nitrogen fixation (c2h2 reduction) by water-restricted conditions1

Plant Physiol. (1990) 92, 595-6010032-0889/90/92/0595/07/$01 .00/00

Received for publication July 18, 1989and in revised form September 29, 1989

Nitrogen Fixation (C2H2 Reduction) byBroad Bean (Vicia faba L.) Nodules and Bacteroids under

Water-Restricted Conditions1

Vincent Guerin, Jean-Charles Trinchant, and Jean Rigaud*Laboratoire de Biologie veg6tale et Microbiologie, URA CNRS 79-Parc Valrose, Universit6 de Nice-Sophia

Antipolis, 06034 Nice Cedex, France

ABSTRACT

Water potentials of leaves and nodules of broad bean (Viciafaba L.) cultivated on a sandy mixture were linearly and highly (r2= 0.99) correlated throughout a water deprivation of plants. Adecrease of 0.2 megapascal of the nodule water potential (I,w)induced an immediate 25% inhibition of the highest level ofacetylene reduction of broad bean nodules attached to roots.This activity continued to be depressed when water stress in-creased, but the effect was less pronounced. Partial recovery ofoptimal C2H2 reduction capacity of mildly water stressed nodules(*,od= -1.2 megapascals) was possible by increasing the exter-nal 02 partial pressure up to 60 kilopascals. The dense packingof the cortical cells of nodules may be responsible for the limita-tion of 02 diffusion to the central tissue. Bacteroids isolated frombroad bean nodules exhibited higher N2 fixation activity withglucose than with succinate as an energy-yielding substrate.Bacteroids from stressed nodules appeared more sensitive to O2,and their optimal activity declined with increasing nodule waterdeprivation. This effect could be partly due to decreased bacter-oid respiration capacity with water stress. Water stress was alsoresponsible for a decrease of the cytosolic protein content of thenodule and more specifically of leghemoglobin. The alteration ofthe bacteroid environment appears to contribute to the decline inN2 fixation under water restricted conditions.

In Western Europe, short periods of water deprivation forbroad bean cultures can occur during the growing season andreduce both growth and yield. Among the numerous studiesdevoted to the plant response to water stress, many werefocused on the limitation of the leaf photosynthetic capacity(1 1). An immediate effect of drought resulted in a limitationof CO2 diffusion due to the stomatal closure (9, 20). Further-more, photosynthetic metabolism would also particularly af-fect the partitioning between sucrose and starch as recentlypointed out by Quick et al. (17) and Vassey and Sharkey (26).The effect of water deprivation has also been investigated

in legumes, specifically in broad beans in direct relation totheir capacity to fix N2 (21). In soybeans, the decrease innitrogenase activity was closely related to the decrease in

' This research was supported by a grant from European Com-munities Coordinated Agricultural Research.

energy charge of the nodules (14) and to the changes inphotosynthate pool sizes (8). However, the decline in N2fixation activity of nodules during stress did not appear cor-related with the availability of carbohydrates, since a largeaccumulation of sucrose was reported in nodules of stressedsoybeans (7). On the other hand, water stress induced a 5%decline in photosynthesis in soybean, while nodule C2H2reduction showed a 70% decrease (6), pointing out the in-volvement of other mechanisms in the drop of N2 fixation.Thus, the possibility for an increase in the resistance to 02diffusion through the nodule cortex to the central tissue hasbeen proposed (6). Little information is available concerningthe effect of water stress on bacteroids ( 13).

In the present paper, the effect of water deprivation wasdetermined on C2H2 reduction by broad bean nodulated rootsexhibiting well defined qw2. The capacity to reduce C2H2 bybacteroids coming from these nodules was also investigatedin relation to increasing PO2 in the incubations. The abilityof the microbial partner to maintain a significant level of N2fixation under water limited conditions in relation to the 02supply and the environment insured by the host cell isdiscussed.

MATERIALS AND METHODS

Nodules

Broad bean ( Viciafaba L. var minor cv Soravi) seeds weresurface-sterilized in 3.5% (w/v) calcium hypochlorite solutionfor 45 min and then rinsed with sterile distilled water. Seedswere germinated on vermiculite for 6 d at 26 'C. Seedlingswere transferred into a sand (particule size: 1.5-3 mm)-vermiculite mixture (3/1, v/v) in plastic vessels (30 x 30 x16 cm). Plants were grown in a glasshouse (temperature range20-27 °C) and received a nitrogen-free medium (18) adjustedto pH 7.5.

Seedlings were inoculated 5 d after transfer to plastic vessels,with a mixture of three strains of Rhizobium leguminosarumbv vicieae (FH 25, FH 34, and FH 20S 1), kindly provided byN. Amarger (INRA Dijon, France) and was repeated twice at2-d intervals. Nodules appeared on the roots 1 week after thefirst inoculation.

2 Abbreviations: '', water potential; In'd, nodule water potential;PO2, 02 partial pressure.

595

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Plant Physiol. Vol. 92,1990

Water Potentials

The leaf '' was determined daily at midday on the young-est completely expanded leaf. It corresponded to the third leaffrom the apex. This potential was measured with a pressurebomb as described by Scholander et al. (19).The 'i'od was determined with a Wescor psychrometer

(Logan, UT) and a C52 sample chamber calibrated withstandard NaCl solutions. Three nodules were bisected, placedin the chamber, and the values ofTw were then checked every15 min until equilibration was achieved (6).

Bacteroid Preparations

About 15 g (fresh weight) of root nodules of V. faba werecrushed with a pestle and mortar in a glove box filled withargon as described elsewhere (22). Bacteroids obtained aftercentrifugation of the homogenates were washed twice in 50mm Na-phosphate buffer (pH 7.4) containing MgSO4 (2 mM)and sucrose (0.3 M). They were resuspended in 25 mm Na-phosphate buffer (pH 7.4) to a final concentration of 40 mg(dry weight)-mL'. Each experiment was in duplicate.

N2 Fixation Assays

N2 fixation activity was determined by C2H2 reductionusing a gas chromatograph (Pye-Unicam 4500, Philips)equipped with a column of Porapak T (80-100 mesh). Incu-bations of nodulated roots were made, in three replicates, in60-mL rubber cap vials which generally contained O2 andC2H2 at the partial pressures of 20 and 10 kPa, respectively,in argon. Some assays were also conducted in the presence ofincreasing O2 tensions ranging from 5 to 80 kPa. Bacteroidincubations (1 mL) were carried out in rubber cap vials (7.2mL) in the presence of glucose or succinate (10 mM). The gasphase contained 5 kPa C2H2 and O2 at different partialpressures in argon (23).

Bacteroid 02 Consumption

Bacteroid suspensions (4 mL) containing 1 to 2 mg (dryweight) were incubated in the chamber of an O2 electrode(Rank Bros., Bottisham, UK) fitted with a recorder. Succinateor glucose was used as energy-yielding substrate (23).

Leghemoglobin and Protein Contents in Nodule Cytosol

The red supernatant obtained by centrifugation of nodulehomogenates was subjected to ammonium sulfate precipita-tions. Leghemoglobin was precipitated between 55 and 80%of (NH4)2 SO4 saturation, and the corresponding pellet wasdissolved in 25 mM Na-phosphate buffer (pH 7.4). The con-centration of the hemoprotein was determined by the pyri-dine-hemochromogen assay (2).

Protein concentration of red supernatants from nodulehomogenates was determined by the method of Bradford (5)with bovine serum albumin as standard.

Microscopy

Immediately after excision, nodules were sliced, fixed withglutaraldehyde, and postfixed with osmium tetroxide. The

segments embedded in epon were stained with methyleneblue, cut with a glass knife in a Reichert Ultracut Ultramicro-tome, and observed with a Reichert-Jung Polyvar microscope.

RESULTS

Water Potentials of Broad Beans under Water Stress

Water potentials were determined at midday for the thirdleaffrom the apex, each measurement requiring no more than1 min. The results of a typical experiment are given in Figure1A, where a water deprivation treatment of 8 d led to a lineardecrease of the 'Iw from -0.6 to -1.4 MPa, under ourexperimental conditions. The well watered plants exhibited aleaf Tw of about -0.5 MPa throughout the same period oftime.Measurements of Tw were also conducted on detached and

bisected broad bean nodules from the upper part of the roots.In the Wescor chamber, 45 to 60 min were necessary to reachequilibrium. As shown in Figure 1B, water deprivation in-duced a regular decrease in the Tw values from -0.7 to -1.8MPa, whereas only slight variations occurred in the control.

Values for Inod were plotted against leaf tw values (Fig. 2).The linear relation (r2 = 0.99) between these two potentialsallowed us to estimate the 'n'd from the values determinedwith leaves, in the range of -0.5 to -1.5 MPa. For the *wless than -1.5 MPa, the Scholander bomb appeared inade-quate because of the high degree of leaf wilting.

Acetylene Reduction by Broad Bean Nodules

Nodulated roots of broad beans exhibited a high rate ofC2H2 reduction activity reaching 22 ,umol.h-'.g-' (freshweight), a value not far from those observed with soybeannodules under the same conditions (24). As shown in Figure3, this activity was immediately depressed by a limitation ofwater availability. For example, a decrease of 0.15 MPa(-0.49 to -0.64 MPa) of TnId was sufficient to cause a 25%inhibition of the highest C2H2 reduction rate. However, afurther decrease of the Tw of 0.15 MPa did not cause a 50%decline in activity, which required a decrease of the 'tw of0.27 MPa. The persistence of water deprivation for 8 d led toa complete disappearance of C2H2 reduction, which occurredat a In',,d of at least -2 MPa.C2H2 reduction of nodulated roots was also examined in

the presence of different 02 tensions in the gas phase ofincubations (Fig. 4). In well watered plants (I'no = -0.5MPa), nodule activity was strongly enhanced by increasingPO2 up to 40 kPa and then sharply declined with increasingPO2. No C2H2 reduction activity occurred with PO2 higherthan 60 kPa. After 5 d of plant water deprivation (In'd =-1.2 MPa), optimal N2 fixation of nodulated roots was 50%inhibited. This level of activity required an 02-enriched en-vironment corresponding to 60 kPa in the gas phase. Increasesin the P02 above 60 kPa caused a steep decline in C2H2reduction rates.

Structure of Broad Bean Nodules

Nodules from well watered plants had a cortex classicallydivided by an endodermis into an outer layer, exhibiting large

596 GUERIN ET AL.



ACETYLENE REDUCTION BY BROAD BEANS UNDER WATER STRESS

m._

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Figure 1. Time course of water potential in well-watered (0) and water-deprived (0) broad bean plants. Measurements were carried out dailyfor leaves (A) and nodules (B). Vertical bars indicate standard deviations (three experiments).

/0

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Figure 2. Relationship between broad bean nodule and leaf waterpotentials.

air spaces, and an inner layer (Fig. 5A). In the central tissue,most of the cells contain bacteroids. In water limited condi-tions, the structure of nodules (T.od = -1.2 MPa) appearedsignificantly modified, most particularly at the cortex level(Fig. 5B). The walls ofthe outer cortical cells were particularlydeformed, and the cells of both layers were densely packed.

Acetylene Reduction by Isolated Bacteroids

Broad beans were subjected to water deprivation and theirnodules, exhibiting decreasing 'I', were harvested and usedfor bacteroid preparations. Since no significant C2H2 reduc-tion activity occurred with the endogenous energy reserves ofbacteroids (data not shown), activity was determined withadded glucose or succinate, two energy-yielding substrateswell known to support nitrogenase activity in different leg-umes (23, 24). For all series of experiments, increasing P02

were added to the gas phase of incubations.In the presence of glucose, high C2H2 reduction rates were

0 -0.5 -1 -1.5 -2

Nodule water potential (MPa)

Figure 3. Effect of water deprivation upon C2H2 reduction activity ofbroad bean nodulated roots. Plants were 5 weeks old when subjectedto water restriction for about 8 to 10 d. C2H2 reduction activity wasmeasured after 30 min at 25 OC. The values are the mean of threereplicates and the bars denote standard deviations. FW - fresh weight.

observed (18 nmol-mg-' dry weight) for bacteroids fromnodules of slightly stressed plants with PO2 values rangingfrom 1.33 to 2.66 kPa (Fig. 6A). The effect of water stress(*od = -0.74 MPa) was detectable at the bacteroid levelwhere a 25% drop of C2H2 reduction occurred at the P02

level required for optimal activity (PO2 = 2.66 kPa). Decreas-ing the *I'd to -0.88 MPa caused both a 60% inhibition ofN2 fixation by bacteroids and a shift toward a lower 02 tension(PO2 = 1.33 kPa) for optimal activity. The lowest 'Inod (-1.6MPa) resulted in residual C2H2 reduction activity of bacter-oids, requiring the lowest 02 tension (PO2 = 0.67 kPa).When succinate replaced glucose as energy-yielding sub-

-2

0.

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Q

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0

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Plant Physiol. Vol. 92, 1990

'N 40

0C

o 30

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E\ - 0

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Figure 4. C2H2 reduction by modulated roots of broad bean in relationto increasing PO2 in the gas phase. Five-week-old plants were well-watered (@) or water deprived (0) during 5 d. Activity was determinedas described in Figure 3. FW - fresh weight.

strate in incubations, C2H2 reduction activity of bacteroidsisolated from regularly watered plants was much less stimu-lated (Fig. 6B). Nitrogenase activity linearly increased withincreasing P02 in the gas phase and reached an optimum of6.7 nmol mg-' (dry weight) for 6.67 kPa PO2. A decrease inT'od (from -0.66 to -0.74 MPa) due to plant water depnva-tion caused a drop in C2H2 reduction activity at all PO2 levels.Under more severe water stress ('I'd = -1.6 MPa), bacteroidsbecame more sensitive to O2; the highest O2 tensions (4-6.67kPa P02) completely inhibited C2H2 reduction activity.

Oxygen Consumption by Bacteroids

Assays were conducted without a gas phase with bacteroidsfrom nodules having variable TIod. Endogenous O2 consump-tion in the control was 2.2- and 6.5-fold stimulated by glucoseand succinate, respectively (Fig. 7). The decrease of T,,.d to-1.3 MPa slightly depressed respiration rates of bacteroids nomatter the substrate. In contrast, bacteroid respiration wasseverely inhibited by 'I'd values of -1.3 up to -1.7 MPa.

Soluble Proteins in Broad Bean Nodules

To avoid the interactions of the Folin-Ciocalteu reagentused in the Lowry method (12) with phenolic compoundslargely present in roots of broad beans, protein content incrude extracts was determined by the Bradford technique (5).Total soluble proteins of nodules harvested from plants sub-jected to increasing water stress regularly declined from 15 to2.5 mg-g-' fresh weight (Fig. 8). During the same period oftime (10-12 d), no significant variation in the level of solubleproteins appeared in the nodules of watered broad beans.Leghemoglobin content of nodules was specifically quanti-

fied throughout the same water stress period (Fig. 8). Thishemoprotein constituted about 16% of the total soluble pro-tein in functional nodules from well watered plants, and itslevel remained stable during the experimental period. In

Figure 5. Light micrographs of sections of broad bean nodules.Sections were from the functional part of nodules from well-watered(A) and water deprived (B) plants. OC, outer cortex; E, endodermis;IC, inner cortex; CT, central tissue. Bars indicate 10 ,im.

GUERIN ET AL.598




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Figure 7. Influence of plant water deprivation upon 02 consumptionA I by bacteroids isolated from broad bean nodules. Experiments were

0 2 3 4 5 6 7 conducted at 25 OC, without a gas phase, in the chamber of an 02

023 electrode (4 mL). Incubation mixtures contained bacteroids (2 mg dryP02 (KPa) weight) and 10 mm succinate (0, 0), glucose (5, U) or no exogenous

substrate (A, A) in 25 mm sodium-phosphate buffer (pH 7.4). OpenFigure 6. Effect of P02 in the gas phase on bacteroid C2H2 reduction.Incubation mixtures (1 mL) contained bacteroids (10 mg dry weight)isolated from broad bean nodules with decreasing water potentialsand 10 mm glucose (A) or succinate (B) in 25 mm sodium-phosphatebuffer (pH 7.4). Assays were at 25 IC for 10 min with shaking (140rpm.). DW - dry weight.

contrast, the leghemoglobin content greatly declined withdecreasing T,,od and represented only 4% of the total solubleprotein for the most water stressed plants.

DISCUSSION

Under our experimental conditions, the water status ofbroad bean nodules during water deprivation was closelyrelated to leafwater status (Fig. 2). This situation was obtainedby the culture of plants on a sandy mixture with a low waterretention capacity. Thus, very negative *I' were obtained forthe stressed nodules which correlated with the wilting of theleaves. This was also observed in soybeans cultivated undercomparable conditions (1). The relatively short time (about 8d) necessary to reach maximal stress status (Fig. 1) avoidedinterference with other phenomena such as nodule senes-cence. In contrast, the use of vermiculite alone to cultivateplants required a longer period of water deprivation to reachthe same level of stress (data not shown). Moreover, the valuesdetermined for leaf and nodule 'I', were largely different.Similarly, Durand et al. (6) reported *Iod three-fold less

symbols represent 02 consumption of bacteroids isolated from nod-ules of well watered plants. DW - dry weight.

negative than those of wilting leaves in soybean. Such differ-ences generally exist under field conditions, preserving a

sufficient level ofN2 fixation and allowing for a rapid recoveryof activity after rewatering.A slight alteration of the broad bean water status has an

immediate effect on the level ofN2 fixation (Fig. 3). However,increasing water deprivation did not continue to depress so

strongly C2H2 reduction, since 50% C2H2 reduction remainedat a relatively low ',,.d (--1 MPa). In this way, soybeansseem more sensitive to water stress, since for the same *Iod a

65 to 75% inhibition of attached nodule activity was observed(6, 10).An important observation reported here concerns the role

played by 02 supply to nodules in relation to water depriva-tion. The loss ofcell turgor ofnodule cortex and the reductionof air spaces (Fig. 5) could contribute to limit the 02 diffusionto the central tissue. Thus, a partial recovery of C2H2 reduc-tion activity can be obtained by increasing the PO2 aroundthe nodulated roots (Fig. 4). However, in spite of the impor-tance of this 02 resistance, as pointed out by Durand et al.

(6) in soybean, other internal factors seem to be involved inthis decline in N2 fixation, since 02 alone was unable tocompletely relieve the inhibition caused by water stress.For the first time, information concerning 02 availability

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Plant Physiol. Vol. 92, 1990

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Figure 8. Total soluble protein (0, 0) and leghemoglobin (A, A)contents of broad bean nodule cytosol in relation to plant waterdeprivation. Open symbols corresponded to nodules from wellwatered plants. FW - fresh weight.

in nodules subjected to increasing water stress was obtainedwith bacteroids isolated from broad bean nodules. Glucosewas chosen as an energy-yielding substrate, since in otherlegumes such as Phaseolus, Sesbania (23), or Glycine (24), itsability to support N2 fixation is known to occur over a narrowrange of low 02 tensions. However, a different situation existsin broad bean bacteroids supplied with this substrate: (a)glucose was able to support optimal bacteroid N2 fixationover a larger range of PO2 (1.5-3 kPa), (b) C2H2 reductionwas 2.5-fold higher with glucose than with succinate, consid-ered as an organic acid commonly used by bacteroids in vivo(3), and (c) during water stress, glucose appeared a moreappropriate substrate than succinate in maintaining the activ-ity of bacteroids, but only over a narrow range of P02 (Fig.6). These effects could be related to a better coupling betweenglucose oxidation and C2H2 reduction in spite of the wellknown capacity for succinate to strongly stimulate the respi-ration of bacteroids isolated from other legumes (Fig. 7) (4,22, 24). The efficiency of glucose must be underlined, sincethis substrate coming from sucrose hydrolysis is certainlylargely available under water stress conditions, known to favorsoluble sugar accumulation in roots and nodules of Glycine(7) as well as in leaves of Phaseolus (26).

For mild water stress (*o,,d = -0.74 MPa), the loss of N2fixation activity reached 25 and 39% for isolated bacteroidsusing glucose for energy and attached nodules, respectively.However, this inhibition was greater at the bacteroid levelwhen water availability decreased. In this way, the capacity ofbacteroids to reduce C2H2 appeared greater inside host cellsof nodules than during incubation after isolation. This obser-vation indicates a protective role played by the bacteroidenvironment, both the bacteroid compartment surroundedby the peribacteroid membrane and the cytosol of the host

cell. From this point of view, the integrity of leghemoglobin,required to provide 02 to the bacteroids, appears to be veryimportant. A drop of 15% in the leghemoglobin content couldbe correlated with a 55% loss ofC2H2 reduction activity when*I,d averaged -1 MPa. The persistence of a substantial levelof leghemoglobin in nodules having a I,,od lower than -2MPa (Fig. 8) could facilitate the large recovery of N2 fixationoccurring after rewatering of broad beans (our unpublisheddata) or of soybeans (7, 14). The disappearance of nodulesoluble proteins induced by plant water stress can be com-pared to the situation naturally occurring during the nodulesenescence (15, 25) or after feeding the plant with nitrate (16).In both cases, the presence ofactive proteases was responsiblefor the protein digestion, and it seems likely that these en-zymes may also be induced under water restricted conditions.

ACKNOWLEDGMENTS

We wish to thank Miss G. Van de Sype for her help with themicroscopy studies and Miss H. Le Bris for assistance in typing themanuscript.

LITERATURE CITED

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7. Fellows RJ, Patterson RP, Raper CD, Harris D (1987) Noduleactivity and allocation of photosyntate of soybean during re-covery from water stress. Plant Physiol 84: 456-460

8. Finn GA, Brun WA (1980) Water stress effects on CO2 assimila-tion, photosynthate partitioning, stomatal resistance, and nod-ule activity in soybean. Crop Sci 20: 431-434

9. Hanson AD, HitzWD (1982) Metabolic responses ofmesophytesto plant water deficits. Annu Rev Plant Physiol 33: 163-203

10. Huang C, Boyer JS, Vanderhoef LN (1975) Limitation of acet-ylene reduction (nitrogen fixation) by photosynthesis in soy-bean having low water potentials. Plant Physiol 56: 228-232

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13. Pankhurst CE, Sprent JI (1975) Effects of water stress on therespiratory and nitrogen-fixing activity of soybean root nod-ules. J Exp Bot 26: 287-304

14. Patterson RP, Rapper CD, Gross HD (1979) Growth and specificnodule activity of soybean during application and recovery ofa leaf moisture stress. Plant Physiol 64: 551-556

15. Pladys D, Rigaud J (1985) Senescence in French-bean nodules:occurrence of different proteolytic activities. Physiol Plant 63:43-48

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0%

A "

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17. Quick P, Siegl G, Neuhaus E, Feil R, Stitt M (1989) Short-termwater stress leads to a stimulation of sucrose synthesis byactivating sucrose-phosphate synthase. Planta 177: 535-546

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19. Scholander PF, Hammel HT, Bradstreet ED, Hemmingsen EA(1965) Sap pressure in vascular plants. Science 148: 339-346

20. Schulze ED (1986) Carbon dioxide and water vapor exchange inresponse to drought in the atmosphere and in the soil. AnnuRev Plant Physiol 37: 247-274

21. Sprent JI (1972) The effects of water stress on nitrogen-fixingroot nodules IV. Effects on whole plants of Vicia faba andGlycine max. New Phytol 71: 603-611

22. Trinchant JC, Rigaud J (1987) Acetylene reduction by bacteroidsisolated from stem nodules of Sesbania rostrata. Specific roleof lactate as energy-yielding substrate. J Gen Microbiol 133:37-43

23. Trinchant JC, Birot AM, Rigaud J (1981) Oxygen supply andenergy-yielding substrates for nitrogen fixation (acetylene re-duction) by bacteroid preparations. J Gen Microbiol 125: 159-165

24. Trinchant JC, Rigaud J (1989) Alternative energy-yielding sub-strates for bacteroids isolated from stem and root nodules ofSesbania rostrata submitted to 02 restricted conditions. PlantSci 59: 141-149

25. Vance CP, Reibach PH, Ellis WR (1986) Proteolytic enzymes oflegumes nodules and their possible role during nodule senes-cence. In MJ Dalling, ed, Plant Proteolytic Enzymes, Vol 2.CRC Press, Bocca Raton, FL, pp 103-124

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