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Nitrogen fixation by tissues of leguminous and nonleguminous plants

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Title:
Nitrogen fixation by tissues of leguminous and nonleguminous plants
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Sloger, Charles, 1938-
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English
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97 leaves : ill. ; 28 cm.

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Subjects / Keywords:
Alkynes ( jstor )
ATMs ( jstor )
Atoms ( jstor )
Flasks ( jstor )
Incubation ( jstor )
Nitrogen ( jstor )
Nitrogen fixation ( jstor )
Nodules ( jstor )
Root nodules ( jstor )
Soybeans ( jstor )
Botany thesis Ph. D
Dissertations, Academic -- Botany -- UF
Nitrogen -- Fixation ( lcsh )
Plants, Effect of nitrates on ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1968.
Bibliography:
Includes bibliographical references (leaves 88-95).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Charles Sloger.

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NITROGEN FIXATION BY TISSUES OF LEGUMINOUS AND NONLEGUMINOUS
PLANTS










By
CHARLES SLOGER


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY










UNIVERSITY OF FLORIDA
1968













ACOWLEDGEMENT S


The author wishes to thank Dr. W. S. Silver, cochairman of the supervisory committee, for his encouragement, guidance, helpful suggestions, and incisive criticisms in the conduct of the research and in the preparation of the manuscript. Dr. G. J. Fritz, chairman of the supervisory committee, provided advice and valuable criticisms in the preparation of the manuscript.

Appreciation is extended to the committee members, Dr. E. S. Ford and Dr. M. Fried, who gave of their time and advice.

The Department of Botany, University of Florida is thanked for an assistantship. The research was made possible by an assistantship supported by the National Institute of Health Research Grant G1-08577. Dr. M. Tyler, chairman of the Department of Bacteriology, University of Florida as well as the faculty of the Department are thanked for use of their facilities.

Marcia Sloger assisted immeasurably in the preparation of the manuscript. Her confidence is gratefully acknowledged.


ii













TABLE OF CONTENTS


Page


ACKNO\LEDGEMENTS . . . . . . . . . . o LIST OF TABLES . . . . . . . . . . . .

LIST OF FIGURES. . . . . . . . . . . .

INTRODUCTION . . . . . . . . . . . . .

REVIEW OF THE LITERATURE . . . . . . .


Origin of the root nodules External appearance of the
nodules . . . . . . .
Internal appearance of the
nodules . . . . . . .


. . .
root . . IrOo


. . . . . . . . . . . .


. . . . . . .


Endophytes of the root nodules . . * . .
Site of N2 fixation . . . . . . . . . .
Hydrogen evolution by the root nodules .
Effect of partial pressure of N2 . . . .
Substrates reduced by the root nodules .
Effect of partial pressure of 02 . . . .
N2 fixation by excised root nodules N2 fixation by homogenates and breis
of root nodules . . . . . . . .

MATERIALS AND METHODS . . .. . . . .

Botanical species . . . . . . . . .
Botanical techniques . . . . . . . . .
Preparation of homogenates and breis Preparation of incubation atmospheres Incubation of samples. . . . . . . . .
Extraction of acid soluble nitrogen . .
KJeldahl method . . . . . . . .
Conv.:rsion of ammonia to nitrogen gas
Colorimetric analysis of total nitrogen.
15
N technices.. . . . . . .
Preparation of 15 . . ... . . .
N method . . . . . . . . . . .
Mass soectromet-v . . . . . . . . . . .
Calculation of I N content . . . . . . .
Gas chromatographic analysis for ethylene
and acetylene . . . . . . . . . . . .


iii


. . . . . . ii


v


. . . . . . vii


1

4

5

7

7
9
10

12 13 13
14

15

17

17
18
20 21 22 22 22 23 23 23 23
24 24 25

25


. .


. .


. .


. . . . . .






TABLE OF 0ONTETS -- Continuca


Page

RESULTS . . . . . . . . . . . . . . . . . . . . . 28

Appearance of root nodules . . . . . . . . 28 N fixation by excised root nodules . . . . 28 ifect of partial pressure of N2 . . . . . 32 Effect of partial pressure of 02 * .* * * 35 Storage of intact nodules . . . . . . . . . 39 Variation among replicate samples . . . . . 39
N2 fixation by nodular homogenates of
1,;. cerifera . . . . . . . . . . . . . . 39
Site of N2 fixation by homogenates. .. . . 47
Acetylene reduction by nodules of M.
cerifera . . . . . . . . . . . - - . . 51
Acetylene reduction by nodules of
various plants . . . . . - .- .a 54
Effect of acetylene concentration on
ethylene production. . . . . . . . . . . 61
N2 inhibition of the reduction of
acetylene. . . . . . . . . . . . . . . . 63
Reduction of acetylene by individual
nodules . . . . . . . . . . . . . . . . 66
Reduction of acetylene by breis . . . . . . 68

DISCUSSION . . . . . . . . . . . . . . . . . . . . 72

SUI7rEARY . . . . . . . . . . . . . . . . . . . . . . 86

LITERATURE CITED . . . . . . . . . . . . . .. . 88

BIOGRAPHICAL SKETCH . . . . . . . . . . . . . . .. 96


iv












LIST OF TABLES
TABLE Page

1 Extent and Distribution of Nodulated
Nonleguminous Angiosperm . . . . . . . . . 6

2 Test for N2 Fixation by Detached Nodules
of 7yrica cerifera . . . . . . . . .. . . 30

3 15N Content of Acid Soluble and Acid
Insoluble Fractions of Nodules 1f
M. cerifera after exposure to N2 . . 31

4 Comparison of N Fixation Between Nodules
with Epidermis Present and Nodules with
Periderm present . . . . . . . . . . . . . 34

5 Studies on Storage of Excised Nodules of
1. cerifera . . . . . . . . . . . . . . . . 40

6 Variability Among Samples Within One
Experiment and Among Several Experiments. . 41

7 Effect of Na S 0 on N2 Fixation by Root
Nodule Holotoates . . . . . . . . . - . - 43

8 Effect of Supplements on N2 Fixation by
Nodular Homogenates . . . . . . . . . . . . 45

9 Effect of Oxygen on N Fixation by Intact
Nodules and Supplevented Nodular
Homogenates . . . . . . . . . . . . . . . . 46

10 Effect of 02 and Na2 S204 on N2 Fixation by Supplemented Homogenates . . . . . . . . . 48

11 Effect of pH on N2 Fixation by Homogenates . . 49

12 N2 Fixation by Supplemented Homogenate Fractions * . - . - . - . - - * . . . . . 50


v








LIST OF TABLES -- Continued
TABLE Page

13 Reduction of Acetylene to Ethylene by Various Plant Tissues . . . . . . . . . . 60

14 Acetylene Reduction by Individual Soybean Nodules . . . . . . . . . . . . . . 67

15 Reduction of Acetylene by Breis of Soybean Nodules Prepared with
Polyvinylpyrrolidone and Ascorbate . . . . . 70

16 Reduction of Acetylene by Breis of Myrica cerifera Prepared with PolyvinylpyrroLidone
and Ascorbate . . . . o . . . . . . . . . 71


vi












LIST OF FIGURES

Figure Page

1 Time course of N2 fixation by excised
nodules of Y. cerifera. . . . . . . . 33

2 Effect of pN on N incorporation by
excised nodules of Yyrica cerifera. . 36

3 Lineweaver-Burk plot to determine the
Michaelis constant for nitrogen fixation by whole nodules of M.
cerifera. . . . . . . . . . .~ . . * 37

4 Effect of 0 on N2 fixation by detached
nodules o Myrica cerifera. . . . . . 38

5 Time course of acetylene reduction to
ethylene by nodules of M. cerifera
at 0.1 atm and 0.2 atm 02 . * * * * * 53

6 Relationship of ethylene production to
acetylene disappearance by nodules
of M. cerifera. * . - . - - . . . . 55

7 Time course of reduction of acetylene
to ethylene by excised nodules of
Casuarina eouisetifolia. . . . . . . 56

8 Relationship of ethylene production to
acetylene disappearance by soybean
nodules. . . . . . . . . * . . . . * 57

9 The effect of partial pressure of acetylene on ethylene production by nodules
of Yyrica cerifera. . . . . . . . . . 62
10 Effect of N2 on ethylene production from
acetylene by nodules of y. cerifera. 64


vii










LIST OF FIGURES -- Continued


Figure Page

11 Effect of pN2 on acetylene reduction by
soybean nodules. . . . . . . . . . . . . 65

12 Comparison of time courses of N fixation
by excised nodules of legumeg and nonlegumes. . . . . . . . . . . . . . . . . 74


viii













INTRODUCTION

Most organisms readily assimilate combined nitrogen, and with it synthesize proteins and other substances vital to their existence. However, the quantity of available combined nitrogen is relatively small, whereas an abundance of nitrogen is present in elemental form in the atmosphere as molecular nitrogen, N2. Thus, a critical step in the nitrogen cycle is the replenishment of the supply of combined nitrogen by nitrogen fixation--the conversion of molecular nitrogen to a fixed form, ammonia.

Both microorganisms and higher plants are involved in nitrogen fixation. Most plants assimilate nitrate and ammonia; only a few, molecular nitrogen. In all authenticated cases in which higher plants assimilate N2' some microorganism is living symbiotically with the higher plant (Bond, 1967). These symbiotic relationships are expressed as leaf nodules, mycorrhizal associations on roots, and commonly as root nodules. Among the angiosperms which can form root nodules, there has been no substantial evidence presented which shows that either of the symbionts, the unnodulated plant or the free living form of the endophyte, have the ability to fix N2 by itself. Because most of the fixed nitrogen in the biosphere is from symbiotic sources






2

(Stewart, 1966), it is important to understand the process of symbiotic nitrogen fixation.

As recently as 1956, Kamen (1956) stated, "Now, there has been a great literature on nitrogen fixation mostly at the physiological level. At the biochemical level there is very little." The information on nitrogen fixation obtained by physiological studies of intact organisms has been limited and difficult to understand. A turning point in the study of nitrogen fixation occurred when Carnahan et al. (1960) reported a method for preparing and maintaining a cell-free N2 fixing extract from a micro-organism. From that time, the elucidation of the biochemistry of nitrogen fixation has rapidly advanced. In contrast, information on symbiotic nitrogen fixation continued to remain at the physiological level.

It is important to point out that knowledge of biochemical aspects of symbiotic nitrogen fixation was almost completely lacking when the present investigation was started in September, 1964. The primary reason is that leguminous nodules lose all capacity to fix N2 when they are fragmented. Even attempts to enhance N2 fixation in sliced soybean nodules by the addition of a variety of substrates, cofactors, crude extracts, and reducing agents were unsuccessful (Aprison et al., 1954). Furthermore, previous to September, 1964, literature on symbiotic nitrogen fixation by nonleguminous angiosperms pertained only to the physiology of the intact root nodule. From the point of view of the








present investigation, it should be stressed that several contributions to our understanding of symbiotic nitrogen fixation by homogenates of root nodules were not available when this investigation was started in September, 1964, but were published after that time by Sloger and Silver (1965), Bergersen (1966b), Bergersen (1966c), Koch et al. (1967a), and Koch et al. (1967b).

The primary objective of the present investigation was the study of nitrogen fixation by excised root nodules and nodular homogenates of a native nonleguminous angiosperm, Yyrica cerifera L. Firstly, a study was made to determine whether the excised nodules of M. cerifera fixed N2, and if so, to determine optimum conditions for nitrogen fixation. Secondly, a study was made to determine requirements for nitrogen fixation by nodular homogenates. Finally, a study was conducted to establish whether the excised nodules would reduce acetylene to ethylene, and to evaluate the acetylene reduction method as an assay for nitrogenase activity.













REVIEW OF THE LITERATURE


The subject of biological nitrogen fixation has

been reviewed recently by Burris (1966). Hardy and Knight

(1967a) have prepared a review pertaining to the biochemistry and postulated mechanisms of nitrogen fixation. Stewart (1966) compiled information on nitrogen fixation by plants. Recently, Stewart (1967) has discussed the role of nitrogen fixing plants. The present discussion will be confined to the literature on symbiotic nitrogen fixation by intact and fragmented root nodules of legumes and nonleguminous angiosperms up to December, 1967. A brief discussion will be given about the development of the root nodules as it relates to the nitrogen fixing symbioses.

The family Leguminosae makes up one group of nodulated plants. Allen and Allen (1958) reported that root nodules have been found on approximately 89 per cent of the species studied, but this represents only a small fraction of the total known species. The endophytes of the root nodules have been identified as members of the bacterial genus Rhizobium.

Nonleguminous plants associated with N2 fixation
are in diverse taxonomic groups. They may be separated on the basis of the nature of the symbionts in the association.


4







5
Bond (1967) suggested the categories nonleguminous angiosperms or actinomycetal associations, mycorrhizal associations, algal-cycad associations, and bacterial-leaf associations. These categories are based upon available evidence and may be changed after further research. In this review and study, the term "nonleguminous" will refer to those angiosperms whose nodules harbor endophytes believed to be actinomycetes. There are 13 genera of plants in this group, as shown in Table 1, and it seems likely that the list of species and genera will be increased.



Origin of the root nodules

Tissues comprising the nonleguminous nodule originated in the pericycle of the root according to Fletcher (1955) and Taubert (1956). Thus, the nodules are considered modified lateral roots. In contrast the tissues comprising the leguminous nodule originate from the inner cortex of the root. Wipf and Cooper (1940) made the observation that within this region, nodule initiation is dependent on the presence of tetraploid cells in a normally diploid plant. Rhizobial infection takes place through a root hair by means of an infection thread. The infection thread penetrates the cortical cells of the root, and as it approaches the tetraploid cells, it stimulates them and adjacent diploid cells into meristematic activity resulting in the formation of a nodule.






6
TABLE 1
EXTENT AND DISTRIBUTION OF NODULATED
NONLEGUMINOUS ANGIOSPERMdS


Number of Present Number of species
Genus species distribution recorded
in genusa -o bear nodules


Coriara Alnus Myricab Casuarina Elaeagnus Hippophae Shepherdia Ceanothus Discaria


15 Mediterranean to Japan,
New Zealand, Chile to Mexico
35 Europe, Siberia, North America,
Japan, Andes

35 Many tropical, subtropical
and temperate regions

45 Australia, tropical Asia,
Pacific Islands


45


Asia, Europe, North America


1 Asia, Europe, from Himalayas
to Arctic Circle
3 North America


55 10


North America Andes, Brazil, New Zealand, Australia


4 Arctic and mountains of
north temperate zone


Purshia Cercocarpus ArctostaphzQjs


2 North America


20

40


North America


Northwest and Central America, Eurbpe, Asia


a Estimates prepared by Mr. H. K. Airy Shaw, Royal Botanic Gardens, Kew for the forthcoming new edition of Willi.'s Dictionary of Flowering Plants and Ferns

b Including Comptonia


From Bond (1967) page 108.


12 25 11


14


9

1

2


30


1


1


2


1







7
External appearance of the root nodules

During early stages of development nonleguminous
nodules resembled those of a typical legume (Bond, 1963). Later, nodule lobes branch frequently and elongate to form a clustered structure. At maturity, the nodule lobes of Yyrica, Casuarina, and Alnus are 2 to 6 mm long, 0.8 to 1 mm wide, and lack root caps and root hairs. The nodules are perennial and may range in diameter from a few mm to 150 mm.

The nodules of most legumes occur as small (less
than 1 cm in diameter), spherical, club-shaped, or branched structures. Leristematic activity is frequently apical in position, but soybean nodules have no distinct growing point.

Both water cultured and field grown Myrica and Casuarina have nodules which differ from those of other plants because of negatively geotropic rootlets extending from each lobe (Bond, 1957).


Internal appearance of the root nodules

The mature nonleguminous nodule shows a number of
differences from the leguminous type. It has an endophytecontaining cortex and a central vascular cylinder (Fletcher, 1955; Furman, 1959; Becking et al., 1964; Silver, 1964). The nodule structure is derived from an enlarged cortical region with certain host cells containing dense growths of endophyte. The smaller, uninfected cortical cells often contain starch and tannins. In contrast, the leguminous nodule has a narrow cortical region and an endophyte-








containing central region (Allen and Allen, 1958). Vascular tissue lies between the cortex and the central region and is surrounded by an endodermis, which may function as a diffusion barrier. The leguminous nodule is soft and is easily crushed. Nonleguminous nodules are woody; only young stages are relatively soft.

The central region of leguminous nodules which are fixing N2 contains a red hemoprotein pigment, hemoglobin (leghemoglobin). Ineffective leguminous nodules or the nodules not fixing N2 do not contain the pigment. Hemoglobin is found within the infection vesicles of infected host cells but outside the bacteroids (Bergersen, 1966a). The recent demonstrations of N2 fixation by isolated bacteroids (Bergersen and Turner, 1967) and by cell-free extracts of bacteroids (Koch et al., 1967a; Koch et al., 1967b) in the absence of hemoglobin have eliminated conclusively hemoglobin as an integral part of nitrogenase. Hemoglobin is said to bind 02 and N2, and therefore may function in the transport of these molecules to bacteroids; also hemoglobin, by binding 02, may prevent 02 inhibition of N2 fixation (Burris, 1966).

Nonleguminous nodules have been shown to contain

hemoglobin by Davenport (1960). He used a microspectroscope to detect hemoglobin in whole nodules of Casuarina cunninghamiana, Alnus glutinosa, and Myrica gale. Also the hematin content of the nodules was eight to eleven times higher than in the root cortex. However, Moore (1964) failed to verify this.







9


Endophytes of the root nodules

The identity of the endophytes in nonleguminous
nodules is unsettled. Most investigators using modern techniaues have concluded that the endophytes were actinomycetes

(Becking et al., 1964; Gardner, 1965). Silver (1964) confirmed the filamentous nature of the endophyte of root nodules

of MIyrica cerifera with an electron microscope and considered it an actinomycete. No confirmed isolation of the infecting endophyte has been achieved. The infected host cells are enlarged and devoid of almost all host cell contents (Silver, 1964). The site of N2 fixation is not known but may be the infected host cell or the endophyte.

Details of the initial infection by rhizobia, development of infection thread, and development of root nodules are well known for legumes. Changes of the fine structure of the host cells which accompany nodule development have been reported by Goodchild and Bergersen (1966), Dart and Mercer (1963) and Jordan et al. (1963). Their work may be summarized as follows. At maturity, infection vesicles each containing four to eight bacteroids fill the host cells of the central region of the nodule. The membrane of the infection vehicle is believed to originate from the host cell. The differentiation of rhizobia to bacteroids is associated with the tissues's ability to fix nitrogen. At this time, the cytoplasm of the host cell loses ribosomes and endoplasmic reticulum, whereas mitochondria and the nucleus remain.






10


Site of N2 fixation

Because only nodulated plants could maintain growth in a nitrogen-free medium, it was assumed that N2 fixation occurred in the nodules. The occurrence of biological nitrogen fixation was proven unequivocally by the use of the 15N method (Burris and Miller, 1941). The first demonstration of consistent 15N enrichment by excised soybean nodules which had been exposed to an atmosphere enriched with 15Nw2 as made by Aprison and Burris (1952). Magee and Burris (1954) demonstrated N2 fixation by excised nodules from nine different legumes representing eight cross inoculation groups by the 15N method.

Bond (1955) showed that nodulated plants of Alnus
glutinosa, Myrica gale, and Hippophae rhamnoides exposed to 15N2 had the greatest enrichment in their nodules and that excised nodules of A. plutinosa fixed 1N2. He also showed that nodules which were attached to native growing A. glutinosa fixed N 2 (Bond, 1956). The 15N method has been used to demonstrate fixation by Qetached nodules of the following species of nonlegumes: Casuarina cunninghamiana, Ceanothus azureus, and Shepherdia canadensis (Bond, 1957); Coriaria arborea (Harris and Morrison, 1958); Discaria toumatou (Morrison, 1961); Comptonia peregrina (Ziegler and Huser, 1963); Myrica cerifera (Sloger and Silver, 1965); 12 species of Ceanothus (Delwiche et al., 1965), and Alnus ;iorullensis (Rodriguez-Barrueco, 1966). It is now accepted that the nodules are the sites of fixation in nonleguminous angiosperms.





11


The site of N2 fixation inside legu:.inous nodules has been suggested. Bergersen (1960) reported that N2 fixation by soybean nodules was located in some component of a membrane fraction which consisted largely of fragments of the intracellular membrane envelopes surrounding the bacteroids. However, R. Klucas repeated these experiments, reported by Delwiche (1966) and Silver (1967), and found that the soluble portion of the soybean nodules consistently contained the highest enrichment after short exposure to 15N2. Kennedy et al. (1966) suggested that bacteroids within serradella nodules incorporated 15T2. Recently, Bergersen and Turner (1967) stated that a bacteroid fraction isolated from soybean nodules had N2 fixing activity. Koch et al. (1967a) and Koch et al. (1967b) reported that cell-free extracts of bacteroids isolated from soybean nodules fixed N2.


Hydrogen evolution by the root nodules

The evolution of hydrogen, H2, by leguminous nodules was detected by Hoch et al. (1957). Later, Hoch et al. (1960) reported that H2 evolution by soybean nodules was inhibited by N2 and nitrous oxide (N20), but was insensitive to carbon monoxide (CO). Also Hoch and co-workers reported that during N2 fixation an exchange occurs between deuterium and hydrogen atoms--this exchange reaction occurs between deuterium and endogenous H donors, and results in the formation of the species HD. They observed that the exchange reaction was inhibited by CO and N20, but







12
was activated by N2. They suggested that hydrogen evolution

and the exchange reaction expressed separate activities of

the enzyme nitrogenase. Bergersen (1963) verified these

observations. Dixon (1967) suggested that pea nodules have1

two hydrogenases, one associated with nitrogenase and one associated indirectly with N fixation. However, nodules did not evolve hydrogen under normal conditions.

Intact nodules of the nonlegumes, Alnus glutinosa,

Alnus rugosa and Elaeagnus commutata did not evolve hydrogen when tested in an atmosphere of 80 per cent helium and 20 per cent oxygen (Moore, 1964).



Effect of partial pressure of N

Nitrogen fixation by nodules is influenced by the availability of N2. Burris et al. (1955) computed the Michaelis constant, the partial pressure of N2 supporting half maximum fixation, for sliced soybean nodules from a thorough statistical analysis of the data obtained in many experiments. The Michaelis constant was 0.02 f 0.004 atm N2 for this leguminous tissue. Maximum fixation was observed above 0.10 atm N2. In contrast, Bond (1959) reported that whole excised nodules of Alnus glutinosa achieved maximum fixation at 0.25 atm N2. It appears that neither the partial pressure of N2 in the atmosphere, nor the partial pressure of N2 within a nodule would be rate limiting.







13


Substrates reduced by the root nodules

N20, which is structurally similar to molecular N2' has been shown to be an inhibitor of N2 fixation by soybean nodules (Hoch et al., 1960). They showed that N20 was reduced to N2 by the intact nodules. Koch and Evans (1966) and Sloger and Silver, reported by Silver (1967),noted that acetylene was reduced to ethylene by nodules of legumes and nonlegumes.



Effect of partial pressure of 0

The effect of oxygen on symbiotic nitrogen fixation has received much attention. Burris et al. (1955) reported that N2 fixation by sliced soybean nodules increased with 02 concentration until maximum fixation was reached at 0.5 atm 02* Above this 02 concentration there was a marked decrease in N2 fixation. The authors believed that these results indicated a very close relation between fixation and respiration, and that oxygen primarily affected the latter process. Bond (1957) showed that oxygen was essential for N2 fixation by nonleguminous nodules. Bond (1961) reported that maximal N, fixation by nonlegwminous nodules was reached at 0.20 to 0.25 atm 02* Above this 02 concentration there was a decrease in N2 fixation. Decreased N2 fixation at high oxygen concentrations appears to be a characteristic of root nodules in general. A possible reason for this was suggested by Bergersen (1962a). He






14


suggested that oxygen competed with nitrogenase for endogenous hydrogen atoms. The competition was possible because hemoglobin in soybean nodules was shown to be completely oxygenated at 0.5 atm 02, the point where inhibition of N2 fixation occurred (Bergersen, 1962b). Bond (1964) suggested that oxygen inhibited N2 fixation by nonleguminous nodules competitively. Bergersen (1963) noted that oxygen affected hydrogen evolution and the exchange reaction by excised soybean nodules in the same manner as for N2 fixation. However, 02 may have little, if any, direct role in the enzymatic process of N2 fixation since some anaerobes fix N2.



N fixation by excised root nodules

Upon excision from the root, nodules lose their capacity for nitrogen fixation within a matter of hours. Aprison and Burris (1952) found that in the time course of N2 fixation by excised soybean nodules the rate was constant for two hours, with only slight fixation occurring thereafter. Dixon (1967) found that excised pea nodules within minutes lost the capacity to fix N2, but when nodules were left attached to a piece of the primary root the time course of fixation was similar to that of excised soybean nodules. The rapid loss of activity by leguminous nodules is a limiting factor in research.

In contrast, nodules of Alnus glutinosa remained

active for 12 hours after detachment, and nodules of Hiprophae rhamnoides remained active for nearly 24 hours,






15


according to Bond (1957). Apparently, fixation is less drastically affected by nodule excision from the root in the nonlegumes than in the legumes. This extended period of activity would be especially desirable in experiments with fragmented nodules.


N fixation by homogenates and breis of root nodules

Only recently have techniques been found which can

stabilize nitrogenase activity in fragmented nodular tissues. Sloger and Silver (1965) noted that anaerobically prepared and buffered homogenates of nodules of M. cerifera supplied with oxygen and Na2204, fixed N2 at over 4 per cent the rate of that of intact nodules. Sloger and Silver (1966) reported that similar nodular homogenates fixed N2 at about 50 per cent the rate of that of intact nodules, when supplied oxygen, an ATP-generating system, and Na2S204. Bergersen (1966b) reported that breis of soybean nodules prepared anaerobically in a buffered sucrose solution would fix N2 only if oxygen were present during incubation. Later Bergersen (1966c) reported that oxygen concentrations up to 0.10 atm stimulated N2 fixation by breis of soybean nodules. He also reported that an exogenous ATP-generating system slightly increased fixation. Bergersen and Turner (1967) showed that nitrogenase activity was present in bacteroids isolated from soybean breis. Another technique for preparing active breis of soybean nodules was reported by Xoch et al. (1967a). These breis, when prepared anaerobically in the presence of






16

buffered ascorbate and insoluble polyvinylpyrrolidone (PVP), fixed N2 and reduced acetylene only when oxygen was present. Koch et al. (1967b) obtained a cell-free extract with nitrogenase activity from bacteroids of active breis. This enzyme system required an exogenous ATP-generating system and Na2 204, but no oxygen.
















Botanical species

Myrica cerifera L., commonly called southern wax

myrtle, was collected from native habits near Gainesville, Florida.

Casuarina equisetifolia J. R. and G. Forst, commonly called Australian pine, was collected by the author at Sanibel Island and Haulover Beach State Park, Florida.

Casuarina cunningiamiana Miq. was collected by Dr. 71. S. Silver from Deland, Florida.

Podocarpus macrophylla D. Don. was grown in the

greenhouse and at the time of the experiment was two years old.

Pinus elliottii Engelm. was obtained as two year

old seedlings from plots in the Austin Cary Memorial Forest, courtesy of Dr. Wayne H. Smith.

Psychotria punctata Vatke, also known as Psychotria bacterioohila (Centifanto and Silver, 1964), belonged to the collection of plants used by Centifanto (1964).

Glycine max Terr. 'Hampton', commonly called soybean, was obtained as seeds coated with a mixed inocultum from Dr. Keull Hinson. Plants from the Uniiversity of Florida


17









Agricultural Experiment Station farm plots were also donated by Dr. Hinson.

C-lycine max Terr. 'Chipewa' was obtained as seeds

from Dr. H. J. Evans, Univobr-ity 3heur, Corvallis, Oreg.

Luzinus albus L. was obtained from the University of Florida Agricultural Experiment Station farm plots.

Crotolaria spectabilis Roth. was collected from native habitats on the campus.

Voucher specimens have been filed in the University of Florida herbarium for the following species: M. cerifera, C. equisetifolia, C. cunninghamiana, P. macrophylla, P. punctata, and C. spectabilis.

Klebsiella rubiacearum (strain K4), a bacterial

endophyte of leaf nodules of P. punctata, was grown on nitrogen-free mineral broth according to the methods of

Neelands (1967).



Botanical techniques

Chipewa soybeans were grown in a greenhouse according to the methods of Koch and Evans (1966) and in a prepared field plot. Hampton soybeans were gro-n in a prepared field nlct in addition to other sources of the plant mentioned earlier. A nitrogen-free nutrient solution described by Centifanto (1964) was supplied to the greenhouse grown Chipewa soybeans.








Nodules of C. ceifera 'ere collected from plants, where they had been exposed to daily weather changes and the annual climatic patterns. The plants were considered heterogeneous because of their varied physiological ages. Environmental conditions affecting the native plants were not controlled. The selection of plants was limited to those 0.5 to 2 meters tall, which represented plants two to ten years old.


All plant material was carefully dug up so as not

to detach nodules from the roots. To facilitate the handling

of Myrica, stems were cut 2 to 3 dm above ground level and discarded, while the remaining portion was placed in appropriate containers for transport to the laboratory. Myrica root systems were washed at the greenhouse first. Roots

with nodules were cut from the plant and then taken to the laboratory. Greenhouse grown plants were removed from pots, washed with tap water, and quickly taken to the laboratory.


At the laboratory nodules were excised, washed in

tap water, and blotted on paper towels. Obviously senescent, dead, and decaying nodules were discarded. Nonleguminous nodules larger than 1 cm in diameter .-;ere divided into two to six pieces. The nodules from one species were lumped together and randomized so that each flask received an equivalent fresh weight of tissue.






20

Preparation of homogenates and breis

Homogenates of nodules of M. cerifera were prepared in an Omnimixer (Servall Corp., Norwalk, Conn.). Nodules (over 10 grams) and buffer solution were placed in a grinding chamber, which had two small holes for gas entrance and exit. The chamber was purged with helium for 10 minutes prior to and during grinding. Nodules were ground for one minute at high speed. Then homogenates were transferred to flasks which were being flushed with helium. Homogenates were kept anaerobic until the flasks were attached to the gassing manifold. For smaller quantities of nodules, homogenates were prepared in a 50 ml cellulose nitrate centrifuge tube, which was attached to the Omnimixer by an adapter. Helium was flushed through the chamber for several minutes prior to grinding. Subsequent handling was the same as that described above.

Breis obtained from nodules of soybean and M. cerifera were prepared according to the method of Koch et al. (1967a).

Cacodylate buffer solutions were adjusted to pH 7 by 1 N KOH and then purged with hydrogen for 15 minutes. The buffer was prepared fresh for each experiment.

Solutions of sodium dithionite, Na2S204, were prepared anaerobically by tipping the chemical from a side arm of a flask into water and a predetermined amount of 1 N KOH to give a solution of pH 7. The water and KOH were purged previously with hydrogen gas. Na 22 4 solutions were prepared immediately prior to use.






1

Preparation of incubation atmospheres

The following procedure was used for 15N experiments. Excised nodules were placed in 50 ml flasks, while homogenates were placed in 70 ml flasks equipped with two side arms. The flasks were then attached to a gassing manifold.

The closed system was evacuated by a vacuum pump and alternately filled with helium three times. Replacement of gases to one atmosphere pressure and to a known composition was by the method of Burris (Umbreit et al., 1951, p. 45). The gases were added in this order: 15N oxygen, and helium.

For the acetylene reduction experiments, test material was placed in 70 ml calibrated flasks equipped with two side arms. Rubber serum stoppers were used in the side arms. The flasks were then attached to a calibrated mercury-filled manometer. Both arms of the manometer were connected by a glass tube in the form of a reverse h with a ball adaptor at the top (Umbreit et al., 1951, p. 45). The adaptors of the manometer were attached to a gassing manifold by a ball and socket connection. With this method a series of eight manometers could be treated at one time. The system was evacuated and filled with helium three times, and then filled in order with acetylene, oxygen, and helium. The composition of gases was determined according to the method of Burris (Tmbreit et al., 1951, p. 45).

The tanks of oxygen, helium, nitrogen, hydrogen, argon, and acetylene were purchased from Yatheson Co.,






22

Atlanta, Ga. Acetylene was slowly sparged through concentrated H2s04 to oxidize acetone and collected in a container for use in preparation of incubation atmospheres.



Incubation of samples

All flasks were incubated in a 30 C Warburg manometer water bath. The shaking rate was 100 oscillations per minute.



Extraction of acid soluble nitrogen

Acid soluble nitrogen was extracted from nodules

and homogenates by grinding the tissue with 10 ml of 3 N HCl for three minutes. The resulting homogenate was centrifuged at 5000 rpm for three minutes in a Clay-Adams clinical centrifuge. The supernate was used for the determination of acid soluble nitrogen; and the residue, for acid insoluble nitrogen.



Kjeldahl method

The method used was adapted from Burris and Wilson (1957). Samples were placed in 100 ml Kjeldahl flasks and

5 ml of concentrated H2so4 was added to each. One glass bead and one mercury catalyst tablet (British Drug House Ltd., Poole, England) were also added. The Kjeldahl flasks were heated gently at first so that charring of the sample was not violent. The digestion proceeded for 16 hours, at which time the solution was clear. Ammonia was distilled by a







23

steam generating apparatus (W. Buchi Manufacture, Flawil, Switzerland) for two minutes into 25 ml of 0.01 N HCl. The distillate was used for conversion of ammonia to nitrogen gas or for determination of ammonia. The mercury catalyst tablets and the prolonged digestion prevented contamination by methylamine (Glover, 1956).



Conversion of ammonia to nitrogen gas

The procedure for the conversion is described in the section Preparation of 15N gas. The distillate was evaporated to 10 ml prior to conversion.



Colorimetric analysis of total nitrogen

The method used was adapted from Burris and Wilson (1957). The distillate was evaporated to 10 ml and then 0.5 ml samples were transferred to test tubes. Water was added to make a total of three ml. Two ml of Nessler's reagent and 2 ml of 2 N NaOH were added. The tubes were allowed to stand for 20 minutes at 20 C, and read in a KlettSummerson colorimeter with a No. 47 filter. A standard curve was prepared for 0 to 40 pag of ammonia N.


15N techniques

Preparation of 15N --15NH NO containing 30 atom 2* 4 3,30ao
per cent 15N excess and 95 atom per cent 15N excess was obtained from the Office National Industriel de l'Azote, Paris, France and from Isomet Co., Palisades Park, N. J.







24
15 ' 5
respectively. The gas N2 was generated from the I NH NO3 by alkaline hypobromite oxidation according to the method of Sims and Cocking (1958) in a Toeppler pump system, as described by Rittenberg (1946). Release of was prevented

by adding 0.1 per cent potassium iodide (KI) to the alkaline hypobromite solution before the addition of bromine. A liquid nitrogen trap was used to condense impurities from the gas before collection in a glass bulb.

15N method.--Four steps are involved in the method:

(1) exposure of the test sample to a gas mixture enriched with 15N2 for a time interval; (2) liberation of the nitrogen in the sample and collection in the form of ammonia by the Kjeldahl procedure; (3) reaction of ammonia with alkaline hypobromite to form gaseous nitrogen, which is collected in mass spectrometer bulbs by the Toeppler pump system; (4) admission of the gas sample to the vacuum chamber in the mass spectrometer and the measurement of isotopic abundance.



Yass spectrometry

Nitrogen gas was analyzed in a Consolidated Electrodynamics Corp. (Pasadena, Calif.), model 21-130 recording

mass spectrometer. Normal operating conditions recommended by the manufacturer ILerc followed: the ioniinn7 current



1Con-olidateC 'lectrodynamics Corp., "Operatio and maintenance manuall for Type 21-130 pass Spectrometer," T, a-de n , tLe.






25

was 20 paa. and the ionizing voltage was 68 volts. In all mass spectrometric analyses, sufficient 12 gas was introduced into the mass spectrometer so that the pressure in the reservoir was 20.46 microns of mercury.



Calculation of 15N content

The abundance of 15N in samples in atom per cent was calculated according to Rittenberg (1946) by the following equation:


atom per cent 15N 100 where R = 28
21H+1 ' -2r

and I28 and I29 represent the intensities of the ion beams of mass 28 and mass 29.

The results of all 15N experiments were expressed as atom per cent 'N excess which was calculated by subtracting the atom per cent 15N for control tissue from the atom per cent 15N for test material. Values greater than

0.015 atom per cent 15NT excess were assumed to indicate that N2 fixation had occurred. This value is the same as that used by Burris and Wilson (1957). A value as high as
1 r
0.015 atom per cent N excess was necessary because of the relatively large variation in results obtained from repeated analyses of samples of tank N2 and from daily analyses of the same gas sample.



Gas chromato,7raphic analysis for ethylene and acetylene

Samples of the gas phase in the incubatin- flasks







26

were taken with 1 ml, polyproplylene syringes (Becton, Dickinson, and Co., Rutherford, N. J.). Since air contamination was undesirable, the "dead space" in the syringe and needle was displaced with a saturated NaCl solution or by flushing with helium. Syringes were placed in a water bath for several minutes, so that all gas samples, including standards, were at the same temperature prior to analysis.

Gas samples, 0.5 ml, were analyzed in either a Loe

Engineering Co. (Pasadena, Calif.), model 15A gas chromatograph, courtesy of Dr. P. H. Smith, or an F and M Scientific Corp. (Avondale, Penn.), model 700 gas chromatograph, courtesy of Dr. D. S. Anthony. Both instruments were equipped

with a 0.63 x 40 cm teflon column containing 80-200 mesh silica gel. The temperature of the column was 90 C and the nitrogen carrier gas flow rate was 15 ml per minute. The model 700 gas chromatograph was equipped with a 0.63 x 60 cm teflon column containing 30-200 mesh silica gel. The temperature of the column was 90 C and the helium carrier gas flow rate was 30 ml per minute. The adsorption of water to the solid phase was minimized by the elevated column temperature. Under these sets of conditions identifications of acetylene and ethylene were made from the retention times, which were five and two minutes respectively.

The sensitivity of each instrument was determined with a 0.1 ml gas sample (assuming 1 ml to be 100 per cent) and







27


was expressed as mm peak height per per cent gas. The height times width at half-height method (McNair and Bonelli, 1967) was used for quantitation of peaks. Linear responses were obtained from standard ethylene samples ranging from 0.002 ml to 0.3 ml. The per cent measured gas in the sample was multiplied by the known volume of the flask to determine the volume of the measured gas in the incubation flask. The pressure and temperature of the gas in the flask at sampling times were used to adjust gases to standard temperature and pressure (STP) values to calculate the quantity of the gas in Imoles.












RESULTS


Avpearance of root nodules

Nodules of M. cerifera were located predominantly

on roots near the soil surface. Nodules are structures which consist of lobes closely clustered to one another. Newly

formed lobes at the periphery of a cluster point upward, because lobes are negatively geotropic. Older, heavily suberized tissues were located inside the perennial nodule. The rootlets on developing lobes were white and 1 cm long. The rootlets on mature nodule lobes from plants growing in wet soil were 2 to 3 cm long, 0.1 cm in diameter and pigmented. The rootlets on mature nodule lobes from plants growing in dry, sandy soil were 1 cm long, less than 0.1 cm in diameter, and brittle.

The heterogeneity of appearance was minimized by

selecting only mature and develo-n..7g nodules for experiments. Those with darkly pigmented and suberized lobes were discarded.



N fixation by excised root nodules
Since nodules of Myrica gale L. were reported by Bond (1957) to fix N2, it was reasonable to assume that nodules of Myrica cerifera 7. would do the same. Excised whole nodules were tested by exposing them to .-s mi:ture


23








containing N2 and 02 in a ra+io similar to that in air. A set of three flasks each containing nodules v.as ex-osed to nitrogen gas which was enriched with 'N while a control set was exposed to nitroen gas of natural 15N abundance. After incubation, the nitrogen of the whole nodules was analyzed by the 15N method described earlier. The results, given in Table 2, showed an increase in atom per cent content in nodules of all flasks which were exposed to the enriched nitrogen gas. There was no increase in atom per
15
cent 1N content in nodules of control flasks. The excised nodules of 1. cerifera fixed N2.

Although results of the analysis of the nitrogen of

whole nodules demonstrated N2 fixation, a more sensitive means of analyzing fixed ~ N was desired. Analysis of the HCl soluble portion of soybean nodules had provided a consistent assay for N2 fixation (Aprison and Burris, 1952). Also, Leaf et al. (1959) had found 15N enrichment in components of the acid soluble portion of nodules of C. tale.

To apply this assay, nodules of Y. cerifera were extracted with Hrl after exposure to 1N enriched nitrogen gas. Nitrogenous compounds of both acid soluble and acid insoluble portions were analyzed, and the results are shown in Table 3. The acid soluble portion contained about tcn times more

enrichment than the acid insoluble portion.

The increased zcnsitivity of the acid soluble extraction method made possible a time course experiment. Nodules







30


TABLE 2

TEST FOR N2 FIXATION BY DETACHED NODULES
OF MYRICA CERIFERA


Flask No. Atom 5 15N excess


1 0.042

2 0.056

3 0.096


Each flask contained 2 g fregh weight of nodules
and 1 ml of N-free nu 7ient solution The incubation gas mixture was 0.80 atm '-'N2 (30 atom % N excess) and 0.20 atm 0 Three control flasks each contained 0.80 atm N (tanks and 0.20 atm 0 All samples were exposed to th3 gas mixtures for 19 h~urs.


1Centifanto (1964)






31


TABLE 3

15N CONTENT OF ACID SOLUBLE AND ACID INSOLUBLE FACTIONS
OF NODULES OF L. CERIFEA AFTER EXPOSURE TO UN2



Flask No. Fraction Atom , 15N excess


1 Acid soluble 1.092

2 " " 0.780

1 Acid insoluble 0.089

2 " " 0.097


Each flask contained The gas mixture was 0.27 atm 0.21 atm 02, and 0.52 atm He.


4 g fresh weight1 f nodules. 15N (30 atom % N excess),
Tqe exposure time was 4 hours.






32

of M. cerifera were excised from roots, washed, and exposed to the incubation gas mixture within 30 minutes. As shown in Figure 1, 15N was rapidly incorporated into the acid soluble fraction, the rate being constant over the first eight hours. The enrichment in the acid insoluble fraction was low. Some variation between replicate flasks at prolonged exposure to the gas was noted. This was not surprising due to the native source of the tissue and was considered minor. It was decided to determine the L5N enrichment in only the acid soluble portion of nodules in further experiments.

Since appearance of nodules varied from young to

aged tissue, the capacity of these tissues to fix nitrogen was checked. Obviously young, unpigmented, unsuberized, and fleshy lobes were placed in one group, while old, darkly pigmented, suberized, and woody nodular parts formed the second group. The latter group was routinely discarded in other experiments. The results of the experiment are shown in Table 4. The young tissue fixed nitrogen at three times the rate of the old tissue. Fixation did occur in both morphologically and physiologically different tissues. Thus, the appearance of the nodule was important and the continued use of the young tissue in experiments was justified.


ffes- of -artia7 pressure of

A study was made to determine the partial pressure of N2 (pN2) supporting the maximum rate of fixation for


























L


00
0



-0





...-.. .. .


0 2 4 6 8 10 12
TiME (HOURS)


Figure 1


14 16 18


Time course of N2 fixation by excised nodules of M. cerifera.

Two experiments wore conducted and the results of analyses of the acid soluble N are denoted for experiment 1 as (A) and for experiment 2 as (o) with each point representing one flask. The average of two flasks is given for the acid insoluble . and is denoted as (0). Each flask contained 4 g fresh weight of nodules and was exposed to a gas mixture containing 0.16 atm 15N2 (30 atom TV excess), 0.22 atm 02, and 0.62 atm He.


33


1.8 r 1.4


.2


0


U
X

Z


0.8
-0
0
2 0.6
0
< 0.4


0.2





34


TABLE 4

COMTPARISON OF N2 FIXATION BETWEEN NODULES WITH EPIDERMIS
PRESENT AND NODULES WITH PERIDERM PRESENT



Flask No. Description Atom ' 1ON excess
of nodules


1 Periderm present (woody) 0.326

2 " " " 0.276

3 Epidermis present (fleshy) 0.858

4 i i V 0.889



Each flask contained 4 g fresh wvei ht of nodules. The gas mixture was 0.23 atm 15N2(30 atom % 1,N excess), 0.21 atm 02, and 0.57 atm He. The exposure time was 4 hours.








excised nodules of Y. cerifera. Two flask were treated identically for each pN2. Each flask contained 0.20 atm 02. Figure 2 represented the results of four experiments conducted in order to determine the effect of varying the p2 on the relative rate of Y2fixation. From the plot, maximum N2 fixation occurred at about 0.10 atm N2 for this tissue.

A Lineweaver-Burk plot (Figure 3) for the data was prepared similar to that described by Burris et al. (1955) for soybean nodules. The average of results from replicate flasks was used in the plot and for the determination of the center line for the data by the method of least squares. The slope of the straight line, K m/V ,ax equaled 0.080. The y intercept, 1/Vmax, equaled 1.150. A Michaelis constant, Km, of 0.069 0.004 atm N2 for N2 fixation by whole nodules of M. cerifera was calculated.



Effect of martial pressure of 02

The effect of 02 concentration on N2 fixation was

considered. The 5 N2 content of the incubation gas mixture was maintained near 0.20 atm to insure saturation of nitrogenase with substrate. As shown in Figurc A, 0, was indispensable for N2 fixation, and maximum fixation occurred at a partial pressure of 02 between 0.20 and 0.30 atm. " fixation was limited at 02 concentrations below 0.20 at, while :> fixation was depressed above 0.3 atm 02-
















1.0


0.9


0.8 '..


0 .7
LU




2 0.5

0.4


0
0- .3


0.2


0.1


0 0.04 0.08 0.12 0.18 0.22 0.24
15
P N2 (ATM) Figure 2 Effect of pN2 on 15N incorporation by excised
nodules of Nyrica cerifera.

Results of three experiments are shown. Each point represented a flash which contained 4 g
fresh weight of nodules and gas mixture
!OFpOSed Of 2 (30 atom / :,, excess) as
indicated above, 0.20 at'm0 , and He to 1 atm.
The Incubation time was fou. hours.







37


7r




5




3








10 20 30 40 50
1IS]

Figure 3 Lineweaver-Burk plot to determine the Michaelis
constant for nitrogen fixation by whole nodules
of 1J. cerifera.

A line has been fitted to the data by the
method of least squares.






38


0.

Li
0.6
X




Z 0.4
0

S0.3
0

<0.2


0.1


0 0.1 0.2 0.3 0.4 0.5
P02 (ATM)

Figure 4 Effect of p02 on N2 fixation by detached nodules
of 71yrica cerifera.

Each point represents a flask which contai d 4 g fresh weight of nodules, and 0.18 atm N (3Q atom I 15N excess), 02 as indicated above
and He to 1 atm. Ti'he incubation time was four
hours.










Storage of intact nodules

The advantage of storing nodules for later experimental use is obvious. Attempts were made to store nodules under helium in a freezer and in liquid nitrogen. Table 5 shows that activity did not survive either type of storage.

Even freshly excised nodules frozen at the temperature of liquid nitrogen and quickly removed failed to have activity. Immersing nodules in 20 per cent glycerol and freezing in

liquid nitrogen was also unsuccessful.



Variation among replicate samples

An experiment was performed treating five samples

identically in order to establish a distribution of measurements of 15N enrichment. The results are given in Table 6. Measures of the central tendency and variability are given as the mean and the standard deviation respectively. For comparison the results from samples in eleven experiments under like conditions were compiled and are shown in the same table. The standard deviation for the one experiment when compared to the corresponding value for the eleven experiments were similar.



L fi2 tion by nodular homogenates of 7J. cerifera
After determining conditions for N2 fixation by whole

nodules of 1. cerifera, the problem of preparing active

homogenates of these nodules was considered. Preparation of

active homogenates had not been reported in the lierture





40


TABLE 5

STUDIES ON STORAGE OF EXCISED NODULES OF Y. CERIFERA



Flask No. Treatment Atom f% 15N excess



1 Under helium in deep freeze 0.016

2 Same as 1 0.003

3 Immersed in liquid N2 0.014

4 Same as 3 0.008

5 No storage 0.732
6 Same as 5 0.632


Each flask contained 5 g fresh weight of nodules which had been stored at each condition indicated for two weeks. The incubation gas mixture contained 0.16 atm 15N2 (30 atom % 15N excess), 0.22 atm 02, and 0.62 He. Incubation time was 4 hours.






41


TABLE 6

VARIABILITY AMONG SAMPLES WITHIN ONE EXPERIMENT
AND AMONG SEVERAL EXPERIMENTS


Range mean SD 90 1 C. I.


5 samples from 0.306-0.893 0.644 0.246 0.410-0.873
1 expt.

15 samples from 0.143-0.975 0.424 0.257 0.307-0.541 11 expt.



Values given for range, mean, and confiIence interval are results of experiments expressed in atom % DN excess.

Each sample of 4 g fresh weight of nodules was gassed with 0.2 atm N2 (30 atom % 15 N excess), 0.2 atm 02, and He
t1at.The incubation time wvas 4 hours.








at this time. Bince the time course of N2 fixation by nodules of N. cerifera was longer than the two hour time course of fixation by soybean nodules reported by Aprison

and 3urris (1952), it seemed possible that the nonlegu7inous nodules might be better experimental material for the preparation of active honogenates. Techniques useful in preparing and handling cell-free extracts of bacteria had been reported by Carnanan et al. (1960). Because of the lack of technical

knowledge about preparing nodular homogenates, techniQues used in bacterial IN2 fixing extracts were applied. The success of preparing active cell-free extracts from bacteria depended partly upon the method of using a low concentration of phosphate buffer ((0.05M) or cacodylate buffer, pH 7.0; also anaerobic manipulation of extracts at room temperature

was necessary.

Nodules of M. cerifera were homogenized anaerobically in buffer at room temperature. Nodules were well fragmented with only some peridermal portions remaining intact. It was assumed that both whole nodules and homogen tes had the same 02 requirements during incubation. Homogenates were tested with and without the electron donor dithionite (Na2ES204,' which had been used by Bulen et al. 7(196) to enhance N2 fixation by cell-free extracts of Azotobacter vinelandii. Homogenates which were supplied Na2 20, and 02 fixed N (Table 7). Although this fixation was much less than that shown by intact nodules used as controls, it was significant since the r -p;Ictos rcQ cicoI-C.





43


TABLE 7

EFFECT OF Na2S204 ON N2 FIXATION BY ROOT
NODULE HONOGENATES


Flask No. Treatment Atom p 15N excess


1 Intact Nodules 0.588

2 Homogenate 0.005

3 Homogenate + Na2S204 0.026
4 Homogenate + Na2S204 0.021

5 Homogenate + Na2S204 0.022


Flask No. 1 contained 5 g fresh weight of nodules.

The homogenates were prepared as follows: 5 g
nodules were homogenized 1 min under He in 5 ml cacodylate buffer, pH 7, 508 Izmoles. 1 ml Na2S204, 240 pmoles, was added aftir gassing. The incubation gas mixture contained
0.20 atm 5N2 (96.6 atom 5 15N excess), 0.22 atm 02, 0.58 atm He. The incubation time was 4 hours.






44


Since others (Mortenson, 1964; Hardy and D'Eustachio, 1964; Bulen et al., 1965) had reported that adenosine triphosphate (ATP) was recutrod for N2 fixation by cell-free extracts of bacteria, the effects of an exogenous AT? generating system on the homogenate were studied. The system according to Bulen et al. (1965) contained AT?, creatine phosphate (CP), and creatine phosphokinase (CPK). Homogenates were prepared as before in the presence of the buffer. Na2s2O4 and the supplements (the AT? generating system and the reduced form of nicotinamide-adenine dinucleotide, NADH) were tipped in after gassing the system. The incubation gas

mixture contained the same proportion of 02 at which maximum N2 fixation by whole nodules occurred. The results are given in Table 8. N2 fixation was substantial for homogenates prepared in the buffer and supplied Na23204, NADH, the ATP generating system, and 02*

With an exogenous ATP generating system supplied, N2 fixation by homogenates may not be dependent upon aerobic respiration. in order to test whether 02 was required in addition to the exogenous ATP generating system, the following experiment was performed. Nodular homogenates of N. cerifera were prepared as described above. The homogenates were exposed to incubation gas mixtures either with 0- or without, and then were sp)lied with supplements as described in the previous experiment. Results of two experiments are shomn in Table 9. Homogenates suslied 'vi t; thiz exogenous ATP gr tin systm fixed N- onS in :n, JresOnce







45


TABLE 8

EFFECT OF SUPPLEMENTS ON N FIXATION
BY NODULAR HOMOGENA .ES


Flask No. Treatment Atom N excess


1 Intact nodules 0.770

2 Buffered homogenate 0.007

3 Same as 2 0.017
4 Buffered homogenate + 0.390
supplements
5 Same as 4 0.117


Flask No. 1 contained 4 g fresh weight of nodules.

The homogenates were prepared as follows: 5.5 g
nodules were homogenized 1 min under He in 5 ml cacodylate buffer, pH 7, 106 Mmoles. The supplements moless per flask), added after gassing, were Na2 204, 50; ATP, 20; NADH, 20; creatine phosphate, 100, and 2 mg of creatine phosphokinase. The final volume of the reaction mixture w s 10 ml. The jjcubation gas mixture contained 0.18 atm 5N2 (96 atom N N excess), 0.18 atm 02, and 0.65 atm He. The incubation
time was 4 hours.






46


TABLE 9

EFFECT OF OXYGEN ON N FIXATION BY INTACT NODULES
AND SUPPLEMENT D NODULAR HOMOGENATES



Tissue 02 Mean Atom %o 15N
excess

Intact nodules - .020 + .026 *

Intact nodules + .079 + .081

Supplemented homogenate - .008 + .003

Supplemented homogenate + .142 + .152


* Standard deviation of the mean.

Values given are the mean of four samples for each treatment.

The homogenates supplemented were as described in Table 8. The incubation gas mixture for -02 was 0.21 atm 15N2 (30 atom 5 15N excess) and 0.79 atm He; for +02 it was 0.21 atm 1N2 0.20 atm 02, and 0.59 atm He. 2
The incubation time was 4 hours.






Z- 7

0 -,LC2 (ee 1ast Iine, 7 le . As shown in Table intact
nodules in the presence of 02 fixed C2 Only to a small extent (0.07? atom rer cent excess), ,,hereas the homogenate in

the presence of 02 fixed much more N2 (0.142 atom per cent 15N excess).

Table 7 shows that the buffered homogenates in the

presence of Na2S2S4 fixed N The addition of other supplements and the presence of 02 enhanced the activity. The necessity of Na a2S204 in the presence of the ATP generating system was tested. The addition of Na2S204 = 02 to the supplemented homogenates was varied as shown in Table 10. IT2 fixation was observed only in one of the supplemented homogenates supplied with Na2S204 and 029

An experiment was performed with supplemented homogenates buffered at three pH levels. The results are shown in Table 11. N2 fixation was observed for one flask in which the homogenate was adjusted to pH 7. Site of 2 fixation by homogenates

In order to determine whether nitrogenase activity occurred in the soluble portion of the homogenate, a homogenate was prepared under conditions equivalent to those described above. A portion of the homogenate was centrifuged at 5000 rpm for three minutes. The pellet and supernate each were exposed to the incubation gas mixture, and then supplements were added. As shown in Table 12, one of the supplemented pellet fractions (Flask 7) fixed N2 C7, 7

significant fixation did not occur in the othor f'c:s. inta-ct rortio-is ofC1 11~






48


TABLE 10

EFFECT OF 02 and Na2O 2A ON N2 FIXATION
BY SUPPLEMENTED HOI0 GENATES


Flask No. Na2S204 02 Atom 1 15N excess


1 - - 0.005

2 - 0.012

3 - + 0.009

4 - + 0.002

5 + 0.003

6 + 0.006

7 + + 1.138

8 + + 0.007


The preparation and supplementation and incubation gas mixtures were the same as Table 8.


of homogenates described in







49


TABLE 11

EFFECT OF pH ON N2 FIXATION BY HOMOGENA"ES


Flask No. pH Atom 15N excess


1 4.5 0.010

2 same as 1 0.006

3 6.0 0.005
4 same as 3 0.007

5 7.0 0.011
6 same as 5 0.035

The buffer was adjusted to pH indicated above. Other conditions were the same as in Table 8, except that 1N2 contained 30 atom ep 15N excess.









TABLE 2.2

N2 FIXATION BY SUP LEBNED HO2COG ENATE FRAOTWONS



Flask :o. Treatment Atom 5 ~5N excess


Intact nodules 0.427

2 Homogenate 0.013

3 Homogenate 0.007

4 Supernate 1 0.009

5 Residue 1 0.007

6 Supernate 2 0.014

7 Residue 2 0.038


Flask No. I contained 4 g fresh weight of nodules.

A sample containing 30 g fresh weight of nodules was homogenized in a 30 ml solution containing cacodylate buffer pH 7, 3 p-moles, and Na ?S20 , 0.3 pmoles. Flasks No. 2 and 3 each received 10 ip1 himogenate. The portion to be fractionated was divided in half and each portion was centrifuged at 5,000 rpm for three minutes. Each pellet fraction was resuspended in 4 ml of buffer. Supplements, I ml, (pmoles per flask) ATP, 20; NADH, 20; creatine phosphate, 100, and 2 mg creatine phosphokinase were added to each fraction and to e homogenate. mhe incubation gas mixture was 0.17 atm N2 (30 atom 6 N excess),
0.20 atm 02, and He to I atm. The incubation time was 4 hours.








In all of the nodular homogenate experiments, the

aim was to determine optimal conditions for N2 fixation. Variations among replicates were observed in many experiments. However, N2 fixation ability was always correlated with conditions which were varied in any given experiment. This type of correlation was considered significant. Because of the existence of variations and the laborious 15N method further investigation into symbiotic nitrogen fixation by homogenates should be modified in future work. The remainder of the present investigation was concerned with the reduction of acetylene by nodules and breis of nodules.



Acetylene reduction by nodules of M. cerifera

The idea that acetylene reduction could be used as

a measure of nitrogenase activity was suggested by Dr. R. V1. F. Hardy (1966, personal communication), who was utilizing cellfree extracts of A. vinelandii and C. pasteurianum in a study of the specificity of nitrogenase for electron acceptors (Hardy and Knight, 1966). Furthermore, Schollhorn and Burris (1966) and Dilworth (1966) showed that extracts of C. pasteurianum which fixed N2 also catalyzed the reduction of acetyene to ethylene. ',hLether the reduction of acetylene might be catalyzed by nitrogenase in nodules of M. cerifera was questioned. Nodules of LI. cerifera were collected and exposed to an incubation gas mixture containing acetylene, oxygen, and helium. Oxygen was supplied at concentrations which supported maximum and half zaxizun








nitrogen fixation (see figure 4). The appearance of ethylene was monitored by flame ionization gas c'romatography. Samples of the incubation gas mixture were analyzed at various times, and a time course was plotted, as shown in Figure 5. The rate of ethylene production at

0.20 atm 02 was constant for the first nine hours. At 0.10 atm 02 the rate of ethylene formation was reduced and was constant for the first five hours only. No ethylene was detected in control flasks gassed with an acetylene-helium mixture without 02. The time course of the reduction of acetylene to ethylene was similar to that of N2 fixation and both processes were dependent upon 02.

The possibility exists that ethylene production was not the consequence of nitrogenase catalyzing the reduction of acetylene. Endogenous ethylene production has been shown to occur in plant tissues, as reviewed by Jansen (1965), and this is unrelated to N2 fixation. In the experiment described above, endogenous ethylene production was not detected in flasks containing excised nodules incubated with air. According to Jansen (1965), ripening orange fruits produced ethylene at a rate of 0.350 pl per gram per hour, which is one of the highest rates observed and is much higher than that for vegetrtivo tis us. From Figure 5, the rate of ethylcne production by nodulec is calculated to be 76.6 pl per gram per hcur--over 200 time






53


40

35


30

25

Lj 20

0



10L
z
LJ
5


Li 0 2 4 6 8 10 12 14 16 18

TIME (HOURS) Figure 5 Time course of acetylene reduction to ethylene
by nodules of I. cerifera at 0.1 atm and 0.2
atm 02*

Each point represents a flask which contained
2 g fresh weight of nodules.
The gas mixture represented by open triangles
was 0.20 atm 02, 0.05 atm C H and He to I atm;
and by closed triangles was M.O atm 02, 0.05
atm C 2 and He to 1 atm.







54

larger than the rate of ethylene production in ripening fruit. It was concluded that if minute endogenous ethylene production did occur, it did not affect the results obtained above.

The possibility that acetylene may have stimulated endogenous ethylene production was tested. The relationship between the production of ethylene and the disappearance of acetylene catalyzed by excised nodules of 7. cerifera was determined (Figure 6). An increase of 5.8 moles or ethylene was detected after four hours, while
5.0 Pmoles of acetylene disappeared during the same time. Thus, about 90 per cent of the ethylene produced could be accounted for by the decrease of acetylene. It was concluded that acetylene is directly reduced to ethylene by excised nodules of V. cerifera.



Acetylene reduction by nodules of various plants

In order to determine whether acetylene reduction was a common property of root nodules which fix N2, nodules from plants other than N. cerifera were tested.

The time course of the reduction of acetylene to ethylene by excised nodules of C. eauisetifolia is shown in Figure 7. The nodules reduced acetylene to ethylene and the rate of ethylene production was constant for six hours, after which activity diminished abruptly.

Nodules from Hampton soybeans reduced acetylene to ethylene as shown in Figure 8. The rate of ethylene






55


8 28




6 26

Cii







z
z2 L22 L






0 12 3 4
TIME (HOURS)

Figure 6 Relationship of ethylene production to
acetylene disappearance by nodules of j.
cerifera.

Each point represents a flask which contained 2 g fresh weight of nodules, gassed with 0.05
atm acetylene, 0.10 atm 02 and H-e to 1 atm.
Ethylene is denoted by an open triangle.
Acetylcne is deno-ed by a closed triangle.






56


2,5




2.0

L

IA

0



-LJ
3 1.5




Z




I >-0.5





0 4 8 12 16 20
TIME (HOURS) Figure 7 Time course of reduction of acetylene to
ethylene by excised nodules of Casuarina
ecuisetifolia.

Each point represents a flask which contained 2 g fresh weight of nodules gassed with 0.05 atm acetylene, 0.20 atm 02, and He to 1 atm.






57


4 -~, 31 C
- -.
4z



3 300





A2-29




28
Z\
Z 2'





0 1 2 3 4
T IME (HOUR S)

Figure 8 Relationship of ethylene production to
acetylene disappearance by soybean
nodules.

Each point represents a flask which contained 2 g fresh weight of nodules gassed with 0.05 atm acetylene, 0.10 atm 02, and
He to 1 atm.
Ethylene is denoted by the open triangle.
Acetylene is denoted by the closed ->a.es.






58

production was constant for the first two hours. During the four hours of the test 2.3 moles of ethylene were

produced while 1.5 Fmoles of acetylene ,,ere reduced.

Symbiotic structures such as leaf nodules and

mycorrhizal associations were tested in addition to root nodules of leguminous and nonleguminous plants. The isolated endophyte of leaf nodules of Psychotria bacteriophila and vegetative plant tissues were included in the acetylene reduction tests. The summary of these data was shown in Table 13. Nodules of six species from five genera supported the reduction of acetylene. Leaves, stems, and roots of M. cerifera and G. max did not catalyze the reduction of acetylene, nor did endogenous ethylene production influence the results. The fresh weight and nitrogen content of nodules was determined and used in the calculation of initial rates of ethylene production from acetylene. A comparison of the rates obtained would not be valid because of the varied ages and environmental conditions of the field tissue prior to analysis. 'Uhat is useful from these data is the knowledge of which tissues exhibit activity and the duration of the activity after excision from the plant. The nodules of the nonleguminous genera, Casuarina and !Jyrica, maintained activity for a longer time after excision than the genera of legumes, Lupinus and Glycine.

Tycorrhizal associations on the roots of Podocarpus macrophylla and Pinus elliottii were tested for the ability to reduce acetylene. These species wer0 thou :ht to :i1









Standard conditions were 0.10 atm acetylene, 0.20 atm 02, and He to 1 atm. Each flask contained
2 crams except for P. macrophylla and P. elliotii which contained 8.3 and 20 grams respectively. The age of plants in months was G. max, 2-3; L. albus, 2; C. soectabilis, 1; P. elliottii, 24.

n.d. means not detectable, (0.005


1 atm. * 0.05 atm acetylene, 0.10 atm 02, and He to

a Plants were collected from Sanibel Island and grown in a greenhouse for one month.
b Plants were collected from Haulover Beach State Park and stored over night in a plastic bag.

c -lleb-iella rubiacearum (strain K4). The
system contained per 5 ml d00 mg dry weight of cells, 250 mg pyruvate, cacodylate buffer pH 7, 0.1N. The incubation gas mixture was 0.05 atm acetylene and He to 1 atm.






60

TABLE 13

REDUCTION OF ACETYLENE TO ETHYLENE
BY VARIOUS PLANT TISSUES


Plant pmoles C2H4/ poles C2H4/ Duration of
g fr wt / hr pmoles N /hr initial rate in hours

Myrica cerifera
nodules 3.4 0.067 9
nodules 1.6* 0.033 5
roots n.d.

Casuarina equisetifolia
nodules e 0.7 0.017 8
nodules b 0.2 0.004 3

Casuarina cunninghamiana
nodules 0.1*

Podocarpus macrophylla
nodulated roots n.d.

Pinus elliottii
mycorrhizal roots n. d.

Psychotria bacteriophila
nodulated leaves n. d.
endophyte C 1.1

Lupinus albus
nodules 2.0 0.080 3
leaves n.d.

Crotolaria spectabilis
nodules 0.8 0.032 5

Glycine max
nodules 1.2* 0.046 2
leaves nod.*
stems nod*
roots nod.*



For legend, see opposite page.






61

nitrogen, but there was no direct evidence. As shorn. in Table 13, no detectable ethylene was formed after 29 hours of incubation, although large amounts of tissue were used.

Leaf nodules of Psychotria bacteriophila are

believed to be a symbiotic N2 fixing association because nodulated plants grew in a nitrogen-free medium for six months without developing symptoms of nitrogen deficiency (Silver et al., 1963). The N2 fixing capacity of the leaf nodules might support the reduction of acetylene. Two grams of young nodulated leaves (less than 3 cm long) were selected from healthy plants, but no detectable ethylene was found after an 18 hour incubation of the nodulated leaves to acetylene (Table 13). However, cell suspensions of isolated endophyte, which had been grown in nitrogen-free mineral broth did form ethylene when the substrate and a source of energy and reducing power were provided.


Effect of acetylene concentration on ethylene production

The effect of acetylene concentration on the reduction of acetylene to ethylene by excised nodules of Y. cerifera was studied. Nodules were incubated with gas mixtures in which the 02 concentration was 0.20 atm and the acetylene concentrations in individual flasks ranged between 0.01 and 0.40 atm. As shown in Figure 9, a concentration of 0.04 atm acetylene saturated the nitrogenase system. For acetylene concentrations above 0.20 atm, activity was one half the maximal value. From these data






62


7

-r



AA

(D
3L
CIO


0

Li








LiJ


0 0.05 0.10 0.15 0.20 0.25 0.30. 0.35 0.40

,C2 H2 (ATM)

Figure 9 The effect of partial pressure of acetylene
on ethylene production by nodules of ;Lyrica
cerifera.

Results of four experiments are shown.
Each point represents a flask containing
1 g of nodules gassed with acetylene as indicated above, 0.20 atm 02, and He to
1 atm. Incubation time was one hour.






63


an apparent Km value of 0.02 atm for acetylene was estimated. The nitrogenase systems's affinity for acetylene appeared to be three times that for N2 (See Figure 2). High acetylene concentrations limited the reduction of acetylene to ethylene to about half the maximum rate.



N inhibition of the reduction of acetylene

If nitrogenase were catalyzing the reduction of

both acetylene and N2, N2 would be expected to inhibit the reduction of acetylene. Excised nodules of M. cerifera were incubated with gas mixtures which included 0.05 atm acetylene and concentrations of N2 ranging from 0 to 0.15 atm. The results of three experiments were plotted as ethylene production (per cent of control) vs. pN2 (Figure 10). As the concentration of N2 increased, the production of ethylene from acetylene decreased. At 0.06 atm N2, acetylene reduction was inhibited more than 60 per cent.

The effect of acetylene concentration on the formation of ethylene by soybean nodules was reported by Koch and Evans (1966). The maximum rate of ethylene production was achieved at approximately 0.1 atm acetylene. Since Koch and Evans did not report the effect of N2 on acetylene reduction in soybean, it was of interest to compare this tissue with that of MDyrica. Various partial pressures of N2 were tested on soybean nodules saturated with 0.1 atm acetylene. As indicated in Figure 11, ethylene production decreased only above a pN2 of 0.30 atm. N2 concentrations






64




100



S8 0F
o


O AA z 60

0
UA
LL40. A



20





0 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.,6

PN2 (ATM)

Figure 10 Effect of N on ethylene production from
acetylene b nodules of T. cerifera.

Results of three experiments are shown.
Each point represents a flask which contained 2 g fresh weight of nodules gassed
with 0.20 atm 02, 0.05 atm acetylene, N2
as indicated above, and He to 1 atm.
The incubation time was one hour.






65


12



10L


8











L3i
5-r








0 0.10 0.20 0.30 0.40 0.50 0.60

PN (ATM)
Figure 11 Bffect of pN2 on acetylene reduction by soybean nodules.

Reul-ts of five experi ments are shown.
Each point represents a flask containing 2 g fresh weight of nodules from Hampton soybean
(66-73 days old) gassed with 0.10 atm
acetylene, 0.20 atm 02, N2 as indicated
above and He to 1 atm. Incubation time was
one hour.






66

of less than 0.30 atm had no apparent effect on ethylene production. At 0.62 atm N2, the rate had decreased to less than 1 per cent of the rate at 0.30 atm N2.



Reduction of acetylene by individual nodules

Routinely, the sensitivity of the gas chromatograph permitted detection of 0.005 poles of ethylene. It was of interest to determine the amount of tissue necessary to produce detectable quantities of ethylene from acetylene. Nodules were collected from M. cerifera. Each of five samples, weighing 1.280 g, 0.353 g, 0.165 g, 0.071 g, and

0.009 g, were exposed to an incubation gas mixture containing 0.05 atm acetylene, 0.20 atm oxygen, and 0.75 atm helium. Acetylene reduction was detected in all five samples. A single young lobe of a nodule, weighing 0.009 g, produced 0.04 poles of ethylene per hour. Thus, the sensitivity of the acetylene reduction method permitted detection of nitrogenase activity in less than a 10 mg sample.

Individual soybean nodules were also tested.

Nodules were collected from Hampton soybean plants which were 40 to 45 days old. The weight, diameter, and location on tap or lateral roots were noted. A total of 28 nodules selected from nine plants were used in the experiment and the results are summarized in Table 14. None of the nodules less than 3 mm in diameter had detectable activity. They were considered immature nodules. Seventy per cent of those with diameters larger than 3 mm reduced acetylene. Activity






67


TABLE 14

ACETYLENE REDUCTION BY INDIVIDUAL
SOYBEAN NODULES


Nodule diameter Total
(mm) tested / Active


< 3.0 5 0

3.0 - 4.0 8 50

4.0 - 5.0 6 83

>5.0 9 77


Each flask contained an individual soybean nodule and were gassed with 0.10 atm acetylene, 0.20 atm 0 , and He to 1 atm. Gas samples were taken after one hour incubation.






68

was detected in four of the five tap root nodules tested and in 60 per cent of the lateral root nodules whose diameters were above 3 mm. Above 3 mm in diameter, active and inactive nodules were indistinguishable with respect to weight, as well as diameter.

The activity of each nodule was determined as pmoles ethylene produced per gram fresh weight per hour. Although there was no absolute correlation between nodule diameter and activity, larger nodules tended to be less active. One exceptionally active nodule with a diameter of 4 mm and a weight of 27.4 mg produced 24.6 moles of ethylene per gram fresh weight.



Reduction of acetylene by breis

The gas chromatographic detection of acetylene

reduction was used as a relatively easy and rapid method for detecting a tissue's ability to fix N2 in further attempts to prepare consistently active nodular breis of M. cerifera. Koch et al. (1967a) had reported that consistent N2 fixation and acetylene reduction by breis of soybean nodules could be maintained by preparation in the presence of polyvinylpyrrolidone (PVP) and buffered ascorbate medium to prevent phenolic oxidation products from inactivating enzymes. Before applying this procedure to nodular breis of M. cerifera, it was decided to prepare active breis from nodules of field grown Hampton and Chipewa soybean plants according to the method of Koch et al. (1967a). Soybean nodules were harvested, washed









and placed in a polyethylene glove bag. The glove bag was evacuated and filled three times with N2 which had been passed through a heated copper column to remove traces of 02. The nodules were macerated in phosphate buffer containing ascorbate, and squeezed through two layers of cheese cloth. The breis were mixed with PVP for ten minutes and again squeezed through cheese cloth. After the breis were exposed to incubation gas mixtures containing acetylene, oxygen, and helium, gas samples were analyzed. The results, given in Table 15, show that breis prepared from nodules of Hampton and Chipewa soybeans reduced only small amounts of acetylene. The results demonstrated that this procedure did provide consistently active breis, but the activity obtained was much lower than that obtained by Koch et al. (1967a). The reasons for the low activity were not known, but they may be due to the fact that these nodules were from field grown soybeans rather than greenhouse plants which were used by Koch and co-workers.


Nodules of M. cerifera were then collected and used in the preparation of nodular breis by the same procedure. However, activity of breis from nodules of 1. cerifera was barely detectable, as shown in Table 16. Activity might be increased by further modifications to protect the enzyme systems.


6 S9





70


TABLE 15

REDUCTION OF ACETYLENE BY BREIS OF SOYBEAN NODULES PREPARED WITH POLYVINYLPYRROLIDONE
AND ASCORBATE


Soybean system


poles of ethylene/
flask


Hampton, intact 10.8

Hampton, brei 0.04

Chipewa, intact 11.2

Chipewa, brei 0.05


Each intact nodule system contained 1 g fresh weight of nodules per flask.

Breis were prepared in a glove bag filled with argon. 18 g of nodules from 40 day old soybean plants were macerated in 27 ml of 0.02 M potassium phosphate buffer pH 7 containing 1 mm MgCl and 200 pMnoles ascorbate. The macerate was squeezed throug cheese cloth and mixed with 9 g of PVP. After 10 min the PVP and macerate were squeezed through cheese cloth and the resulting brei was gassed with 0.10 atm acetylene, 0.20 atm 0 , and He to
1 atm. Gas samples were taken after one h~ur.






71


TABLE 16

REDUCTION OF ACETYLENE BY BREIS OF MYICA CERIFERA
PREPARED WITH POLYVINYLPYRROLIDONE AND AS&ORBATE


Myrica system


moles ethylene
per flask


Intact nodules 3.5

Brei 0.01

Brei 0.01


The intact nodule system was 1 g fresh weight nodules per flask.

Breis were prepared as described in Table 15. 7 g of nodules were macerated in 10.5 ml of potassium phosphate buffer. 3.5 g of PVP was used. The gas mixture was 0.05 atm acetylene, 0.20 atm 02, and He to 1 atm. Samples were taken after one hour.













DISCUSSION

The results of the 15N method provided the first demonstration that excised nodules of native M. cerifera fixed N2. This contribution extended the list of nodulated nonleguminous angiosperms which have proven to fix N2.

The 15N method has been used as a sensitive assay for N2 fixation. The lack of any detectable exchange reaction between fixed nitrogen and molecular nitrogen and of any detectable preference by an organism for the isotopes of nitrogen established the validity of the 15N method (Burris and Miller, 1941). The 15N method detected changes in the ratio of 15N/14N and not changes in absolute nitrogen content. However, the initial incorporation of the 15N tracer into fixed nitrogen is confined to the metabolically active portion of cells, and analysis of the total nitrogen would dilute the tracer enrichment.

The mass spectrometric analysis of the acid soluble nitrogenous compounds of nodules of M. cerifera increased the sensitivity of detecting 15N incorporation. Since newly fixed 15N accumulated in the acid soluble fraction, the analysis of this fraction minimized the dilution effect of the fixed tracer by protein nitrogen.


72






73


The demonstration that the fixed tracer accumulated in the acid soluble fraction of nodules of M. cerifera confirms and extends the results of previous investigators. Aprison et al. (1954) reported that the fixed tracer was found in amino acids and ammonia, which were part of the acid soluble fraction of soybean nodules. Leaf et al. (1959) found that after one hour exposure of nodules of M. gale to 15N2, 82 per cent of the tracer fixed by the nodules was in the acid soluble fraction. Bergersen (1965) provided evidence that ammonia is the primary stable intermediate in N2 fixation by soybean nodules. Kennedy (1966a) and Kennedy (1966b) presented more evidence to confirm the formation of amino acids and amides from ammonia in leguminous nodules.


The present investigation has shown that the nodules of M. cerifera were good experimental material for research on symbiotic nitrogen fixation. This conclusion was included in the preliminary report of this study by Sloger and Silver (1965). The reasons for this conclusion may be summarized as follows. Since the rate of N2 fixation by nodules was constant for seven hours after detachment from roots, the time required to prepare and perform an experiment was not very critical. The results of experiments on the time course of N 2 fixation with nodules of I. cerifera can be compared to similar experiments reported for other nodules (see Figure 12). Nodules of M. cerifera as well as the






74
1.0




.8h Myrica
Acid soluble N

U
X
LrJ


.



Hippophae
Whole nodule N wO~o

0



Soybean
a.2 -Acid soluble NA
Alnus
Whole nodule N



4 8 12 16
TIME (HOURS)

Figure 12 Comparison of time courses of N9 fixation by
excised nodules of legumes and Lnlegumes.

Whole nodule analysis: Alnus glutinosa and
Hippophae rhamnoides 1.5 9 of nodzles
exposed to 0.10 atm 15N (36 atom % -5N excess),
0.20 atm 02, and 0.70 a m Argon. The results
were taken from Bond (1957).

Acid soluble analysis: Spybean nodules, 2 g, were exposed to 0.10 atm IDN2 (20 atom % 15N excess), 0.20 atm 02, 0.05 atm CO , and 0.65 atm He. The results were taken f om Aprison
and Burris (1952). M. cerifera data were from
Figure 1.









other nonleguminous nodules reported by Bond (1957) all have a prolonged activity after excision of the nodule from the root--a characteristic of nonleguminous nodules. Nodules of M. cerifera are better experimental material than leguminous nodules because the time course of N2 fixation is longer than that for soybean nodules reported by Aprison and Buris (1952). The initial rate of 15N incorporation in the acid soluble fraction was similar for L. cerifera and soybean, but the greater enrichment achieved by the nonlegume was due largely to prolonged ability for N2 fixation by the nodules after detachment from the plant.

In this study, the apparent Michaelis constant, Kim' of 0.069 + 0.004 atm N2 obtained for whole nodules of M. cerifera is greater than 0.02 + 0.004 atm N2 reported by Burris et al. (1955) for sliced soybean nodules.

The higher apparent Km for nodules of 1. cerifera may reflect differences in the permeability of gases into the sliced and intact tissues and not differences in affinities for substrate. Burris et al. (1955) suggested that the nitrogenases of various organisms were similar with respect to their affinity for substrate. 'he results of this study do not discount this hypothesis, but the use of intact tissues and organisms rather than soluble enzyme preparations hinders the verification of this hypothesis.

Recently, Strandberg and Wilson (1967) reported that from a comparison of Km values for N2 fixation .y intact







76


bacterial cells, a disparity of values was found, depending upon the method used. They also noted that using soluble N2 fixing enzyme preparations from several bacteria led to Km values which were three times higher than those obtained for the intact organisms.

The Michaelis constant and the pN2 function reported in this study represents the first evidence which suggests that nonleguminous and leguminous nodules are similar in respect to substrate affinity. In contrast to the results of this study, Bond (1959) reported that maximum fixation was observed at 0.25 atm N2 for intact nodules of Alnus glutinosa, a nonlegume. The lower optimal substrate concentrations for nodules of M. cerifera may be due to nodule size or lack of diffusional barriers.

The optimal 02 concentration for N2 fixation by nodules of M. cerifera noted in this study agreed with results obtained by Bond (1961), who reported that the optimal 02 concentration for fixation was between 12 and 25 per cent (0.12 to 0.25 atm) for the nonleguminous nodules examined, and the higher 02 concentrations depressed N2 fixation. Since this p02 is similar to that of air, it may be concluded that 02 is not a limiting factor for N2 fixation under natural conditions.

From the study of homogenates prepared from nodules of M. cerifera, it was concluded that active preparations required anaerobic homogenization with a buffer at pH 7







77


and incubation with Na2 S204, an ATP generating system, and 02. Although N2 fixing activity varied among preparations, when activity was obtained it was always associated with these conditions. The requirements for maintaining consistently active preparations may be related directly to the prevention of enzyme inactivation.

Chronologically, these results represented the

first demonstration that Na2S204, an exogenous ATP generating system and 02 were requirements for active fragmented nodular systems, and it has been the only report of a study of homogenates from nonleguminous nodules. Preliminary reports of these studies with homogenates were made by Sloger and Silver (1965) and by Sloger and Silver at the colloquium on biological nitrogen fixation at Sagehen, Calif., September, 1965, reported by Delwiche (1966). Independently, Bergersen (1966b) reported a preliminary study which demonstrated that breis of soybean nodules fixed N2 in the presence of 02. Subsequently, reports of studies on homogenates and breis were presented by Sloger and Silver (1966), Bergersen (1966c), Bergersen and Turner (1967), and Koch et al. (1967a), and Koch et al. (1967b).
The results obtained in the present study of nodular homogenates demonstrated that both endogenous and exogenous sources of energy, as well as an electron donor, were required for N2 fixation. In the intact nodule, respiration provides the energy sources required for N2 fixation. In cell-free






78


enzyme systems, an exogenous ATP source would serve the same role. Since 02 and exogenous ATP together maintained fixation by nodular homogenates of M. cerifera, both a soluble nitrogenase system and intact portions of nodules may have been fixing N2.


The 02 requirement for N2 fixation by nodular homogenates of N. cerifera has been confirmed and extended by others. Bergersen (1966b) and Bergersen (1966c) reported that although active breis of soybean nodules must be prepared anaerobically, 02 was essential in the incubation gas mixture for N2 fixation. Koch et al. (1967a) confirmed that N2 fixation by anaerobically prepared breis of soybean nodules was dependent upon 02. Furthermore, Bergersen and Turner (1967) showed that intact bacteroids from active breis of soybean nodules fixed N2 in the presence of 02*


Although 02 supported N2 fixation in intact and fragmented nodules, N2 fixation is considered to be a reductive process. In fact, 02 inactivated highly purified components of nitrogenase as shown by Bulen and LeComte (1966) and Mortenson et al. (1967). Recently, Koch et al. (1967b) demonstrated that cell-free extracts prepared from active bacteroids of soybean nodules fixed N2 without 02* Therefore, from the reports cited above and the results of the present study, it is suggested that 02 maintained respiration within intact portions of the filamentous endophyte and host cells and thereby stimulated N2 fixation.









Since N2 fixation by nodular homogenates of 1.

cerifera required an exogenous source of ATP in addition to 02, these data differed from observations made by other investigators. Bergersen (1966b) found that 02 was essential for N2 fixation by breis of soybean nodules, while the addition of an ATP generating system had little effect on the fixation rate. Active breis from soybean nodules prepared by Koch et al. (1967a) fixed N2 in the presence of 02 and no exogenous ATP was needed. In contrast, the nitrogenase system isolated from bacteroids fixed N2 with the addition of only ATP and an electron donor (Xoch et al., 1967b). Therefore, it is suggested that an exogenous ATP source and electron donor were requirements for N2 fixation by soluble nitrogenase which was released due to fragmentation of the filaT.mntous-endophyte of the nodules of M. cerifera. If this is so, the method of preparing homogenates should be modified in order to collect intact filaments of the endophyte. Then it may be possible to prepare cell-free extracts from the er.dophyte with consistent activity.

Although the roles of Na2S2O4 and ' ATP generating system in stimulating fixation by nodular homogenates of M. cerifera are not kmown with certainty, similar observations on cell-free extracts of bacteria have been made. Carnahan et al. (1960) found that added pyruvate was required to obtain fixation by zaxtracts of C. oasteurianum. Pyruvate metabolism was sho,.rn to serve both as an






80


ATP source and as an electron donor for C. pasteurianum (Mortenson, 1964; Hardy and D'Eustachio, 1964; and D'Eustachio and Hardy, 1964) and for A. vinelandii (Bulen et al., 1965). The requirements for symbiotic nitrogen fixation might be similar to those of free-living bacteria.


Preparation of consistently active nodular homogenates of M. cerifera would depend probably upon preventing enzyme inactivation. Phenolic oxidation products inactivated enzymes and their removal by polyvinylpyrrolidone (PVP) insured consistent activity in breis of soybean nodules, as shown by Koch et al. (1967a). However, when PVP was added to nodular breis of M. cerifera, only marginal activity was observed. The reasons for this are unknown at present. The search for compounds which will stabilize enzyme activity should be continued.


The results of this study have demonstrated that

only nodules with N2 fixing capacity reduced acetylene and that N2 inhibited the reduction of acetylene by nodules of M. cerifera and soybean. This evidence suggested that both N2 and acetylene reductions were catalyzed by nitrogenase. Molecular nitrogen and acetylene may be competing for the same binding site or for electrons. Clarification of these

mechanisms can be done with purified nitrogenase preparations. However, it was concluded that the reduction of acetylene may be used to measure the capacity of a tissue to fix N2#






81

That the acetylene assay is a valid and useful

adjunct to the 15N method in studies of symbiotic nitrogen fixation is based on several observations. All symbiotic N2 fixing tissues (root nodules and an isolated endophyte), except those of equivocal N2 fixing potential, reduced acetylene to ethylene. There existed a close correlation between the time course of acetylene reduction and N2 fixation by excised nodules of M. cerifera. The ability of excised nodules of legumes to reduce acetylene was short lived, as was the capacity for N2 fixation. There was a stoichiometric relationship between the production of ethylene and the disappearance of acetylene which suggested formation of ethylene from acetylene. Like N2 fixation, the reduction of acetylene was detected only when 02 was present and was stimulated by an increased 02 concentration. Furthermore, as cited above, N2 inhibited the reduction of acetylene by excised leguminous and nonleguminous nodules.


Preliminary results of these studies on the reduction of acetylene by nodular tissues were presented by Sloger and Silver (1967) and by Sloger and Silver at a colloquium on biological nitrogen fixation held at Sanibel Island, 71a., reported by Silver (1967). Chronologically, the latter report was the first demonstration that acetylene reduction was characteristic of a number of nonleguminous and leguminous plants. This report extended the previous data by Koch and. Evans (1966), who demonstrated reduction of acetylene by

soybean nodules. Additional observations by Stewart et al.






82


(1967) indicated that nodules of Alnus, Comptonia, and bluegreen algae in lake and soil habitats reduced acetylene.


From a comparison of data on optimal conditions for acetylene and N2 reductions by excised nodules of M. cerifera, it was observed that 0.04 atm acetylene and 0.08 atm N2 supported maximum reduction. This suggested that the affinity of nitrogenase for acetylene was about two to three times that for N2. The permeability characteristics of acetylene compared to N2 for nodular tissue may effect the apparent high affinity for acetylene.


Although the apparent Km values for N2 and acetylene were different for nodules of M. cerifera, they were similar for soybean nodules as shown by Koch and Evans (1966). In contrast, Hardy and Knight (1967b) presented data which showed that the nitrogenase of heated cell-free extracts of A. vinelandii appeared to bind N2 ten times as well as acetylene. More data are required to establish whether the mechanics of binding and reduction of molecules by the nitrogenases of various organisms are similar.


The evidence for acetylene and N2 reduction by nitrogenase of nodules in these studies suggests that the active site of the enzyme is not specific for a single substrate. That acetylene and N2 reductions were analogous processes, catalyzed by nitrogenase from C. pasteurianum was shown by Dilworth (1966). Hardy and Knight (1967b), after studying the reduction of various molecules by cell-free extracts







83


of A. vinelandii and C. pasteurianum, suggested that the N2 fixing system had a versatile substrate binding site.

Acetylene appeared to have interfered with enzyme activity because nodules of M. cerifera were affected by concentrations of acetylene above 0.2 atm. A decreased rate of ethylene production was observed. This result differed from the data for soybean nodules reported by Koch and Evans (1966). Their data showed no adverse effect of acetylene up to a concentration of 0.4 atm. A second interesting observation was made from the results which were obtained for N2 inhibition of acetylene reduction by soybean nodules and nodules of N. cerifera. Acetylene and N2 appeared to have equal access to the enzyme site within nodules of M. cerifera, whereas the data from soybean nodules suggested that the gases were not equally available to the enzyme. If both N2 and acetylene molecules reached the bacteroids of soybean nodules by diffusion, then the observed results should be like that found for nodules of M. cerifera. However, if N2 was binding to a carrier substance (Burris, 1966), and subsequently transported to the bacteroids, then acetylene may compete for the carrier. The high affinity of acetylene for the carrier may explain the fact that a N2 to acetylene concentration ratio of three to one did affect the reduction of acetylene.

The feasibility of using the acetylene reduction method as a measure of N2 fixation is demonstrated by the






84


present studies. The method has numerous advantages over the 15N method. The cost of gas chromatographic apparatus is relatively inexpensive compared to a mass spectrometer and highly enriched nitrogen. The sensitivity of the acetylene reduction method is about 10 to 100 times greater than the 'N method used in these studies. Ethylene production could be detected with as little as 10 mg of tissue-an individual soybean nodule or a small lobe from a nodule of 1. cerifera. In contrast, about one gram of nodules is required for the 15N method. Furthermore, analysis of gas samples can be made in one hour or less after incubating the nodules with acetylene. In investigations using the 15N method, exposure lasts for hours or days and is followed by laborious digestion, distillation and conversion of ammonia to molecular nitrogen for analysis. On the basis of these factors, the acetylene reduction method is a practical means of determining N2 fixation by various organisms.

The usefulness of the acetylene reduction method demonstrated in these studies confirms the application of the method by Koch and Evans (1966) for the assay of activity of nitrogenase in cell-free extracts of nodules. Furthermore, Stewart et al. (1967) showed that the method can be employed as an index of N2 fixation in aquatic environments, in soils, and by modulated plants.







85

With acetylene reduction as a simple assay for N 2 fixing ability and with further work resulting in preparations of cell-free extracts of nodules of Tr,. cerifera, the roles of the endophyte and plant proteins in symbiotic nitrogen fixation by nonlegumes may be elucidated.












SUMMARY


Excised intact root nodules from native Myrica

cerifera were shown by the 15N method to fix molecular nitrogen. The 15N method involves mass spectrometric analyses for 15N enrichment, after nodules are exposed to 15N2' Limiting the analyses to the acid soluble nitrogenous compounds of the nodules increased the sensitivity of detecting N2 fixation. The fact that the newly fixed N2 accumulated in the metabolically active portion of the nodules supports the idea that N2 is reduced to ammonia.

The time course of N2 fixing activity by nodules after detachment as well as the optimal N2 and 0 concentrations were determined. Nodules of M. cerifera were good experimental material for studies of symbiotic nitrogen fixation because they had several physiological aspects which were typical of nonleguminous nodules and because they had prolonged activity after detachment.

Active nodular homogenates of M. cerifera were prepared by a procedure involving anaerobic homogenization in a buffered cacodylate medium and later supplementing with Na2S204, an ATP generating system, and 020 In this system Na2S204 served as an electron source and the ATP generating system served as an energy source. The requirements for ATP and electrons were consistent with findings


86






87


for other nitrogenase systems. The required 02 stimulated endogenous respiration necessary for N2 fixation by intact portions of the tissue.


Nodules of several species of leguminous and nonleguminous plants were shown to reduce acetylene to ethylene. Since the requirements for acetylene reduction and N2 fixation were similar, and since N2 inhibited acetylene reduction, nitrogenase was assumed to catalyze both reactions.


The ability of nodules to reduce extremely small amounts of acetylene to ethylene could be detected by gas chromatography. By this sensitive analysis it was possible to detect activity by individual nodular lobes of M. cerifera and single soybean nodules. Therefore, acetylene reduction by nodules, and its detection by gas chromatography, is considered to be a valid and rapid means of measuring the N2 fixing ability of nodules.


The advantages of the acetylene reduction method compared to the 15N method were discussed.













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Bergersen, F. J. 1962a. The effects of partial pressure
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00


Bulen, W. A. and J. R. LeComte. 1966. The nitrogenase system from Azotobacter: two-enzyme requirement
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pN2 and the p02 function for nitrogen fixation by
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Carnahan, J. E., L. E. Mortenson, H. F. Mower, and J. E.
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Centifanto, Y. M. 1964. Leaf nodule symbiosis in Psychotria
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Dart, P. J. and F. V. Mercer. 1963. Development of the
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Delwiche, C. C., P. J. Zinke, and C. M. Johnson. 1965.
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Furman, T. E. 1959. The structure of the root nodules of
Ceanothus sanguineus and Ceanothus velutinus, with special reference to the endophyte. Amer. J. Bot.
46: 698-708.

Gardner, I. C. 1965. Observations on the fine structure
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Hardy, R. W. F. and E. Knight, Jr. 1966. Reduction of N 0
by biological N2 fixing systems. Biochem. BiophyK.
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Hardy, R. W. F. and E. Knight, Jr. 1967a. The biochemistry
and postulated mechanisms of nitrogen fixation.
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Hardy, R. W. F. and E. Knight, Jr. 1967b. ATP-dependent
reduction of azide and HCN by N2 fixing enzymes of
Azotobacter vinelandii and Clostridium pasteurianum.
Biochim. Biophys. Acta 139: 69-90.

Harris, G. P. and T. Y. Morrison. 1958. Fixation of
nitrogen by excised nodules of Coriaria aborea
Lindsay. Nature 182: 1812.

Hoch, G. E., H. N. Little, and R. H. Burris. 1957. Hydrogen
evolution from soybean root nodules. Nature 179:
430-431.

Hoch, G. E., K. C. Schneider, and R. H. Burris. 1960.
Hydrogen evolution and exchange, and conversion of N20 to N2 by soybean root nodules. Biochim.
Biophys. Acta 37: 273-279.

Jansen, E. F. 1965. Ethylene and polyacetylenes. In:
Plant Biochemistry. J. Bonner and J. E. Varner,
eds. Academic Press, New York. p. 641-664.

Jordan, D. C., I. Grinyer, and W. H. Coulter. 1963.
Electron microscopy of infection threads and
bacteria in young root nodules of Kedicago sativa.
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Kamen, M. 1956. Introductory Remarks. In: A Symposium
on Inorganic Nitrogen Metabolism. W. D. YcElroy
and B. Glass, eds. John Hopkins Press, Baltimore,
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Kennedy, I. R. 1966a. Primary products of symbiotic nitrogen fixation. I. Short-term exposure of serradella
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Acta 130: 295-303.




Full Text

PAGE 1

NITROGEN FIXATION BY TISSUES OF LEGUMINOUS AND NONLEGUMINOUS PLANTS By CHARLES SLOGER A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1968

PAGE 2

ACKNOWLEDGEMENTS The author wishes to thank Dr. W. S. Silver, cochairman of the supervisory committee, for his encouragement, guidance, helpful suggestions, and incisive criticisms in the conduct of the research and in the preparation of the manuscript. Dr. G. J. Fritz, chairman of the supervisory committee, provided advice and valuable criticisms in the preparation of the manuscript. Appreciation is extended to the committee members, Dr. E. S. Ford and Dr. M. Pried, who gave of their time and advice. The Department of Botany, University of Florida is thanked for an assistantship. The research was made possible by an assistantship supported by the National Institute of Health Research Grant GM-08577. Dr. M. Tyler, chairman of the Department of Bacteriology, University of Florida as well as the faculty of the Department are thanked for use of their facilities. Llarcia Sloger assisted immeasurably in the preparation of the manuscript. Her confidence is gratefully ackno wl e dge d . ii

PAGE 3

TABLE OP CONTENTS Page ACKNOWLEDGEMENTS ii LIST OP TABLES v LIST OP FIGURES vii INTRODUCTION 1 REVIEW OP THE LITERATURE 4 Origin of the root nodules 5 External appearance of the root nodules 7 Internal appearance of the root nodules 7 Endophytes of the root nodules 9 Site of N 2 fixation 10 Hydrogen evolution by the root nodules ... 11 Effect of partial pressure of N2 12 Substrates reduced by the root nodules ... 13 Effect of partial pressure of O2 13 N2 fixation by excised root nodules .... 14 N2 fixation by homogenates and breis of root nodules 15 MATERIALS AND METHODS ..... 17 Botanical species 17 Botanical techniques 18 Preparation of homogenates and breis .... 20 Preparation of incubation atmospheres ... 21 Incubation of samples 22 Extraction of acid soluble nitrogen .... 22 IQ'eldahl method 22 Conversion of ammonia to nitrogen gas ... 23 Colorimetric analysis of total nitrogen. . . 23 "^N techniques 23 Preparation of -^jj 23 15 N method 24 Mass spectrometry 24 Calculation of -^N content 25 Gas chromatographic analysis for ethylene and acetylene 25 iii

PAGE 4

TABLE OF CONTENTS — Continued Page RESULTS 28 Appearance of root nodules 28 No fixation by excised root nodules .... 28 Effect of partial pressure of Ng 32 Effect of partial pressure of 35 Storage of intact nodules 39 Variation among replicate samples 39 N2 fixation by nodular homogenates of M. cerifera 39 Site of N2 fixation by homogenates. .... 47 Acetylene reduction by nodules of M. cerifera 51 Acetylene reduction by nodules of various plants 54 Effect of acetylene concentration on ethylene production 61 N 2 inhibition of the reduction of acetylene 63 Reduction of acetylene by individual nodules 66 Reduction of acetylene by breis 68 DISCUSSION 72 SUMMARY 86 LITERATURE CITED 88 BIOGRAPHICAL SKETCH 96 iv

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LIST OF TABLES TABLE Page 1 Extent and Distribution of Nodulated Nonleguminous Angio sperms 6 2 Test for N 2 Fixation by Detached Nodules of IJyrica cerifera 30 15 3 N Content of Acid Soluble and Acid Insoluble Fractions of Nodules., of M. cerifera after exposure to .... 31 4 Comparison of N ? Fixation Between Nodules with Epidermis Present and Nodules with Periderm present 34 5 Studies on Storage of Excised Nodules of M. cerifera 40 6 Variability Among Samples Within One Experiment and Among Several Experiments. . 41 7 Effect of Na 2 S 2 0, on N« Fixation by Root Nodule Homogenates 43 8 Effect of Supplements on Np Fixation by Nodular Homogenates . 45 9 Effect of Oxygen on N ? Fixation by Intact Nodules and Supplemented Nodular Homogenates 46 10 Effect of 0 2 and Na^O^ on N 2 Fixation by Supplemented Homogenates 48 11 Effect of pH on N 2 Fixation by Homogenates . . 49 12 Np Fixation by Supplemented Homogenate Fractions ..... 50

PAGE 6

LIST OF TABLES — Continued TABLE Page 13 Reduction of Acetylene to Ethylene by Various Plant Tissues 60 14 Acetylene Reduction by Individual Soybean Nodules 67 15 Reduction of Acetylene by Breis of Soybean Nodules Prepared with Polyvinylpyrrolidone and Ascorbate . . 70 16 Reduction of Acetylene by Breis of Myrica vi

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LIST OF FIGURES Figure Page 1 Time course of Np fixation by excised nodules of M. cerif era 33 15 2 Effect of pNp on K incorporation by excised nodules of Myrica cerifera . . 36 3 Line weave r-Burk plot to determine the Michaelis constant for nitrogen fixation by whole nodules of cerifera 37 4 Effect of pOp on Np fixation by detached nodules or Myrica cerifera 38 5 Time course of acetylene reduction to ethylene by nodules of M. cerifera at 0.1 atm and 0.2 atm O2 53 6 Relationship of ethylene production to acetylene disappearance by nodules of M. cerifera 55 7 Time course of reduction of acetylene to ethylene by excised nodules of Casuarina equisetif olia 56 8 Relationship of ethylene production to acetylene disappearance by soybean nodules 57 9 The effect of partial pressure of acetylene on ethylene production by nodules of I-Tyrica cerifera 62 10 Effect of Np on ethylene production from acetylenS by nodules of 11. cerifera . 64 vii

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LIST OP FIGURES — Continued Figure 11 12 Page Effect of pN 2 on acetylene reduction by soybean nodules. . . . Comparison of time courses of N„ fixation by excised nodules of legume! and nonlegumes 65 74 viii

PAGE 9

INTRODUCTION Most organisms readily assimilate combined nitrogen, and with it synthesize proteins and other substances vital to their existence. However, the quantity of available combined nitrogen is relatively small, whereas an abundance of nitrogen is present in elemental form in the atmosphere as molecular nitrogen, N 2 . Thus, a critical step in the nitrogen cycle is the replenishment of the supply of combined nitrogen by nitrogen fixation — the conversion of molecular nitrogen to a fixed form, ammonia. Both microorganisms and higher plants are involved in nitrogen fixation. Most plants assimilate nitrate and ammonia; only a few, molecular nitrogen. In all authenticated cases in which higher plants assimilate N,,, some microorganism is living symbiotically with the higher plant (Bond, 1967). These symbiotic relationships are expressed as leaf nodules, mycorrhizal associations on roots, and commonly as root nodules. Among the angiosperms which can form root nodules, there has been no substantial evidence presented which shows that either of the symbionts, the unnodulated plant or the free living form of the endophyte, have the ability to fix I\ T 2 by itself. Because most of the fixed nitrogen in the biosphere is from symbiotic sources 1

PAGE 10

2 (Stewart, 1966), it is important to understand the process of symbiotic nitrogen fixation. As recently as 1956, Kamen (1956) stated, "Now, there has been a great literature on nitrogen fixation mostly at the physiological level. At the biochemical level there is very little." The information on nitrogen fixation obtained by physiological studies of intact organisms has been limited and difficult to understand. A turning point in the study of nitrogen fixation occurred when Carnahan et al. (I960) reported a method for preparing and maintaining a cell-free fixing extract from a micro-organism. Prom that time, the elucidation of the biochemistry of nitrogen fixation has rapidly advanced. In contrast, information on symbiotic nitrogen fixation continued to remain at the physiological level. It is important to point out that knowledge of biochemical aspects of symbiotic nitrogen fixation was almost completely lacking when the present investigation was started in September, 1964. The primary reason is that leguminous nodules lose all capacity to fix when they are fragmented. Even attempts to enhance fixation in sliced soybean nodules by the addition of a variety of substrates, cof actors, crude extracts, and reducing agents were unsuccessful (Aprison et al., 1954). Furthermore, previous to September, 1964, literature on symbiotic nitrogen fixation by nonleguminous angiosperms pertained only to the physiology of the intact root nodule. Prom the point of view of the

PAGE 11

3 present investigation, it should be stressed that several contributions to our understanding of symbiotic nitrogen fixation by homogenates of root nodules were not available when this investigation was started in September, 1964, but were published after that time by Sloger and Silver (1965), Bergersen (1966b), Bergersen (1966c), Koch et al. (1967a), and Koch et al. (1967b). The primary objective of the present investigation was the study of nitrogen fixation by excised root nodules and nodular homogenates of a native nonleguminous angiosperm, Myrica cerifera L. Firstly, a study was made to determine whether the excised nodules of M. cerifera fixed N^, and if so, to determine optimum conditions for nitrogen fixation. Secondly, a study was made to determine requirements for nitrogen fixation by nodular homogenates. Finally, a study was conducted to establish whether the excised nodules would reduce acetylene to ethylene, and to evaluate the acetylene reduction method as an assay for nitrogenase activity.

PAGE 12

REVIEW OP THE LITERATURE The subject of biological nitrogen fixation has been reviewed recently by Burris (1966). Hardy and Knight (1967a) have prepared a review pertaining to the biochemistry and postulated mechanisms of nitrogen fixation. Stewart (1966) compiled information on nitrogen fixation by plants. Recently, Stewart (l 9 6 7 ) has discussed the role of nitrogen fixing plants. The present discussion will be confined to the literature on symbiotic nitrogen fixation by intact and fragmented root nodules of legumes and nonleguminous angiosperms up to December, 1967. A brief discussion will be given about the development of the root nodules as it relates to the nitrogen fixing symbioses. The family Leguminosae makes up one group of nodulated plants. Allen and Allen (1958) reported that root nodules have been found on approximately 8 9 per cent of the species studied, but this represents only a small fraction of the total known species. The endophytes of the root nodules have been identified as members of the bacterial genus Rhizobium. Nonleguminous plants associated with ^ fixation are in diverse taxonomio groups . They may be separated on the basis of the nature of the symbionte in the association.

PAGE 13

5 Bond (1967) suggested the categories nonleguminous angiosperms or actinomycetal associations, mycorrhizal associations, algal-cycad associations, and bacterial -leaf associations. These categories are based upon available evidence and may be changed after further research. In this review and study, the term "nonleguminous" will refer to those angiosperms whose nodules harbor endophytes believed to be actinomycetes. There are 13 genera of plants in this group, as shown in Table 1, and it seems likely that the list of species and genera will be increased. Origin of the root nodules Tissues comprising the nonleguminous nodule originated in the pericycle of the root according to Fletcher (1955) and Taubert (1956). Thus, the nodules are considered modified lateral roots. In contrast, the tissue g^nr>^^7 a«ieg™inous nodule , or iglnate_f £aBL the inner coi^JTTthT root,., Wipf and Cooper (1940) made the observation that within this region, nodule initiation is dependent on the presence of tetraploid cells in a normally diploid plant. Rhizobial infection takes place through a root hair by means of an infection thread. The infection thread penetrates the cortical cells of the root, and as it approaches the tetraploid cells, it stimulates them and adjacent diploid cells into meristematic activity resulting in the formation of a nodule.

PAGE 14

6 TABLE 1 EXTENT AND DISTRIBUTION OP NODULATED NONLEGUMINOUS ANGIOSPERMS Genus Number of species in genus 3. Present distribution Number of spe recorded to bear nodul Coriara 15 Mediterranean to Jp^nn New Zealand, Chile to Mexico 12 Alnus 35 Europe, Siberia, North America. Japan, Andes 25 Myrica b 35 Many tropical, subtropical and temperate regions 11 Casuarina 45 Australia, tropical Asia, Pacific Islands 14 Elaea^us 45 Asia, Europe, North America 9 Hipponhae 1 Asia, Europe, from HimsT a troo 7 r^i xj. uiu xiinicixayas to Arctic Circle 1 Shepherdia 3 North America 2 Ceanothus 55 North America 30 1 Discaria 10 Andes, Brazil, New Zealand, Australia ' Dryas Arctic and mountains of north temperate zone 1 Purshia 2 North America 2 Cercocarpun 20 North America 1 40 SWLS 4 C9ntral 1 Botanic tarXnt^w f or^hf^tf" H " K " Air ^ Shaw Hoyal b Including Comptonia From Bond (l 9 67) page 108.

PAGE 15

7 External appearance of the root nodules During early stages of development nonleguminous nodules resembled those of a typical legume (Bond, 1963). Later, nodule lobes branch frequently and elongate to form a clustered structure. At maturity, the nodule lobes of feica, Casuarina, and Alnus are 2 to 6 mm long, 0.8 to 1 mm wide, and lack root caps and root hairs. The nodules are perennial and may range in diameter from a few mm to 150 mm. The nodules of most legumes occur as small (less than 1 cm in diameter), spherical, club-shaped, or branched structures. Lleristematic activity is frequently apical in position, but soybean nodules have no distinct growing point. Both water cultured and field grown Myrica and Casuarina have nodules which differ from those of other plants because of negatively geotropic rootlets extending from each lobe (Bond, 1957). Internal appearance of the root nodules The mature nonleguminous nodule shows a number of differences from the leguminous type. It has an endophytecontaining cortex and a central vascular cylinder Fletcher, 1955; Furman, 1959; Becking et al., l 9 64; Silver, 1 9 64). The nodule structure is derived from an enlarged cortical region with certain host cells containing dense growths of endophyte. The smaller, uninfected cortical cells often contain starch and tannins. In contrast, the leguminous nodule has a narrow cortical region and an endophyte-

PAGE 16

8 containing central region (Allen and Allen, 1958). Vascular tissue lies between the cortex and the central region and is surrounded by an endodermis, which may function as a diffusion barrier. The leguminous nodule is soft and is easily crushed. Nonleguminous nodules are woody; only young stages are relatively soft. The central region of leguminous nodules which are fixing K 2 contains a red hemoprotein pigment, hemoglobin (leghemoglobin) . Ineffective leguminous nodules or the nodules not fixing Np do not contain the pigment. Hemoglobin is found within the infection vesicles of infected host cells but outside the bacteroids (Bergersen, 1966a). The recent demonstrations of fixation by isolated bacteroids (Bergersen and Turner, 1967) and by cell-free extracts of bacteroids (Koch et al., 1967a; Koch et al., 1967b) in the absence of hemoglobin have eliminated conclusively hemoglobin as an integral part of nitrogenase. Hemoglobin is said to bind 0 2 and K 2 , and therefore may function in the transport of these molecules to bacteroids; also hemoglobin, by binding 0 ? , may prevent 0 2 inhibition of N p fixation (Burris, 1966). Nonleguminous nodules have been shown to contain hemoglobin by Davenport (i960). He used a microspectroscope to detect hemoglobin in whole nodules of Casuarina cunnin ghamiana , Alnus glutinosa , and Myrica gale . Also the hematin content of the nodules was eight to eleven times higher than in the root cortex. However, Moore (1964) failed to verify this.

PAGE 17

Endophytes of the root nodules The identity of the endophytes in nonleguminous nodules is unsettled. Most investigators using modern techniques have concluded that the endophytes were actinomycetes (Becking et al., l 9 64; Gardner, 1965). Silver (1964) confirmed the filamentous nature of the endophyte of root nodules of Myrica cerifer a with an electron microscope and considered it an actinomycete. No confirmed isolation of the infecting endophyte has been achieved. The infected host cells are enlarged and devoid of almost all host cell contents (Silver, 1964). The site of N 2 fixation is not known but may be the ' infected host cell or the endophyte. Details of the initial infection by rhiaobia, development of infection thread, and development of root nodules are well known for legumes. Changes of the fine structure of the host cells which accompany nodule development have been reported by Goodchild and Bergersen (1966), Bart and Kercer (1963) and Jordan et al. (1963). Their work may be summarized as fellows. At maturity, infection vesicles each containing four to eight bacteroids fill the host cells of the central region of the nodule. The membrane of the infection vesicle ' is believed to originate from the host cell. The differentiate of rhizobia to bacteroids is associated with the tissues . B ability to fix nitrogen. At this time, the cytoplasm of the host cell loses ribosomes and endoplasmic reticulum, whereas mitochondria and the nucleus remain.

PAGE 18

10 Site of N c fixation Because only nodulated plants could maintain growth in a nitrogen-free medium, it was assumed that N 2 fixation occurred in the nodules. The occurrence of biological nitrogen fixation was proven unequivocally by the use of the 15 N method (Burr is and Miller, 1941). The first demonstration of consistent 15 N enrichment by excised soybean nodules which had been exposed to an atmosphere enriched 15 with N 2 was made by Aprison and Burris (1952). Magee and Burris (1954) demonstrated N 2 fixation by excised nodules from nine different legumes representing eight cross inoculation groups by the 15 N method. Bond (1955) showed that nodulated plants of Alnus glutinosa , Myrica gale, and Hippophae rhamnoidp. exposed to N 2 had the greatest enrichment in their nodules and that excised nodules of A. glutinosa fixed 15 N,>. He also showed that nodules which were attached to native growing A. glutinosa fixed N 2 (Bond, 1956). The 15 N method has been used to demonstrate fixation by detached nodules of the following species of nonlegumes: Casuarina eurmin^^,.., Ceanothus azureus > and Shepherdia canadensis (Bond, 1957); Coriaria arborea (Karris and Morrison, 1958); Mscaria toun.pt™, (Morrison, 1 9 61); Comptonia peregrin, (Ziegler and Huser, 1963); Myrica cerifer a (Sloger and Silver, 1 9 6 5 ); 12 species of Peanothus (Delwiche et al., 1 9 6 5 ), and Alnus .-ferula. (Rodriguez-Barrueco, 1966). It is now accepted that the nodules are the sites of fixation in nonle^uminous angiosperms

PAGE 19

11 The site of fixation inside leguminous nodules has been suggested. Bergersen (I960) reported that ^ fixation by soybean nodules was located in some component of a membrane fraction which consisted largely of fragments of the intracellular membrane envelopes surrounding the bacteroids. However, R. Klucas repeated these experiments, reported by Delwiche (1966) and Silver (1967), and found that the soluble portion of the soybean nodules consistently contained the 15 highest enrichment after short exposure to Kennedy et al. (1966) suggested that bacteroids within serradella nodules 15 incorporated ^N^. Recently, Bergersen and Turner (1967) stated that a bacteroid fraction isolated from soybean nodules had N 2 fixing activity. Koch et al. (1967a) and Koch et al. (1967b) reported that cell-free extracts of bacteroids isolated from soybean nodules fixed Np. Hydrogen evolution by the root nodules The evolution of hydrogen, H 2 , by leguminous nodules was detected by Hoch et al. (1957). Later, Hoch et al. (I960) reported that H^ evolution by soybean nodules was inhibited by and nitrous oxide (N^O), but was insensitive to carbon monoxide (CO). Also Hoch and co-workers reported that during fixation an exchange occurs between deuterium and hydrogen atoms — this exchange reaction occurs between deuterium and endogenous H donors, and results in the formation of the species HD. They observed that the exchange reaction was inhibited by CO and N«0, but

PAGE 20

12 was activated by N,,. They suggested that hydrogen evolution and the exchange reaction expressed separate activities of the enzyme nitrogenase. Bergersen (l 9 63) verified these observations . Mxon^lgS?) sugg ested that pea no dule^^ ^hydrogenases , one associated wij^rfcro^^ .^gggHigjSjjg^^ N 2 fixation . Howeve^^^H ^^l^Bl^^eJ ^ogen under .normal condition*'. Intact nodules of the nonlegurr.es, Alnus glutinn™ . Alnus rugosa and Elaeagnus commute, did not evolve hydrogen when tested in an atmosphere of 80 per cent helium and 20 per cent oxygen (Moore, 1964 ). Effect o f partial pressure of n Nitrogen fixation by nodules is influenced by the availability of N,. Burris et al. (1955) C0I3puted the Michaelis constant, the partial pressure of ^ supporting half maximum fixation, for sliced soybean nodules from a thorough statistical analysis of the data obtained in many experiments. The Michaelis constant was 0.02 i 0.004 atm N 2 for this leguminous tissue. Maximum fixation was observed above 0.10 atm N g . m contrast, Bond (1 959) reported that whole excised nodules of Alnus glutinosa achieved maximum fixation at 0.2 5 atm ^ It appears ^ neither ^ ^ tial pressure of N g in the atmosphere, nor the partial Pressure of within a nodule would be rate limiting

PAGE 21

13 Substrates reduced by the root nodules N ? 0, which is structurally similar to molecular N ? , has been shown to be an inhibitor of N ? fixation by soybean nodules (Hoch et al., I960). They showed that N 2 0 was reduced to N ? by the intact nodules. Koch and Evans (1966) and Sloger and Silver, reported by Silver (1967), noted that acetylene was reduced to ethylene by nodules of legumes and nonlegumes. Effect of partial pressure of O p The effect of oxygen on symbiotic nitrogen fixation has received much attention. Burris et al. (1955) reported that N« fixation by sliced soybean nodules increased with 0 ? concentration until maximum fixation was reached at 0.5 atm Op. Above this Op concentration there was a marked decrease in Np fixation. The authors believed that these results indicated a very close relation between fixation and respiration, and that oxygen primarily affected the latter process. Bond (1957) showed that oxygen was essential for Np fixation by nonleguminous nodules. Bond (1961) reported that maximal N ? fixation by nonleguminous nodules was reached at 0.20 to 0.25 atm Op. Above this 0 2 concentration there was a decrease in Np fixation. Decreased Np fixation at high oxygen concentrations appears to be a characteristic of root nodules in general. A possible reason for this was suggested by Bergersen (1962a). He

PAGE 22

14 suggested that oxygen competed with nitrogenase for endogenous hydrogen atoms. The competition was possible because hemoglobin in soybean nodules was shown to be completely oxygenated at 0.5 atm Og, the point where inhibition of fixation occurred (Bergersen, 1962b). Bond (1964) suggested that oxygen inhibited fixation by nonleguminous nodules competitively. Bergersen (1963) noted that oxygen affected hydrogen evolution and the exchange reaction by excised soybean nodules in the same manner as for fixation. However, may have little, if any, direct role in the enzymatic process of fixation since some anaerobes fix N^. No fixation by excised root nodules Upon excision from the root, nodules lose their capacity for nitrogen fixation within a matter of hours. Aprison and Burris (1952) found that in the time course of N 2 fixation by excised soybean nodules the rate was constant for two hours, with only slight fixation occurring thereafter. Dixon (1967) found that excised pea nodules within minutes lost the capacity to fix N«, but when nodules were left attached to a piece of the primary root the time course of fixation was similar to that of excised soybean nodules. The rapid loss of activity by leguminous nodules is a limiting factor in research. In contrast, nodules of Alnus glutinosa remained active for 12 hours after detachment, and nodules of Hipjoophae rha mnoides remained active for nearly 24 hours,

PAGE 23

15 according to Bond (1957). Apparently, fixation is less drastically affected by nodule excision from the root in the nonlegumes than in the legumes. This extended period of activity would he especially desirable in experiments with fragmented nodules. fixation by homogenates and breis of root nodules Only recently have techniques been found which can stabilize nitrogenase activity in fragmented nodular tissues. Sloger and Silver (1965) noted that anaerobically prepared and buffered homogenates of nodules of M. cerifera supplied with oxygen and NagSgO^, fixed at over 4 per cent the rate of that of intact nodules. Sloger and Silver (1966) reported that similar nodular homogenates fixed at about 50 per cent the rate of that of intact nodules, when supplied oxygen, an ATP-generating system, and Na^O^. Bergersen (1966b) reported that breis of soybean nodules prepared anaerobically in a buffered sucrose solution would fix only if oxygen were present during incubation. Later Bergersen (1966c) reported that oxygen concentrations up to 0.10 atm stimulated fixation by breis of soybean nodules. He also reported that an exogenous ATP-generating system slightly increased fixation. Bergersen and Turner (1967) showed that nitrogenase activity v/as present in bacteroids isolated from soybean breis. Another technique for preparing active breis of soybean nodules was reported by Koch et al. (1967a). These breis, when prepared anaerobically in the presence of

PAGE 24

16 "buffered ascorbate and insoluble polyvinylpyrrolidone (PVP), fixed N 2 and reduced acetylene only when oxygen was present. Koch et al. (1967b) obtained a cell-free extract with nitrogenase activity from bacteroids of active breis. This enzyme system required an exogenous ATP-generating system and Na 9 S 9 0 A , hut no oxygen.

PAGE 25

MATERIALS AND METHODS Botanical species Myrica cerifera L., commonly called southern wax myrtle, was collected from native habits near Gainesville, Florida. Casuarina eguisetif olia J. R. and G. Forst, commonly called Australian pine, was collected by the author at Sanibel Island and Haulover Beach State Park, Florida. Casuarina cunninghamiana Miq. was collected by Dr. W. S. Silver from Deland, Florida. Podocarpus macrophylla D. Don. was grown in the greenhouse and at the time of the experiment was two years old. Pinus elliottii Engelm. was obtained as two year old seedlings from plots in the Austin Cary Memorial Forest, courtesy of Dr. Wayne H. Smith. Psycho tria punctata Vatke, also known as Psychotria bacteriophila (Centifanto and Silver, 1964), belonged to the collection of plants used by Centifanto (1964). Glycine max Merr. 'Hampton' , commonly called soybean, was obtained as seeds coated with a mixed inoculum from Dr. Keull Hinson. Plants from the University of Florida 17

PAGE 26

18 Agricultural Experiment Station farm plots were also donate by Dr. Hinson. Glycine max Kerr. 'Chipewa' was obtained as seeds Or <.«y>-. SV-cf«U*vlvtr5i!W^ from Dr. H. J. Evans, Univ o rsil^y o£ Orison , Corvallis, Oreg Luoinus albus L. was obtained from the University of Florida Agricultural Experiment Station farm plots. Crotolaria spectabilis Roth, was collected from native habitats on the campus. Voucher specimens have been filed in the University of Florida herbarium for the following species: M. cerifera C. equisetifolia , C. cunninghamiana , P. macrophylla , P. punctata , and C. spectabilis . Klebsiella rub i ace arum (strain K4), a bacterial endophyte of leaf nodules of P. punctata , was grown on nitrogenfree mineral broth according to the methods of Ne elands (1967). Botanical techniques Chipewa soybeans were grown in a greenhouse according to the methods of Koch and Evans (1966) and in a prepared field plot. Hampton soybeans were grown in a prepared field plot in addition to other sources of the plant mentioned earlier. A nitrogen-free nutrient solution described by Centifanto (1964) was supplied to the greenhouse grown Chipewa soybeans.

PAGE 27

13 Nodules of I", cerifera were collected from plants, where they had he en exposed to daily weather changes and the annual climatic patterns. The plants were considered heterogeneous because of their va.ried physiological ages. Environmental conditions affecting the native plants were not controlled. The selection of plants was limited to those 0.5 to 2 meters tall, which represented plants two to ten years old. All plant material was carefully dug up so as not to detach nodules from the roots. To facilitate the handling of Myrica , stems were cut 2 to 3 dm above ground level and discarded, while the remaining portion v/as placed in appropriate containers for transport to the laboratory. Myrica root systems were washed at the greenhouse first. Roots with nodules were cut from the plant and then taken to the laboratory. Greenhouse grown plants were removed from pots, washed with tap water, and quickly taken to the laboratory. At the laboratory nodules were excised, washed in tap water, and blotted on paper towels. Obviously senescent, dead, and decaying nodules were discarded. Nonleguminous nodules larger than 1 cm in diameter were divided into two to six pieces. The nodules from one species were lumped together and randomized so that each flask received an equivalent fresh weight of tissue.

PAGE 28

20 Preparation of homogenates and breis Homogenates of nodules of M. cerifera were prepared in an Onmimixer (Servall Corp., Norwalk, Conn.). Nodules (over 10 grams) and buffer solution were placed in a grinding chamber, which, had two small holes for gas entrance and exit. The chamber was purged with helium for 10 minutes prior to and during grinding. Nodules were ground for one minute at high speed. Then homogenates were transferred to flasks which were being flushed with helium. Homogenates were kept anaerobic until the flasks were attached to the gassing manifold. For smaller quantities of nodules, homogenates were prepared in a 50 ml cellulose nitrate centrifuge tube, which was attached to the Omnimixer by an adapter. Helium was flushed through the chamber for several minutes prior to grinding. Subsequent handling was the same as that described above. Breis obtained from nodules of soybean and M. cerifera were prepared according to the method of Koch et al. (1967a). Cacodylate buffer solutions were adjusted to pH 7 by 1 N KOH and then purged with hydrogen for 15 minutes. The buffer was prepared fresh for each experiment. Solutions of sodium dithionite, Na 2 S 2 0^, were prepared anaerobically by tipping the chemical from a side arm of a flask into water and a predetermined amount of 1 N KOH to give a solution of pH 7. The water and KOH were purged previously with hydrogen gas. Na 2 S 2 0 4 solutions were prepared immediately prior to use.

PAGE 29

21 Preparation of incubation atmospheres 15 The following procedure was used for N experiments. Excised nodules were placed in 50 ml flasks, while homogenates were placed in 70 ml flasks equipped with two side arms. The flasks were then attached to a gassing manifold. The closed system was evacuated by a vacuum pump and alternately filled with helium three times. Replacement of gases to one atmosphere pressure and to a known composition was by the method of Burr is (Umbreit et al., 1951, p. 45). 15 The gases were added in this order: N 2 , oxygen, and helium. For the acetylene reduction experiments, test material was placed in 70 ml calibrated flasks equipped with two side arms. Rubber serum stoppers were used in the side arms. The flasks were then attached to a calibrated mercury-filled manometer. Both arms of the manometer were connected by a glass tube in the form of a reverse h with a ball adaptor at the top (Umbreit et al., 1951, p. 45). The adaptors of the manometer were attached to a gassing manifold by a ball and socket connection. With this method a series of eight manometers could be treated at one time. The system was evacuated and filled with helium three times, and then filled in order with acetylene, oxygen, and helium. The composition of gases was determined according to the method of Burris (Umbreit et al., 1951, p. 45). The tanks of oxygen, helium, nitrogen, hydrogen, argon, and acetylene were purchased from Katheson Co.,

PAGE 30

22 Atlanta, Ga. Acetylene was slowly sparged through concentrated H^SO^ to oxidize acetone and collected in a container for use in preparation of incubation atmospheres. Incubation of samples All flasks were incubated in a 30 C Warburg manometer water bath. The shaking rate was 100 oscillations per minute. Extraction of acid soluble nitrogen Acid soluble nitrogen was extracted from nodules and homogenates by grinding the tissue with 10 ml of 3 N HC1 for three minutes. The resulting homogenate was centrifuged at 5000 rpm for three minutes in a Clay-Adams clinical centrifuge. The supernate was used for the determination of acid soluble nitrogen; and the residue, for acid insoluble nitrogen. Kjeldahl method The method used was adapted from Burris and Wilson (1957). Samples were placed in 100 ml Kjeldahl flasks and 5 ml of concentrated H 2 S0 4 was added to each. One glass bead and one mercury catalyst tablet (British Drug House Ltd. Poole, England) were also added. The Kjeldahl flasks were heated gently at first so that charring of the sample was not violent. The digestion proceeded for 16 hours, at which time the solution was clear. Ammonia was distilled by a

PAGE 31

23 steam generating apparatus (W. Buchi Manufacture, Flawil, Switzerland) for two minutes into 25 ml of 0.01 N HC1. The distillate was used for conversion of ammonia to nitrogen gas or for determination of ammonia. The mercury catalyst tablets and the prolonged digestion prevented contamination by methylamine (Glover, 1956). Conversion of ammonia to nitrogen gas The procedure for the conversion is described in 15 the section Preparation of N g gas . The distillate was evaporated to 10 ml prior to conversion. Colorimetric analysis of total nitrogen The method used was adapted from Burris and Wilson (1957). The distillate was evaporated to 10 ml and then 0.5 ml samples were transferred to test tubes. Water was added to make a total of three ml. Two ml of Nessler's reagent and 2 ml of 2 N NaOH were added. The tubes were allowed to stand for 20 minutes at 20 C, and read in a KlettSummerson colorimeter with a No. 47 filter. A standard curve was prepared for 0 to 4-0 jig of ammonia N. 15 N techniques Preparation of 15 N g .-^NE^NO-, containing 30 atom 15 1^ per cent N excess and 95 atom per cent N excess was obtained from the Office National Industriel de 1' Azote, Paris, France and from Isomet Co., Palisades Park, N. J.

PAGE 32

24 15 15 respectively. The gas y N 2 was generated from the NH^NO^ by alkaline hypobromite oxidation according to the method of Sims and Cocking (1958) in a Toeppler pump system, as described by Rittenberg (1946). Release of 0^ was prevented by adding 0.1 per cent potassium iodide (KI) to the alkaline hypobromite solution before the addition of bromine. A liquid nitrogen trap was used to condense impurities from the gas before collection in a glass bulb. 15 N method . — Four steps are involved in the method: (1) exposure of the test sample to a gas mixture enriched 15 with for a time interval; (2) liberation of the nitrogen in the sample and collection in the form of ammonia by the Kjeldahl procedure; (3) reaction of ammonia with alkaline hypobromite to form gaseous nitrogen, which is collected in mass spectrometer bulbs by the Toeppler pump system; (4) admission of the gas sample to the vacuum chamber in the mass spectrometer and the measurement of isotopic abundance. Mass spectrometry Nitrogen gas was analyzed in a Consolidated Electrodynamics Corp. (Pasadena, Calif.), model 21-130 recording mass spectrometer. Normal operating conditions recommended by the manufacturer 1 were followed: the ionizing current Consolidated Electrodynamics Corp., "Operation and Maintenance Manual for Type 21-130 Mass Spectrometer , " Pasadena, Calif.

PAGE 33

25 was 20 ua. and the ionizing voltage was 68 volts. In all mass spectrometric analyses, sufficient N ? gas was introduced into the mass spectrometer so that the pressure in the reservoir was 20.46 microns of mercury. 15 Calculation of "IT content 15 The abundance of N in samples in atom per cent was calculated according to Rittenberg (1946) by the following equation: atom per cent "^N 100 where R = 2R+1 ' Z 2S 23 2 Q and I and I represent the intensities of the ion beams of mass 28 and mass 29. 15 The results of all N experiments were expressed ] e; as atom per cent excess which was calculated by sub15 tracting the atom per cent N for control tissue from the 15 atom per cent N for test material. Values greater than 15 0.015 atom per cent N excess were assumed to indicate that N 2 fixation had occurred. This value is the same as that used by Burris and Wilson (1957). A value as high as 15 0.015 atom per cent excess was necessary because of the relatively large variation in results obtained from repeated analyses of samples of tank I\ T 2 and from daily analyses of the same gas sample. Gas chromatographic analysis for ethylene and acetylene Samples of the gas phase in the incubating flasks

PAGE 34

26 were taken with 1 ml, polyproplylene syringes (Bee ton, Dickinson, and Co., Rutherford, N. J.). Since air contamination was undesirable, the "dead space" in the syringe and needle was displaced with a saturated NaCl solution or by flushing with helium. Syringes were placed in a water bath for several minutes, so that all gas samples, including standards, were at the same temperature prior to analysis. Gas samples, 0.5 ml, were analyzed in either a Loe Engineering Co. (Pasadena, Calif.), model 15A gas chromatographic ourtesy of Dr. P. H. Smith, or an F and M Scientific Corp. (Avondale, Perm.), model 700 gas chromatograph, courtesy of Dr. D. S. Anthony. Both instruments were equipped with a 0.63 x 40 cm teflon column containing 80-200 mesh silica gel. The temperature of the column was 90 C and the nitrogen carrier gas flow rate was 15 ml per minute. The model 700 gas chromatograph was equipped with a 0.63 x 60 cm teflon column containing 30-200 mesh silica gel. The temperature of the column was 90 C and the helium carrier gas flow rate was 30 ml per minute. The adsorption of water to the solid phase was minimized by the elevated column temperature. Under these sets of conditions identifications of acetylene and ethylene were made from the retention times, which were five and two minutes respectively. The sensitivity of each instrument was determined with a 0.1 ml gas sample (assuming 1 ml to be 100 per cent) and

PAGE 35

27 was expressed as mm peak height per per cent gas. The height times width at half -height method (McNair and Bonelli, 1967) was used for quantitation of peaks. Linear responses were obtained from standard ethylene samples ranging from 0.002 ml to 0.3 ml. The per cent measured gas in the sample was multiplied by the known volume of the flask to determine the volume of the measured gas in the incubation flask. The pressure and temperature of the gas in the flask at sampling times were used to adjust gases to standard temperature and pressure (STP) values to calculate the quantity of the gas in umoles.

PAGE 36

RESULTS Appearance of root nodules Nodules of M. cerifera were located predominantly on roots near the soil surface. Nodules are structures which consist of lobes closely clustered to one another. Newly formed lobes at the periphery of a cluster point upward, because lobes are negatively geotropic. Older, heavily suberized tissues were located inside the perennial nodule. The rootlets on developing lobes were white and 1 cm long. The rootlets on mature nodule lobes from plants growing in wet soil were 2 to 3 cm long, 0.1 cm in diameter and pigmented. The rootlets on mature nodule lobes from plants growing in dry, sandy soil were 1 cm long, less than 0.1 cm in diameter, and brittle. The heterogeneity of appearance was minimized by selecting only mature and developing nodules for experiments. Those with darkly pigmented and suberized lobes were discarded. ?U fixation by excised root nodules Since nodules of Myrica ffale L. were reported by Bond (1957) to fix N ? , it was reasonable to assume that nodules of Myrica cerifera L. would do the same. Excised whole nodules were tested by exposing them to gas mixtures 23

PAGE 37

29 containing and 0 2 in a ratio similar to that in air. A set of three flasks each containing nodules was exposed to 15 nitrogen gas which was enriched with K while a control 15 set was exposed to nitrogen gas of natural N abundance. After incubation, the nitrogen of the whole nodules was 15 analyzed by the N method described earlier. The results, given in Table 2, showed an increase in atom per cent content in nodules of all flasks which were exposed to the enriched nitrogen gas. There was no increase in atom per 15 cent ~ N content in nodules of control flasks. The excised nodules of M. cerifera fixed N ? . Although results of the analysis of the nitrogen of whole nodules demonstrated N ? fixation, a more sensitive 15 means of analyzing fixed 'N was desired. Analysis of the KC1 soluble portion of soybean nodules had provided a consistent assay for N 2 fixation (Aprison and Burris, 1952). Also, Leaf et al. (1959) had found 15 N enrichment in components of the acid soluble portion of nodules of M. gale . To apply this assay, nodules of M. cerifera were extracted with HC1 after exposure to N enriched nitrogen gas. Nitro genous compounds of both acid soluble and acid insoluble portions were analyzed, and the results are shown in Table 3 The acid soluble portion contained about ten times more enrichment than the acid insoluble portion. The increased sensitivity of the acid soluble extrac tion method made possible a time course experiment. Nodules

PAGE 38

30 TABLE 2 TEST FOR N ? FIXATION BY DETACHED NODULES OF MYRICA CERIFERA Flask No. Atom 4> 15 N excess 1 2 3 0.042 0.056 0.096 Each flask contained 2 g fresh weight of nodules and 1 ml of N-free nutrient solution 1 ,,The incubation ras mixture was 0.80 atm l >d„ (30 atom $ A5 N excess) and 0.20 atm Oj. Three control flasks each contained 0.80 atm N 0 (tankj and 0.20 atm 0 p . All samples were exposed to thi gas mixtures for 19 hours. Centifanto (1964)

PAGE 39

31 TABLE 3 N CONTENT OF ACID SOLUBLE AND ACID INSOLUBLE FRACTIONS OF NODULES OF M. CERIFERA AFTER EXPOSURE TO i5 N 2 Flask No. Fraction Atom fo ^N excess 1 Acid soluble 1.092 2 " " 0.780 1 Acid insoluble 0.089 2 " " 0.097 Each flask contained 4 g fresh weight of nodules. The gas mixture was 0.27 atm ^N« (30 atom fo "^^N excess), 0.21 atm 0 0 , and 0.52 atm He. The exposure time was 4 hours.

PAGE 40

32 of M. cerifera were excised from roots, washed, and exposed to the incubation, gas mixture within 30 minutes. As shown in Figure 1, was rapidly incorporated into the acid soluble fraction, the rate being constant over the first eight hours. The enrichment in the acid insoluble fraction was low. Some variation between replicate flasks at prolonged exposure to the gas was noted. This was not surprising due to the native source of the tissue and was considered minor. It was decided to determine the x N enrichment in only the acid soluble portion of nodules in further experiments. Since appearance of nodules varied from young to aged tissue, the capacity of these tissues to fix nitrogen was checked. Obviously young, unpigmented, unsuberized, and fleshy lobes were placed in one group, while old, darkly pigmented, suberized, and woody nodular parts formed the second group. The latter group was routinely discarded in other experiments. The results of the experiment are shown in Table 4. The young tissue fixed nitrogen at three times the rate of the old tissue. Fixation did occur in both morphologically and physiologically different tissues. Thus, the appearance of the nodule was important and the continued use of the young tissue in experiments was justified. Effect of partial pressure of T' ^ A study was made to determine the partial pressure of (pNg) supporting the maximum rate of fixation for

PAGE 41

33 6 8 10 12 TIME (HOURS) 14 IS 13 Figure 1 Time ^course ^ of N 2 fixation by excised nodules of Two experiments were conducted and the results of analyses of the acid soluble N are denoted for Sfeacf i< a ? (A) aM f ° r experiment as n? ? P^ 111 ^ re P^senting one flask. The averfresh weight of nodule^an^i f exp 0 .e C ^^o^ ned 4 S mixture containing 0.16 a?m %L (fo £ol < iff excess), 0.22 atm Q and 0.62 atm HeT

PAGE 42

34 TABLE 4 COMPARISON OF N 2 FIXATION BETWEEN NODULES WITH EPIDERMIS PRESENT AND NODULES WITH PERIDERM PRESENT Flask No. Description Atom # 15 N excess of nodules Periderm present (woody) 0.326 0.276 Epidermis present (fleshy) O.858 0.889 Each flask contained 4 g fresh weight of nodules. The gas mixture was 0.23 atm 15 N 2 (30 atom % ^N excess), 0.21 atm 0 2 , and 0.57 atm He. The exposure time was 4 hours. 1 2 3 4

PAGE 43

35 excised nodules of M. cerifera . Two flasks were treated identically for each pN«. Each flask contained 0.20 atm 0^ Figure 2 represented the results of four experiments conducted in order to determine the effect of varying the pNp on the relative rate of N« fixation. From the plot, maximum fixation occurred at about 0.10 atm Np for this tissue . A Lineweaver-3urk plot (Figure 3) for the data was prepared similar to that described by Burr is et al. (1955) for soybean nodules. The average of results from replicate flasks was used in the plot and for the determination of the center line for the data by the method of least squares. The slope of the straight line, K /V to ' m / max' equaled 0.080. The y intercept, l/V_ . equaled 1.150. A IucL-X. Michaelis constant, X. of 0.069 ± 0.004 atm N 0 for N„ m c 2 fixation by whole nodules of M. cerifera was calculated. Effect of partial pressure of O p The effect of Op concentration on I\ T p fixation was 15 considered. The ^Np content of the incubation gas mixture was maintained near 0.20 atm to insure saturation of nitrogenase with substrate. As shown in Figure 4, 0 ? was indispensable for K 2 fixation, and maximum fixation occurred at a partial pressure of 0 ? between 0.20 and 0.30 atm. N~ fixation was limited at 0 9 concentrations below 0.20 atm, while N 2 fixation was depressed above 0.3 atm 0~.

PAGE 44

36 1.0 p i A 0.9 0.8 r A 10 I 1 i ' ' ' ' ' i i i i \ 0 0.04 0.08 0.12 0.18 0.2 2 0-24 p N 2 (ATM) Figure 2 Effect of pl\ T 2 on incorporation by excised nodules of Myrica cerifera . Results of three experiments are shown. Each point represented a flask which contained 4 g fresh weight of nodules and a gas mixture composed of ^ 2 (30 atom jfi X 5 N ' excess) as indicated above, 0.20 atm 0 P , and He to 1 atm. Tne incubation time was four hours.

PAGE 45

37 7 r > 1/CS Figure 3 Lineweaver-Burk plot to determine the Michaeli constant for nitrogen fixation by whole nodule of III, cenfera. A line has been fitted to the data by the method of least squares.

PAGE 46

38 Q9 0.1 0.2 0.3 P 0 2 (ATM) 0.4 J 0.5 Figure 4 Effect of p0 2 on N 2 fixation by detached nodules of Myrica cerifera .. Each point represents a flask which contained 4 g fresh weight of nodules, and 0.18 atm I5 N (3Q atom % ±5$ excess), 0 2 as indicated above 2 and He to 1 atm. The incubation time was four hours.

PAGE 47

39 Storage of intact nodules The advantage of storing nodules for later experimental use is obvious. Attempts were made to store nodules under helium in a freezer and in liquid nitrogen. Table 5 shows that activity did not survive either type of storage. Even freshly excised nodules frozen at the temperature of liquid nitrogen and quickly removed failed to have activity. immersing nodules in 20 per cent glycerol and freezing in liquid nitrogen was also unsuccessful. Variation among r eplicate samples An experiment was performed treating five samples identically in order to establish a distribution of measurements of 15 N enrichment. The results are given in Table 6. Measures of the central tendency and variability are given as the mean and the standard deviation respectively. For comparison the results from samples in eleven experiments under like conditions were compiled and are shown in the same table. The standard deviation for the one experiment when compared to the corresponding value for the eleven experiments were similar. fixation by nodular homogenates of M. cerifera After determining conditions for N 2 fixation by whole nodules of M. cerifera , the problem of preparing active homogenates of these nodules was considered. Preparation of active homogenates had not been reported in the literature

PAGE 48

40 TABLE 5 STUDIES ON STORAGE OF EXCISED NODULES OF M. CERIFERA Flask No. Treatment Atom ^ 15 v N excess I Under helium in deep freeze 0.016 2 Same as 1 u .003 3 Immersed in liquid N 2 0.014 4 Same as 3 0.008 5 No storage 0.732 6 Same as 5 0.632 Each flask contained 5 g fresh weight of nodules which had been stored at each condition indicated for two weeks. The incubation gas mixture contained 0.16 atm N 2 (30 atom
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41 TABLE 6 VARIABILITY AMONG SAMPLES WITHIN ONE EXPERIMENT AND AMONG SEVERAL EXPERIMENTS Range Mean SD 90 # C. I. 5 samples from 0.306-0.893 0.644 0.246 0.410-0.873 I expt. 15 samples from 0.143-0.975 0.424 0.257 0.307-0.541 II expt. Values given for range, mean, and confidence interval are results of experiments expressed in atom
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42 at this tine. Since the time course of I\ T p fixation by nodules of M. cerifera was longer than the two hour time course of fixation "by soybean nodules reported by Aprison and Burr is (1952), it seemed possible that the nonleguminous nodules might be better experimental material for the preparation of active homogenates. Techniques useful in preparing and handling cell-free extracts of bacteria had been reported by Carnahan et al. (i960). Because of the lack of technical knowledge about preparing nodular homogenates, techniques used in bacterial K« fixing extracts were applied. The success of preparing active cell-free extracts from bacteria depended partly upon the method of using a low concentration of phosphate buffer (<0.05M) or cacodylate buffer, pH 7.0; also anaerobic manipulation of extracts at room temperature was necessary. Nodules of M. cerifera were homogenized anaerobically in buffer at room temperature. Nodules were well fragmented with only some peridermal portions remaining intact. It was assumed that both whole nodules and homogenates had the same 0 2 requirements during incubation. Homogenates were tested with and without the electron donor dithionite (Na 2 S 2 0 ), which had been used by Bulen et al. (1965) to enhance N 2 fixation by cell-free extracts of Azotobacter vinelandii . Homogenates which were supplied Na o S o 0, and 0 o fixed N (Table 7). Although this fixation was much less than that shown by intact nodules used as controls, it was significant since the replicates agreed closely.

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43 TABLE 7 EFFECT OF Na 2 S 2 0 4 ON N g FIXATION BY ROOT NODULE HOMOGENATES Flask No. Treatment Atom io ^N excess 1 Intact Nodules 0.588 2 Hornogenate 0.005 3 Homogenate + Na^O^ 0.026 4 Hornogenate + Na^O^ 0.021 5 Homogenate + Na^O^ 0.022 Flask No. 1 contained 5 g fresh weight of nodules. The homogenates were prepared as follows: 5 g nodules were homogenized 1 min under He in 5 ml cacodylate buffer, pH 7, 508 umoles. 1 ml Na S 0,, 240 umoles, was added after gassing. The incubation gas mixture contained 0.20 atm ^n 2 (96.6 atom * 1% excess), 0.22 atm 0„, 0.53 atm He. The incubation time was 4 hours. 1

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44 Since others ("ortenson, 1964; Hardy and D'Eustachio, 1964; Bulen et al., 1965) had reported that adenosine triphosphate ( ATP) was required for N« fixation by cell-free extracts of bacteria, the effects of an exogenous ATP generating system on the homogenate were studied. The system according to Bulen et al. (1965) contained ATP, creatine phosphate (CP), and creatine phosphokinase (CPK). Homogenates were prepared as before in the presence of the buffer. Na 2 S 2 0^ and the supplements (the ATP generating system and the reduced form of nicotinamide-adenine dinucleotide , NADH) were tipped in after gassing the system. The incubation gas mixture contained the same proportion of 0 2 at which maximum N fixation by whole nodules occurred. The results are given in Table 8. Ng fixation was substantial for homogenates prepared in the buffer and supplied Na^O^, NADH, the ATP generating system, and 0 2 « With an exogenous ATP generating system supplied, N 2 fixation by homogenates may not be dependent upon aerobic respiration. In order to test whether 0 2 was required in addition to the exogenous ATP generating system, the following experiment was performed. Nodular homogenates of M. cerifera were prepared as described above. The homogenates were exposed to incubation gas mixtures either with 0 2 or without 0 2 , and then were supplied with supplements as described in the previous experiment. Results of two experiments are shown in Table 9. Homogenates supplied with the exogenous ATP generating system fixed N g only in the presence

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45 TABLE 8 EFFECT OF SUPPLEMENTS ON N 9 FIXATION BY NODULAR HOMOGENATES Flask No. Treatment 1 5 Atom f~ ^N excess I Intact nodules 0.770 2 Buffered homogenate 0.007 3 Same as 2 0.017 4 Buffered homogenate + supplements 0.390 5 Same as 4 0.117 Flask No. 1 contained 4 g fresh weight of nodules. The homogenates were prepared as follows: 5.5 g nodules were homogenized 1 min under He in 5 ml cacodylate -J , 'Jl H 7 > 106 F^les. The supplements (umoles per flask), aadea after gassing, were Na 2 S 2 0 4 , 50; ATP, 20; NADH, 20; creatine phosphate, 100, and 2 mg of creatine pho spho kinase . ine iinal volume of the reaction mixture was 10 ml. The jncubation gas mixture contained 0.18 atm ^ 2 (96 atom % N excess), 0.13 atm 0 and 0.65 atm He. The incubation time was 4 hours. *

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46 TABLE 9 EFFECT OF OXYGEN ON N~ FIXATION BY INTACT NODULES AND SUPPLEMENTED NODULAR H0M0GENATE3 Tissue 0 2 Mean Atom
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47 of C2 (see last line, Table 9). As shown in Table 9, intact nodules in the presence of fixed N« only to a small extent (0.079 atom per cent K excess), whereas the homogenate in the presence of 0^ fixed much more (0.142 atom per cent 15 N excess). Table 7 shows that ' the buffered homogenates in the presence of Na 2 S 2 0^ fixed N^. The addition of other supplements and the presence of 0 o enhanced the activity. The necessity of N&pSpO/i in ' fclie presence of the ATP generating system was tested. The addition of NagS^O^ and 0^ to the supplemented homogenates was varied as shown in Table 10. IL, fixation was observed only in one of the supplemented homogenates supplied with UstpSpO^ and 0^. An experiment was performed with supplemented homogenates buffered at three pH levels. The results are shown in Table 11. fixation was observed for one flask in which the homogenate was adjusted to pK 7. Site of fixation by homogenates In order to determine whether nitrogenase activity occurred in the soluble portion of the homogenate, a homogenate was prepared under conditions equivalent to those described above. A portion of the homogenate was centrifuged at 5000 rpm for three minutes. The pellet and supernate each were exposed to the incubation gas mixture, and then supplements were added. As shown in Table 12, one of the supplemented pellet fractions ( Flask 7) fixed N 2 , whereas significant fixation did not occur in the other flasks. Intact portions of nodules may have fixed N~.

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48 TABLE 10 EFFECT OF 0 2 and Na^O. ON Np FIXATION BY SUPPLEMENTED HOMOGENATES Flask No. Na 2 S 2 0 4 0, Atom fo ^N excess 1 2 3 4 5 6 7 8 + + + + 0.005 0.012 0.009 0.002 0.003 0.006 1.138 0.007 The preparation and supplementation of homogenates and incubation gas mixtures were the same as described in Table o •

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49 TABLE 11 EFFECT OP pH ON N 0 FIXATION BY HOMOG-ENATES Flask No. PH Atom ^ "^N excess 1 4.5 2 same as 1 0.006 3 6.0 0.005 4 same as 3 0.007 5 7.0 0.011 6 same as 5 0.035 The buffer was adjusted to pH indicated above. Other conditions were the same as in Table 8, except that -'Np contained 30 atom
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50 TABLE 12 N 2 FIXATION 3Y SUPPLEMENTED HOMOGENATE FRACTIONS Flask No. Treatment Atom $ 15 N excess 1 Intact nodules 0.427 2 Homogenate 0.013 3 Homogenate 0.007 4 Supernate 1 0.009 5 Residue 1 0.007 6 Supernate 2 0.014 7 Residue 2 0.038 Flask No. 1 contained 4 g fresh weight of nodules. A sample containing 30 g fresh weight of nodules w^ m °i e ? 1Z f d i3 \ a 30 solution containing cacodylate buffer pH 7, 3 poles, and Na S 0 0.3 umoles. Flasks No. 2 ano. 3 each received 10 ^homogenate. The portion uo be xractionated was divided in half and each "coition was centnfuged at 5,000 rpm for three minutes. Each pellet fraction was resuspended in 4 ml of buffer. Supplements, 1 ml, (umoles per flask) ATP, 20: NADH. 20creatine phosphate 100, and 2 mg creatine 'phos^okinase ? were acaea to each fraction and to the homogenate. , The incubation gas mixture was 0.17 atm (30 atom * ^N excess) hours? 2 ' K9 t0 1 atau Thi incub atiok time was 4

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51 In all of the nodular homogenate experiments, the aim was to determine optimal conditions for N 2 fixation. Variations among replicates were observed in many experiments. However, N 2 fixation ability was always correlated with conditions which were varied in any given experiment. This type of correlation was considered significant. Because of 15 the existence of variations and the laborious K method further investigation into symbiotic nitrogen fixation by homogenates should be modified in future work. The remainder of the present investigation was concerned with the reduction of acetylene by nodules and breis of nodules. Acetylene reduction by nodules of M. cerifera The idea that acetylene reduction could be used as a measure of nitrogenase activity was suggested by Dr. R. W. P. Kardy (1966, personal communication), who was utilizing cellfree extracts of A. vinelandii and C. pasteurianum in a study of the specificity of nitrogenase for electron acceptors (Hardy and Knight, 1966). Furthermore, Schollhora and Burris (1966) and Dilworth (1966) showed that extracts of C. pasteurianum which fixed N 2 also catalyzed the reduction of acetylene to ethylene. Whether the reduction of acetylene might be catalyzed by nitrogenase in nodules of M. cerifera was questioned. Nodules of M. cerifera were collected and exposed to an incubation gas mixture containing acetylene, oxygen, and helium. Oxygen was supplied at concentrations which supported maximum and half maximum

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52 nitrogen fixation (see Figure 4). The appearance of ethylene was monitored by flame ionization gas chromatography. Samples of the incubation gas mixture were analyzed at various times, and a time course was plotted, as shown in Figure 5. The rate of ethylene production at 0.20 atm 0 2 was constant for the first nine hours. At 0.10 atm 0 2 the rate of ethylene formation was reduced and was constant for the first five hours only. No ethylene was detected in control flasks gassed with an acetylene-helium mixture without 0 2 . The time course of the reduction of acetylene to ethylene was similar to that of N 2 fixation and both processes were dependent upon 0 2 . The possibility exists that ethylene production was not the consequence of nitrogenase catalyzing the reduction of acetylene. Endogenous ethylene production has been shown to occur in plant tissues, as reviewed by Jansen (1965), and this is unrelated to N 2 fixation. In the experiment described above, endogenous ethylene production was not detected in flasks containing excised nodules incubated with air. According to Jansen (1965), ripening orange fruits produced ethylene at a rate of 0.350 ul per gram per hour, which is one of the highest rates observed and is much higher than that for vegetative tissues. From Figure 5, the rate of ethylene production by nodules is calculated to be 76.6 ul per gram per hour — over 200 times

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53 40 r TIME (HOURS) Figure 5 Time course of acetylene reduction to ethylene by nodules of M. cerifera at 0.1 atm and 0.2 atm 0 2 . Each, point represents a flask which contained 2 g fresh weight of nodules. The gas mixture represented by onen triangles was 0.20 atm 0 2 , 0.05 atm C ? H ? and He to 1 atm and by closed triangles was 6.10 atm 0 9 , 0.05 atm ^2^2 an ^ ^ e ^° ^ a ^ m *

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54 larger than the rate of ethylene production in ripening fruit. It was concluded that if minute endogenous ethylene production did occur, it did not affect the results obtained above. The possibility that acetylene may have stimulated endogenous ethylene production was tested. The relationship between the production of ethylene and the disappearance of acetylene catalyzed by excised nodules of M. cerifera was determined (Figure 6). An increase of 5.3 umoles or ethylene was detected after four hours, while 5.0 umoles of acetylene disappeared during the same time. Thus, about 90 per cent of the ethylene produced could be accounted for by the decrease of acetylene. It was concluded that acetylene is directly reduced to ethylene by excised nodules of M. cerifera. Acetylene reduction by nodules of various plants In order to determine whether acetylene reduction was a common property of root nodules which fix Ng, nodules from plants other than M. cerifera were tested. The time course of the reduction of acetylene to ethylene by excised nodules of C. eauisetif olia is shown in Figure 7. The nodules reduced acetylene to ethylene and the rate of ethylene production was constant for six hours, after which activity diminished abruptly. Nodules from Hampton soybeans reduced acetylene to ethylene as shown in Figure 3. The rate of ethylene

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55 h2 3 TIME (HOURS) Figure 6 Relationship of ethylene production to acetylene disappearance by nodules of M. cerifera . Each point represents a flask which contained 2 g fresh weight of nodules, gassed with 0.05 atm acetylene, 0.10 atm 0 ? and He to 1 atm. Ethylene is denoted by an open triangle. Acetylene is denoted by a closed triangle.

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56 2.5 rLL o 2.0 1.5 o 1 3" 1,0 u z u _J > 0.5 I — LU 12 16 TIME (HOURS) 20 Figure 7 Time course of reduction of acetylene to ethylene by excised nodules of Casuarina equisetifolia . ~ Each point represents a flask which contained 2 g fresh weight of nodules gassed with 0.05 atm acetylene, 0.20 atm 0 and He to 1 atm.

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57 4 r2 3 TIME (HOURS) Figure 8 Relationship of ethylene production to acetylene disappearance by soybean nodules. Each point represents a flask which conlil^n t reSh We 7 Sht of ^dules gassed with 0.05 atm acetylene, 0.10 atm Op, and . ne to 1 atm. Ethylene is denoted by the open triangles. Acetylene is denoted by the closed triangles.

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58 production was constant for the first two hours. During the four hours of the test 2.3 umoles of ethylene were produced while 1.5 proles of acetylene were reduced. Symbiotic structures such as leaf nodules and mycorrhizal associations were tested in addition to root nodules of leguminous and nonleguminous plants. The isolated endophyte of leaf nodules of Psychotria bacterio phila and vegetative plant tissues were included in the acetylene reduction tests. The summary of these data was shown in Table 13. Nodules of six species from five genera supported the reduction of acetylene. Leaves, stems, and roots of M. cerifera and G. max did not catalyze the reduction of acetylene, nor did endogenous ethylene production influence the results. The fresh weight and nitrogen content of nodules was determined and used in the calculation of initial rates of ethylene production from acetylene. A comparison of the rates obtained would not be valid because of the varied ages and environmental conditions of the field tissue prior to analysis. What is useful from these data is the knowledge of which tissues exhibit activity and the duration of the activity after excision from the plant. The nodules of the nonleguminous genera, Casuarina and Mvrica, maintained activity for a longer time after excision than the genera of legumes, Lupinus and Glycine . Itycorrhizal associations on the roots of Podocarpus aacrophylla and Pinus elliottii were tested for the ability to reduce acetylene. These species were thought to fix

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Standard conditions were 0.10 atm acetylene, 0.20 atm Op, and He to 1 atm. Each flask contained 2 grams except for P. macrophylla and P. eliiotii which contained 8.3 and 20 grams respectively. The age of plants in months was G. max , 2-3; I. albus , 2; C. spectabilis , 1; P. elliottii , 24. n.d. means not detectable, < 0.005 * 0.05 atm acetylene, 0.10 atm 0 2 » and He to 1 atm. a Plants were collected from Sanibel Island and grown in a greenhouse for one month. b Plants were collected from Haulover Beach State Park and stored over night in a plastic bag. c Klebsiella rubiacearum (strain K4). The system contained per 5 ml 800 mg dry weight of cells, 250 mg pyruvate, cacodylate buffer pH 7, 0.1N. The incubation gas mixture was 0.05 atm acetylene and He to 1 atm.

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60 TABLE 13 REDUCTION OP ACETYLENE TO ETHYLENE BY VARIOUS PLANT TISSUES Plant umoles OJl^/ umoles CpH,/ Duration of g fr wt / hr umoles N /hr initial rate in hours Myrica cerifera nodules 3.4 0.067 9 nodules 1.6* 0.033 5 roots n.d. Casuarina equisetif olia nodules a 0.7 0.017 8 nodules ° 0.2 0.004 3 Casuarina cunninghamiana nodules 0.1* 8 Podocarpus macrophylla nodulated roots n.d. Pinus elliottii mycorrhizal roots n. d. Psychotria bacteriophila nodulated leaves n. d. endophyte c 1.1 1 Lupinus albus nodules 2.0 0.080 3 leaves n.d. Crotolaria spectabilis nodules 0.8 0.032 5 glycine max nodules 1.2* 0.046 leaves n.d.* stems n.d.* roots n.d.* 2 Por legend, see opposite page

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61 nitrogen, but there was no direct evidence. As shown in Table 13, no detectable ethylene was formed after 29 hours of incubation, although large amounts of tissue were used. Leaf nodules of Psychotria bacteriophila are believed to be a symbiotic N 2 fixing association because nodulated plants grew in a nitrogen-free medium for six months without developing symptoms of nitrogen deficiency (Silver et al., 1963). The N 2 fixing capacity of the leaf nodules might support the reduction of acetylene. Two grams of young nodulated leaves (less than 3 cm long) were selected from healthy plants, but no detectable ethylene was found after an 18 hour incubation of the nodulated leaves to acetylene (Table 13). However, cell suspensions of isolated endophyte, which had been grown in nitrogen-free mineral broth did form ethylene when the substrate and a source of energy and reducing power were provided. Effect of acetylene concentration on ethylene production The effect of acetylene concentration on the reduction of acetylene to ethylene by excised nodules of M. cerifera was studied. Nodules were incubated with gas mixtures in which the 0 2 concentration was 0.20 atm and the acetylene concentrations in individual flasks ranged between 0.01 and 0.40 atm. As shown in Figure 9, a concentration of 0.04 atm acetylene saturated the nitrogenase system. For acetylene concentrations above 0.20 atm, activity was one half the maximal value. From these data

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62 5 _ o \ 3 CO LU — I O =J_2 LU LU _J >LU A A A A-Ar A AAA A -A_A — i 1 i i i 0 0.05 0.10 0.15 0.20 0.25 0.30. 0.35 0.40 C 9 h' (ATM) Figure 9 The effect of partial pressure of acetylene on ethylene production by nodules of I.Iyrica cerif era . Results of four experiments are shown. Each point represents a flask containing 1 g of nodules gassed with acetylene as indicated above, 0.20 atm 0 2 , and He to 1 atm. Incubation time res one hour.

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63 an apparent Km value of 0.02 atra for acetylene was estimated. The nitrogenase systems' s affinity for acetylene appeared to be three times that for N 2 (See Figure 2) . High acetylene concentrations limited the reduction of acetylene to ethylene to about half the maximum rate. inhibition of the reduction of acetylene If nitrogenase were catalyzing the reduction of both acetylene and rT 2 , N 2 would be expected to inhibit the reduction of acetylene. Excised nodules of M. cerifera were incubated with gas mixtures which included 0.05 atm acetylene and concentrations of N 2 ranging from 0 to 0.15 atm. The results of three experiments were plotted as ethylene production (per cent of control) vs. pN 2 (Figure 10). As the concentration of N 2 increased, the production of ethylene from acetylene decreased. At 0.06 atm N 2 , acetylene reduction was inhibited more than 60 per cent. The effect of acetylene concentration on the formation of ethylene by soybean nodules was reported by Koch and Evans (1966). The maximum rate of ethylene production was achieved at approximately 0.1 atm acetylene. Since Koch and Evans did not report the effect of N 2 on acetylene reduction in soybean, it was of interest to compare this tissue with that of Myrica . Various partial pressures of N 2 were tested on soybean nodules saturated with 0.1 atm acetylene. As indicated in Figure 11, ethylene production decreased only above a pN 2 of 0.30 atm. N 0 concentrations

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64 0 0.02 004 0.06 0.08 0.10 0.12 0.14 0.16 P N 2 (ATM) Figure 10 Effect of N« on ethylene production from acetylene by nodules of M. cerifera . Results of three experiments are shown. Each point represents a flask which contained 2 g fresh weight of nodules gassed with 0.20 atm 0 2 , 0.05 atm acetylene, N 0 as indicated above, and He to 1 atm. The incubation time was one hour.

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65 Results of five expsriraents are shown, jach point represents a flask containing 2 afresh weight of nodules from Hampton soybean (66-73 days old) gassed with 0.10 atm acetylene 0.20 atm o 2 , N ? as indicated one hour! 1 1§cuba *i°n time was

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66 of less than 0.30 atm had no apparent effect on ethylene production. At 0.62 atm N T 2 , the rate had decreased to less than 1 per cent of the rate at 0.30 atm N 2 » Reduction of acetylene by individual nodules Routinely, the sensitivity of the gas chroma to graph permitted detection of 0.005 umoles of ethylene. It was of interest to determine the amount of tissue necessary to produce detectable quantities of ethylene from acetylene. Nodules were collected from M. cerifera . Each of five samples, weighing 1.280 g, 0.353 g, 0.165 g, 0.071 g, and 0.009 g, were exposed to an incubation gas mixture containing 0.05 atm acetylene, 0.20 atm oxygen, and 0.75 atm helium. Acetylene reduction was detected in all five samples. A single young lobe of a nodule, weighing 0.009 g, produced 0.04 umoles of ethylene per hour. Thus, the sensitivity of the acetylene reduction method permitted detection of nitrogenase activity in less than a 10 mg sample. Individual soybean nodules were also tested. Nodules were collected from Hampton soybean plants which were 40 to 45 days old. The weight, diameter, and location on tap or lateral roots were noted. A total of 23 nodules selected from nine plants were used in the experiment and the results are summarized in Table 14. None of the nodules less than 3 mm in diame ter had detectable activ ity. They were considered immature nodules. Seventy per cent of those with diameters larger than 3 mm reduced acetylene. Activity

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67 TABLE 14 ACETYLENE REDUCTION BY INDIVIDUAL SOYBEAN NODULES Nodule diameter Total , f \ . % Active (mm) tested ' <3.0 5 0 3.0 4.0 8 50 4.0 5.0 6 83 > 5.0 9 77 Each flask contained an individual soybean nodule and were gassed with 0.10 atm acetylene, 0.20 atm Op, and He to 1 atm. Gas samples were taken after one hour incubation.

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68 was detected in four of the five tap root nodules tested and in 60 per cent of the lateral root nodules whose diameters were above 3 nun. Above 3 mm in diameter, active and inactive nodules were indistinguishable with respect to weight, as well as diameter. The activity of each nodule was determined as umoles ethylene produced per gram fresh weight per hour. Although there was no absolute correlation between nodule diameter and activity, larger nodules tended to be less active. One exceptionally active nodule with a diameter of 4 mm and a weight of 27.4 mg produced 24.6 umoles of ethylene per gram fresh weight. Reduction of acetylene by breis The gas chromatographic detection of acetylene reduction was used as a relatively easy and rapid method for detecting a tissue's ability to fix N g in further attempts to prepare consistently active nodular breis of K. cerifera . Koch et al. (1967a) had reported that consistent N 2 fixation and acetylene reduction by breis of soybean nodules could be maintained by preparation in the presence of polyvinylpyrrolidone (PVP) and buffered ascorbate medium to prevent phenolic oxidation products from inactivating enzymes. Before applying this procedure to nodular breis of M. cerifera , it was decided to prepare active breis from nodules of field grown Hampton and Chipewa soybean plants according to the method of Koch ' et al. (1967a). Soybean nodules were harvested, washed

PAGE 77

69 and placed in a polyethylene glove bag. The glove bag was evacuated and filled three times with ^ which had been passed through a heated copper column to remove traces of 0^. The nodules were macerated in phosphate buffer containing ascorbate, and squeezed through two layers of chees cloth. The breis were mixed with PVP for ten minutes and again squeezed through cheese cloth. After the breis were exposed to incubation gas mixtures containing acetylene, oxygen, and helium, gas samples were analyzed. The results given in Table 15, show that breis prepared from nodules of Hampton and Chipewa soybeans reduced only small amounts of acetylene. The results demonstrated that this procedure did provide consistently active breis, but the activity obtained was much lower than that obtained by Koch et al. (1967a). The reasons for the low activity were not known, but they may be due to the fact that these nodules were from field grown soybeans rather than greenhouse plants which were used by Koch and co-workers. Nodules of K. cerifera were then collected and used in the preparation of nodular breis by the same procedure. However, activity of breis from nodules of M. cerifera was barely detectable, as shown in Table 16. Activity might be increased by further modifications to protect the enzyme systems.

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70 TABLE 15 REDUCTION OF ACETYLENE BY BREIS OF SOYBEAN NODULES PREPARED WITH POLYVINYLPYRROLIDONE AND ASCORBATE Soybean system umoles of ethylene/ flask Hampton, intact 10.8 Hampton, brei 0.04 Chipewa, intact 11.2 Chipewa, brei 0.05 Each intact nodule system contained 1 g fresh weight of nodules per flask. Breis were prepared in a glove bag filled with argon. 13 g of nodules from 40 day old soybean plants were macerated in 27 ml of 0.02 M potassium phosphate buffer pH 7 containing 1 mm MgCl p and 200 umoles" ascorbate . The macerate was squeezed througn cheese cloth and mixed with 9 g of PVP. After 10 min the PVP and macerate were squeezed through cheese cloth and the resulting brei was gassed with 0.10 atm acetylene, 0.20 atm 0 P , and He to 1 atm. Gas samples were taken after one hour.

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71 TABLE 16 REDUCTION OE ACETYLENE BY BREIS OF MYRICA CERIESRA PREPARED WITH POLYVINYLPYRROLIDONE AND ASCORBATE „ ^ Tro . umoles etnylene Mynca system r— _«/ J per flask Intact nodules 3.5 Brei O.Ol Brei 0.01 The intact nodule system was 1 g fresh weight nodules per flask. Breis were prepared as described in Table 15. 7 g of nodules were macerated in 10.5 ml of potassium phosphate buffer. 3.5 g of PVP was used. The gas mixture was 0.05 atm acetylene, 0.20 atm 0 ? , and He to 1 atm. Samples were taken after one hour.

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DISCUSSION 15 The results of the N method provided the first demonstration that excised nodules of native M. cerifera fixed N 2 . This contribution extended the list of nodulated nonleguminous angiosperms which have proven to fix 15 The N method has been used as a sensitive assay for N 2 fixation. The lack of any detectable exchange reaction between fixed nitrogen and molecular nitrogen and of any detectable preference by an organism for the 15 isotopes of nitrogen established the validity of the method (Burris and Miller, 1941). The method detected changes in the ratio of "^n/^N and not changes in absolute nitrogen content. However, the initial incorporation of 15 the N tracer into fixed nitrogen is confined to the metabolically active portion of cells, and analysis of the total nitrogen would dilute the tracer enrichment. The mass spectrome trie analysis of the acid soluble nitrogenous compounds of nodules of M. cerifera increased 15 the sensitivity of detecting incorporation. Since 15 newly fixed •'N accumulated in the acid soluble fraction, the analysis of this fraction minimized the dilution effect of the fixed tracer by protein nitrogen. 72

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73 The demonstration that the fixed tracer accumulated in the acid soluble fraction of nodules of M. cerifera confirms and extends the results of previous investigators. Aprison et al. (1954) reported that the fixed tracer was found in amino acids and ammonia, which were part of the acid soluble fraction of soybean nodules. Leaf et al. (1959) found that after one hour exposure of nodules of 15 M. gale to ^N 2 , 82 per cent of the tracer fixed by the nodules was in the acid soluble fraction. Bergersen (1965) provided evidence that ammonia is the primary stable intermediate in N~ fixation by soybean nodules. Kennedy (1966a) and Kennedy (1966b) presented more evidence to confirm the formation of amino acids and amides from ammonia in leguminous nodules. The present investigation has shown that the nodules of M. cerifera were good experimental material for research on symbiotic nitrogen fixation. This conclusion was included in the preliminary report of this study by Sloger and Silver (1965). The reasons for this conclusion may be summarised as follows. Since the rate of N 2 fixation by nodules was constant for seven hours after detachment from roots, the time required to prepare and perform an experiment was not very critical. The results of experiments on the time course of N 2 fixation with nodules of K, cerifera can be compared to similar experiments reported for other nodules (see Figure 12). Nodules of M. cerifera as well as the

PAGE 82

74 TIME (HOURS) Figure 12 Comparison of time courses of N 2 fixation byexcised nodules of legumes and nonlegumes. Whole nodule analysis: Alnus glutinosa and Hippophae rhamnoides . 175 g of nodules exposed to 0.10 atm 1 5n 2 (36 atom excess), 0.20 atm 0 2 , and 0.70 atm Argon. The results were taken from Bond (1957). Acid soluble analysis: Soybean nodules, 2 g, were exposed to 0.10 atm 1 ^N 2 (20 atom $5 1 5jf excess), 0.20 atm 0 2 , 0.05 atm C0 ? , and 0.65 atm He. The results were taken from Aprison and Burris (1952). M. cerifera data were from Figure 1.

PAGE 83

75 other nonleguminous nodules reported by Bond (1957) all have a prolonged activity after excision of the nodule from the root — a characteristic of nonleguminous nodules. Nodules of M. cerifera are better experimental material than leguminous nodules because the time course of N2 fixation is longer than that for soybean nodules reported by Aprison and Burris (1952) . The initial rate of **N incorporation in the acid soluble fraction was similar for M. cerifera and soybean, but the greater enrichment achieved by the nonlegume was due largely to prolonged ability for N2 fixation by the nodules after detachment from the plant. In this study, the apparent Michaelis constant, K^, of 0.069 + 0.004 atm N 2 obtained for whole nodules of M. cerifera is greater than 0.02 + 0.004 atm N 2 reported by Burris et al. (1955) for sliced soybean nodules. The higher apparent for nodules of M. cerifera may reflect differences in the permeability of gases into the sliced and intact tissues and not differences in affinities for substrate. Burris et al. (1955) suggested that the nitrogenases of various organisms were similar with respect to their affinity for substrate. The results of this study do not discount this hypothesis, but the use of intact tissues and organisms rather than soluble enzyme preparations hinders the verification of this hypothesis. Recently, Strandberg and Wilson (1967) reported that from a comparison of values for ]\ T 2 fixation by intact

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76 bacterial cells, a disparity of values was found, depending upon the method used. They also noted that using soluble fixing enzyme preparations from several bacteria led to K_ values which were three times higher than those obtained m for the intact organisms. The Michaelis constant and the pN 2 function reported in this study represents the first evidence which suggests that nonleguminous and leguminous nodules are similar in respect to substrate affinity. In contrast to the results of this study, Bond (1959) reported that maximum fixation was observed at 0.25 atm N 2 for intact nodules of Alnus glutinosa , a nonlegume. The lower optimal substrate concentrations for nodules of K. cerifera may be due to nodule size or lack of diffusional barriers. The optimal 0 2 concentration for N 2 fixation by nodules of M. cerifera noted in this study agreed with results obtained by Bond (1961) , who reported that the optimal 0 2 concentration for fixation was between 12and 25 per cent (0.12 to 0.25 atm) for the nonleguminous nodules examined, and the higher 0 2 concentrations depressed N 2 fixation. Since this p0 2 is similar to that of air, it may be concluded that 0 2 is not a limiting factor for N 2 fixation under natural conditions. From the study of homogenates prepared from nodules of M. cerifera , it v/as concluded that active preparations required anaerobic homogenization with a buffer at pH 7

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77 and incubation with Na 2 S 2 0 4 , an ATP generating system, and 0 2 . Although N 2 fixing activity varied among preparations, when activity was obtained it was always associated with these conditions. The requirements for maintaining consistently active preparations may be related directly to the prevention of enzyme inactivation. Chronologically, these results represented the first demonstration that Na-^O^, an exogenous ATP generating system and 0 2 were requirements for active fragmented nodular systems, and it has been the only report of a study of homogenates from nonleguminous nodules. Preliminary reports of these studies with homogenates were made by Sloger and Silver (1965) and by Sloger and Silver at the colloquium on biological nitrogen fixation at Sagehen, Calif., September, 1S65, reported by Delwiche (1S66). Independently, Bergersen (1966b) reported a preliminary study which demonstrated that breis of soybean nodules fixed N 2 in the presence of 0 2 . Subsequently, reports of studies on homogenates and breis were presented by Sloger and Silver (1966), Bergersen (1966c), Bergersen and Turner (1967), and Koch et al. (1967a), and Koch et al. (1967b). The results obtained in the present study of nodular homogenates demonstrated that both endogenous and exogenous sources of energy, as well as an electron donor, were required for N 2 fixation. In the intact nodule, respiration provides the energy sources required for N~ fixation. In cell-free

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78 enzyme systems , an exogenous ATP source would serve the same role. Since 0 2 and exogenous ATP together maintained fixation by nodular homogenates of M. cerifera , both a soluble nitrogenase system and intact portions of nodules may have been fixing N 2 . The C>2 requirement for fixation by nodular homogenates of M. cerifera has been confirmed and extended by others. Bergersen (1966b) and Bergersen (1966c) reported that although active breis of soybean nodules must be prepared anaerobically, 0 2 was essential in the incubation gas mixture for N 2 fixation. Koch et al. (1967a) confirmed that N 2 fixation by anaerobically prepared breis of soybean nodules was dependent upon 0 2 . Furthermore, Bergersen and Turner (1967) showed that intact bacteroids from active breis of soybean nodules fixed N 2 in the presence of (L,. Although 0 2 supported N 2 fixation in intact and fragmented nodules, N 2 fixation is considered to be a reductive process. In fact, 0 2 inactivated highly purified components of nitrogenase as shown by Bulen and LeComte (1966) and Mortenson et al. (1967). Recently, Koch et al. (1967b) demonstrated that cell-free extracts prepared from active bacteroids of soybean nodules fixed N 2 without 0 2 . Therefore, from the reports cited above and the results of the present study, it is suggested that 0 2 maintained respiration within intact portions of the filamentous endophyte and host cells and thereby stimulated TS 0 fixation.

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79 Since N 2 fixation by nodular homogenates of Mj. cerifera required an exogenous source of ATP in addition to 0 2 , these data differed from observations made by other investigators. Bergersen (1966b) found that 0 2 was essential for N 2 fixation by breis of soybean nodules, while the addition of an ATP generating system had little effect on the fixation rate. Active breis from soybean nodules prepared by Koch et al. (1967a) fixed N g in the presence of 0 2 and no exogenous ATP was needed. In contrast, the nitrogenase system isolated from bacteroids fixed N 2 with the addition of only ATP and an electron donor (Koch et al. , 1967b). Therefore, it is suggested that an exogenous ATP source and electron donor were requirements for N 2 fixation by soluble nitrogenase which was released due to fragmentation of the filamentous -endophyte of the nodules of M. cerifera . if this is so, the method of preparing homogenates should be modified in order to collect intact filaments of the endophyte. Then it may be possible to prepare cell-free extracts from the endophyte with consistent activity. Although the roles of Na^O^ and the ATP generating system in stimulating fixation by nodular homogenates of M. cerifera are not known with certainty, similar observations on cell -free extracts of bacteria have been made. Carnahan et al. (I960) found that added pyruvate was required to obtain fixation by extracts of C. -oasteurianum. Pyruvate metabolism was shown to serve both as an

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80 ATP source and as an electron donor for C. pasteurianum (Mortenson, 1964; Hardy and D'Eustachio, 1964; and D'Eustachio and Hardy, 1964) and for A. vinelandii (Bulen et al., 1965). The requirements for symbiotic nitrogen fixation might be similar to those of free-living bacteria. Preparation of consistently active nodular homogenates of M. cerifera would depend probably upon preventing enzyme inactivation. Phenolic oxidation products inactivated enzymes and their removal by polyvinylpyrrolidone (PVP) insured consistent activity in breis of soybean nodules, as shown by Koch et al. (1967a). However, when PVP was added to nodular breis of M. cerifera . only marginal activity was observed. The reasons for this are unknown at present. The search for compounds which will stabilize enzyme activity should be continued. The results of this study have demonstrated that only nodules with N 2 fixing capacity reduced acetylene and that N 2 inhibited the reduction of acetylene by nodules of M. cerifera and soybean. This evidence suggested that both N 2 and acetylene reductions were catalyzed by nitrogenase. Molecular nitrogen and acetylene may be competing for the same binding site or for electrons. Clarification of these mechanisms can be done with purified nitrogenase preparations. However, it was concluded that the reduction of acetylene may be used to measure the capacity of a tissue to fix N 0 .

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81 That the acetylene assay is a valid and useful 15 adjunct to the N method in studies of symbiotic nitrogen fixation is based on several observations. All symbiotic fixing tissues (root nodules and an isolated endophyte), except those of equivocal N 2 fixing potential, reduced acetylene to ethylene. There existed a close correlation between the time course of acetylene reduction and N 2 fixation by excised nodules of M. cerifera . The ability of excised nodules of legumes to reduce acetylene was short lived, as was the capacity for N 2 fixation. There was a stoichiometric relationship between the production of ethylene and the disappearance of acetylene which suggested formation of ethylene from acetylene. Like N 2 fixation, the reduction of acetylene was detected only when 0 2 was present and was stimulated by an increased 0 2 concentration. Furthermore, as cited above, N 2 inhibited the reduction of acetylene by excised leguminous and nonleguminous nodules. Preliminary results of these studies on the reduction of acetylene by nodular tissues were presented by Sloger and Silver (1967) and by Sloger and Silver at a colloquium on biological nitrogen fixation held at Sanibel Island, Fla., reported by Silver (1967). Chronologically, the latter report was the first demonstration that acetylene reduction was characteristic of a number of nonleguminous and leguminous plants. This report extended the previous data by Koch and Evans (1966), who demonstrated reduction of acetylene by soybean nodules. Additional observations by Stewart et al.

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82 (1967) indicated that nodules of Alnus , Comptonia , and "bluegreen algae in lake and soil habitats reduced acetylene. From a comparison of data on optimal conditions for acetylene and N2 reductions by excised nodules of M. cerifera , it was observed that 0.04 atm acetylene and 0.08 atm supported maximum reduction. This suggested that the affinity of nitrogenase for acetylene was about two to three times that for Ng. The permeability characteristics of acetylene compared to N2 for nodular tissue may effect the apparent high affinity for acetylene. Although the apparent values for and acetylene were different for nodules of M. cerifera , they were similar for soybean nodules as shown by Koch and Evans (1966). In contrast, Hardy and Knight (1967b) presented data which showed that the nitrogenase of heated cell-free extracts of A. vinelandii appeared to bind Ng ten times as well as acetylene. More data are required to establish whether the mechanics of binding and reduction of molecules by the nitrogenases of various organisms are similar. The evidence for acetylene and reduction by nitrogenase of nodules in these studies suggests that the active site of the enzyme is not specific for a single substrate. That acetylene and N 2 reductions were analogous processes, catalyzed by nitrogenase from C. pasteurianum was shown by Dilworth (1966). Hardy and Knight (1967b), after studying the reduction of various molecules by cell-free extracts

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83 of A. vinelandii and C. pasteurianura , suggested that the N 2 fixing system had a versatile substrate "binding site. Acetylene appeared to have interfered with enzyme activity because nodules of M. cerifera were affected by concentrations of acetylene above 0.2 atm. A decreased rate of ethylene production was observed. This result differed from the data for soybean nodules reported by Koch and Evans (1966). Their data showed no adverse effect of acetylene up to a concentration of 0.4 atm. A second interesting observation was made from the results which were obtained for N 2 inhibition of acetylene reduction by soybean nodules and nodules of M. cerifera . Acetylene and N 2 appeared to have equal access to the enzyme site within nodules of M. cerifera , whereas the data from soybean nodules suggested that the gases were not equally available to the enzyme. If both N 2 and acetylene molecules reached the bacteroids of soybean nodules by diffusion, then the observed results should be like that found for nodules of M. cerifera . However, if N 2 was binding to a carrier substance (Burris, 1966), and subsequently transported to the bacteroids, then acetylene may compete for the carrier. The high affinity of acetylene for the carrier may explain the fact that a N 2 to acetylene concentration ratio of three to one did affect the reduction of acetylene. The feasibility of using the acetylene reduction method as a measure of N p fixation is demonstrated by the

PAGE 92

84 present studies. The method has numerous advantages over the **N method. The cost of gas chromatographic apparatus is relatively inexpensive compared to a mass spectrometer and highly enriched nitrogen. The sensitivity of the acetylene reduction method is about 10 to 100 times greater than the V N method used in these studies. Ethylene production could he detected with as little as 10 mg of tissuean individual soybean nodule or a small lobe from a nodule of M. cerifera . In contrast, about one gram of nodules is 15 required for the N method. Furthermore, analysis of gas samples can be made in one hour or less after incubating the nodules with acetylene. In investigations using the method, exposure lasts for hours or days and is followed by laborious digestion, distillation and conversion of ammonia to molecular nitrogen for analysis. On the basis of these factors, the acetylene reduction method is a practical means of determining N 2 fixation by various organisms. The usefulness of the acetylene reduction method demonstrated in these studies confirms the application of the method by Koch and Evans (1966) for the assay of activity of nitrogenase in cell-free extracts of nodules. Furthermore, Stewart et al. (1967) showed that the method can be employed as an index of N 2 fixation in aquatic environments, in soils, and by nodulated plants.

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85 With, acetylene reduction as a simple assay for N« fixing ability and with, further work resulting in prepara tions of cell-free extracts of nodules of M. cerifera , th roles of the endophyte and plant proteins in symbiotic nitrogen fixation by nonlegumes may be elucidated.

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SUMMARY Excised intact root nodules from native Myrica 15 cerifera were shown by the method to fix molecular nitrogen. The "^N method involves mass spectrometric analyses 15 15 for enrichment, after nodules are exposed to N 2 . Limiting the analyses to the acid soluble nitrogenous compounds of the nodules increased the sensitivity of detecting fixation. The fact that the newly fixed N 2 accumulated in the metabolically active portion of the nodules supports the idea that N 2 is reduced to ammonia. The time course of N 2 fixing activity by nodules after detachment as well as the optimal N 2 and Op concentrations were determined. Nodules of M. cerifera were good experimental material for studies of symbiotic nitrogen fixation because they had several physiological aspects which were typical of nonleguminous nodules and because they had prolonged activity after detachment. Active nodular homogenates of M. cerifera were prepared by a procedure involving anaerobic homogenisation in a buffered cacodylate medium and later supplementing with Na 2 S 2 0^, an ATP generating system, and 0 2 , In this system Na-jSgO^ served as an electron source and the ATP generating system served as an energy source. The requirements for ATP and electrons were consistent with findings 86

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87 for other nitrogenase systems. The required 0 2 stimulated endogenous respiration necessary for ^ fixation by intact portions of the tissue. Nodules of several species of leguminous and nonleguminous plants were shown to reduce acetylene to ethylene. Since the requirements for acetylene reduction and fixation were similar, and since inhibited acetylene reduction, nitrogenase was assumed to catalyze "both reactions. The ability of nodules to reduce extremely small amounts of acetylene to ethylene could be detected by gas chromatography. By this sensitive analysis it was possible to detect activity by individual nodular lobes of M. cerifera and single soybean nodules. Therefore, acetylene reduction by nodules, and its detection by gas chromatography, is considered to be a valid and rapid means of measuring the N 2 fixing ability of nodules. The advantages of the acetylene reduction method 15 compared to the method were discussed.

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LITERATURE CITED Allen, E. K. and 0. N. Allen. 1958. Biological aspects of symbiotic nitrogen fixation. In: Encyclopedia of Plant Physiology. Vol. 8. W. Ruhland, ed. Springer-Verlag, Berlin, p. 4-8-118. Aprison, M. H. and R. H. Burris. 1952. Tine course of fixation of N« by excised soybean nodules. Science 115: 264-2657 Aprison, M. H., W. E. Magee, and R. H. Burris. 1954. Nitrogen fixation by excised soybean root nodules. Jour. Biol. Chem. 208: 29-39. Becking, J. H., W. E. de Boer, and A. L. Houwink. 1964. Electron microscopy of the endophyte of Alnus glutinosa . Antonie van Leeuv/enhoek, J. Microbiol. Serol. 30: 343-376. 15 Bergersen, P. J. I960. Incorporation of ^N 2 into various fractions of soybean root nodules. J. Gen. Microbiol. 22: 671-677. Bergersen, P. J. 1962a. The effects of partial pressure of oxygen upon respiration and nitrogen fixation in soybean root nodules. J. Gen. Microbiol. 29: 113-125. Bergersen, P. J. 1962b. Oxygenation of leghaemoglobin in soybean root nodules in relation to the external oxygen tension. Nature 194: 1059-1061. Bergersen, P. J. 1963. The relationship between hydrogen evolution, hydrogen exchange, nitrogen fixation, and applied oxygen tension in soybean root nodules. Aust. J. Biol. Sci. 16: 669-680. Bergersen, F. J. 1965. Ammonia — an early stable product of nitrogen fixation by soybean root nodules. Aust. J. Biol. Sci. 18: 1-9. Bergersen, P. J. 1966a. Nitrogen fixation in the legume root nodule. In: IX International Congress of Microbiology. Moscow, p. 97-101. 88

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89 Bergersen, F. J. 1966b. Nitrogen fixation in breis of soybean root nodules. Biochim. Biophys. Acta 115: 247-249. Bergersen, P. J. 1966c. Some properties of nitrogenfixing breis from soybean root nodules. Biochim. Biophys. Acta 130: 304-312. Bergersen, F. J. and G. L. Turner. 1967. Nitrogen fixation by the bacteroid fraction of breis of soybean root nodules. Biochim. Biophys. Acta 141: 507515. Bond, G. 1955. An isotopic study of the fixation of nitrogen associated with nodulated plants of Alnus, Ivlyrica , and Hippophae . J. exp. Bot. 6: 303-311. ' Bond, G. 1956. Evidence for fixation of nitrogen by root nodules of alder ( Alnus ) under field conditions. New Phyt. 55: 147-153. Bond, G. 1957. Isotopic studies of nitrogen fixation in non-legume root nodules. Ann. Bot. n. s. 21: 511521. Bond, G. 1959. Fixation of nitrogen in non-legume rootnodule plants. Symp. Soc. exp. 3iol. 13: 59-72. Bond, G. 1961. The oxygen relation of nitrogen fixation in root nodules. Zeits. Allg. Hikrobiol. 1: 93-99. Bond, G. 1963. The root nodules of non-leguminous angiosperms. Symp. Soc. Gen. Microbiol. 13: 72-91. Bond, G.^_1964. Isotopic investigations of nitrogen fixation in non-legume root nodules. Nature 204: 600-601. Bond, G. 1967. Fixation of nitrogen by higher plants other than legumes. Ann. Rev. Plant Physiol. 18: 107—126 . Bulen, W. A. , R. C. Burns, and J. R. LeComte. 1965. l^ 0se ^ f i x&tion: Hydrosulfite as electron donor y\>? e i ree P re P ara ^i°ns of Asotobac ter vinelandii and Rhodosprillum rubrum. Proc . Uatl. Acad. Li? J » ^« OS'. 532-539.

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90 Bulen, V7. A. and J. R. LeComte. 1966. The nitrogenase system from Azotobacter : two-enzyme requirement for N 2 reduction, ATP-dependent H 2 evolution, and ATP hydrolisis. Proc. Natl. Acad. Sci. U. S. 56: 979-986. Burris, R. H. 1965. Nitrogen fixation. In: Plant Biochemistry. J. Bonner and J. E. Varner, eds. Academic Press, New York. p. 961-979. Burris, R. H. 1966. Biological nitrogen fixation. Ann. Rev. Plant Physiol. 17: 155-184. Burris, R. H. , W. E. Magee, and M. K. Bach. 1955. The pN 2 and the p0 2 function for nitrogen fixation by excised soybean nodules. Ann. Acad. Sci. Perm. 60: 190-199. Burris, R. H. and C. E. Miller. 1941. Application of N 15 to the study of biological nitrogen fixation. Science 93: 114-115. Burris, R. H. and P. Y7. Wilson. 1957. Methods for measurement of nitrogen fixation. In: Methods in Enzymology. S. P. Colowick and N. 0. Kaplan, eds. Academic Press, New York. 4: 355-366. Carnahan, J. E. , L. E. Mortenson, H. P. Mower, and J. E. Castle. I960. Nitrogen fixation in cell-free extracts of Clostridium pasteurianum . Biochim. Biophys. Acta 44: 520-535. Centifanto, Y. M. 1964. Leaf nodule symbiosis in Psychotria bacteriophila . Ph. D. Dissertation, University of Florida, Gainesville. Centifanto, Y. M. and W. S. Silver. 1964. Leaf-nodule symbiosis. I. Endophyte of Psychotria bacterio phila . J. Bacteriol. 88: 77fa-701. Dart, P. J. and P. V. Mercer. 1963. Development of the bacteroids in the root nodules of barrel medic ( Medicago tribuloides Desr.) and subterranean clover ( Triioiium subterraneum L.). Arch. Mikrobi ol . 4b! 382-401. Davenport, H. E. I960. Haemoglobin in the root nodules of Casuarina cunninghamiana . Nature 186: 653-654.

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91 Delwiche, C. C. , P. J. Zinke, and C. M. Johnson. 1965. Nitrogen fixation by Ceanothus . Plant Phvsiol. 40: 1045-1047. Delwiche, C. C. 1966. Biological nitrogen fixation. Science 151: 1565-1566. D'Eustachio, A. J. and R. W. F. Hardy. 1964. Reductants and electron transport in nitrogen fixation. Biochera. Biophys. Res. Comm. 15: 319-323. Dilworth, M. J. 1966. Acetylene reduction by nitrogen fixing preparations from Clostridium pasteurianum . Biochim. Biophys. Acta 1271 265-294. Dixon, R. 0. D. 1967. Hydrogen uptake and exchange by pea root nodules. Ann. Bot. n.s. 31: 179-188. Fletcher, W. W. 1955. The development and structure of the root-nodules of Myrica gale L. with special reference to the nature of the endophvte. Ann. Bot. n. s. 19: 501-513. Furman, T. E. 1959. The structure of the root nodules of Ceanothus sanguineus and Ceanothus velutinus , with special reference to the endophyte. Amer. J. Bot. 46: 698-708. Gardner, I. C. 1965. Observations on the fine structure of the endophyte of the root-nodules of Alnus glutinosa (L.) Gaertn. Arch. Mikrobiol. 51: 365-^3. Glover, J. 1956. Methods of involving labelled atoms. In: Modern Methods of Plant Analysis. K. Paech and M. V. Tracey, eds. Springe r-Verlag, Berlin. 1: 325-374. Goodchild, D. J. and F. J. Bergersen. 1966. Electron microscopy of the infection and subsequent development of the soybean nodule cells. J. Bacteriol. 92: 204-213. Hardy, R. V/. F. and A. J. D'Eustachio. 1964. The dual role of pyruvate and the energy requirement in nitrogen fixation. Biochem. Biophys. Res. Comm. 15: 314318.

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92 Hardy, R. W. F. and E. Knight, Jr. 1966. Reduction of N p 0 by biological N2 fixing systems. Biochem. Biorhys. Res. Comraun. 23: 409-414. Hardy, R. W. P. and E. Knight, Jr. 1967a. The biochemistry and postulated mechanisms of nitrogen fixation. In: Progress in Phyto chemistry. L. Reinhold, ed. John Wiley and Sons, London, p. 387-469. Hardy, R. W. P. and E. Knight, Jr. 1967b. ATP-dependent reduction of azide and HON by N2 fixing enzymes of Azotobacter vinelandii and Clostridium pasteurianum . Biochim. Biophys. Acta 139: 69-90. Harris, G. P. and T. M. Morrison. 1958. Fixation of nitrogen by excised nodules of Coriaria aborea Lindsay. Nature 182: 1812. Hoch, G. E. , H. N. Little, and R. H. Burris. 1957. Hydrogen evolution from soybean root nodules. Nature 179: 430-431. Hoch, G. E. , K. C. Schneider, and R. H. Burris. I960. Hydrogen evolution and exchange, and conversion of N2O to N2 by soybean root nodules. Biochim. Biophys. Acta 37: 273-279. Jansen, E. P. 1965. Ethylene and polyacetylenes. In: Plant Biochemistry. J. Bonner and J. E. Varner, eds. Academic Press, New York. p. 641-664. Jordan, D. C. , I. Grinyer, and W. H. Coulter. 1963. Electron microscopy of infection threads and bacteria in young root nodules of Medicago sativa . J. Bacteriol. 86: 125-137. Kamen, M. 1956. Introductory Remarks. In: A Symposium on Inorganic Nitrogen Metabolism. W. D. McElroy and B. Glass, eds. John Hopkins Press, Baltimore, P. 295. ' Kennedy, I. R. 1966a. Primary products of symbiotic nitrogen fixation. I. Short-term exposure of serradella nodules to 15 N?. Biochim. Biophys. Acta 130: 285-294. Kennedy, I. R. 1966b. Primary products of symbiotic nitrogen fixation. II. Pulse -labelling of serradella nodules with 15n 2 . Biochim. Biophys. Acta 130: 295-303.

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93 Kennedy, I. R. , C. A. Parker, and D. K. Kiddy. 1966. The probable site of nitrogen fixation in root nodules of ornithonus sativus. Biochim. Biophys. Acta 130: Koch, B. and H. J. Evans. 1966. Reduction of acetylene to ethylene by soybean root nodules. Plant Physiol. 41: 1748-1750. Koch, 3., H. J. Evans, and S. Russell. 1967a. Reduction of acetylene and nitrogen gas by breis and cell-free extracts of soybean root nodules. Plant Physiol. 42: 466-468. Koch, 3., H. J. Evans, and S. Russell. 1967b. Properties of the nitrogenase system in cell-free extracts of bacteroids from soybean root nodules. Proc. Natl. Acad. Sci. U. S. 58: 1343-1350. Leaf, G., I. C. Gardner, and G. Bond. 1959. Observations on the composition and metabolism of the nitrogenfixing root nodules of Myrica . Biochem. J. 72: 662-667. Magee, W. E. and R. H. Burris. 1954. Fixation of N2 1 ^ by excised nodules. Plant Physiol. 29: 199-200. McNair, H. M. and E. J. Bonelli. 1967. Basic Gas Chromatography. Varian Aerograph, Walnut Creek, Calif. 248 p. Moore, A. W. 1964. Note on non-leguminous nitrogen-fixing plants in Alberta. Can. J. Bot. 42: 952-955. Morrison, T. M. 1961. Fixation of nitrogen-15 by excised nodules of Discaria toumatou . Nature 189 : 945. Mortenson, L. E. 1964. Ferredoxin and ATP, requirements for nitrogen fixation in cell-free extracts of Clostridium pasteurianum . Proc. Natl. Acad. Sci. tJTX 52! 272-279. Mortenson, 1. E. , J. A. Morris, and D. Y. Jeng. 1967. Purification, metal composition and properties of molybdof erredoxin and azoferredoxin, two of the components of the nitrogen-fixing system of Clostridium pasteurianum . Biochim. Biophys. Acta I4H 516-522.

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94 Neelands, M. 1967. Mutants of Klebsiella rubiacerura unable to fix atmospheric nitrogen. Master's Thesis, University of Florida, Gainesville. Rittenberg, D. 1946. The preparation of gas samples for mass-spectrographic isotope analysis. In': Preparation and measurement of isotopic tracer. D. W. Wilson, A. 0. Nier, and S. P. Reiman, eds.
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95 Stewart, W. D. P. 1967. Nitrogen-fixing plants. Science 158: 1426-1432. Stew-art, iff. D. P., G. P. Fitzgerald, and R. H. Burris. 1967. In Situ studies on N 2 fixation using the acetylene reduction technique. Proc. Natl. Acad. Sci. U. S. 58: 2071-2078. Strandberg, G. W. and P. W. Wilson. 1967. Molecular N„ and the pN p function of Azotobacter . Proc. Natl. lead. Sci. UT S. 58: 1404-1409. Taubert, H. 1956. Uber den inf ektionsvorgan und die entwicklung der knollchen bei Alnus gluti nosa Gaertn. Planta 48: 135-15 6. Umbreit, W. W. , R. H. Burris, and J. ?. Stauffer. 1951. Manometric Techniques and Tissue Metabolism. Burgess Publishing Co. Minneapolis, Minn. 227 p. Wipf, L. and D. C. Cooper. 1940. Some doubling of chromosomes and nodular infection in certain Leguminosae. Amer. J. Bot. 27: 821-824. Ziegler, H. and H. Huser. 1963. Fixation of atmospheric nitrogen by root nodules of Comptonia oerearrina. Nature 199: 508. a

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BIOGRAPHICAL SKETCH Charles Sloger was born December 22, 1938, at Albany, New York. In June, 1957, he was graduated from Berne-Knox Central High School. In June, 1961, he received the degree of Bachelor of Science with a major in Biology from the State University of New York at Albany. He continued in the Graduate School at the State University of New York at Albany and held a teaching assistantship in the Department of Biology. He received the degree of Maste of Science in June, 1963. In September, 1963, he enrolled in the Graduate School of the University of Florida. He worked as a teaching assistant in the Department of Botany until June, 1964. From September, 1964, until the present time he has pursued his work toward the degree of Doctor of Philosophy. Charles Sloger is married to the former Marcia Alic Schekter. He is a member of the Society of the Sigma Xi and the American Institute of Biological Sciences. 96

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This dissertation was prepared under the direction of the chairman of the candidate's supervisory committee and has been approved by all members of that committee. It was submitted to the Dean of the College of Agriculture and to the Graduate Council, and was approved as partial fulfillment of the requirements for the degree of Doctor of Philosophy. March, 1968 ^^/Dean, College of Agriculture Dean, Graduate School Supervisory Committee: ' Co-chairman