Skip to main content
PMC home page
Plant Physiology logoLink to Plant Physiology
. 2006 May;141(1):280–287. doi: 10.1104/pp.106.077552

Adenylate-Coupled Ion Movement. A Mechanism for the Control of Nodule Permeability to O2 Diffusion1,[OA]

Hui Wei 1,2, David B Layzell 1,*
PMCID: PMC1459327  PMID: 16531483

Abstract

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.

To control the O2 supply and O2 concentration to and within the infected cells, the nodule permeability to O2 diffusion varies in response to a variety of treatments. This study is focused on improving our understanding of the mechanism by which various treatments alter nodule permeability, including the nature of the sensor and transducer that link treatments to the permeability change.

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

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 (Inline graphic) or CZ Inline graphic tissues (Fig. 1B). However, the ratio of Inline graphic 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).

Figure 1.

Figure 1.

Open in a new tab

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.

In contrast, no significant differences were observed in the cortex or CZ concentrations of Ca2+ Inline graphic or Mg2+ (Inline graphic) in response to 10% O2. Neither was there an effect of 10% O2 on the cortex to CZ ratio of ion concentrations (i.e. Inline graphic or Inline graphic; Fig. 1, C and D).

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.

After 1 h of 30% O2, the cortex K+ concentration Inline graphic had increased by 10% (P < 0.01) compared to control, and the CZ K+ concentration Inline graphic declined by 14% (P < 0.05) relative to the control nodules (Fig. 1F). Therefore, this treatment increased (P < 0.05) the Inline graphic ratio from 1.8 in the control nodules to 2.2 after 1 h of 30% O2 treatment, respectively (Fig. 1F).

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

When the nodulated roots were exposed to Ar:O2 (80:20) for 1 h, no significant changes were observed in the cortex K+ concentration Inline graphic. However, the CZ K+ concentration Inline graphic declined by 20% (P < 0.01) relative to the control nodules (Fig. 1J). Therefore, the Ar:O2 treatment increased (P < 0.01) the Inline graphic ratio from 2.0 in the control nodules to 2.6 after 1 h of exposure to Ar:O2 (Fig. 1J).

In contrast, no significant differences were observed in the cortex or CZ concentrations of Ca2+ Inline graphic or Mg2+ Inline graphic 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. Inline graphic or Inline graphic; 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 Inline graphic was decreased slightly by 6% (not statistically significant) compared to the control plants, whereas the CZ K+ concentration Inline graphic had decreased by 18% (P < 0.01) compared to control. Therefore, a significant 17% increase (P < 0.01) was observed in the Inline graphic 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+ Inline graphic or Mg2+ Inline graphic in response to stem girdling. Neither was there an effect of stem girdling on the cortex to CZ ratio of ion concentrations (i.e. Inline graphic or Inline graphic; Fig. 1, O and P).

DISCUSSION

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 Inline graphic 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 Inline graphic 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 Inline graphic 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 Inline graphic declined much more than the Inline graphic.

Consequently, in all treatments, the observed changes in the ratio of Inline graphic 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.

Figure 2.

Figure 2.

Open in a new tab

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.

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

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

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.

Figure 3.

Figure 3.

Open in a new tab

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.

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.

1

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

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@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.

References

  1. Becker D, Hoth S, Ache P, Wenkel S, Roelfsema MR, Meyerhoff O, Hartung W, Hedrich R (2003) Regulation of the ABA-sensitive Arabidopsis potassium channel gene GORK in response to water stress. FEBS Lett 554: 119–126 [DOI] [PubMed] [Google Scholar]
  2. Briskin DP, Gawienowski MC (1996) Role of the plasma membrane H+-ATPase in K+ transport. Plant Physiol 111: 1199–1207 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brown SM, Chudek JA, Hunter G, Sprent JI, Walsh KB, Wurtz G (1997) Proton density and apoplastic domains within soybean nodules in relation to the oxygen diffusion barrier. Plant Cell Environ 20: 1019–1029 [Google Scholar]
  4. Davidson AL, Newcomb W (2001) Organization of microtubules in developing pea root nodule cells. Can J Bot 79: 777–786 [Google Scholar]
  5. de Lima ML, Oresnik IJ, Fernando SM, Hunt S, Smith R, Turpin DH, Layzell DB (1994) The relationship between nodule adenylates and the regulation of nitrogenase activity by O2 in soybean. Physiol Plant 91: 687–695 [Google Scholar]
  6. Denison RF, Hunt S, Layzell DB (1992) Nitrogenase activity, nodule respiration and O2 permeability following detopping of alfalfa and birdsfoot trefoil. Plant Physiol 98: 894–900 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Denison RF, Kinraide TB (1995) Oxygen-induced membrane depolarizations in legume root nodules (possible evidence for an osmoelectrical mechanism controlling nodule gas permeability). Plant Physiol 108: 235–240 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Diaz del Castillo L, Hunt S, Layzell DB (1994) The role of oxygen in the regulation of nitrogenase activity in drought-stressed soybean nodules. Plant Physiol 106: 949–955 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gálvez S, Hirsch AM, Wycoff KL, Hunt S, Layzell DB, Kondorosi A, Crespi M (2000) Oxygen regulation of a nodule-located carbonic anhydrase in alfalfa. Plant Physiol 124: 1059–1068 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Goh CH, Jung KH, Roberts SK, McAinsh MR, Hetherington AM, Park YI, Suh K, An G, Nam HG (2004) Mitochondria provide the main source of cytosolic ATP for activation of outward-rectifying K+ channels in mesophyll protoplast of chlorophyll-deficient mutant rice (OsCHLH) seedlings. J Biol Chem 279: 6874–6882 [DOI] [PubMed] [Google Scholar]
  11. Gordon AJ (1991) Enzyme distribution between the cortex and the infected region of soybean nodules. J Exp Bot 42: 961–967 [Google Scholar]
  12. Hartwig UA, Boller BC, Nosberger J (1987) Oxygen supply limits nitrogenase activity of clover nodules after defoliation. Ann Bot (Lond) 59: 285–291 [Google Scholar]
  13. Hosy E, Vavasseur A, Mouline K, Dreyer I, Gaymard F, Poree F, Boucherez J, Lebaudy A, Bouchez D, Very AA, et al (2003) The Arabidopsis outward K+ channel GORK is involved in regulation of stomatal movements and plant transpiration. Proc Natl Acad Sci USA 100: 5549–5554 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hunt S, King BJ, Layzell DB (1989) Effects of gradual increases in O2 concentration on nodule activity in soybean. Plant Physiol 91: 315–321 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hunt S, Layzell DB (1993) Gas exchange of legume nodules and the regulation of nitrogenase activity. Annu Rev Plant Physiol Plant Mol Biol 44: 483–511 [Google Scholar]
  16. Iannetta PPM, James EK, McHardy PD, Sprent JI, Minchin FR (1993) An ELISA procedure for quantification of relative amounts of intercellular glycoprotein in legume nodules. Ann Bot (Lond) 71: 85–90 [Google Scholar]
  17. Jacobsen KR, Rousseau RA, Denison RF (1998) Tracing the path of oxygen into birdsfoot trefoil and alfalfa nodules using iodine vapor. Bot Acta 111: 193–203 [Google Scholar]
  18. James EK, Iannetta PPM, Deeks L, Sprent JI, Minchin FR (2000) Detopping causes production of intercellular space occlusions in both the cortex and infected region of soybean nodules. Plant Cell Environ 23: 377–386 [Google Scholar]
  19. James EK, Sprent JI, Minchin FR, Brewin NJ (1991) Intercellular location of glycoprotein in soybean nodules — effect of altered rhizosphere oxygen concentration. Plant Cell Environ 14: 467–476 [Google Scholar]
  20. Jeschke WD, Atkins CA, Pate JS (1985) Ion circulation via phloem and xylem between root and shoot of nodulated white lupin. J Plant Physiol 117: 319–330 [DOI] [PubMed] [Google Scholar]
  21. King BJ, Hunt S, Weagle GE, Walsh KB, Pottier RH, Canvin DT, Layzell DB (1988) Regulation of O2 concentration in soybean nodules observed by in situ spectroscopic measurement of leghemoglobin oxygenation. Plant Physiol 87: 296–299 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. King BJ, Layzell DB (1991) Effect of increases in O2 concentration during the argon-induced decline in nitrogenase activity in root nodules of soybean. Plant Physiol 96: 376–381 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kuzma M, Hunt S, Layzell DB (1993) Role of oxygen in the limitation and inhibition of nitrogenase activity and respiration rates in individual soybean nodules. Plant Physiol 101: 161–169 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kuzma M, Layzell DB (1994) Acclimation of soybean nodules to changes in temperature. Plant Physiol 106: 263–270 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kuzma MM, Winter H, Storer P, Oresnik I, Atkins CA, Layzell DB (1999) The site of oxygen limitation in soybean nodules. Plant Physiol 119: 399–407 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Layzell DB (1998) Oxygen and the control of nodule metabolism and N2 fixation. In C Elmerich, A Kondorosi, WE Newton, eds, Biological Nitrogen Fixation for the 21st Century. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 435–440
  27. Minchin FR (1997) Regulation of oxygen diffusion in legume nodules. Soil Biol Biochem 29: 881–888 [Google Scholar]
  28. Minchin FR, Iannetta PPM, James EK, Sprent JI, Thomas BJ, Witty JF (1995) Mechanisms for short-term (15 min) changes in oxygen diffusion barrier of legume nodules. In IA Tikhonovich, NA Provorov, VI Romanov, WE Newton, eds, Nitrogen Fixation: Fundamentals and Applications. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 600
  29. Minchin FR, Thomas BJ, Mytton LR (1994) Ion distribution across the cortex of soybean nodules: possible involvement in control of oxygen diffusion. Ann Bot (Lond) 74: 613–617 [Google Scholar]
  30. Moloney AH, Guy RD, Layzell DB (1994) A model of the regulation of nitrogenase electron allocation in legume nodules (II. Comparison of empirical and theoretical studies in soybean). Plant Physiol 104: 541–550 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Oresnik IJ, Layzell DB (1994) Composition and distribution of adenylates in soybean (Glycine max L.) nodule tissue. Plant Physiol 104: 217–225 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Parsons R, Day DA (1990) Mechanism of soybean nodule adaptation to different oxygen pressures. Plant Cell Environ 13: 501–512 [Google Scholar]
  33. Purcell LC, Sinclair TR (1994) An osmotic hypothesis for the regulation of oxygen permeability in soybean nodules. Plant Cell Environ 17: 837–843 [Google Scholar]
  34. Rao IM, Sharp RE, Boyer JS (1987) Leaf magnesium alters photosynthetic response to low water potentials in sunflower. Plant Physiol 84: 1214–1219 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Spalding EP, Goldsmith M (1993) Activation of K+ channels in the plasma membrane of Arabidopsis by ATP produced photosynthetically. Plant Cell 5: 477–484 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Streeter JG (1992) Analysis of apoplastic solutes in the cortex of soybean nodules. Physiol Plant 84: 584–592 [Google Scholar]
  37. Thomas BJ, Minchin FR (1992) X-ray microanalysis of ion distribution across the cortex of soybean nodules. Micron Microsc Acta 23: 389–390 [Google Scholar]
  38. Thumfort PP, Layzell DB, Atkins CA (2000) A simplified approach for modeling diffusion into cells. J Theor Biol 204: 47–65 [DOI] [PubMed] [Google Scholar]
  39. Vessey JK, Walsh KB, Layzell DB (1988) Oxygen limitation of N2 fixation in stem-girdled and nitrate-treated soybean. Physiol Plant 73: 113–121 [Google Scholar]
  40. Walsh KB (1995) Physiology of the legume nodule and its response to stress. Soil Biol Biochem 27: 637–655 [Google Scholar]
  41. Walsh KB, Vessey JK, Layzell DB (1987) Carbohydrate supply and N2 fixation in soybean: the effect of varied daylength and stem girdling. Plant Physiol 85: 137–144 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wei H, Atkins CA, Layzell DB (2004. a) Adenylate gradients and Ar:O2 effects on legume nodules. I. Mathematical models. Plant Physiol 134: 801–812 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wei H, Atkins CA, Layzell DB (2004. b) Adenylate gradients and Ar:O2 effects on legume nodules. II. Changes in the subcellular adenylate pools. Plant Physiol 134: 1775–1783 [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES