NITROGEN FIXATION IN THE CORALLOID ROOTS OF
MACROZAMIA COMMUNIS L. JOHNSON
By F. J. BERGERSEN,* G. S. KENNEDy,t and W. WITTMANNt
[Manuscript received August 4, 1965J
1965]
Summary
Coralloid roots of Macrozamia communis have been shown by the isotopic
method to fix nitrogen when they contain the endophytic blue·green algae. Immature
coralloid roots devoid of the endophyte did not fix nitrogen. Coralloid roots from
glasshouse·grown
2·77 times as much nitrogen when illuminated than
glasshouse-grown plants fixed 2·
they did in the dark and the 15N
IfiN excess was about equally divided between fractions
soluble or insoluble in 3N HCI. Coralloid roots excavated from beneath large fieldgrown plants were opaque and did not fix more nitrogen when illuminated than they
did in the dark. Most of the newly fixed nitrogen was found in the buffered sucrose
extract of crushed tissue. When an intact plant bearing coralloid roots was exposed
to an atmosphere containing a large excess of 15N.
IfiN. for 48 hr the 15N
IfiN was found to be
distributed through the plant parts. Nitrogen fixed in the coralloid roots is thus
available for the growth of the plant. The coralloid roots evolved small amounts of
hydrogen.
hydrogen_
1. INTRODUCTION
The symbiotic association of blue-green algae in the apogeotropic coralloid
roots of cycads was first described by Reinke (1872, quoted by Thieret 1958). The
endophytes isolated by Winter (1935) from Cycas circinalis, Encephelartos altensteinii,
and E.
E_ cycadifolius fixed nitrogen in culture and this was confirmed for the endophyte
of C. circinalis by Douin (1953). Bond (reported by Fowden 1958) obtained nitrogen
fixation with detached coralloid roots of species of Ceratozamia and Encephelartos
using 15N techniques. Watanabe and Kiyohara (1963) obtained 15N incorporation by
the cultured endophytes of two species of M acrozamia and three other species of cycads
but the coralloid roots failed to fix nitrogen. There is thus some confusion about the
nitrogen-fixing ability of the coralloid roots of these plants and there is no clear
of the cycad_
cycad.
evidence that any nitrogen fixed by the endophyte is available for growth ofthe
The present work was undertaken to ascertain whether the coralloid roots of
Macrozamia communis L. Johnson were capable of fixing nitrogen and to investigate
the availability
a vaila bility of any fixed nitrogen for the growth of the host plant. This particular
species of cycad is a prominent understorey plant of forests in coastal south-eastern
Australia (Fig. 1) and nitrogen fixation in its coralloid roots would be a very significant
aspect of the nitrogen economy of these ecosystems. A description of the anatomy
of the coralloid roots of M. communis is given by Wittmann, Bergersen, and
Kennedy (1965).
* Division of Plant Industry, CSIRO, Canberra.
t Department of Botany, Australian National University, Canberra.
Aust. J. Bioi.
Biol. Sci., 1965, 18, 1135-42
1136
F. J. BERGERSEN, G. S. KENNEDY, AND W. WITTMANN
II.
MATERIALS AND METHODS
(a) Coralloid
Ooralloid Roots
Field-grown material was brought from the vicinity of Bateman's Bay, N.S.W.
Glasshouse-grown material was gathered from plants which had been brought from
the same area and grown for some months in pots of soil. Where possible the
Fig. l.-M. communis understorey to Eucalyptus maculata Hook growing adjacent to Clyde
Olyde
Mountain Road, Bateman's Bay, N.S.W.
coralloid roots were detached from plants immediately prior to the commencement
of the experiments but in experiment 2 the plants were too big to transport intact
and the material was detached, packed in plastic bags, cooled in ice, and transported
as quickly as possible (within 3-4 hr) to Canberra.
NITROGEN FIXATION IN MACROZAMIA COMMUNIS
1137
(b) Apparatus
Incubations with atmospheres containing 15N2 were done in 100-ml flasks
containing 12-16 g (fresh weight) of coralloid roots. The flasks were immersed in a
Perspex water-bath controlled at 23 or 25°0 and were attached to a glass manifold
equipped with a mercury manometer and gas sampling points and attached to a
reservoir containing the gas mixture. The flasks were evacuated, flushed twice with
argon, and filled with the gas mixture to a pressure of 700 mmHg. Illumination was
provided by a 500-W
500-W Philips Altrilux lamp placed 1 ft from the flasks and shining
through the side of the water-bath. Flasks in dark treatments were wrapped in
aluminium foil. Heat from the lamp was dissipated from the water-bath by means
of a copper coil carrying cold water.
(c) Gas Mixture
These were prepared from good quality commercial gases and 15N2 was prepared
from 15NHa3 by oxidation with copper oxide at 600°0. The percentage composition
of the gas mixtures was measured mass spectrometrically before and after experiments,
in samples taken from each flask.
(d) Mass Spectrometer Methods
In all experiments the methods used were those described previously (Bergersen
1962, 1963, 1965).
(e) Analytical
Nitrogen was determined by Kjeldahl digestion of tissue, tissue fractions, or
extracts, and distillation and titration of the resulting ammonia (Paech and Tracey
1956). 15N content of this ammonia was measured using standard methods (Bergersen
1962, 1963, 1965).
III.
EXPERIMENTAL
(a) Experiment 1
Juvenile coralloid roots containing no endophyte were obtained from young
field-grown plants. These roots were a yellow-brown colour with pale tips. Mature
coralloid roots with well-developed endophyte were obtained from glasshouse material.
These roots had grown near the soil surface, were pale-coloured externally, and the
green of the endophyte was visible through the surface cell layers. After 4 hr incubation in the labelled gas mixture at 23°0 the tissues were ground in 3N HOI and
extracted for 1 hr at room temperature. Both extract and residue were analysed
for 15N.
(b) Experiment 2
Ooralloid roots from large field-grown plants were excavated from a depth of
about 8-12 in., detached, and brought to the laboratory. These roots were darker in
colour, and had opaque periderm layers. Although a well-developed endophyte zone
was present, this was not visible through the root surface. After incubation with 15N2
the tissues were gently crushed in 0 ·lM
·IM potassium phosphate, pH 7 ·0,
'0, containing 0O··3M
3M
F. J. BERGER
BERGERSEN,
SEN, G. S. KENNEDY, AND W. WITTMANN
1138
sucrose, so that a minimum of osmotic damage should be done to algae and plant
particles. The brei was strained through organdie and centrifuged at 25,000 g
(J for
10 min in a refrigerated Servall centrifuge. Three layers were obtained in the deposit.
The lowermost was a small white deposit which was discarded and above this was a
thick deposit of algal cells. The top layer was a thick buff-coloured deposit overlayed
by a slightly opalescent supernatant. The algal layer and the buff-coloured layer
were separated, washed twice with buffered sucrose, and, together with the supernatant, were made to 3N with HCl.
HOI. After 1 hr the acid extracts were analysed for 15N.
(c) Experiment 3
A healthy young plant bearing a number of coralloid roots and with its tap root
wrapped in moistened cotton-wool was enclosed in a glass tube 50 cm long and 5 cm
in diameter equipped with a glass stopcock at one end. The lower part of the tube was
wrapped in aluminium foil. The assembly was evacuated and filled with a gas mixture
TABLE
1
FIXATIO" OF ATMOSPHERIC NITROGEN BY JUVENILE AND GLASSHOUSE-GROWN CORALLOID ROOTS
FIXATION
15N
InN content of 13 g (fresh weight) of coralloid roots after 4 hr at 23°C'in
23°C 'in an atmosphere composed
of 13% nitrogen (52·4
(52· 4 atoms % I5N), 18% oxygen, and 69% argon
1 ____
H~I
3N HCI Extract
Extract______
3N
3N HCI Residue
Coralloid
Roots
Used
Usod
Growth
Conditions
Total
I Total
Nitrogen
(mg)
I5N
(atoms %
excess)
(I'-g
(fJ-g
excess)
Total
Nitrogen
(mg)
I5N
(atoms %
excess)
(p,g
(I'-g
excess)
Glasshousegrown
Dark
Illuminated
III uminated
11·68
13,99
13·99
0,259
0·259
0·552
30·25
77·22
73·00
78·00
0·032
0·082
23·36
63,96
63·96
Juvenilo
Juvenile
Dark
Illuminated
4·23
4·16
0
0
0
0
25·25
26·00
0
0
0
0
- -
- ----- -- -----------
I5N
-------
I
I5N
---
----
------~
composed of 12% nitrogen (44 atoms % 15N), 25% oxygen, and 63% argon. It was
then placed in an illuminated growth cabinet at 24°0
24°C for 48 hr during which time
there were two 6-hr dark periods at 18°0.
18°C. The plant was then dissected into its parts
which were
wcre dried, milled, and analysed for 15N.
IV.
RESULTS
(a) Experiment 1
Ooralloid roots containing endophytic blue-green algae were clearly capable of
Coralloid
fixing quite large amounts of atmospheric nitrogen while young roots with no
endophyte fixed none (Table 1). Illuminated coralloid roots (13 g fresh weight)
fixed 141 p..g 15N in 4 hr from an atmosphere containing 52 atoms % 15N. This
corresponds to a total fixation of about 5·2 p..g N
N/g/hr,
fgfhr, a rate comparable with that of
excised soybean root nodules (Bergersen 1965), and 10 times greater than that of
NITROGEN FIXATION IN MACROZAMIA
MAOROZAMIA COMMUNIS
OOMMUNIS
1139
nodulated roots of Podocarpus
Podocarpu8 lawrencei (Bergersen and Costin
Oostin 1964). Darkened
coralloid roots fixed less than half as much nitrogen as did the illuminated ones.
(b) Experiment 2
In experiment 1 the 3N RCI-soluble
HOI-soluble fraction contained the highest enrichment
of15N.
(atoms % excess) of
15N. In experiment 2 it was shown that this 15N
15N was concentrated
in the soluble material of the coralloid roots, only small amounts being found in the
algal cells (Table 2). With this sample of coralloid roots there was no effect of
illumination in the total amount of 15N
15N excess obtained but the algal fraction of the
illuminated roots contained almost twice as much 15N
15N excess as did that of the
darkened roots. The total amount of 3N RCI-extractable
HOI-extractable 15N
15N fixed per hour in this
experiment was very similar to that of the dark treatment of experiment 1. The
buff-coloured deposit which overlaid the algal fraction in the centrifuge tube
contained no RCI-soluble
HOI-soluble nitrogen.
TABLE 2
DISTRIBUTION OF i5N IN SOLUBLE AND ALGAL FRACTIONS OF CORALLOID ROOTS
i5N content of 3N HOI
HCI extracts from the soluble and algal fractions of 16 g (fresh weight) of
detached, field-grown coralloid roots after 2 hr at 25°0
25°C in an atmosphere composed of 23%
23 %
nitrogen (69'5
(69·5 atoms % i5N), 23% oxygen, and 54% argon
Algal Fraction
Soluble Fraction
----
Growth
Oonditions
Conditions
Dark
Illuminated
I
i5N
i5N
(p,g
{fJ-g
excess)
Total
Nitrogen
(mg)
i5N
l5N
{atoms
(atoms %
excess)
(p,g
(fLg
excess)
0·116
0·105
13·35
13·28
0·35
0·21
0·062
0·091
0·217
0·191
0·103
0·096
11·48
13·30
0·49
0·38
0·081
0·080
0·437
0·304
Total
Nitrogen
(mg)
{atoms
(atoms %
excess)
11·50
12·65
11·15
13·85
I
I
I
I
i
I
i5N
In this experiment evidence was obtained for hydrogen evolution from the
coralloid roots. The initial gas mixture contained a trace of hydrogen (hydrogen :
argon ratio == 32·4 X 10-6 ). Mter
After the incubation period the gas in the flasks contained
an average hydrogen: argon ratio of 209·7 X 10-6 for the illuminated and 189·4 X 10-6
for the dark treatment, while changes in the other gas components (022 and CO
002)
2 ) were
of the order of 2% due to the respiration of the tissue.
(c) Experiment 3
Experiment 2 showed that most of the 3N RCI
HOI extractable 15N
15N excess was found
eX'~ract of the crushed tissue. This suggested that
in solution in the buffered sucrose extract
it would be mobile and was probably available to the plant. The next experiment
showed that this was indeed so since 15N
15N2 fixed in the coralloid roots became distributed throughout the plant with the exception of the leaves (Fig. 2 and Table 3).
Apart from the coralloid roots themselves (109/Lg
(109 fLg 15N
15N excess), the bulbous storage
organ (swollen leaf bases and stem) contained the most 15N
15N (66/Lg).
(66 fLg).
1140
F. J. BERGERSEN, G. S. KENNEDY, AND W. WITTMANN
v.
V.
DISCUSSION
Detached
Dctached coralloid roots of Macrozamia communis have been shown by the
results presented to fix atmospheric nitrogen in amounts comparable with detached
soybean root nodules, provided that the coralloid roots contained the endophytic
blue-green algae. Fixation by glasshouse-grown material was greater when illuminated
than in the dark. These observations constitute strong evidence that the algal endo-
LEAVES
SWOLLEN LEAF
BASES AND STEM
HYPOCOTYL AND
SWOLLEN TAP ROOT
Fig. 2.-Various plant parts of M. communis
analysed for distribution of 15N.
l5N.
phyte is the agent of nitrogen fixation and not nitrogen-fixing bacteria as suggested
by Spratt (1915) and McLuckie (1922). This conclusion supports the work of Schaede
(1944) with M. spiralis (syn. M. communis). In this laboratory a pure culture of the
endophyte of M. communis has not yet been obtained although cultures have been
maintained on nitrogen-free salts media for many transfers.
The absence of the light stimulation of fixation in experiment 2 may have been
due to one or more of three factors. In this experiment the coralloid roots were
detached from the plants several
scveral hours before the 15N tests began and changes in the
1141
NITROGEN FIXATION IN MAOROZAMIA OOMMUNIS
tissue may have occurred. Secondly, these roots came from rather deep in the soil
and would not previously have been exposed to light, suggesting that the photosynthetic pathways in the algae may not have been functioning. Thirdly, the
periderm of these coralloid roots was dark with tannins and may have been almost
completely opaque to light.
It is significant that hydrogen evolution from nitrogen-fixing coralloid roots
has been detected. The connection between thc
the possession of hydrogenases and the
ability to fix nitrogen has been recognized for some time and has been elucidated
further with recent studies of cell-free extracts from nitrogen-fixing bacteria (Bulen,
Burns, and LeComte 1964, 1965; D'Eustachio and Hardy 1964). Soybean root
nodules (Hoch, Schneider, and Burris 1960; Bergersen 1962) and nodulated roots of
Podocarpus
Podocarpu8 lawrencei (Bergersen and Costin 1964) have also been shown to evolve
hydrogen.
TABLE
TABLE 3
DISTRIBUTION
DISTRIBUTION OF
I5N
PLANT PARTS
PARTS (SEE
(SEE FIG.
IN THE
THE VARIOUS
VARIOUS PLANT
2)
The plant was exposed to an atmosphere containing 12% nitrogen (44 atoms % I5N), 25% oxygen,
63%
and 63
% argon, for 48 hr in a growth cabinet
"~-----
I5N
Dry
Weight
(g)
Total
Nitrogen
(mg)
(atoms %
excess)
Leaves
i
Petioles
I
Swollen leaf bases and stem I
I
Coralloid roots
Hypocotyl and swollen
tap root
Roots
3·82
2·12
10·20
0·81
60·2
15·2
101·9
15·3
0
0·065
0·057
0·716
O· 716
0
9·88
66·24
109·55
1·68
l·lO
1·10
21·7
13·4
0·064
0·113
13·89
15·14
Total
19·73
Plant
Part
-----"------,--
--
I
---".------~--
227·7
I5N
15N
(p,g
(fLg excess)
I
-"""--""--_ _ _ _ _ _
1 ____ -
--
214·70
----
---
...
_-
The movement of the fixed nitrogen from the coralloid roots into the plant was
clearly established in experiment 3. In the period of the experiment, however, the
15N did not move into the leaves. This may have been due to restricted transpiration
in the closed system. The relatively large proportion of the fixed nitrogen found in
the swollen leaf bases may also indicate that this provides a relatively large sink for
the fixed nitrogen which is only slowly released to the leaves. The growing point of
the plant is also situated within the swollen leaf bases and fixed nitrogen may be
preferentially used there for protein synthesis.
VI.
ACKNOWLEDGMENTS
Miss Heather Cartwright and Mr. P. L. Kempe provided skilled assistance with
the nitrogen and 15N analyses.
1142
F. J. BERGERSEN, G. S. KENNEDY, AND W. WITTMANN
VII.
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