex is coupled to a decrease in nodule permeability to O₂ diffusion, and (2) how the environmental and physiological treatments employed in this study induce the observed movement of K⁺.

Linking K⁺ Movement to Changes in Nodule Permeability to O₂ Diffusion

Previous studies have linked K⁺ to the regulation of nodule O₂ permeability. Purcell and Sinclair (1994) observed a decline and recovery of nodule O₂ permeability in nodules infiltrated with KCl. Minchin and coworkers found a loss of vacuolar K⁺ in the cells of the inner cortex within 15 min of nodule disturbance by root shaking (Minchin et al., 1995), a treatment known to induce a decrease on nodule permeability. A change in inorganic osmoticants like K⁺ has also been proposed to accompany by membrane depolarization in legume nodule (Denison and Kinraide, 1995).

Changes in the permeability of the nodule diffusion barrier have been compared (Gálvez et al., 2000) to stomatal opening and closing or to leaf movements controlled by a pulvinus. Stomatal closure and leaf movements are both associated with the movement of K⁺ out of the cells into the apoplast, a phenomenon that draws water from the cell, causing a decrease in turgidity. In these systems, as with the control of nodule diffusion permeability, the changes that occur in one direction (leaf folding, stomatal closure, and permeability decrease) can occur very quickly (seconds), whereas changes in the opposite direction (leaf opening, stomatal reopening, and permeability increase) are often much slower, generally taking many minutes.

It is possible that the nodule treatments involving 30% O₂, Ar:O₂, or stem girdling induce the movement of K⁺ out of the bacteria-infected cells, drawing water with the K⁺, effectively flooding the intercellular spaces with water. Since the spaces between the cells of the inner cortex are smaller than the spaces in the central, infected zone of the nodules (Parsons and Day, 1990; Jacobsen et al., 1998), capillary action would draw the water (and the K⁺ it contains) into these spaces. As the water replaces the gas within these spaces, the permeability of the nodule to O₂ diffusion would decline sharply since diffusivity of O₂ in water is about 350,000 times lower than that in air (Thumfort et al., 2000).

On the other hand, a treatment such as nodule exposure to 10% O₂ may induce the bacteria-infected cells to take up K⁺ from the apoplast, thereby drawing water into the cells. This would tend to replace gas with water in the intercellular spaces, thereby increasing the nodule's permeability.

An alternative mechanistic explanation is consistent with earlier suggestions that an intercellular glycoprotein may be associated with diffusion barrier control in nodules (James et al., 1991; Iannetta et al., 1993; James et al., 2000). K⁺ movement into the spaces may increase the hydrophilic nature of the protein, thereby displacing gas spaces with water and reducing nodule permeability to O₂ diffusion.

Linking the Treatment Effects to K⁺ Movement

Understanding how the environmental and physiological treatments employed in this study induce the observed movement of K⁺ is more complex, but may be explained by incorporating insights from a variety of recent studies, including studies that have reported on how these treatments affect adenylate (ATP, ADP, and AMP) pools, the infected cell oxygen concentration, electron allocation and total electron flow through nitrogenase, and the O₂ limitation status of the nodules.

The following paragraphs will draw on Figure 2 to propose a possible mechanism linking each of the four treatments to K⁺ movement that ultimately alters nodule permeability to O₂ diffusion.

The 30% O₂ Treatment
When the nodulated roots of legumes are transferred from air to 30% O₂, a rapid rise in the infected cell O₂ concentration (measured as the fractional oxygenation of leghemoglobin) coincides with a drop in nitrogenase activity (King et al., 1988). Within minutes, the infected cell O₂ concentration recovers but nodule activity recovers more slowly (King et al., 1988). Within minutes of the 30% O₂ treatment, the whole nodule ATP/ADP ratio increases sharply, but declines again over the recovery period (de Lima et al., 1994). Since infected cell mitochondria are clustered around the intercellular spaces (Davidson and Newcomb, 2001), whereas the bacteroids are distributed in the center of the cell, the mitochondria are likely to have priority access to the O₂ that diffuses into the cells, and a significant part of the change in ATP/ADP ratio is probably associated with the plant fraction (Fig. 2). Models predicting steep gradients in O₂ concentration across the radius of the infected cell (Thumfort et al., 2000) support this suggestion. If the resultant rise in cytosolic ATP/ADP ratio increases the activity of a K⁺ pump (i.e. a K⁺-ATPase) or closes down an adenylate-gated K⁺ channel in the plasma membrane, there would be increased K⁺ efflux into the apoplast that could reduce nodule permeability to O₂ diffusion through the mechanism described above. Elevated O₂ is known to reduce permeability and serves to return the infected cell O₂ concentration to its initial value, while stabilizing the intercellular ATP/ADP ratio (de Lima et al., 1994).

Ar:O₂ Treatment
Changing the atmosphere around a nodulated root from N₂:O₂ to Ar:O₂ prevents N₂ fixation, and the initial response of the nitrogenase enzyme is to maintain its activity, but reduce protons to H₂ gas (King and Layzell, 1991). Within 5 to 10 min of Ar:O₂ exposure, nitrogenase declines as a result of an O₂ 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, 2004b) linking the reduction in NH₃ production to reduced cytosolic ATP demand in support of Gln synthetase activity (Fig. 2), resulting in an increase in the cytosolic ATP/ADP ratio. If the elevated cytosolic ATP/ADP ratio results in the observed K⁺ efflux from the cell (Fig. 1J), the change in nodule permeability would be similar to that proposed for the 30% O₂ treatment as described above.

This proposed mechanism is consistent with the studies of Brown et al. (1997), who used electron spin resonance to observe the ¹H-NMR image of soybean nodules. Their results showed that 1 h of Ar:O₂ exposure treatment caused a dramatic decrease in the proton intensity (i.e. loss of water mobility) in the region of the inner cortex and the outer part of the CZ.

Stem-Girdling Treatment
The stem-girdling treatment is not as clean a treatment as 30% O₂ or Ar:O₂, since it disrupts the supply of all phloem constituents to the nodule, including water, carbohydrates, and ions, such as K⁺. However, stem girdling has no direct impact on the export of xylem that carries water, fixed N, and ions back to the plant. Previous studies have shown that 2 to 3 h of stem girdling reduces nodule metabolism and nitrogenase activity (Walsh et al., 1987), decreases nodule permeability and infected cell O₂ concentration, and causes a sharp decline in the whole nodule ATP/ADP ratio (de Lima et al., 1994). The decline in ATP/ADP ratio coupled with the movement of K⁺ out of the CZ (Fig. 1N), positions the stem-girdling treatment as mechanistically different from either the 30% O₂ or the Ar:O₂ treatments. The simplest explanation may be that stem girdling first impacts carbohydrate and K⁺ pools in the cortical tissues, resulting in a loss of cell turgidity and water movement into the apoplast, thereby reducing nodule permeability to O₂ diffusion (Fig. 2). A secondary effect of carbohydrate deprivation in the infected cells seems to be overridden by a severe O₂ limitation in the infected cells, as evidenced by low fractional oxygenation of leghemoglobin (D.B. Layzell, unpublished data) and the low ATP/ADP ratio (de Lima et al., 1994). What is unclear is why the CZ lost 18% of its K⁺ pool when the whole nodule ATP/ADP ratio was so low. It is possible that stem girdling may result in large differences in the ATP/ADP ratio between the bacteroid and cytosolic pools. A severe O₂ and carbohydrate limitation in the bacteroid fraction could account for the low whole nodule ATP/ADP ratio while the cytosolic ATP/ADP ratio may be much higher. A nonaqueous fractionation study of adenylate pool changes following stem girdling needs to be carried out to test this hypothesis.

The 10% O₂ Treatment
When nodulated roots of a legume are transferred from air to 10% O₂, there is a sharp drop in the infected cell O₂ concentration that inhibits nodule metabolism and N₂ fixation and reduces whole nodule ATP/ADP ratios. However, over the next hour, a gradual recovery in nodule activity and ATP/ADP ratio has been reported (de Lima et al., 1994) as the nodule's O₂ permeability increases in response to the 10% O₂ treatment. A nonaqueous fractionation study of the initial response to a 10% O₂ treatment (Kuzma et al., 1999) has shown that the bacteroid adenylate pools are most severely impacted, although there is evidence for a reduction in the cytosolic ATP/ADP ratio. If an adenylate-regulated K⁺ pump or channel is located on the plasma membrane, the change in cytosolic ATP/ADP ratio could explain the observed accumulation of K⁺ in the infected cells (Fig. 1B). This may result in water flowing from the apoplast into the infected cells, thereby opening up gas-filled spaces in the apoplast and increasing the permeability of the nodule to O₂ diffusion. With a higher permeability to O₂ diffusion, the infected cell O₂ concentration and whole nodule ATP/ADP would partially recover until a new homeostasis occurs.

The mechanisms proposed here to explain the effects of the four treatments all involved linking observed changes in the cytosolic ATP/ADP ratio, to the efflux of K⁺ across the plasma membrane of the infected cells, and the subsequent apoplastic movement between the CZ and cortex tissues. Similar mechanisms of cytosolic ATP in control of K⁺ efflux have been reported in other plant cells, in which the outward-rectified K⁺ channels were activated or completely abolished within 15 min in the presence or absence of cytosolic ATP (Spalding and Goldsmith, 1993; Briskin and Gawienowski, 1996; Goh et al., 2004). This response period is similar to that observed for changes in nodule permeability to oxygen diffusion, providing further support for the suggestion that cytosolic ATP-coupled K⁺ movement is involved in the control of oxygen diffusion in legume nodules.

	CONCLUSION

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
The results from this study show that perturbations known to alter the permeability of the nodule to O₂ diffusion change the distribution of K⁺ between the CZ and cortex tissues. Treatments (30% O₂, Ar:O₂, and stem girdling) that result in a the reduction of nodule permeability are associated with an increase in the ratio of cortical K⁺ to CZ K⁺, whereas the one treatment employed here (10% O₂) that caused an increase in nodule permeability was associated with a decrease in the ratio of cortical K⁺ to CZ K⁺.

The fact that so many diverse treatments all affect K⁺ movement in a predictable fashion supports the suggestion that K⁺ movement between tissues is a critical and fundamental component in the regulation of nodule permeability to O₂ diffusion. Based on current understanding of the role of K⁺ transport in the regulation of stomata and leaf movements, we propose that the decrease in nodule permeability is associated with K⁺ movement from the infected cell cytoplasm to the apoplast, thereby drawing water out of the cells. We propose that this water will carry the K⁺ to the narrower network of intercellular spaces in the inner cortex of the nodules where it creates a barrier to the diffusion of O₂.

This study also proposed a series of mechanisms to explain the linkage between nodule perturbations and K⁺ transport. It should be possible to test these hypotheses through a variety of strategies, including the use of infected cell protoplasts. A recent report used the patch clamp technique with rice (Oryza sativa) mesophyll cell protoplasts to investigate the role of cytosolic ATP in regulating outward-rectifying K⁺ channels (Goh et al., 2004). Their results demonstrated that cytosolic ATP, derived from mitochondrial respiration, activates the plasma membrane outward-rectifying K⁺ channels. A similar approach could be used to explore the role of cytosolic ATP in the control of K⁺ efflux in the infected cells of legume nodules.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Culture and Treatments

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:O₂ (80:20), 30% O₂ (balance N₂), or 10% O₂ (balance N₂) to the nodulated roots for 1 h.

All experiments were repeated twice, and there were at least six replicate plants per treatment. To avoid or minimize diurnal fluctuations, the plants were subjected to treatments between 10 AM and 3 PM local time.

Nitrogenase Activity Assay

Before commencing any of the treatments, the intact, nodulated roots of all plants were first flushed with N₂:O₂ (80:20) for 60 min and assayed for ANA (H₂ evolution in air) as described previously (Hunt et al., 1989), to ensure that they were at a similar physiological state before entering their respective treatments. The plants designated for the control treatment were immediately harvested, while the other treatments were imposed.

Nodule Harvest and Dissection

After each treatment, the nodulated roots were rapidly (<2 s) removed from the pots and plunged into liquid N₂ (–196°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 N₂. They were then weighed and stored in liquid N₂ 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. 3) before each fraction was weighted with a Cahn C-31 microbalance to 0.1 mg precision, before being stored separately in an Eppendorf tube at –20°C.

Extraction and Analysis of Ca²⁺, K⁺, and Mg²⁺ in the Cortex and CZ Tissues

Concentrations of Ca²⁺, K⁺, and Mg²⁺ were measured in single, hydrated nodules (approximately 30–40 mg fresh weight), lyophilized cortex tissues (approximately 2–3 mg dry weight), and lyophilized CZ (4–6 mg dry weight) tissue from nodules exposed to each of the experimental treatments.

Each sample was ground and digested in 0.4 mL 25% (v/v) nitric acid (trace metal grade, Fisher Scientific; with Ca²⁺, K⁺, and Mg²⁺ 0.5 nL L^–1) at 70°C, overnight. After digestion, the extract was diluted to 5 mL with fresh Millipore (Synergy water purification system) deionized water (the final nitric acid concentration was 2%), and analyzed for the concentration of Ca²⁺, K⁺, and Mg²⁺ using inductively coupled plasma-optical emission spectroscopy in the Analytical Service Unit at Queen's University.

Trial experiments indicated that the recovery rates for the ions during the trial nitric acid extractions were between 97% to 102%. Since all of the dissected samples were extracted and analyzed in one trial run with inductively coupled plasma-optical emission spectroscopy, the original data have been presented with no adjustments.

	ACKNOWLEDGMENTS

 
We are grateful to Dr. Allison Rutter and Ms. Mary Andrews for assistance with the ion measurements, to Ms. Sara Porter and Li Sun for help in nodule dissection, and to Dr. Stephen Hunt for stimulating discussions.

Received January 22, 2006; returned for revision January 22, 2006; accepted February 20, 2006.

	FOOTNOTES

 
¹ This work was supported by grants to D.B.L. from the National Science and Engineering Research Council of Canada.

² Present address: Section on Developmental Genetics, Heritable Disorders Branch, NICHD, National Institutes of Health, Bethesda, MA 20892–1830.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: David B. Layzell (layzelld{at}biology.queensu.ca).

^[OA] Open Access articles can be viewed online without a subscription.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.077552.

^* Corresponding author; e-mail layzelld{at}biology.queensu.ca; fax 613–542–0045.

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TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
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