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Planta (1982)154:352-360
9 Springer-Verlag 1982
Morphogenesis of lucerne root nodules incited by Rhizobium meliloti
in the presence of combined nitrogen
Georges L. Truchet* and Frank B. Dazzo**
Department of Microbiologyand Public Health, Michigan State University, East Lansing, MI 48824, USA
Abstract. Combined light and transmission electron
microscopy were used to examine the effect of nitrate
on the development of root nodules in lucerne (alfalfa,
M e d i c a g o sativa L.) following induction by the nitrogen-fixing symbiont, R h i z o b i u m meliloti. The timing
of NO~ addition was varied in order to study its
effect on all of the recognized morphogenetic steps
of nodule formation. Roots of plants inoculated in
the presence of 18 m M NO~ had straight root hairs
which were devoid of adherent rhizobia and infection
threads, and developed no nodules. However, nodules
were formed on roots if 18 mM NO~- was added
5 d after inoculation. At this time, the initiation of
nodule primordia had already commenced in the root
cortex. The histology and ultrastructure of young
nodules which had developed for 5 d in the absence
of NO~ and another 5 d in the presence of 18 m M
NO~- resembled nodules developing under N-free
conditions, except that in the infection threads within
the infection zone of the nodule 1) some bacteria
tended to loose their normal shape and gain more
electron density, indicating premature degradation,
and 2) the matrix of the infection threads was abnormally enlarged. In the presence of high NO~ levels
in the medium, lysis and degeneration of the bacteria
released from the infection threads were observed in
the infection and bacteroid zones of developing nodules, indicative of premature senescence. On the other
hand, the nodule meristems continued to proliferate
even after 12d of exposure of 18 m M NO~. This
was the only morphogenetic step of root nodulation
which was insensitive to levels of combined nitrogen
that completely prevented infection if present at the
time of inoculation. These data indicate that all of
the recognized steps of root nodule morphogenesis
in which the bacteria play a key role are sensitive
to the inhibitory effect of combined nitrogen.
Key words: M e d i c a g o
Nitrogen, combined
b i u m - Root nodule formation.
Rhizo-
Introduction
The development of the nitrogen-fixing R h i z o b i u m legume symbiosis is suppressed if the roots of the
host plant are grown in the presence of combined
nitrogen (for early literature, see Fred et al. 1932).
The symbiotic events of root hair attachment, root
hair infection, and root nodulation by R h i z o b i u m are
completely inhibited by NOa- at concentrations well
below the level necessary to cause injury to the bacterial symbiont or the host plant (Dazzo and Brill 1978 ;
Gibson and Pagan 1977; Munns 1968; Thornton
1936). Studies using split root systems showed that
inhibition of nodulation by NO~ is localized, affecting only those roots which have grown in the NO3
solution (Wilson 1916).
Ultrastructural studies have been performed on
mature nodules of inoculated Trifolium and M e d i c a g o
plants developing for 35 d under N-free conditions
before the addition of ammonium nitrate (Dart and
Mercer 1964). The purpose of the present study was
to examine the histological developmental sequence
of very young nodules of lucerne roots exposed to
a concentration of NO~- which inhibits nodulation.
* Permanent address, to which reprint requests should be sent:
Institut de Cytologic et de Biologic Cellulaire, Facult6 des
Sciences Marseille-Luminy, L.A.C.N.R.S. 179, Fq3288 Marseille Cedex 2, France
** To whom all correspondenceshould be addressed prior to July,
1982
0032-0935/82/0154/0352/$01.80
Materials and methods
Lucerne (Medicago sativa L. cv. DuPuits) and Rhizobium meliloti
strain L5-30 (both from J. D~nari~, CNRA, Toulouse, France)
G.L. Truchet and F.B. Dazzo: Nitrate effect on early nodulation in lucerne
were used. Seeds were surface-sterilized with saturated calcium
hypochlorite for 30 rain (Truchet 1978), rinsed with sterile water,
and germinated for 36 h at room temperature in the dark on
Wright's agar (Vincent 1970). Sterile seedlings were transferred
to glass tubes (20 m m diameter, 200 m m along) containing 20 mi
of nitrogen-free mineral solution (Fahraeus 1957) supplemented
as indicated with KNO3. The seedlings were then incubated in
a plant growth r o o m p r o g r a m m e d for 16 h of light from fluorescent
lamps (27,000 lux) and 8 h of darkness, 85% relative humidity,
and 24 ~ C. The bacteria were grown on agar slants of Wright's
medium (Vincent 1970) for 3 d, suspended in N-free medium, and
then inoculated on the roots of plants grown for 8 d under the
above conditions. Each plant received the same (approx. l0 s cells)
inoculum size.
The sequence of the exposure of the plants roots to K N O 3
and the bacteria was varied before the roots were examined. In
the first experiment, the plants were maintained in the N-free mediu m and examined after various periods of incubation with the
bacteria. In the second experiment, plants were transferred at the
time of inoculation to media containing various concentrations
of KNO3 (0 18 m M ) and then examined for nodulation after various periods of incubation. In the third experiment, plants were
inoculated, grown in the N-free medium for 5 d, transferred to
a m e d i u m containing 18 m M KNO3, and examined after another
5 and 12 d of development, i.e. the nodules were examined after
the plants had been incubated with the bacteria for 10 or 17 d.
After incubation, the plants were removed from the culture
medium, and the roots were examined directly by phase-contrast
353
microscopy, or fixed for 90 min in 4% glutaraldehyde in 0.2 M
cacodylate buffer (pH 7.0), post-fixed for 1 h in 1% OsO,, dehydrated in an ethanol series, and embedded in araldite. Very young
nodules were located inside the cortex after clearing the root tissue
with methyl benzoate as described by Truchet (i978)..Ultrathin
sections were stained by the basic fuchsin-methylene blue method
(Huber et al. 1968), and examined by light microscopy, or poststained for 10 rain each with 2.5% uranyl acetate and 0.5% lead
citrate. Post-stained sections were examined by transmission electron microscopy using a H U l l electron microscope (Hitachi,
Tokyo, Japan).
Results
The first experiment was designed to examine the nodulation process using the specific combination of symbionts under N-free conditions. Many root hairs of
inoculated plants grown in the N-free medium were
associated with adherent bacteria, were markedly deformed, and contained infection threads (Fig. 1). The
first nodules were visible on roots no earlier than
6 d after inoculation. Nodules examined 10 and 17 d
after inoculation had the typical organization of the
meristematic-type nodule (Kijne 1975; Libbenga and
Harkes 1973; Newcomb 1980; Truchet 1978), namely
Fig. 1. Root hairs of lucerne seedlings inocuIated with Rhizobium meliloti L 530 and grown in N-free medium for 5 d. Marked deformed
root hairs and infection thread. The arrow points to attached bacteria, x 480; b a r = 10 g m
Fig. 2. As Fig. 1 but grown in 18 m M NO~. Note the straight root hairs, x 920; b a r = 10 g m
354
G.L. Truchet and F.B. Dazzo: Nitrate effect on early nodulation in lucerne
Table 1. Nodulation of lucerne by Rhizobium meliloti L5-30 in
the presence of increasing concentrations of KNO3
Conc. of KNO3
(mM) ~
No. of nodules emerged b
0
1.5
3
6
12
18
116
58
16
13
7
0
a In the medium at the time of seed germination
b Total from 12 plants, three weeks after inoculation
a distal meristematic zone 1 free of bacteria, an infection zone 2, a bacteroid zone 3, and a proximal senescence zone 4, each of which was surrounded by a
layer of uninfected cortical tissue.
The second experiment was designed to determine
what concentration of KNO3 available to the plant
at the time of inoculation completely inhibited nodulation of lucerne by R. meliloti L5-30. Progressively
fewer nodules developed as the concentration of NO~was increased, and nodule formation was completely
inhibited by 18 mM NO;- (Table 1). Root hairs of
inoculated plants growing in the presence of 18 mM
NO;- remained straight and were devoid of adherent
rhizobia and infection threads (Fig. 2). All nodules
that did develop in the presence of lower concentrations of NO;- had an histological organization similar
to that found under N-free conditions (Figs. 3-6).
The third experiment was designed to examine the
effect of different timings of application of 18 mM
NO;- on the development of nodules previously initiated under N-free conditions. Nodules formed if
addition of 18 mM NO~- was delayed for 5 d after
inoculation of the plant with the bacteria. After 5 d
of exposure to 18 m M NO;-, i.e. 10 d after inoculation, fully initiated nodule primordia were found in
the root cortex at different stages of development
(Figs. 7, 8). These primordia had no zonation and
consisted only of meristematic cells. At the same time
of observation, other nodules were more developed
and had emerged from the root (Fig. 9). The latter
had the classic organization of zones of dividing meristematic cells (Figs. 10, 11), infected cells (Fig. 12),
and cells filled with bacteroids (Figs. 13, 14). The
only exceptions at this stage of development were
found in the infection threads in zone 2, where some
bacteria tended to loose their normal shape and gain
more electron density, indicating an early degeneration (Figs. 15, 16), and where the lumen of the infection thread was abnormally enlarged and full of rodshaped bacteria (Fig. 17).
Nodules which had developed after 5 d on N-free
medium and then 12 d on medium with 18 m M NO;had a normal meristematic zone 1 (Fig. 18) and a
distal infection zone 2 (Fig. 19) similar in appearance
to the corresponding zones after only 5 d of development in the presence of NO;-, except an increased
tendency of bacteria to degenerate in the infection
thread matrix (Fig. 19). The major effect of NO~
on the ultrastructure of infected cells observed at this
time was apparent in the center of the nodule where
the proximal zone 2, the bacteroid zone 3, and the
senescent zone 4 were cytologically indistinguishable.
This is in contrast to control nodules developing
under N-free conditions were zonation is very distinct.
The main features unique to this central region of
nodules developing in the presence of NO;- were (1)
an increased electron density and pleomorphism of
bacteria in the infection threads, indicating their premature degeneration (Fig. 20), and (2) the presence
of both enlarged bacteroids and degenerative forms
of the bacteria in the cytoplasm of the same host
cells (Fig. 21).
Discussion
Our study was focused specifically on the identification of those morphogenetic events in root nodule
development in lucerne after inoculation with rhizobia (R. meliloti) that are subject to inhibition by combined nitrogen. Increasing the concentration of NO;reduced the number of nodules formed. However,
nodules which did develop under these conditions
had an histological organization similar to that of
nodules developing under N-free conditions. The addition of high levels of NO3 (18 mM) to the roots
of lucerne seedlings at the time of inoculation with
R. meliloti completely inhibited infection of root
hairs, and hence, root nodulation. When inoculated
plants were grown first for 5 d under N-free conditions and then in the presence of 18 m M NO3 for
5 d more, two major symbiotic steps were restricted:
(1) the release of bacteria from the infection threads
into the cytoplasm of the host cells, and (2) the conversion of released vegetative bacteria into enlarged
bacteroids. Premature degeneration of bacteria in the
matrix of hypertrophied infection threads was observed after 5 d exposure to 18 m M NO;-. After another 7 d on NOa--medium, bacteria both inside the
infection threads and in the host cytoplasm were undergoing premature degeneration. The premature degeneration of bacteria was particularly apparent in
the region of the nodule where differentiation of the
microsymbiont occurs (proximal zone 2 and the entire
zone 3). This effect results in an abnormally developed
senescent zone in nodules exposed for 12 d to NO~
Figs. 3-6. Light micrographs of lucerne nodules developed with different concentrations of N O 3 - a d d e d at the time of inoculation
Figs. 3 and 4. Longitudinal sections of nodules obtained respectively in presence of 3 or 6 m M NO~-. The four principal zones are
labeled (Z1, Z2, Z3, Z4). Fig. 3, • 200; Fig. 4, x 155; b a r s = 100 g m
Fig. 5. Interzone 1-2 of a nodule grown with 3 m M NO3-. Host cells are invaded by infection threads (it) and released bacteria (arrows).
x 1,800; b a r = 10 Izm
Fig. 6. Part of the third zone of a nodule grown in the presence of 12 m M NO~. Arrows point to enlarged bacteroids.
b a r = 10 g m
• 1,800;
Figs. 7-9. Nodules of different age of lucerne seedlings which have grown 5 d after inoculation in N-flee conditions and then 5 d
with 18 m M NOa-. B a r s = 100 p.m
Figs. 7 and 8. Very young nodules are distinguishable by their round form and enhanced stainability. In Fig. 7 the nodule is located
in the root cortex, whereas in Fig. 8, the nodule is emerging at the root surface. C, root cortex; R, root. Fig. 7, x 400; Fig. 8, x 620
Fig. 9. Longitudinal section of a well-developed nodule. The vascular bundles (VB) and the different zones (Z1, Z2, Z3, Z4) are labeled;
NC, nodular cortex. The appearance of the vascular bundles in the apical part of the nodule indicates that this section is partially
oblique, x 250
Figs. 10-14. Details of the different zones of nodules developed in the same conditions as described in Figs. 7-9; b a r s - i0 ~tm (Fig. 13);
1 ~tm (other Figures)
Fig. 10. Apical cells of the first zone presenting all the features of the meristematic cells, i.e., isodiametric form, central large nucleus
(N), unenlarged vacuolar system (I0, numerous cytoplasmic organelles (m, mitochondria, p, plastids), granular cytoplasm (CY), plamodesmata (arrows) in the plant cell wall (CW). x 6,500
Fig. 11. Mitotic figure (metaphase~me) in a nodular meristematic cell. x 12,000
Fig. 12. Part of an infected cell of the second zone. b, bacteria; it, infection thread; CW, plant cell wall; CY, plant cell cytoplasm;
V, vacuole, x 6,500
Figs. 13 and 14. Light and electron micrographs of the third zone where cells are filled with bacteroids (arrows, Fig. 13; b, Fig. 14).
Fig. 13 x 1,250; Fig. 14, x 19,000
Figs. 15-17. Details of infection threads in the second zone of nodules developed under the conditions described in Figs. 7 9. In some
infection threads, bacteria (b) are released (Fig. 16) or are degenerated inside the matrix of the infection thread (arrows, Fig. 15, 16).
The highly electron-dense degenerative bacteria in the infection thread (it) have a cytoplasm which separates from the cell wall (arrows,
Fig. 15). This degeneration of bacteria in the infection threads contrasts with the well-preserved host cytoplasm. The lumen of another
infection thread is particularly enlarged as in Fig. 17 and filled with rod-shaped bacteria containing granules of poly-/Lhydroxybutyrate
(arrows). rna, matrix of the infection thread; CW, infection thread cell wall; CY, plant cell cytoplasm. Fig. 15, x 18,000; Fig. 16,
x 19,200; Fig. 17, x 21,000; b a r s = 1 gm
Figs. 18-21. Ultrastructure of nodules of lucerne seedlings which have grown 5 d after inoculation in N-free conditions and then 12 d
with 18 m M N O 3 ; b a r s = l gm
Fig. 18. Meristematic cells. N, nucleus; CY, plant cell cytoplasm; CW, plant cell wall; V, vacuole; p, undifferentiated plastids; m,
mitochondria, x 10,500
Fig. 19. Distal part of the second zone. Intact bacteria (b) surrounded by a membrane envelope (arrows) are seen in the plant cytoplasm.
Other bacteria remain in the infection thread (it). Their electron-density suggests a premature degeneration, x i0,500
Figs. 20 and 21. Central degenerative zone. In this region, all bacteria in the infection thread matrix (ma) (Fig. 20) and many in the
plant cytoplasm (CY) (Figs. 20 and 21) are generally highly degenerative. Few of the bacteria have differentiated to the bacteroid
form (b) and remain enclosed in the peribacteroid membrane (arrow). V, vacuole. Fig. 20, x 16,500; Fig. 21, x 16,500
360
G.L. Truchet and F.B. Dazzo: Nitrate effect on early nodulation in lucerne
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host-symbiont recognition in the Rhizobium-clover symbiosis.
Plant Physiol. 62, 18-21
Fahraeus, G. (1957) The infection of clover root hairs by nodule
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and leguminous plants. University of Wisconsin Press, Madison, Wis.
Gibson, A.H., Pagan, J.D. (1977) Nitrate effects on the nodulation
of legumes inoculated with nitrate-reductase-deficient mutants
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(1973) The role of hormones and gradients in the initiation
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Received 16 October, accepted 28 December 1981
a n d d i s o r g a n i z a t i o n of the classical i n t e r n a l z o n a t i o n
o f the m e r i s t e m a t i c nodule.
By contrast, the a c t i v a t i o n of the m e r i s t e m o f
y o u n g d e v e l o p i n g nodules, i.e. their c a p a c i t y to proliferate, was preserved at least d u r i n g 12 d following
the a d d i t i o n o f N O ; - . W e d i d n o t observe the cytological changes in the m e r i s t e m a t i c p a r t of n o d u l e s exp o s e d to c o m b i n e d n i t r o g e n r e p o r t e d b y T h o r n t o n
a n d R u d o r f (1936) a n d D a r t a n d M e r c e r (1965). This
can p r o b a b l y be e x p l a i n e d b y the difference in age
a n d d e v e l o p m e n t of the n o d u l e s s t u d i e d (developing
versus old nodules).
Using the m e t h o d o l o g y d e s c r i b e d here, it was n o t
possible to observe the specific step o f n o d u l e initiation in the inner r o o t cortex ( L i b b e n g a et al. 1973;
Phillips 1971 ; T r u c h e t et al. 1980). H o w e v e r , the dev e l o p m e n t o f n o d u l e p r i m o r d i a at the stages illust r a t e d by Figs. 7 a n d 8 occurs a p p r o x . 3-4 d after
i n o c u l a t i o n with R . m e l i l o t i ( T r u c h e t 1978). Based
on this timing and the size o f the uninfected n o d u l e
p r i m o r d i a in Figs. 7 a n d 8, we h y p o t h e s i z e t h a t n o d u l e - m e r i s t e m initiation can c o m m e n c e in the presence
o f c o m b i n e d nitrogen.
O u r results, in their entirety, show t h a t those recognized steps of n o d u l e m o r p h o g e n e s i s which are
sensitive to c o m b i n e d n i t r o g e n seem to be those where
the m i c r o s y m b i o n t is directly involved. In c o n t r a s t ,
the step k n o w n n o t to require the presence o f bacteria,
i.e., the a c t i v a t i o n of the n o d u l e m e r i s t e m (Truchet
et al. 1980), is n o t i n h i b i t e d b y N O ~ a d d i t i o n . W e
also c o n f i r m some o f the previous r e p o r t s which indicate t h a t the early s y m b i o t i c events, all o f which require bacteria, are specifically i n h i b i t e d b y critical
levels o f c o m b i n e d n i t r o g e n : a d h e s i o n of r h i z o b i a to
r o o t hairs ( D a z z o a n d Brill 1978); m a r k e d d e f o r m a tion o f r o o t hairs ( T h o r n t o n 1936; M u n n s 1968);
a n d i n f e c t i o n - t h r e a d d e v e l o p m e n t ( T h o r n t o n 1936;
M u n n s 1968).
This research was supported by a grant to G.L.T, from the European Molecular Biology Organization, and grants to F.B,D. from
the U.S. Department of Agriculture, Science and Education Administration (Competitive Grant No. 78-59-2261-0-1-050-2) and
the National Science Foundation (PCM 80-21906). This is Michigan Agricultural Experiment Station Article No. 10104.