This article was downloaded by: [Uniwersytet Warszawski]On: 15 October 2014, At: 00:18Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Soil Science and Plant NutritionPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/tssp20

Respiratory Metabolism ofMitochondria in Soybean RootNodulesNorio Suganuma a & Yukio Yamamoto aa Department of Agronomy, School of Agriculture , NagoyaUniversity , Chikusa-ku , Nagoya , 464 , JapanPublished online: 30 Oct 2012.

To cite this article: Norio Suganuma & Yukio Yamamoto (1987) Respiratory Metabolism ofMitochondria in Soybean Root Nodules, Soil Science and Plant Nutrition, 33:1, 93-101, DOI:10.1080/00380768.1987.10557555

To link to this article: http://dx.doi.org/10.1080/00380768.1987.10557555

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information(the “Content”) contained in the publications on our platform. However, Taylor& Francis, our agents, and our licensors make no representations or warrantieswhatsoever as to the accuracy, completeness, or suitability for any purpose of theContent. Any opinions and views expressed in this publication are the opinions andviews of the authors, and are not the views of or endorsed by Taylor & Francis. Theaccuracy of the Content should not be relied upon and should be independentlyverified with primary sources of information. Taylor and Francis shall not be liablefor any losses, actions, claims, proceedings, demands, costs, expenses, damages,and other liabilities whatsoever or howsoever caused arising directly or indirectly inconnection with, in relation to or arising out of the use of the Content.

This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,systematic supply, or distribution in any form to anyone is expressly forbidden.Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

Soil Sci. Plant Nutr., 33 (I), 93-101, 1987

RESPIRATORY METABOLISM OF MITOCHONDRIA IN SOYBEAN ROOT NODULES

Norio SUGANUMA* and Yukio YAMAMOTO

Department 0/ Agronomy. School of Agriculture. Nagoya University. Chikusa-ku. Nagoya. 464 Japan

Received February 13, 1987

Bacteroid-free mitochondria exhibiting respiratory control were prepared from soybean root no~ules using Percoll discontinuous gradient centrifugation. Though the activities of the enzymes for ethanol fermentation in the nodule cytosol fraction were higher than those in the root cytosol fraction, the mitochondria isolated from the nodules also displayed higher oxidative and phosphorylative activities than those isolated from the roots. During the nodule development, the acetylene reducing activity of detached nodules and leghemoglobin content in the nodules varied nearly in parallel, increasing up to 6 weeks from sowing and then de­creased. However, the amount of mitochondrial protein recovered from the nodules and the oxidative and phosphorylative activities in the nodule mitochondria hardly changed during the nodule development, except that the respiratory control ratio with malate as a substrate varied. The activities of the malic enzyme and isocitrate dehydrogenase in the nodule mito­chondria were also constant throughout the development. Malate dehydrogenase and cyto­chrome c oxidase activities in the nodule mitochondria varied showing sharp peaks, without being closely correlated with the acetylene reducing activity.

The mechanisms underlying the higher oxidative and phosphorylative activities of mito­chondria in soybean root nodules were analyzed in relation to nitrogen fixation.

Key Words: mitochondria, nitrogen fixation, respiration, soybean nodule.

The root nodule formed by the infection of bacteria has a compartmentalized metabolism. Nodule cells contain leghemoglobin, resembling hemoglobin or myo­globin observed in animals, which induces a very Iow concentration of free oxygen in the nodule cell (about 10 nM) (l). Enzymatic studies (2, 3) have suggested that ethanol fermentation is active in the nodule cytosol and related to nitrogen fixation. Bacter­oids, nitrogenase of which can be damaged by oxygen, require oxygen to maintain

This work was presented at the annual meeting of the Japanese Society of Soil Science and Plant Nutrition

in 1985 . • Present address: Department of Biology, Aichi University of Education, Kariya, Aichi, 448 Japan. Abbreviations: Tris, tris(hydroxymethyl)aminomethane; EDTA, ethylenediaminetetraacetic acid; BSA, bovine serum albumin; ADP, adenosine diphosphate; RCR, respiratory control ratio; NAD, nicotinamide adenine dinucleotide; NADH, reduced form ofNAD; NADPH, reduced form of nicotin­amide adenine dinucleotide phosphate.

93

Dow

nloa

ded

by [

Uni

wer

syte

t War

szaw

ski]

at 0

0:18

15

Oct

ober

201

4

94 N. SUGANUMA and Y. YAMAMOTO

oxidative phosphorylation which supplies energy for nitrogen fixation. Transport of 02 to bacteroids is mediated by leghemoglobin (4).

On the other hand, the function of mitochondria in the nodule ceIls has not been fully investigated. The contribution of leghemoglobin to the mitochondrial function is unknown. Microscopic observation showed that mitochondria in the infected cells are locates in the cytosol surrounding the bacteroids, especially in the cytosol adjacent to intracellular air spaces (1, 5). MUECKE and WISKICH (6) prepared mitochondria from soy bean root nodules, but these exhibited a relatively low respiratory control.

Bacteroids need carbon compounds to maintain nitrogen fixation, and also the host plant cells consume carbon compounds for ammonia assimilation and nodule growth. Mitochondria produce not only energy but also substrates for several bio­synthetic reactions, especially a-ketoglutarate for the synthesis of glutamic acid and glutamine which are essential amino acids in ammonia assimilation of the nodule (7, 8). Accordingly, the function of mitochondria in the nodule may be closely related to the nitrogen fixing activity.

In the present study, bacteroid-free mitochondria were isolated from soybean root nodules at different stages of plant development and the respiratory activities and enzyme activities of these mitochondria were compared.

MATERIALS AND METHODS

Plant materials. Soybean seeds (Glycine max: A62-1) were inoculated with Rhizobium japonicum strain 009 and sown in vermiculite. After 7 days, plants were grown in N-free liquid culture media in a greenhouse under natural daylight conditions (3). Only the nodules formed on the upper basal portion of the tap root were used for the aging experiments.

Preparation of mitochondria. Freshly harvested nodules or roots were homog­enized in four times the weight of a grinding medium [0.05 M Tris-HCI buffer pH 7.5, 0.5M sucrose, 10 mM EDTA, 1% (w/v) Na-isoascorbate, 0.1% (w/v) BSA] with 30% of the weight of Polyclar AT powder. The nodules or roots were at first cut with a razor blade and then ground using a mortar and pestle. The homogenate was squeezed through four layers of gauze and the filtrate centrifuged at 1,000 x g for 10 min. The supematant fraction was centrifuged at 10,000 x g for 15 min. The pellet obtained was suspended in washing medium [0.05 M Tris-HCI buffer pH 7.5,0.4 M sucrose, 0.1% (w/v) BSA] and centrifuged again at 1,000 x g for 10 min followed by final pelletting at 10,000 x g for 15 min.

The purification of the mitochondria was carried out essentially according to the method of JACKSON et al. (9). The washed mitochondrial pellet resuspended in 5 rn1 of washing medium was layered on the top of a discontinuous Percoll gradient com­posed of 12 ml 45% Percoll and 12 ml 21 % Percolllayers. Each solution contained 10 mM Tris, 0.25 M sucrose, and 0.1 % (w/v) BSA, adjusted to pH 7.5 with HCt. The gradient was centrifuged at 7,500 x g for 30 min utilizing an angle rotor. The mito-

Dow

nloa

ded

by [

Uni

wer

syte

t War

szaw

ski]

at 0

0:18

15

Oct

ober

201

4

Mitochondria in Soybean Nodules 95

chondrial fraction which appeared at the interface of the 45 and 21 % Percoll layers ~as collected with a Pasteur pipette and washed two times with washing medium.

O2 uptake measurement. O2 uptake was measured polarographically at 25°C using a Clark-type electrode (Yanaco Co., Ltd.) for 2.8 ml of reaction medium [20 mM K­PO, buffer pH 7.2, 0.4 M mannitol, 0.5 mM EDTA, 5 mM MgClz, 0.1% (w/v) BSA]. The final concentrations of the substrate were 7 mM for succinate, 7 mM for malate, and 1.7 mM for NADH. In the aging experiments, the malate concentration was raised to 30 lIlM. The O2 concentration in the air-saturated medium was estimated at 237 pM. The ADP to 0 ratio and the respiratory control (RC) ratio were measured by the addition of a known amount of ADP (125 nmol) to the reaction medium.

Enzyme assays and analytical methods. The activities of enzymes were determined spectrophotometrically at 30°C. .B-Hydroxybutyrate dehydrogenase (.aHBDH) activity was assayed by the method of WONG and EVANS (10) after incubation of the extract with 10% Triton X-lOO for 2 min. Isocitrate dehydrogenase (ICDH) activity was measured in the presence of 50 mM Tris-HCI (pH 7.6), 10 mM MgCI2, 0.5 mM NAD, 4 mM isocitrate, 1.7 mM KCN, and 0.02% Triton X-lOO in final concentrations. Other enzyme activities were determined as described in the literature (parentheses): i.e. fumarase (11), NADPH-cytochrome c reductase (12), catalase (13), malate dehydro­genase (MDH) (14), cytochrome c oxidase (Cyt oxi) (14), malic enzyme (ME) (15), alcohol dehydrogenase (3), and pyruvate decarboxylase (16).

The nodule or root cytosol fraction was prepared according to the method of TAJIMA and LARuE (2), under aerobic conditions. One g of nodule or root was homogenized in 0.2 M K-PO, buffer (pH 7.5) containing 0.3 M sucrose, 5 mM dithio­threitol, 1 mM MgClz, 0.4 mM EDTA, and 0.3 g Polyclar AT using a mortar and pestle. The homogenate was squeezed through four layers of gauze and the filtrate centrifuged at 16,000 x g for 30 min. The supernatant was used as the cytosol fraction. Acetylene reducing activity was measured using detached nodules. Gas-samples were collected and analyzed by gas chromatography on a Unibeads A column. Leghemoglobin con­centration of the nodule cytosol fraction was determined by the pyridine hemochro­mogen method (17). Protein was determined by the modified Lowry procedure (18).

RESULTS

Table 1 shows marker enzyme activities measured in the crude mitochondrial fraction and also in the purified mitochondrial fraction obtained by Percoll density gradient centrifugation. Approximately 55% of the fumarase activity was recovered in the purified mitochondria. .e-Hydroxybutyrate dehydrogenase activity (marker of bacteroids) was barely detectable in the purified mitochondria, indicating that most of the bacteroids had been removed by the purification. On the other hand, the puri­fied mitochondrial fraction contained about 40% of NADPH-cytochrome c reductase activity [marker of endoplasmic reticulum (19)] and about 26% of catalase activity [marker of peroxisomes (19)] originally present in the crude mitochondrial fraction,

Dow

nloa

ded

by [

Uni

wer

syte

t War

szaw

ski]

at 0

0:18

15

Oct

ober

201

4

96 N. SUGANUMA and Y. YAMAMOTO

Table 1. Marker enzyme activities recovered in the crude mitochondrial fraction and in the purified mitochondrial fraction after Percoll discontinuous gradient centrifugation.

Fraction

Crude mitochondria

Purified mitochondria

Fumarase (pmol/min)

0.594

0.327

fJHBDH (pmol/min)

0.035

0.0005

NADPH-Cyt c reductase (pmol/min)

0.064

0.026

Catalase (mmol/min)

0.234

0.062

Enzyme activities are expressed as pmol or mmol per min per fraction. Abbreviations of enzyme names are presented in Materials and Methods.

Table 2. Oxidative and phosphorylative activities of the purified mitochondria isolated from the nodules and roots.

Fraction Substrate Respiratory ADP/O RCR rate

Nodule mitochondria Succinate 179±49 1.24±0.18 1. 87±0.27

Malate 56±13 2.20±O.19 3. 82±O.95

NADH 90±33 1.15±0.29 1. 51 ±0.10

Root mitochondria Succinate 84±32 1.09±O.18 1. 49±0.13

Malate 33±21 1. 92±0.26 2.60±O.62

NADH 211±124 0.87±0.60 1.26±0.16

Respiratory rates which refer to state 3 respirations are expressed as nmol O2 per min per mg mito­chondrial protein. Values are the averages±standard deviations of at least 3 experiments.

indicating the contamination by the endoplasmic reticulum and peroxisomes. Per­centages of the activities recovered in the purified mitochondria were, however, lower than the percentage of the fumarase activity recovered.

The activities of the enzymes for ethanol fermentation in the cytosol fraction and of respiratory metabolism of the purified mitochondria, from soybean nodules and roots, respectively, were compared. The activity of pyruvate decarboxylase, a key enzyme for ethanol fermentation, which was 0.161 pmol per min per mg protein in the nodule cytosol fraction could not be detected in the root cytosol fraction. The activities of alcohol dehydrogenase in the nodule cytosol and in the root cytosol were 0.538 and 0.185 pmol per min per mg protein, respectively.

The respiratory rates, the ADP to 0 ratios, and the RC ratios of mitochondria isolated from these metabolically different tissues are shown in Table 2. The purified mitochondria prepared from the nodules were able to oxidize succinate, malate, and NADH, with higher values for the ADP to 0 ratios as well as the RC ratios than those of the mitochondria prepared from roots. The respiratory rates with different sub­strates were in the decreasing order of succinate> NAD H > malate in the nodule mito­chondria, and the rates of succinate- and malate-oxidation in the nodule mitochondria

Dow

nloa

ded

by [

Uni

wer

syte

t War

szaw

ski]

at 0

0:18

15

Oct

ober

201

4

Mitochondria in Soybean Nodules 97

were about two times higher than those of the root mitochondria. The root mito­chondria oxidized NADH significantly, though the values fluctuated to a large extent. Table 3 shows the enzyme activities of the mitochondria purified from both nodules and roots. The isocitrate dehydrogenase activity of the nodule mitochondria was slightly lower than that of the root mitochondria, but the nodule mitochondria ex­hibited higher activities of fumarase, malate dehydrogenase, and cytochrome c oxidase.

Changes in the acetylene reducing activity, leghemoglobin content, and the amount of mitochondrial protein recovered during the nodule development are indicated in

Table 3. Enzyme activities of the purified mitochondria isolated from the nodules and roots.

Specific activity Enzyme

Isocitrate dehydrogenase

Fumarase

Malate dehydrogenase

Cytochrome c oxidase

Nodule mitochondria

0.085 0.202

2.583 0.634

Specific activities are expressed as pmol per min per mg mitochondrial protein.

~

~ ul;; 16 'C.!!I e"' ~iII12 'O'~ =~ § "; 8 --~~ :: ~ ,. ~.g

0.4,. ':: ..... .0: .... ~ .. ..

0.3 ~ ;a co ..., ... '" ..

D.2~ .::: co .. - ...... ... -co co

0.1 !l a. .c: ......

~ NE U... 0 !--L..--L_.l-.-L..-..l_..L--J......J1...-.j0 = 0.6 ... ~; 0.5 .... oC IS l': 0.4 -::;.:: ~ at 0.3 e ...... ... El' 0.2 ........ ~ ..

• • ~ .. • • • •

~ 0; 0.1 ... c; ~a 01~-L..-..l __ ..L--J.._~-L..--L--.l-.~

23" 5 6 7 8 9 Plant age [weeks Iller sowIng J

Root mitochondria

0.097 0.137 0.815 0.321

Fig. 1. Acetylene reducing activity of detached nodules, leghemoglobin content in the nodule and amount of mitochondrial protein recovered at different stages of the nodule development. The leghemoglobin content was determined in the nodule cytosol fraction and is expressed as pmol per g fresh weight of nodules. The amount of recovered mitochondrial protein is ex­pressed as mg protein per g fresh weight of nodules and the value of 3 separate experiments is plotted.

Dow

nloa

ded

by [

Uni

wer

syte

t War

szaw

ski]

at 0

0:18

15

Oct

ober

201

4

N. SUGANUMA and Y. YAMAMOTO

Fig. 1. The specific activity of acetylene reduction by the detached nodules increased rapidly as the nodule on the mother plant developed, and decreased sharply after 6 weeks, Similarly the leghemoglobin content in the nodule increased, but declined slightly compared with the acetylene reducing activity. On the other hand, the amount of mitochondrial protein recovered per g fresh weight of nodule hardly changed or seemed to slightly decrease during the nodule development.

Figures 2 and 3 show the respiratory rates, ADP to 0 ratios, and RC ratios as measured in the presence of succinate and malate, respectively, supplied as a substrate for the mitochondria prepared from the nodules at different stages of plant develop­ment. Almost all the values remained constant throughout the nodule development, except for the RC ratio with malate as a substrate. The rate of NADH oxidation showed a similar variation to that of succinate oxidation (data not shown). The activities of isocitate dehydrogenase and malic enzyme in the nodule mitochondria were also kept nearly constant (Fig. 4). However, the malate dehydrogenase activity increased rapidly in the early stage, and decreased gradually after 3 weeks. The varia-

300

!!! ... .. 200 t:' ~ :! =- 100 tU Ill:

Cl

ii:: "" cc

Cl: ... Cl:

o 2

2

-

~ 0

~

~

23456 789 Plant age [weeks alter sowing]

Fig. 2.

-

300

!!! :! 200 t:' ~ :! =- 100 Cl> Cl:

Cl .....

o 3

~ 2 cc

7

~ 6 Cl:

5

f-

~

I-~

I-

I-~ 2 3 4 5 8 7 8 9 Plant age [weeks after sowing]

Fig. 3.

-

Fig. 2. Oxidative and phosphorylative activities of the mitochondria prepared from nodules at different stages of plant development with succinate as a substrate. Respiratory rates are expressed as nmo] 0 , per min per mg mitochondrial protein.

Fig. 3. Oxidative and phosphorylative activities of the mitochondria prepared from nodules at different stages of plant development with malate as a substrate. Respiratory rates are ex­pressed as nmol 0 1 per min per mg mitochondrial protein.

Dow

nloa

ded

by [

Uni

wer

syte

t War

szaw

ski]

at 0

0:18

15

Oct

ober

201

4

,...., c::: B f! co.

~ ... c::: ...

.&: ... ~ e ... IS -c::: e -C; IS =-...... !: ~ ... ... ~ "" .... c::: ....

Mitochondria in Soybean Nodules

8.0

7.0

6.0

5.0

4.0

3.0

2.0

1.0

0.10

0.05

0

~, MOH : ''Q , \ , \

: \ It , " , , \,.!t : l,.~'" \, , " ,

I' " \ b.. ... , , : It. ; \ 00;'0 , l \ " , / 6/ '. / '" • ~ ' .. ' j.."

Cyt oxl

~ "--... .. -

ME

2 3 456 7 8 9 Plant age [weeks alter sowing]

Fig. 4. Enzyme activities of mitochondria prepared from nodules at different stages of plant development. Abbreviations of enzyme names are presented in Materials and Methods.

99

tion in the cytochrome c oxidase activity was irregular, showing a maximum peak in 6 week~old nodules.

DISCUSSION

The crude mitochondrial fraction prepared by differential centrifugation contained large quantities of bacteroids. Most bacteroids were removed by Percoll discontinuous gradient centrifugation using 45 and 21 % PercoIllayers (Table 1).

The activities of pyruvate decarboxylase and alcohol dehydrogenase mentioned above suggest that ethanol production is active in the nodules, but not in the roots. The presence of acetaldehyde and ethanol in soybean nodules also indicates that ethanol fermentation operates in vivo (2). On the other hand, mitochondria prepared from the nodules displayed higher oxidative and phosphorylative activities than those pre­pared from the roots (Tables 2 and 3), with the same isolation procedure. The prepa~ ration of mitochondria from soybean nodules exhibiting respiratory control was re­ported by MUECKE and WISKICH (6). They showed that the ratios of ADP to 0 ranged from 0.9 to 1.15 with succinate as a substrate, and from 1.55 to 1.95 with malate. The higher values obtained in the present study in either case (Table 2) nearly coincided or were slightly lower as compared with those obtained in spinach leaf mitochondria (9, 19, 20). This observation confirms that the substrate oxidation by the mitochon-

Dow

nloa

ded

by [

Uni

wer

syte

t War

szaw

ski]

at 0

0:18

15

Oct

ober

201

4

100 N. SUOANUMA and Y. YAMAMOTO

dria isolated from soy bean root nodues is closely coupled to phosphorylation even though anaerobic metabolism operates in the nodule cytosol.

Although the acetylene reducing activity and leghemoglobin content changed appreciably during the nodule development, the protein content and respiratory ac­tivities of the nodule mitochondria hardly changed with some exceptions (Figs. 1-4). The changes in the acetylene reducing activity and leghemoglobin content may affect the carbon and nitrogen metabolism, and the microenvironment in the nodule, for example, O2 tension. Nevertheless, the respiratory activities of the mitochondria prepared were not influenced by these changes. The results obtained suggest that mitochondria operate in the periphery of the infected cells where the free O2 tension would be highest (1). However, it remains to be determined whether all the mito­chondria were recovered in the present experiments and whether mitochondria actually operate in vivo. Furthermore, there is a possibility that mitochondria can use the oxygen bound to leghemoglobin like bacteroids (4).

The CO2 dark fixation catalyzed by the phosphoenolpyruvate carboxylase is active in legume root nodules (21). GADAL (22) suggested that oxaloacetate or malate pro­duced by the CO2 dark fixation offers a carbon skeleton for amino acids, in particular, asparagine biosynthesis and is a source of reducing power to bacteroids, and a sub­strate for the tricarboxylic acid cycle in the mitochondria. Mitochondria prepared from soy bean nodules exhibited malic enzyme activity and was able to oxidize malate (Fig. 4 and Table 2). Even if pyruvate is converted to ethanol in the plant cytosol of nodule, the tricarboxylic acid cycle in the mitochondria is able to operate if malate is supplied. PETERSON and EVANS (23) indicated that the activity of pyruvate kinase in soybean nodule cytosol is inhibited by NH, +, resulting in a higher accumulation of phosphoenolpyruvate available for phosphoenolpyruvate carboxylase. Their results further suggest that the CO2 dark fixation is more accelerated than ethanol fermentation when NH,+ is accumulated by nitrogen fixation. Accordingly, the malate produced by the CO2 dark fixation can be used as a carbon source for the operation of the mito­chondrial tricarboxylic acid cycle and is in turn converted to a-ketoglutarate followed by the amino acid biosynthesis.

The current work presented evidence that the enzymes for ethanol fermentation are active in the soy bean nodule cytosol and at the same time that the mitochondria in the soybean root nodules have an oxidati\'e and phosphorylative capacity similar to that in the roots with aerobic metabolism.

REFERENCES

1) ROBERTSON, J.O. and FARNDEN, K.J.F., Ultrastructure and metabolism of the developing legume root nodule, In The Biochemistry of Plants, Vo!. 5, ed. by B.J. Miflin, Academic Press, New York, 1980. pp.~65-113

2) TAJIMA, S. and LARuE, T.A., Enzymes for acetaldehyde and ethanol formation in legume nodules, Plant Physiol., 70, 388-392 (1982)

3) SUOANUMA, N. and YAMAMOTO, Y., Carbon metabolism related to nitrogen fixation in soybean

Dow

nloa

ded

by [

Uni

wer

syte

t War

szaw

ski]

at 0

0:18

15

Oct

ober

201

4

Mitochondria in Soybean Nodules 101

root nodules, Soil Scl. Plant Nutr., 33, 79-91 (1987) 4) ApPLEBY, C.A., Leghemoglobin and Rhizobium respiration, Annu. Rev. Plant Physiol., 35, 443-478

(1984) 5) GOODCHILD, D.J. and BERGERSEN, F.J., Electron microscopy of the infection and subsequent de­

velopment of soybean nodule cells, J. Bacterial., 92, 204-213 (1966) 6) MUECKE, P.S. and WISKICH, J.T., Respiratory activity of mitochondria from legume root nodules,

Nature, 221, 673-675 (1969) 7) RAWSTHORNE, S., MINCHIN, F.R., SUMMERFlELD, R.J., CooKSON, C., and CooMBS, J., Carbon and

nitrogen metabolism in legume root nodules, Phytochemistry, 19, 341-355 (1980) 8) SCHRAMM, R.W., Carbon assimilation and flux connected with nitrogen fixation in legumes, Isr. J.

Bot., 31, 131-139 (1982) 9) JACKSON, C., DENCH, J.E., HALL, D.O., and MooRE, A.L., Separation of mitochondria from

contaminating subcellular structures utilizing silica sol gradient centrifugation, Plant Physiol., 64, 150-153 (1979)

10) WONG, P.P. and EVANs, H.J., Poly-p-hydroxybutyrate utilization by soybean (Glycine max Merr.) nodules and assessment of its role in maintenance of nitrogenase activity, Plant Physiol, 47, 750-

755 (1971) 11) BOLAND, M.J., HANKS, J.F., REYNOLDS, P.H.S., BLEVINS, D.G., TOLBERT, N.E., and SCHUBERT,

K.R., Subcellular organization of ureide biogenesis from glycolytic intermediates and ammonium in nitrogen-fixing soybean nodules, Planta, 155, 45-51 (1982)

12) NISHIMURA, M. and BEEVERS, H., Hydrolases in vacuoles from castor bean endosperm, Plant Physiol.,

62, 44-48 (1978) 13) TAJIMA, S., YATAZAWA, M., and YAMAMOTO, Y., Allantoin production and its utilization in relation

to nodule formation in soybeans. Enzymatic studies, Soil Sci. Plant Nutr., 23, 225-235 (1977) 14) NAWA, Y. and ASAHI, T., Rapid development of mitochondria in pea cotyledons during the early

stage of germination, Plant Physiol., 48, 671-674 (1971) 15) MACRAE, A.R., Isolation and properties of a 'malic' enzyme from cauliflower bud mitochondria,

Biochem. I., 122, 495-501 (1971) 16) JOHN, C.D. and GREENWAY, H., Alcoholic fermentation and activity of some enzymes in rice roots

under anaerobiosis, Aust. I. Plant Physiol., 3, 325-336 (1976) 17) DILWORTH, M.J., Leghemoglobins, In Methods in Enzymology, Vol. 69, ed. by A.S. Pietro, Academic

Press, New York, 1980. pp. 812-823 18) BENSADOUN, A. and WEINSTEIN, D., Assay of proteins in the presence of interfering materials,

Anal. Biochem., 70, 241-250 (1976) 19) NISHIMURA, M., DOUCE, R., and AKAZAWA, T., Isolation and characterization of metabolically

competent mitochondria from spinach leaf protoplasts, Plant Physiol., 69, 916-920 (1982) 20) BERGMAN, A., GARDESTRBM, P., and ERICSON, I., Method to obtain a chlorophyll-free preparation

of intact mitochondria from spinach leaves, Plant Physiol., 66, 442-445 (1980) 21) DERocHE, M.E., CARRAYOL, E., and JOLlVET, E., Phosphoenolpyruvate carboxylase in legume

nodules, Physiol. veg., 21, 1075-1081 (1983) 22) GADAL, P., Phosphoenolpyruvate carboxylase and nitrogen fixation, Physiol. Veg., 21, 1069-1074

(1983) 23) PETERSON, J.B. and EVANS, H.J., Properties of pyruvate kinase from soybean nodule cytosol, Plant

Physiol., 61, 909-914 (1978)

Dow

nloa

ded

by [

Uni

wer

syte

t War

szaw

ski]

at 0

0:18

15

Oct

ober

201

4