The role of diffusion in oxygen protection of nitrogenase in nodules of Alnus rubra

The role of diffusion in oxygen protection of nitrogenase in nodules of Alnus rubral

LAWRENCE J. WINS HIP^ A N D JOHN D. TJEPKEMA~ Hnrvnrd Forest, Hnrvnrd University, Petersham, MA, U.S.A. 01366

Received December 1, 1982

WINSHIP, L. J . , and J. D. TJEPKEMA. 1983. The role of diffusion in oxygen protection of nitrogenase in nodules of Alnus rubrn. Can. J. Bot. 61: 2930-2936.

Endophyte respiration in actinorhizal nodules has two main functions: to provide ATP and reductant for nitrogenase and associated enzymes and to protect nitrogenase from inactivation by oxygen. Combined with a diffusion path which limits the flow of oxygen, the consumption of oxygen by respiration maintains a very low internal Po,. We have used a continuous-flow root-nodule gas-exchange system to investigate the properties of such a diffusion path in intact, attached nodules of Aln~ts rubrn infected with Frnnkia strain ArI3. When nodules were exposed to gas mixtures containing argon, oxygen, and acetylene, the resulting curves of ethylene production versus acetylene concentration showed diffusion-limitedenzyme kinetics. Consequently, ethylene production at very low acetylene concentrations (<0.2% v/v) was used to characterize the diffusion path that both acetylene and oxygen must traverse to reach their active sites in the endophyte. The permeability of this path changed markedly with temperature and can account for the shape of the temperature response curve for total nitrogenase activity. Owing to the temperature-dependent barrier, energy production remains limited by oxygen diffusion, and nitrogenase can function over a wide temperature range at a relatively constant energy cost without inactivation by excess oxygen.

WINSHIP, L. J . , et J . D. TJEPKEMA. 1983. The role of diffusion in oxygen protection of nitrogenase in nodules of Alrzus rubrn. Can. J. Bot. 61: 2930-2936.

La respiration de l'endophyte dans les nodules actinorhiziens a deux fonctions principales: fournir de 1'ATP et des rkducteurs a la nitrogCnase et aux enzymes qui lui sont associCes et protCger la nitrogknase contre I'inactivation par l'oxygkne. CombinCe B une voie de diffusion qui limite le flux de l'oxygkne, la consommation d'oxygkne par la respiration maintient une trks faible Po? interne. Nous avons utilisC un systkme d'kchanges gazeux B flux continu pour Ctudier les propriCtCs de cette voie de diffusion dans des nodules intacts et attaches d'Alnus rubrn infect& par la souche ArI3 de Frc~nkin. Lorsque les nodules sont exposCs des mClanges gazeux contenant de l'argon, de l'oxygkne et de I'acCtylene, les courbes rksultantes de production d'Cthylkne et de concentration d'acktylkne montrent une cinCtique enzymatique IimitCe par la diffusion. Par consCquent, la production d'Cthylkne

de trks faibles concentrations d3acCtylkne (<0,2% v/v) a CtC utilisCe pour caractkriser la voie de diffusion que I'acCtylkne et I'oxygkne doivent tous les deux emprunter pour atteindre leurs sites actifs dans l'endophyte. La permCabilitC de cette voie change fortement avec la temperature et peut expliquer la forme de la courbe de rCponse la temperature que prCsente I'activitC nitrogenasique totale. A cause des barrikres likes a la temperature, la production d'Cnergie demeure IimitCe par la diffusion de l'oxygkne; de plus, la nitrogCnase peut fonctionner dans une gamme Ctcndue de tempkratures, a un coClt CnergCtique relativement constant, sans Ctre inactivCe par un excks d'oxygkne.

[Traduit par le journal]

Introduction Nitrogenase (N2ase) activity is very oxygen labile,

yet the most efficient pathway for the production of the large amounts of ATP needed for nitrogen fixation is oxidative electron transport. Each aerobic nitrogen- fixing system has evolved a mechanism for providing sufficient oxygen flow to energy-producing pathways, while maintaining a very low internal oxygen concentration around N2ase. In each case, the protection-supply mechanism involves a relatively impermeable barrier to oxygen diffusion combined with an oxygen uptake enzyme with a very low K, (Michaelis-Menten dis-

'Supported by United States Department of Agriculture grant No. 78-59-2252-0-1-005-1.

'present address: School of Natural Science, Hampshire College, Amherst, MA, U.S.A. 01002.

3~resen t address: Department of Botany and Plant Pathol- ogy, University of Maine at Orono, Orono, ME, U.S.A. 04469.

sociation constant) for oxygen (e.g., cytochrome oxidase). For example, the multilaminar heterocyst wall in nitrogen-fixing cyanobacteria, exemplified by Anab- aena, may function as such a diffusion barrier (Hasel- korn 1978). In nodules of soybeans (Tjepkema and Yocum 1974) and other legumes and of Parasponia (Tjepkema and Cartica 1982), a layer of host cortical cells lacking air spaces surrounds the inner bacteroid zone and limits oxygen diffusion. Actinorhizal nodules do not appear to have this kind of specialized host morphological structure, yet they are able to fix nitrogen over a broad range of oxygen conditions at specific activities as high as those of legume nodules and with equivalent efficiencies (Tjepkema and Winship 1980). It has been hypothesized that since vesiculated cultures of Frankia are able to fix nitrogen under aerobic conditions, the vesicle envelope may provide the necessary diffusion barrier (Tjepkema et al. 1980).

If diffusion-limited oxidative energy metabolism and

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nitrogen fixation occur in the infected tissue of actinorhizal nodules, then kinetics characteristic of diffusion limitation should be evident in the response of endophyte respiration and N2ase activity to external substrate concentration. The properties of such metabolism can be succinctly described by combining the equations for Michaelis-Menten (M-M) enzyme kinetics and for molecular diffusion as was done by Lommen et al. (1971) for photosynthetic leaf gas exchange. For N2ase or for cytochrome oxidase,

where V is specific activity in micromoles per hour, Vmax is the maximum enzyme activity at saturating substrate levels in micromoles per hour, K,, is the M-M dissociation constant in micromoles per millilitre, and Ci is the concentration of substrate at the active site in micromoles per millilitre.

For steady-state diffusion of substrates into a compartment surrounded by a diffusion barrier,

where F is the flux of substrate in micromoles per hour, P is the permeability of the diffusion barrier in millilitres per hour, and C, is the concentration of the substrate external to the diffusion barrier and Ci is the concentration inside the compartment in micromoles per millilitre. At steady state, these two functions will balance each other such that V is equal to F and Ci will become stable. Simultaneous solution of the two equations yields expressions for both Ci and V as a function of C, for a given set of parameters.

C41 V = P((C0 + Km + (VmaxIP)) - ((Co + Km + (Vmaxlp)) - 4Co(VmaxlP))0~5)/2

Alternatively, activity can be measured at a series of external concentrations (C,) and the data fitted to the equations to estimate the parameters.

Measuring the parameters of this model for intact nodules of Alnus or any other symbiotic association is complicated by the heterogeneity of the tissue. Since oxygen is also being taken up by nonfixing cortical cells, which are likely to be aerobic (as shown for Myrica gale by oxygen microelectrode (Tjepkema 1979)), the kinetics of oxygen uptake or of carbon dioxide evolution is not a good indicator of the properties of the diffusion path to N2ase. Acetylene uptake (and hence ethylene evolution), on the other hand, is a highly specific measure of N2ase activity. By measuring the rate of acetylene reduction by entire, intact nodules at a series

of external concentrations, in the absence of nitrogen so that electron flow is solely to acetylene or to hydrogen, it should be possible to measure the average permeability characteristics for the combined nitrogen-fixing com- partments of that nodule.

The initial objectives of this study were to test the applicability of a combined diffusion-enzyme kinetics model to gas exchange by Alnus root nodules and to assess the validity of measurements of N2ase activity at very low concentrations as estimates of nodule gas permeability. Measurements of this kind, when combined with studies of variation in nodule cytology, could help elucidate the roles of host and endophyte adapta- tions in oxygen protection and energy supply.

This method was then applied to the temperature dependence of nodule permeability. Changes due to temperature could be an important feature of nodule adaptation to low temperature. Since enzyme activity generally has a stronger temperature dependence (Qlo approximately 2) than molecular diffusion (Qlo = 1.0-1.3), it is possible that oxygen influx to the nitrogen-fixing regions of the nodule might exceed uptake by respiration at low temperatures, resulting in internal concentrations of oxygen high enough to inactivate N2ase. Alternatively, the permeability of the fixing zones to oxygen could decrease more rapidly than simple diffusion physics would predict, perhaps via changes in the molecular organization of surrounding membrane layers. Nitrogenase activity would still decline, but not as sharply and not by inactivation, but as a result of limited energy production, thereby maintaining energy cost at a minimum. This hypothesis was tested by using low levels of acetylene to measure nodule permeability over a range of temperatures.

Materials and methods Seeds of Alnus rubra Bong. (Clatsop County, OR) were

germinated in sand flats in a growth chamber. When the seedlings were 3-5 cm tall, they were washed free of sand and transferred to a modified aeroponics box (Zobel et al. 1976) in the greenhouse. Supplemental lighting was provided by 1000-W high-pressure sodium vapor lamps (Sylvania Luma- lux, LU1000). They were grown on %-strength Hoagland's solution supplemented with iron (11) phosphate, crushed oyster shells (which maintained the pH between 6.5 and 7.0), and 2 m M nitrate. When the plants had reached 10-15cm in height, they were inoculated with a homogenized suspension of Frankia strain ArI3 (Berry and Torrey 1979) and the nitrate supplement was discontinued. Nodules formed in 2-3 weeks. Nodules used in these experiments were 8 to 12 weeks old and ranged in size from 8 to 15 mm in diameter.

Assay system Rates of carbon dioxide, ethylene, and hydrogen production

were measured using a root-nodule gas-exchange system as previously described (Winship and Tjepkema 1982). An individual nodule, attached to an actively growing plant, was

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enclosed in a temperature-controlled, gastight cuvette and a stream of gas of known composition was continuously passed over the nodule at a measured flow rate. Activity was calculated from the difference in the composition of the incoming and outgoing gas. The outgoing gas stream was sampled automatically every 4.2 min and concentrations of carbon dioxide, ethylene, acetylene, hydrogen, oxygen, and nitrogen were measured by gas chromatography.

Data analysis After any change in conditions, steady-state activities for

respiration and nitrogenase were calculated as the means of several determinations when rates had become constant. When a complete set of data for a specific set of conditions and range of acetylene concentrations had been obtained, parameters were fitted as follows. An initial value for V,,, was taken as the ethylene production rate at 10% acetylene. P was estimated as the initial slope of the concentration curve below 0.2% acetylene. K, was set to 0.01 atm (1 atm = 101.325 Pa). The concentrations of acetylene used in the experiment were substituted into the model equations. The calculated activities were then compared with the experimental rates. Error was calculated as the difference between the calculated and measured rates divided by the measured rate. These values were squared and summed. V,,,, P, and K,, were then adjusted manually to minimize this value. V,,, was not allowed to vary from the measurement at 10% acetylene by more than 5%.

Results and discussion Acetylene reduction and hydrogen evolution

When Alnus nodules were first exposed to 10% acetylene for 30-60min and then placed in an atmo- sphere containing 80% argon and 20% oxygen, they evolved large amounts of hydrogen. Nodules which had never been exposed to a~et~lenehroduced little or no net hydrogen. This is evidence that acetylene inhibits the activity of the uptake hydrogenase in Frankia strain Ar13 as has been reported for the uptake hydrogenase of Azotobacter chroococcum (Yates and Walker 1980). When nodules were returned to an acetylenefree at- mosphere, net hydrogen evolution slowly decreased eve; several days, as hydrogen uptake activity was reestablished.

As shown in Fig. 1, net hydrogen evolution was increasingly inhibited by higher acetylene concentrations, as in other N2ase systems. Total electron flow, estimated as the sum of ethylene and hydrogen production, remained relatively constant over the range of acetylene concentrations tested. This indicated that nitrogenase turnover was virtually insensitive to acetylene concentration and that "unused" reducing power in the absence of acetylene or dinitrogen went to the production of hydrogen. The small decrease in total electron flow at low acetylene concentrations might have been due to residual uptake hydrogenase activity.

Substrate response kinetics Although the response of N2ase activity to external

acetylene concentration appears very similar to typical

" 0 1 2 3 4 5 6

ACETYLENE CONCN , % v / v

FIG. 1. Effect of acetylene concentration on acetylene reduction, hydrogen evolution, and total electron flow through nitrogenase. Gas composition was acetylene as shown and 20% v/v oxygen, and the balance was argon. Temperature was 24°C. The nodule had been exposed to 10% v/v acetylene for 1-2 h to inactivate uptake hydrogenase.

0 1 2 3 4 5 6

ACETYLENE CONCN . % V / V

FIGS. 2 and 3 . Effect of temperature on the response of nitrogenase activity to acetylene concentration. The symbols represent data obtained at two temperatures (20% v/v oxygen, balance argon) with a representative nodule, while the lines are calculated best fits to Michaelis-Menten enzyme kinetics only (Fig. 2) and M-M plus diffusion limitation (Fig. 3). Each point represents the mean of several determinations at steady state. Standard deviations for each point were smaller than the diameter of the symbol.

Michaelis-Menten enzyme kinetics (equation I), it was not possible to derive values for K, and V,,, that would allow the curve to pass through all the data points at either nodule temperature, as shown in Fig. 2. The initial slope of the data was too shallow and the plateau too flat. Lineweaver-Burke plots (not shown) were curvilinear. When the diffusion equation (equation 2) was included, a very close fit was obtained at both temperatures tested (Fig. 3). The effect of diffusion limitation at low acetylene concentration was to straighten the initial portion of the curve and reduce the slope. The best fit using Michaelis-Menten kinetics alone was even less satisfactory for the response curves at different.oxygen concentrations (Figs. 4 and 5) .

The values for K, used in the fitted curves (Table 1) are all within the range reported for other N2ase systems (Hardy et al. 1973). Those derived from Michaelis-

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Figure

WINSHIP AND TJEPKEMA

TABLE 1. Fitted parameters for the data from Figs. 2-5

i t ? U

0 1 2 3 4 5 6

A C E T Y L E N E CONCN , % V / V

Fig. 3.

0 1 2 3 4 5 6

ACETYLENE CONCN. .% v / v

FIGS. 4 and 5 . Effect of external oxygen concentration on the response of nitrogenase activity to acetylene concentration. Temperature was 24°C. Legend as in Figs. 2 and 3.

Menten kinetics alone, however, differed greatly among nodules and under differing environmental conditions (from 0.12 to 0.4 pmol/mL or 0.003 to 0.01 atm in these and other replicate experiments). When the diffusion model was used, the same K, (0.07-0.085 pmol/ mL or 0.00175-0.0022 atm) provided the best fit in all cases. It is unlikely that the true K, of N2ase for acetylene would vary widely among nodules infected with the same bacterial strain. The lower K, values (0.001 -0.002 atm), as used for the diffusion model, are also consistent with results obtained from in vitro N2ase preparations (Hollenbeck et al. 1979).

t-

2 o 0 1 2 3 4 5 6

A C E T Y L E N E CONCN.. % V / V

Fig. 5

Consequently the combined diffusion-enzyme kinet- ic model appears to provide a better description of alder nodule gas exchange than does simple enzyme kinetics.

Parameter estimation and temperature effects It was important to determine how valid the initial

slope of the concentration dependence curve, i.e. C,/V, was as a measure of P . One possible drawback of the proposed method was that as V,,, was reduced, due to oxygen, temperature, or other effects, enzyme activity might no longer keep up with diffusion. Ci would become a significant fraction of C,, reducing the influence of P on the shape of the curve.

Using fitted parameters, the internal acetylene concentrations (Ci) were calculated for the data from Figs. 2 and 3 (Table 2). As a result of the relatively high K, of N2ase for acetylene, these values are a significant fraction of the external acetylene concentrations (C,). This results in an underestimation of the permeability parameter when Ci is assumed to be zero; i.e., when the initial slope of the response curve (V/C,) is taken as an estimate of gas permeability, P.

The effect of changes in total N2ase activity (V,,,) on the magnitude of this underestimate is illustrated by the data in Fig. 6. Measurements of acetylene reduction were made on a single nodule at four different temperatures, first at saturating concentrations (10%) to obtain

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2934 CAN. J. BOT. VOL. 61. 1983

TABLE 2. Effect of internal acetylene concentration on the estimation of P from the data of Figs. 2 and 3

Temp., Vmax, Co, V, C, , Co/V, Ci/V, 1/P, Error, "C kmol/h kmol/mL kmol/h krnol/mL h/mL h/mL h/mL %

TEMPERATURE, "C

FIG. 6. Effect of temperature on the permeability of a nodule to acetylene. Gas composition was 20% v/v oxygen and 0.10% acetylene and the balance argon. Upper curve represents data corrected for the internal concentration of acetylene at the active site (Ci), while the lower curve represents the uncorrected initial slopes of the concentration curves.

V,,,, then at 0.10%. This latter rate was used to obtain a best-fit permeability parameter in the following fashion.

K, was assumed to be 0.08 pmol/mL (0.002 atm) since this was the average value obtained in all experiments with alder nodules. Since V,,, was experimen- tally determined, P could be first estimated as the initial slope of the concentration curve, then adjusted until the activity predicted by the model at 0.10% acetylene was equal to the measured value. The difference between this corrected permeability factor and the initial slope became a greater percentage of that factor as temperature was lowered, but the shape of the curve remained the same. The initial slope of the concentration dependence curve provided an accurate qualitative picture of variation in permeability, while assays of V,,, were needed to obtain a true quantitative estimate of P. By using several measurements at different concentrations, however, the true diffusion gradient and hence P were determined.

A more detailed example of the dependence of P upon temperature (estimated as V/C,) is shown in Fig. 7. Permeability fell almost linearly above 20°C with a Qlo of about 1.4, then with increasing rapidity below 15°C. This permeability change corresponds to the range of temperatures where total acetylene reduction (substrate saturated) also drops rapidly, indicating that a limitation of energy production caused by a reduced flux of oxygen could be the cause of the decline. At temperatures below

TEMPERATURE ,'C

FIG. 7. Effect of temperature on the permeability of a nodule to acetylene, based upon the uncorrected initial slope of the concentration dependence curves.

10°C, some inactivation of nitrogenase must also occur, since activity ultimately ceases altogether at 5-7°C. At some point near this temperature, respiration may decrease more rapidly than permeability, increasing internal oxygen concentrations to toxic levels.

Response to rapid shifts in temperature and Po, Perturbation experiments, using step shifts in tem-

perature and external oxygen concentration, were also used to distinguish between inactivation of Nzase at low temperatures and diffusion limitation of activity. If N2ase was being largely inactivated by high internal oxygen, the time course of recovery from low temperatures should resemble that following inactivation attrib- utable directly to oxygen toxicity.

As shown in Fig. 8 , N2ase activity and total nodule respiration recover very rapidly from low temperatures (9°C) when restored to 20°C. The recovery of respiration to full activity lagged behind the rise of nodule temperature by only 16-20 min. Acetylene reduction regained 90% of its previous value during this time. The slower recovery of the remaining 10% of activity may represent the reactivation of enzyme inhibited by oxygen in parts of the nodule less well protected against oxygen diffusion (higher permeability or lower specific activity).

The time courses resulting from shifts in oxygen concentration were markedly different. When a nodule was equilibrated at 5% oxygen, then step shifted to 20% oxygen, acetylene reduction plunged immediately to zero (Fig. 9). The inrush of oxygen diffusion down

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WINSHIP AND TJEPKEMA 2935

O l i 1 1 ' 1 0 1 2 3 4 5

T IME, h

FIG. 8. Time course of nodule response to step changes in temperature. Gas composition was 20% oxygen, 10% acetylene, balance argon. Each point represents one activity measurement.

FIG. 9. Time course of nodule response to a step shift in external oxygen concentration from 5 to 20% v/v. Tempera- ture was 24°C. Legend as in Fig. 8.

the fourfold increase in diffusion gradient evidently caused a spike of high internal oxygen concentration before respiration could increase to accommodate the new levels. Respiration began to increase in response to the higher oxygen levels, then fell again, perhaps due to a buildup of unused reductant and ATP due to the lack of N2ase activity. As acetylene reduction slowly reap- peared, respiration also increased. In 90 min, acetylene reduction rose only to 40% of its rate in 5% oxygen. In 20% oxygen, the eventual maximum velocity should have reached at least twice the rate at 5%.

The recovery of activity following a temperature shift appears to involve a much more rapid mechanism than that following oxygen inactivation and further impli- cates nodule permeability as a controlling factor in N2ase activity.

Conclusions A combined diffusion-enzyme kinetics model was

tested as a description of gas exchange by nodules of the actinorhizal plant Alnus rubra. The model accounted for the observed behavior of such nodules under a variety of

temperature and oxygen conditions. At very low concentrations of acetylene, ethylene production by nitrogenase was shown to be largely diffusion limited. Since oxygen is much less soluble than acetylene in water and many other materials and hence less permeable, this diffusion limitation is evidence that oxygen protection of nitrogenase in Frankia may indeed involve a permeability barrier.

By using this analysis scheme and model, it was possible to follow the effect of temperature on the permeability of the diffusion path to nitrogenase. The permeability changed rapidly at lower temperatures (less than 15°C). This lends support to the hypothesis that diffusion-limited respiration both limits nitrogenase activity and protects nitrogenase from inactivation at these temperatures. The shape of the temperature dependence also suggests that the barrier is undergoing some structural change. This concept was further supported by the difference between recovery from low temperature (very rapid) and oxygen shock (very slow), indicating that inactivation by oxygen is not a major factor in the decline of nitrogenase activity between 10 and 15°C. Any change in structure must be rapidly reversible.

The nature of this oxygen protection mechanism also points out a difference between rhizobial and actinorhizal systems. The temperature response of legume nodules has been found to consist of a relatively temperature-independent region from 15 to 30°C and a rapid decline to virtually zero activity below 10°C (Pankhurst and Sprent 1976). This decline has been interpreted as oxygen inactivation of nitrogenase due to the inability of the permeability barrier to continue to exclude oxygen at low temperatures (Tjepkema and Cartica 1982). Frankia associations, on the other hand, appear to have a mechanism which increases the diffusion resistance at lower temperatures. Respiration remains limited by the amount of oxygen which can diffuse into the active site and the internal oxygen concentration does not increase enough to inactivate nitrogenase. Further work, perhaps with isolated vesi- cles or nitrogen-fixing cultures of Frankia, may determine if the vesicle envelope has the necessary properties to provide this mechanism. Comparisons of plants with radically different endophyte and host morphologies may also provide more insight into the relationship between structure and function in oxygen protection and adaptability of actinorhizal nodules.

Acknowledgements We thank R. Lundquist and S. LaPointe for growing

the plant material for this study, A. Beny for Ar13 cultures and helpful discussion, and M. Mattmuller and M. Courtney for help with the figures. Seed was provided by B. Rottink of Crown Zellerbach.

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HARDY, R. W. F., R. C. BURNS, and R. D. HOLSTEN. 1973. Application of the acetylene reduction assay for measurement of nitrogen fixation. Soil Biol. Biochem. 5: 47-8 1.

HASELKORN, R. 1978. Heterocysts. Annu. Rev. Plant Physiol. 29: 319-344.

HOLLENBECK, P. C. , T. J. KOSTEL, and J. R. BENEMANN. 1979. Purification and properties of nitrogenase from the cyanobacterium Anabaerla cylindricurn. Eur. J. Biochem. 98: 275-284.

LOMMEN, P. W., C. R. SCHWINTZER, C. S. YOCUM, and D. M. GATES. 197 1. A model describing photosynthesis in terms of gas diffusion and enzyme kinetics. Planta, 98: 192-220.

PANKHURST, C . E., and J. I. SPRENT. 1976. Effects of temperature and oxygen tension on the nitrogenase and respiratory activities of turgid and water-stressed soybean and French bean root nodules. J. Exp. Bot. 27: 1-9.

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TJEPKEMA, J. D., and R. J. CARTICA. 1982. Diffusion limitation of oxygen uptake and nitrogenase activity in the root nodules of Parasponia rigida Merr. and Perry. Plant Physiol. 69: 728-733.

TJEPKEMA, J. D., W. ORMEROD, and J. G. TORREY. 1980. Vesicle formation and acetylene reduction in Frankia sp. CpI 1 cultured in a defined nutrient media. Nature (London), 287: 633-635.

TJEPKEMA, J. D., and L. J. WINSHIP. 1980. Energy require- ment for nitrogen fixation in actinorhizal and legume root nodules. Science (Washington D.C.), 209: 279-28 1 .

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The role of diffusion in oxygen protection of nitrogenase in nodules of Alnus rubra