Journal of Experimental Botany, Vol. 50, No. 339, pp. 1577–1585, October 1999
Light microscopy of early stages in the symbiosis of
soybean with a delayed-nodulation mutant of
Bradyrhizobium japonicum
Laura S. Green1 and David W. Emerich
Biochemistry Department, 117 Schweitzer Hall, University of Missouri-Columbia, Columbia, MO 65211,
USA
Received 18 March 1999; Accepted 25 June 1999
Abstract
An a-ketoglutarate dehydrogenase mutant (LSG184) of
Bradyrhizobium japonicum USDA110 has a delayed
nodulation phenotype when inoculated onto soybean
(Glycine max L.). To pinpoint the defective stage of
symbiotic development, light microscopic techniques
were used to monitor early responses of soybean to
inoculation with the mutant as compared to the wildtype strain. Methylene blue was used to visualize
curled root hairs and a convenient haematoxylin staining method was developed that could detect nodule
primordia as early as 2 d after inoculation. The results
demonstrate that early symbiotic events occur with
normal timing after inoculation with LSG184 and that
its developmental delay is first evident during the progression of nodule primordia into emergent nodules.
The timing of this delay suggests that LSG184 is not
deficient in Nod factor production, at least during the
early stages of symbiosis, but rather may have a defect
in infection thread initiation or elongation. The results
further imply that the rate of development of advanced
soybean nodule primordia is, in part, dependent on
the metabolic capabilities of the invading bacterium.
Key words: Soybean, nodulation, light microscopy, haematoxylin, symbiosis.
Introduction
The successful development of nitrogen-fixing nodules
requires ongoing communication between the plant host
and the endosymbiotic rhizobium. Some signals involved
in controlling symbiotic development, in particular the
lipochito-oligosaccharide nodulation factors (Nod factors), have been subject to intensive study over the past
decade and their structure, if not their exact mode of
action, is well-characterized. Nod factors are diffusible
molecules produced by rhizobia in response to chemical
signals from the host plant and are required for inducing
many of the earliest responses of the host plant to
inoculation (Long, 1996). The communication that occurs
at subsequent stages of symbiotic development is less well
understood. A classic approach to this question has been
to analyse mutants of the bacterial partner that show
defects in the developmental step of interest (reviewed in
Niner and Hirsch, 1998). Such an approach has revealed,
for example, the importance of rhizobial extracellular
oligo/polysaccharides, lipopolysaccharides, and cyclic
b-glucans in infection thread growth and in the suppression of host defence reactions (Stacey et al., 1991; Parniske
et al., 1994; Dunlap et al., 1996; Eggleston et al., 1996;
Cheng and Walker, 1998).
Careful and often painstaking analysis of symbiotic
development is required to pinpoint the exact stage that
is most affected by a particular rhizobial mutation. A
wide range of plant responses have been used as developmental markers for this purpose including root hair
curling (van Workum et al., 1998), infection thread
formation and morphology (Cheng and Walker, 1998),
the synthesis of fluorescent flavonoids (Mathesius et al.,
1998), nodule emergence, and the induction of nodulin
gene expression (Dunlap et al., 1996). One important
event in symbiotic development is the induction of mitotic
activity in the cortex of the host root, giving rise to new
cells that will eventually form the nodule primordium.
The induction of new cell divisions, especially in soybean,
has generally been monitored by light microscopic ana-
1 To whom correspondence should be addressed. Fax: +1 314 882 5635. E-mail: [email protected]
© Oxford University Press 1999
1578 Green and Emerich
lysis of serial sections of fixed and embedded plant roots
(Calvert et al., 1984; Mathews et al., 1989; Chatterjee
et al., 1990; Gerahty et al., 1992). However, the timeconsuming nature of the serial-sectioning technique has
meant that very few rhizobial mutants have been analysed
for their ability to induce new cell divisions on soybean.
As part of a study of bacteroid carbon metabolism, a
mutant of Bradyrhizobium japonicum, the endosymbiont
of soybean, that is missing the tricarboxylic acid cycle
enzyme a-ketoglutarate dehydrogenase (Green and
Emerich, 1997a) was isolated. Surprisingly, this mutant
(LSG184), despite its metabolic defect, is capable of
forming normal nitrogen-fixing bacteroids when inoculated onto soybean (Green and Emerich, 1997b).
However, nodulation by the mutant is delayed by about
5 d relative to the wild type and the nodules formed by
the mutant contain a reduced number of infected cells.
These results raise the question of why a-ketoglutarate
dehydrogenase should be more important during symbiotic development than it is to the subsequent functioning
of mature bacteroids. To determine the cause of the
delayed nodulation phenotype, an attempt has been made
to define more precisely the stage at which the delay first
appears. In the present study methylene blue has been
used to monitor the formation of curled root hairs and a
simple and convenient haematoxylin staining method
used to visualize new mitotic activity in the soybean root
cortex. The results demonstrate that these early symbiotic
events occur with normal timing after inoculation with
LSG184 and that the developmental delay is first evident
during the transition from nodule primordia to emergent
nodules. The timing of this delay is consistent with
LSG184 having a defect in infection thread initiation or
elongation.
Materials and methods
Plant material and bacterial strains
Soybeans (Glycine max L. cv. Williams 82) were surfacesterilized and sprouted on 1% (w/v) agar/water at 28 °C in
covered glass trays. After 2–3 d, when roots were 2–4 cm long,
the seedlings were dipped in inoculum and placed into sterile
growth pouches (Mega International, Minneapolis, MN ). The
position of each root tip was marked on the outside of the
pouch with an indelible marker at the time of planting. The
pouches were watered initially with 20 ml of sterile, nitrogenfree plant nutrient solution (Ahmed and Evans, 1960) and
thereafter with sterile water as required. Pouches were incubated
in a growth cabinet with a 16 h photoperiod and a day/night
temperature of 27/24 °C. On average, the roots grew 2.5 cm in
the first 24 h after transfer into the growth pouches.
Inoculum was prepared by growing the bacterial strains to
an A
of 1.0 to 2.0 in defined media with 20 mM arabinose
630
as the carbon source, ammonium as the nitrogen source, and
antibiotics added as appropriate (Green and Emerich, 1997a).
Cells were harvested by centrifugation (7 000 g, 5 min, room
temperature), washed once with, and finally resuspended in,
nitrogen-free plant nutrient solution. Unless otherwise specified,
cells were resuspended to an A
of 0.1, approximately
630
108 cells ml−1. Strains used were: Bradyrhizobium japonicum
USDA110, B. japonicum LSG184 (sucA::Tn10-minikan, a-ketoglutarate dehydrogenase mutant; Green and Emerich, 1997a),
B. japonicum nodA2 (nodA::Tn5 mutant; Lamb and Hennecke,
1986), and Sinorhizobium fredii USDA191 and USDA257
(Heron and Pueppke, 1984).
Methylene blue staining of curled root hairs
Curled root hairs were stained with methylene blue using a
procedure modified from Stokkermans and Peters (Stokkermans
and Peters, 1994). Root segments of 3 cm, starting from 0.5 cm
above the root tip mark, were cut from pouch-grown seedlings.
Root segments were soaked in 1% (w/v) sodium hypochlorite
(a 153 dilution of commercial bleach, Novel Wash Co. Inc.,
Dupo, Illinois, in water) for 2 h. Roots were rinsed briefly in
deionized water and then incubated for 1 h in 0.01% (w/v)
methylene blue in water. Root segments were destained by
incubating for 1 h in 35% (w/v) ethanol and 1.5 h to overnight
in 70% ethanol, and then observed using a dissecting microscope
and bright field illumination.
Haematoxylin staining of soybean nodules and nodule primordia
A 40× haematoxylin stock (Dudley et al., 1987) was prepared
as follows: 2 g of haematoxylin (Sigma catalogue number
H-9627) was dissolved in 50 ml of 45% (v/v) acetic acid, filtered
and stored at room temperature in a brown glass bottle. The
stain gave best results when freshly made but could be used for
several weeks. A precipitate gradually formed during storage
and staining was greatly improved if the stock was refiltered
(or centrifuged) directly before use.
Soybean root segments, harvested as described for methylene
blue staining, were stained with haematoxylin using a method
modified from Dudley et al. (Dudley et al., 1987). Root
segments were boiled for 1.5–2 min in lactophenol (32%
glycerol, 16% lactic acid, 20% phenol, by vol. in water), the
shorter length of time being used for younger roots. Root
segments were then blotted on tissue, rinsed in a Petri dish of
water for 2 min, and stained overnight in haematoxylin (40×
stock diluted 1540 in water just before use). The roots were
protected from light during the staining step. After staining,
the root segments were destained in water for 1–2 h and then
observed under a dissecting microscope using dark field
illumination with additional top lighting as needed. Additional
destaining was sometimes necessary to improve the contrast
between the nodule primordia and the surrounding root tissue,
especially if the haematoxylin stain was several weeks old.
In this case the root segments were soaked in 20 mM HCl
for 0.5–1 h and then returned to water for observation.
Haematoxylin-stained structures were counted on one side of
each root segment, with eight segments in each sample. For
observations at higher magnification, 100 mm thick Vibratome
sections were prepared from haematoxylin-stained root segments, mounted in water, and viewed under dark field or
Nomarski optics.
Results
Methylene blue staining of curled root hairs
One of the earliest morphological changes to follow
inoculation is the induction of root hair curling. To
determine whether the delay in nodulation by LSG184 is
accompanied by a delay in root hair curling, the root
Delayed nodulation mutant 1579
hairs of soybean seedlings were examined at various
intervals after inoculation with the mutant or the wildtype parental strain, USDA110. Curled root hairs were
very difficult to see on unstained roots, but stained deep
blue and were readily observable after treating the roots
with methylene blue ( Fig. 1A). Similar methylene blue
staining of curled root hairs was previously reported for
Glycine soja inoculated with B. japonicum ( Eskew et al.,
1993). The stained, curled root hairs occurred in dense
patches at irregular intervals along the root ( Fig. 1A)
and were present by 48 h after inoculation with USDA110
or LSG184 ( Fig. 1A, B). There was no observable delay
in the induction of curled root hairs by the mutant,
although the response, in terms of the number and size
of the stained root hair patches, appeared to be slightly
weaker. Methylene blue-stained root hairs never occurred
on roots inoculated with a nodA mutant of B. japonicum
or on uninoculated controls (Fig. 1C, D). The fact that
staining was never observed after inoculation with the
nodA mutant suggests that methylene blue stained the
curled root hairs themselves rather than films of bacteria
on the surface of the root or on the root hairs.
Haematoxylin staining of soybean nodule primordia
Soybean root segments inoculated with USDA110 and
stained with haematoxylin after 3 d showed small, darkly
stained patches of cells distributed over their surface
(Fig. 2A). At higher magnification these patches appeared
to be made up of small, presumably newly-divided cells
in the outermost layers of the cortex. Small patches of
these darkly stained cells could be observed as early as
2 d after inoculation (Fig. 2B) and were never observed
on uninoculated roots. At longer time intervals after
inoculation a small subset of the darkly stained patches
of cells enlarged and developed into emergent nodules
( Fig. 2C–F ). Initially the nodule primordium cells stained
dark brown, but as the primordia assumed a more
rounded shape the cells in the interior took on a purplish
hue. When present on the root segments, the root tip and
lateral root meristems also stained deep purple ( Fig. 2A).
The lateral root meristems could be distinguished from
the nodule primordia by their emergence from deeper
within the root cortex and by having a more pointed apex.
To view the presumptive nodule primordia at higher
magnification, 100 mm Vibratome sections were cut from
haematoxylin-stained root segments (Fig. 3). The haematoxylin-stained cells are clearly smaller than those in the
surrounding tissue, implying that they had undergone
recent cell divisions ( Fig. 3A, C ). In agreement with
earlier studies (Calvert et al., 1984), the new crosswalls
in these young primordia were predominantly anticlinal
( Fig. 3B). Even as early as 2 d after inoculation some
primordia showed anticlinal divisions extending several
layers down into the root cortex (Fig. 3B).
These observations validated haematoxylin staining as
a method to monitor the formation of nodule primordia.
For the purposes of further experiments the developing
nodules were divided into three broad categories: foci,
domes, and nodules. ‘Foci’ were defined as patches of
darkly stained, newly-divided cells that had not yet begun
to protrude from the surface of the root (Fig. 2B, C ).
‘Domes’ were defined as primordia that had begun to
bulge from the root surface, but had not yet assumed a
spherical shape (Fig. 2D, E). ‘Nodules’ were defined as
Fig. 1. Methylene blue-stained root hairs. Soybean roots were harvested 2 d after inoculation with various strains of B. japonicum and stained with
methylene blue as described in Materials and methods. Roots were inoculated with USDA110 (A), LSG184 (B), nodA2 (C ) or left uninoculated
(D). Clusters of curled root hairs (black arrows) and emerging lateral roots (white arrows) are indicated. Scale bars=100 mm.
1580 Green and Emerich
roots could not readily be observed, no attempt was
made to distinguish between true infections and pseudoinfections (Calvert et al., 1984).
Time-course of nodule development after inoculation with
wild-type and mutant B. japonicum
Fig. 2. Haematoxylin-stained soybean root segments. Soybean roots
were inoculated with USDA110 and stained with haematoxylin at
various times after inoculation. Roots were harvested 2 d (B), 3 d (A,
C ), 4 d (D), 6 d ( E ) or 8 d (F ) after inoculation. Haematoxylin-stained
foci ( large white arrows) and an emerging lateral root (small white
arrow) are indicated. Scale bars=1 mm (A) or 200 mm (B–F ).
primordia that had become spherical and emerged more
than half-way from the surface of the root ( Fig. 2F ).
Because haematoxylin staining of whole roots does not
reveal structural changes deep within the root cortex, the
nodule primordia could not be staged as precisely as
allowed by the serial sectioning technique. However, our
foci category roughly coincides with stages I–V as defined
by Calvert et al. (Calvert et al., 1984), and domes and
nodules correspond approximately to developmental
stages VI–IX and X–XX, respectively. As curled root
hairs or infection threads on the haematoxylin-stained
Soybean root segments inoculated with USDA110 showed
a large number of haematoxylin-stained foci by 48 h after
inoculation ( Fig. 4A). There were still a large number of
foci evident at 3 d, but thereafter the numbers declined
until only about a third of the peak number remained at
7 d after inoculation. From 7 to 14 d after inoculation
there was little further decline in the number of foci. By
4 d after inoculation a small proportion (approximately
10%) of the foci had enlarged enough to swell above the
surface of the root and be scored as domes (Fig. 4B). By
9 d after inoculation the total number of domes declined,
as most of the domes continued to develop into nodules.
The first nodules were observed at 5 d after inoculation
( Fig. 4C ), increasing in number over the next few days
to an average of about five nodules per segment.
Similar to observations with USDA110, root segments
inoculated with LSG184 also showed large numbers of
haematoxylin-stained foci. The number of foci reached
its peak by 2 d after inoculation, then declined sharply
between days 5 and 7 to about 50% the peak value
( Fig. 4A). Although the peak number of foci formed by
the mutant was only 60–65% of the wild-type peak, the
time-course of appearance and subsequent regression of
the foci showed no delay relative to the wild type. In
general, the foci formed by LSG184 appeared to be
somewhat smaller and more faintly stained than those
formed by the wild type.
As with the wild-type inoculations, a small proportion
(about 10%) of the foci induced by LSG184 developed
further into domes and nodules. The first domes were
evident within 4 d, but the first nodules did not appear
until 9 d after inoculation ( Fig. 4B, C ). Thus the delay
in nodulation observed with LSG184 became most evident
after 4–5 d of symbiotic development. The number of
days between the appearance of the first foci to the
appearance of the first domes was 2 d for both USDA110
and LSG184 inoculations. In contrast, the time between
the appearance of the first domes and the appearance of
the first nodules was only 1 d for USDA110, but 5 d for
LSG184.
Reduced number of foci does not necessarily cause a delay
in nodulation
The number of foci induced by LSG184 was significantly
lower than was observed after inoculation with the wild
type. To determine whether a reduced number of foci
could cause delayed nodulation, foci and nodule development were monitored after inoculating soybean seedlings
Delayed nodulation mutant 1581
Fig. 3. Haematoxylin-stained cell division foci. Soybean roots were harvested 2 d (B) or 6 d (A, C ) after inoculation with USDA110 and stained
with haematoxylin. Vibratome sections were prepared from the stained roots and viewed under dark field (B) or Nomarski (A, C ) optics. Scale
bars=50 mm (A, B) or 100 mm (C ).
with reduced concentrations of USDA110. Reducing the
inoculum density from 108 to 105 cells ml−1 dramatically
reduced the number of foci observed at 3 d after inoculation ( Table 1). Nevertheless, roots inoculated at the lower
density had still developed nodules by 5 d. Even roots
inoculated at 102 cells ml−1, which had no haematoxylinstained foci at 3 d, showed occasional nodules at 5 d.
Therefore a reduced number of foci does not necessarily
lead to a delay in nodulation.
Distribution of foci and nodules along the root
The foci induced by USDA110 and LSG184 were not
evenly distributed along the root segments, but were
concentrated in the region at or above the root tip at the
time of inoculation (Fig. 5). There was also about a 1 d
delay, relative to the top part of the root (section 1), in
the appearance of foci in the youngest region of the
standard root segment (section 4), corresponding, on
average, to the position of the root tip 1 d after inoculation. This delay was somewhat more pronounced in root
segments inoculated with LSG184. By 7 d, after the
majority of the foci had regressed, the remaining foci
were evenly distributed along the root segments. Although
more foci initially formed in the upper region of the root
segments, nodules (scored at 14 d) were evenly distributed
( Fig. 6).
The ability to induce foci correlates with the ability to
produce Nod factors
The induction of foci by several strains of B. japonicum
and Sinorhizobium fredii that vary in their ability to
nodulate the cultivar of soybean used in this study
( Williams 82) was monitored next. The nodA2 mutant of
B. japonicum fails to produce any Nod factors and has a
Nod− phenotype on soybean (Lamb and Hennecke,
1986). No haematoxylin-stained foci were ever observed
after inoculation with this strain. Two strains of S. fredii
were compared next: USDA191, which produces Nod
factors and nodulates Williams 82, and USDA257, which
produces the appropriate Nod factors, but has other
genetic determinants that prevent it from nodulating
improved cultivars of soybean (Heron et al., 1989;
Becferte et al., 1994). As expected, USDA191 was able
to induce haematoxylin-stained foci on Williams 82 (an
average of 17.9±3.4 per root segment at 3 d after inoculation, on eight roots). USDA257, despite its inability to
1582 Green and Emerich
Fig. 4. Time-course of nodule development after inoculation with
USDA110 and LSG184. Soybean roots were inoculated with USDA110
or LSG184, harvested at various time intervals, stained with haematoxylin, and scored for the presence of foci (A), domes (B) and nodules
(C ). Each point represents the average from 7–8 roots from a single
experiment, except for those for days 3 and 6 which represent the
mean±SE of two experiments containing eight roots each.
Abbreviation: DAI, days after inoculation.
nodulate Williams 82, also induced a similar number of
foci (an average of 13.0±1.7 per root segment at 3 d
after inoculation, on eight roots). Neither S. fredii strain
induced as many foci as USDA110 or LSG184 ( Table 1).
Taken together, these results indicate that the induction
of foci was correlated with the ability of the inoculant to
produce Nod factors rather than with its nodulation
phenotype.
Fig. 5. Spatial distribution of foci. Data from the same root segments
used for Fig. 4 were analysed to show the spatial distribution of foci
after inoculation with USDA110 (#) or LSG184 ($). Each 3 cm root
segment was divided into four 0.75 cm sections, each of which was
scored separately, from section 1 at the top (oldest) to section 4 at the
bottom (youngest) of the root segment. Abbreviation: DAI, days after
inoculation.
Discussion
Haematoxylin staining has been used as a simple and
convenient method to localize new cortical cell divisions
in soybean roots after inoculation with B. japonicum and
S. fredii. The response is specific to rhizobial strains
producing Nod factors and is never seen on uninoculated
roots. Using this method it was possible to score new
Table 1. The effect of inoculum density on the formation of nodule primordia
Soybean roots were inoculated with USDA110 or LSG184 at the indicated cell density, harvested after 3 d or 5 d, stained with haematoxylin, and
scored for the presence of foci, domes, and nodules. Values represent the mean±SE for each sample, with the sample size given in parentheses.
Strain
Experiment 1
Experiment 2
USDA110
LSG184
USDA110
USDA110
USDA110
Inoculum
density
(cells ml−1)
3d
5d
Foci
No. per root
Foci+domes
No. per root
Nodules
No. per root
108
108
108
105
102
57.6±5.1 (16)
33.7±4.5 (16)
45.4±13.0 (5)
5.6±2.0 (5)
0 (5)
39.7±5.2 (8)
33.8±3.5 (8)
48.7±5.2 (3)
11.8±3.9 (5)
0.8±0.5 (4)
2.5±1.6
0 (8)
8.3±0.3
3.8±1.0
0.2±0.2
(8)
(3)
(5)
(4)
Delayed nodulation mutant 1583
Fig. 6. Spatial distribution of foci versus nodules. Data from the same experiments presented in Fig. 4, analysed to show the spatial distribution of
foci on day 3 as compared to nodules on day 14 (±SD), after inoculation with USDA110 or LSG184.
mitotic activity on much larger sample sizes than is
practical using serial sectioning methods (Mathews et al.,
1989; Takats, 1990; Gerahty et al., 1992). For example,
20 root segments were routinely scored in under 2 h,
allowing the analysis of more than 250 root segments for
this study. Large sample sizes are important in quantifying
cell division foci in soybean, to compensate for the high
variability between roots (Mathews et al., 1989; Gerahty
et al., 1992). Nodule primordia in soybean have also been
stained with nile blue (O’Hara et al., 1988), but to our
knowledge not within the first few days after inoculation.
Methylene blue stains very young primordia on Glycine
soja, but not on the larger roots of Glycine max ( Eskew
et al., 1993; Stokkermans and Peters, 1994; our own
observations). Fluorescence of novel flavonoids has also
recently been proposed as a convenient developmental
marker for very young nodule primordia in several
legumes, but excessive background autofluorescence
obscures the signal in soybean (Mathesius et al., 1998).
In contrast, soybean cell division foci as early as 3 d after
inoculation have been localized with eriochrome black
(Hacin et al., 1997) and this technique may be a suitable
alternative to haematoxylin. Soybean is particularly
amenable to the haematoxylin staining method because,
unlike legumes forming indeterminate nodules, the new
cortical cell divisions induced by inoculation take place
close to the surface of the root. Nevertheless, the haematoxylin staining method should also work well for any
other legumes, such as Lotus and Phaseolus species, that
initiate nodule primordia in a manner similar to soybean.
The total number of haematoxylin-stained foci that
formed after inoculation of soybean was dependent on
both the rhizobial strain used and the inoculum density
( Table 1). Since the formation of foci on soybean is a Nod
factor-dependent response (this study; Stokkermanns and
Peters, 1994), the total number formed may be determined
by the overall concentration of Nod factors in contact
with the root, which in turn would be affected by both
inoculum strain and density. The foci induced after inoculation with B. japonicum were also unevenly distributed
along the 3 cm root segments analysed in this study, with
many more forming at or above the position of the root
tip at the time of inoculation. The uneven distribution
may be a consequence of the inoculation method, in that
the top part of the root segment, containing the tissue
most susceptible to infection at the time of inoculation
(Bhuvaneswari et al., 1980), was exposed directly to the
inoculum. In contrast, the bottom part of the segment,
not formed until 1 d after inoculation, may have come
into contact with fewer bacteria. Despite the uneven
distribution of foci, mature nodules were evenly distributed along the root segments, confirming that the number
of nodules eventually formed is regulated independently
of the number of foci present early in the symbiosis.
Most of the foci present on day 3 after inoculation
regressed rather than developing further into domes and
nodules. This result confirms the findings of several earlier
studies using the serial-sectioning technique (Calvert et al,
1984; Mathews et al., 1989; Gerahty et al., 1992), that
inoculation of soybean induces many more foci than ever
develop into nodules. The transiently stained foci probably correspond to what has been termed psuedoinfections (Calvert et al., 1984), so called because they
were not closely associated with curled root hairs or
infection threads and were blocked prior to the formation
of a nodule meristem. Although the majority of the
haematoxylin-stained foci apparently regressed within 7 d,
a small but stable number remained even after 14 d. These
foci may represent a reservoir of primordia that can be
used by the plant to make additional nodules should the
need arise. The existence of such reserve primordia was
suggested by Caetano-Anollés et al., who showed that
when soybean roots were stripped of their nodules, new
nodules developed from primordia that had been formed
1584 Green and Emerich
at the time of initial inoculation and not from new
infections (Caetano-Anollés et al., 1991).
Roots inoculated with the tricarboxylic acid cycle
mutant LSG184 showed no delay relative to the wild type
in the formation of curled root hairs or the induction
of new cortical cell divisions. Since these responses are
both Nod factor-dependent (this study; Stokkermans and
Peters, 1994; Minami et al., 1996), these results imply
that LSG184 is not grossly deficient in Nod factor production. The earliest stage at which the mutant showed a
pronounced delay relative to the wild type was in the
development of domes (stages VI–IX ) into nodules (stages
X and beyond). This transition began within 5 d after
inoculation with the wild type, but took an additional
4–5 d after inoculation with the mutant. This delay shows
that nodule development beyond stages VI–IX is dependent in part on the phenotype of the invading bacterium.
This phase of soybean nodule development is normally
when infection threads are progressing toward the newly
formed nodule primordia ( Turgeon and Bauer, 1982).
Based on these observations, it is proposed that
LSG184 may have a defect in infection thread initiation
or growth and that normal development of soybean
nodules past stage VI may depend in part on the proximity
of an infection thread.
Beyond its delayed nodulation phenotype, LSG184
shows an additional delay in later symbiotic events.
Although there was just a 5 d delay in nodule emergence
there was a 12–14 d delay in release of bacteria into the
nodule cells and the subsequent onset of nitrogen fixation
(Green and Emerich, 1997b). However, once the bacteria
were released into the host cells, symbiotic development
proceeded normally. This observation lends further support to the notion that the symbiotic defect of LSG184
manifests itself mainly in the initiation or growth of
infection threads.
An alternative explanation for the delayed nodulation
phenotype of LSG184 is that the mutant, although producing enough Nod factor to initiate the early steps of
symbiosis is, nevertheless, deficient in Nod factor production at later stages. The development of soybean nodule
primordia beyond the initial stages probably depends on
continuous exposure to Nod factor from the invading
bacteria. A latent deficiency in Nod factor production by
LSG184, sufficient to cause delayed nodulation, could
become manifest if: (1) the mutant produces less Nod
factor once inside the host; (2) the mutant continues to
produce Nod factor at the same rate on an individual cell
basis, but there is a delay, because of growth defects (see
below), in the accumulation of a sufficient number of
mutant bacteria near the developing nodule primordia;
or (3) a higher threshold concentration of Nod factor is
required for advanced nodule development than for root
hair curling or stimulation of cortical cell divisions.
The basis of LSG184’s defect may relate to its altered
use of carbon sources. Because LSG184 grows more
slowly than the wild type on some substrates in culture
(Green and Emerich, 1997a), it may proliferate poorly
on the root surface or inside infection threads. The
nutrients used by rhizobia during invasion of the host
plant are not yet known. However, evidence derived from
studies of mutants with defects in hexose or dicarboxylate
utilization imply that multiple carbon substrates are available within the infection thread (McDermott et al., 1989;
O’Brian and Maier, 1989). LSG184 grows poorly on
glutamate, pyruvate, or acetate as a carbon source and is
also inhibited by the latter compound even in the presence
of more favourable carbon substrates. If acetate accumulated within the infection thread, growth of the mutant
could be impaired. LSG184 also grows poorly on glutamate as a nitrogen source and infection thread function
could be impaired if this amino acid is an important
source of nitrogen during invasion of the host.
Alternatively, LSG184’s lesion in central carbon metabolism could have downstream effects on EPS or LPS
quantity or structure, and thus influence symbiotic
development.
The delay in nodule development seen after inoculation
with LSG184 is later than the stages usually implicated
in the autoregulatory response of soybean, the mechanism
by which the host plant controls the number of primordia
allowed to develop into nodules (Calvert et al., 1984;
Mathews et al., 1989; Gerahty et al., 1992). Studies of
supernodulating mutants of soybean, in which autoregulation is defective, suggest that the most advanced
nodule primordia induce the formation of a shoot-derived
signal that inhibits the development of further nodules
(Gresshoff, 1993; Sheng and Harper, 1997). In preliminary experiments, the supernodulating mutants nts382 and
nts1007 showed both supernodulation and delayed emergence of nodules when inoculated with LSG184 (data not
shown). Thus the delay in nodule development conditioned by LSG184 does not interfere with expression of
the supernodulating phenotype, but is sufficient to counteract the postulated accelerated growth of primordia on
supernodulating soybean (Gresshoff, 1993). Further study
of the nodulation kinetics of specific strain/cultivar combinations, facilitated by the haematoxylin-staining technique, should shed more light on the regulation of soybean
nodule development.
Acknowledgements
We are grateful to Tobias I Baskin and the members of his
laboratory for allowing us free use of their microscopes and for
generous assistance with optimizing our methods. We also
thank Bruce McClure for the use of his dissecting microscope
during the early stages of the project. We are grateful to HansMartin Fischer (Eidgenössische Technische Hochschule,
Zürich), Hari Krishnan and Peter Gresshoff ( University of
Tennessee, Knoxville) for providing Bradyrhizobium japonicum
Delayed nodulation mutant 1585
nodA2, Sinorhizobium fredii USDA191 and USDA257, and
nts382 and nts1007 soybean seed, respectively. David Day
(Australian National University, Canberra) generously provided computer facilities during preparation of the manuscript.
This research was supported by USDA-NRICGP grant
95–37305–2112 to DWE.
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