Journal of Experimental Botany, Vol. 48, No. 316, pp. 1935-1941, November 1997
Journal of
Experimental
Botany
Root system growth and nodule establishment
on pea (Pisum sativum L.)
Frederique Tricot1, Yves Crozat2 and Sylvain Pellerin3'4
1
ESA, Laboratoire de Biotechnologie des Sols, 55 rue Rabelais, F-49007 Angers, France
CIRAD-CA, Avenue du Val de Montferrand, F-34032 Montpellier, France
2
3
INRA, Laboratoire d'Agronomie, 71 avenue Edouard-Bourleaux, BP81, F-33883 Villenave d'Ornon, France
Received 10 June 1997; Accepted 8 July 1997
Abstract
Development of the root system, appearance of nodules, and relationships between these two processes
were studied on pea {Pisum sativum L., cv. Solara).
Plants were grown in growth cabinets for 4 weeks on
a nitrogen-free nutrient solution inoculated with
Rhizobium leguminosarum. Plant stages, primary root
length, distance from the primary root base to the
most distal first-order lateral root, and distance from
the root base to the most distal nodule, were recorded
daily. Distribution of nodules along the primary root
and distribution of laterals were recorded by sampling
root systems at two plant stages. Primary root elongation rate was variable, and declined roughly in conjunction with the exhaustion of seed reserves. First-order
laterals appeared acropetally on the primary root. A
linear relationship was found between the length of
the apical unbranched zone and root elongation rate,
supporting the hypothesis of a constant time lag
between the differentiation of first-order lateral's
primordia and their emergence. Decline of the primary
root elongation rate was preceded by a reduction in
density and length of first-order laterals. Nodules
appeared not strictly but roughly acropetally on the
primary root. A linear relationship was found between
the length of the apical zone without nodule and root
elongation rate, supporting the hypothesis of a constant time lag between infection and appearance of a
visible nodule. A relationship was found between the
presence/absence of nodules on a root segment and
the root elongation rate between infection and appearance of nodules on the considered root segment.
Regulation of both processes by carbohydrate availability, as a causal mechanism, is proposed.
* To whom correspondence should be addressed. Fax: +33 5 56 84 30 54.
O Oxford University Press 1997
Key words: Pisum sativum L, root system, nodules.
Introduction
Pea (Pisum sativum L.), as most legumes, establishes in
root nodules a symbiotic association with Rluzobium
leguminosarum bacteria. Atmospheric N 2 is reduced to
NH^ within nodule cells and then incorporated into
amino acids before being released into the phloem, thus
providing assimilated nitrogen to the whole plant.
At the root segment level, much has been achieved in
recent years in understanding the steps which are essential
for the formation of a functional nodule (see review
articles from Sprent, 1989; Hirsch, 1992; Mylona et ai,
1995). Infections by Rhizobium bacteria leading to nodule
formation are restricted to only a narrow band of cells
above the zone of root elongation and just below the
smallest emergent root hairs (Bhuvaneswari, 1981;
Bhuvaneswari et ai, 1980, 1981; Pueppke, 1986). The
interaction of Rhizobia and legumes involves signal
exchange and recognition of the symbiotic partners, followed by attachment of the Rhizobia to the plant root
hairs. The root hair deforms, and the bacteria invade the
plant by a newly formed infection thread growing through
it. Simultaneously, cortical cells are mitotically activated,
giving rise to the nodule primordium. Nodule morphogenesis is therefore elicited by at least two distinct signals:
one from Rhizobium, a product of the nod genes (Nod
factor), and a second signal, which is generated within
plant tissues after treatment with Nod factor (Hirsch,
1992).
At the whole-root system level, the number of nodules
depends on both internal and environmental factors (see
review article from Caetano-Anolles and Gresshoff, 1991;
1936
Tricot et al.
Mengel, 1994). Regulation by internal factors has often
been termed autoregulation. It is manifested as inhibition
of nodule formation on one part of the root by prior
initiation elsewhere on the root. Environmental factors
known to affect the number of nodules are nitrate concentration in the growing medium (Macduff et al., 1996),
soil compaction (Katoch et al., 1983), air and soil temperature (Munevar and Wollum, 1981; Rawsthorne et al.,
1985), air CO2 concentration (Phillips et al., 1976;
Murphy, 1986), and light intensity (Kosslak and Bohlool,
1984). The mechanism by which these environmental
factors affect nodulation and interact with the process of
autoregulation is not understood. Investigations at this
whole-plant level require a good preliminary knowledge
of the dynamics of nodule establishment, which is in
strong interaction with root system formation.
Unexpectedly, relationships between these two processes
apparently have been rarely investigated. The objective
of this work was to study the development of the root
system, the appearance of nodules, and relationships
between these two processes for pea plants grown in
hydroponic conditions.
flask. The number of nodules carried by the primary root, and
distance between the most distal nodule and the root apex,
were also recorded daily. Nodules were recorded as soon as
they became visible (>0.5 mm). All plant manipulations were
performed under a laminar flow hood to avoid contamination.
At the 6 leaf stage and at the beginning of flowering, 10 plants
were harvested, and the positions of first-order laterals and
nodules along the primary root were recorded.
Assumptions tested and calculations
Differentiation of lateral roots occurs within the pericycle of
the mother root, a few millimetres behind the root tip, whereas
emergence of laterals occurs several centimetres behind, thus
leading to the existence of an apical unbranched zone (KJepper,
1990). Data were used to test the assumption of a constant
time lag between differentiation and emergence of first-order
laterals. Figure 1 shows the theoretical relationship between the
length of the apical unbranched zone, the root elongation rate,
and the time required from primordia formation to appearance
of laterals on a root segment. If the duration required from
primordia formation to appearance of laterals is constant, then
the relationship between the length of the apical unbranched
zone (LAUZ) and the root elongation rate (RER) is expected
to be linear. A similar assumption can be tested for the
appearance of nodules. If the time lag between Rhizobium
infection and nodule appearance is constant, then the relationship between length of the apical zone without nodule (LAZWN)
and root elongation rate is expected to be linear.
Materials and methods
Growth of seedlings
Seeds of pea (cultivar Solara) were weighed and only those of
similar weight (between 0.277 and 0.283 g) were used in order
to reduce plant-to-plant variability. Healthy, viable seeds were
surface-sterilized with a 0.2% mercuric chloride solution for
5 min, and then rinsed well in sterile distilled water to remove
traces of the sterilizing chemicals. They were then transferred
aseptically into Petri dishes on blotting paper moistened with
sterile distilled water for germination in darkness at a constant
temperature (20 °C). During germination, seeds were also
inoculated by scattering a suspension of Rhizobium leguminosarum P221 at 2.106 cells ml" 1 . After 5 d, 20 seedlings were
transferred in aseptic conditions on a wine mesh support at the
top of sterilized flasks (0.18 m depth, 0.5 1 volume). The flasks
were kept filled with a nitrogen-free nutrient solution so that
the whole root system was under submerged conditions. This
solution was inoculated with a suspension of Rhizobium
leguminosarum to provide a concentration of 107 Rhizobium
ml" 1 . It was completely replaced twice during the experiment
and reinoculated. Each flask was covered with a nontransparent plastic sheet to keep roots in darkness. Air was
bubbled through each flask to provide aeration and mixing of
the nutrient solution. Plants were grown for 6 weeks in a
growth chamber with the following conditions: 16/8 h day/
night; 18 °C day/18 °C night; 90% air relative humidity. Light
was provided by cool-white fluorescent tubes (58 W, Sylvania,
Germany). The photosynthetically active radiation at the top
of the plants was 14.1 J m " 2 s" 1 .
Measurements on shoots, roots and nodules
Developmental plant stages were recorded daily using the
measurement scale proposed by Maurer et al. (1966). Length
of the primary root, length of the basal unbranched zone, and
length of the apical unbranched zone were measured daily by
very carefully removing each plant and its root system from the
Results
Elongation of the primary root
Figure 2 shows the average length of the primary root
versus time after the beginning of seed imbibition. The
elongation curve was almost linear during the first 18 d,
with an average root elongation rate of 22 mmd" 1 .
\
LAUZ,
i
LAUZ
\±
1
Fig. 1. Theoretical relationship between length of the apical unbranched
zone, parent root elongation rate and the time required from pnmordia
formation to appearance of laterals on a root segment. /,: date at which
lateral primordia differentiated in the considered root segment; l2: date
at which laterals emerged on this root segment. Length of the apical
unbranched zone (LA UZ) is the sum of the distance from the root tip
to the root segment when lateral primordia differentiated (LAUZ0)
and the root length increase between tl and t2. Thus, LAUZ =
LAUZ0 + RERy.(t1 — tl), where RER is the average root elongation
rate between t, and t2 (adapted from Pellerin and Tabourel, 1995).
Root growth and nodulation on pea
200
500
500
1937
180
T •
4.07 X + 10.6
r" = 0 . 8 7
160
400
400
140
300
E300
I
I
1200
120
„
200
E
S
100
80
60
100
100
Primary root length
Lateral front
10
20
30
40
Days after seed Imbibition
Fig. 2. Length of the primary root, and position of the front of firstorder lateral emergence, versus the number of days after the beginning
of seed imbibition. Vertical bars represent ± one standard deviation.
Arrows indicate plant stages: lL: one leaf; 4L: four leaves; 6L: six
leaves; BF. beginning of flowering.
Elongation rate drastically decreased between 18 and 25
d, and started again thereafter. It definitely stopped after
35 d. Such an elongation curve was observed on all
individual primary roots.
Emergence of first-order laterals
First-order laterals appeared acropetally on the primary
root. Basal and apical unbranched zones were clearly
visible on all primary roots, and at all sampling dates.
Since laterals appeared acropetally, the length of the basal
unbranched zone did not vary during the experiment
(data not shown). Its average length was 3 mm. By
contrast, length of the apical unbranched zone varied
widely between sampling dates. Values ranged between 5
and 185 mm.
Figure 3 shows the relationship between the length of
the apical unbranched zone and the root elongation rate
calculated as proposed in Fig. 1. A rough linear relationship was observed, thus supporting the hypothesis of a
constant time lag required from primordia formation to
40
20
10
20
30
40
RER (mm per day)
Fig. 3. Length of the apical unbranched zone (LAUZ) versus primary
root elongation rate (RER).
appearance of laterals. According to Fig. I, this delay is
given by the slope of the regression line (about 4 d under
our experimental conditions). The intercept of the regression line was statistically different from zero (T=3.22,
p > | 7 | = 0.0017). It measures the distance between the
most apical lateral and the root tip when primary root
elongation rate is zero. It also shows the distance between
the root tip and the root segment where lateral primordia
differentiate. In our set of data it was about 11 mm.
Density and length of first-order laterals
Figure 4 shows the density of first-order laterals (number
of laterals mm" 1 of primary root) and their average
length versus the distance from the root base. Only plants
harvested at the beginning of flowering were used for
these calculations. Both curves have a similar shape, with
the density of laterals and their average length being
minimal between 160 and 200 mm from the root base.
Considering the position of the front of emergence of
laterals, it appears that laterals on this less densely
branched root segment were produced just before the
decline of the primary root elongation rate (Fig. 2).
1938
Tricot eta\.
Density of first-order laterals
-0,1
50
100
150
200
250
300
350
400
450
500
Distance from the root base (mm)
Fig. 4. Number of first-order laterals per millimetre of primary root and average length of first-order laterals versus distance from the pnmary root
base Counting of first-order laterals was performed on root segments of 20 mm. Vertical bars represent!one standard deviation
Appearance of nodules
Nodules appeared on the root systems of all plants
studied, thus confirming that these conditions were suitable for nodulation. Nodules were found mainly on the
primary root, but some first-order laterals also were
nodulated (Fig. 5). Nodules appeared roughly but not
strictly acropetally. As for root branching, basal and
apical non-nodulated zones were visible. Average length
of the basal zone without nodules was constant throughout the experiment (data not shown). By contrast, length
of the apical zone without nodules varied between observation dates.
Figure 6 shows the relationship between the length of
the apical zone without nodules and root elongation rate,
calculated as indicated in Fig. 1. Only roots whose
nodulated zone was elongating were considered for this
calculation. A linear relationship exists between the length
of the apical zone without nodules and the root elongation
rate. This supports the hypothesis of a constant lag time
required from Rhizobium infection to the appearance of
a macroscopic nodule. This delay is given by the slope of
11
I
10
m Number of laterals per root segment of 20 mm
a Number of laterals carrying nodules
|
CM
°
i:
e
I
I
1
z
L
li..ll
p
in
1
CM
1
.ill . 1
Distance from the root base (mm)
Fig. 5. Total number of first-order laterals per segment of 20 mm, and number of them carrying nodules, versus distance from the primary root base.
Roof growth and nodulation on pea 1939
100
250
90 >
dott«d linei
y-7.71jc r'«0.47
80
200
« 70
c
O)
150
W
/
I
| 50
100
D
/
•S40
D
|
30
20
60
10
5
10
15
20
25
30
0-10
Fig. 6. Length of the apical zone without nodules {LAZWN) versus
primary root elongation rate. The dotted line is the regression line
obtained when the intercept is imposed to zero (y = 7.7l.v; ^ = 047).
the regression line. The distance between the root tip
and the root segment where infections occur is
theoretically given by the intercept. However, because
experimental points were missing at low elongation rates,
the intercept could not be estimated with accuracy.
According to the literature, infections mainly occur at a
short distance behind the root tip (about 10 mm)
(Bhuvaneswari, 1981; Bhuvaneswari et ai, 1980, 1981;
Pueppke, 1986). The delay between infection and the
appearance of a macroscopic nodule can, therefore, be
approximated by the slope of the regression line passing
through the origin, which is 7.7 d for our experimental
conditions.
Density of nodules
At the beginning of flowering, the average density of
nodules within the nodulated segment of the primary root
(i.e. the root segment between the most proximal and the
most distal nodule) ranged between 0.04 and 0.25 nodules
mm" 1 , depending on the plant. Nodules, however, were
unevenly distributed within this nodulated segment. Some
10-20
20-30
30-40
>40
Class of root elongation rate (mm/day)
Root elongation rate (mm per day)
Fig. 7. Percentage of root segments carrying nodules per class of root
elongation rate.
non-nodulated root segments were found within the nodulated zone. The question thus arises as to the reason for
this. In Fig. 7, the assumption of a link between the
presence or absence of nodules and primary root elongation rate was tested. Each root segment was characterized
by (i) presence or absence of nodules at the beginning of
flowering and (ii) root elongation rate between infection
and actual (or theoretical) emergence of nodules on the
root segment under consideration. Calculations were performed using the delay between infection and emergence
described above. Apical root segments, on which nodules
could not have appeared because the time elapsed since
infection was lower than the time required for nodule
appearance, were discarded. The percentage of root segments carrying nodules was plotted for each root elongation rate class (Fig. 7). Percentage of root segments
carrying nodules was found to increase with root elongation rate.
Discussion
Strong relationships were found between nodule establishment and root system growth, thus emphasizing the
1940
Tricot et al.
importance of considering the latter in nodulation studies.
An unexpected decline in primary root elongation rate
was observed between 18 and 25 d after seed imbibition
which could not be attributed to an artefact. Growing
conditions were constant during the experiment, and this
decline did not occur when primary roots reached the
bottom of the flasks. Bourdu and Gregory (1983),
Deleens et al. (1984) and Derieux et al. (1989) have
observed a decline of maize root elongation rate at the
beginning of the translocation of photosynthates from
leaves to roots. A possible explanation is that the reduction of primary root elongation rate observed in this
experiment corresponded to the exhaustion of seed
reserves. Actually weighing seeds has shown that, at this
moment, the percentage of utilization of seed reserves
was 86%. Carbon shortage due to the transition from the
heterotrophic to the autotrophic phase is very likely to
occur in growth chambers because of the low radiation,
compared to field conditions. The decline of primary root
elongation rate was preceded by a reduction in branching
rate and elongation of first-order laterals, suggesting a
link between these processes. Carbon shortage at the time
of transition from the heterotrophic to the autotrophic
phase, which may be involved in the decline of primary
root elongation rate, may also explain a lower branching
rate and elongation of laterals. In this regard, Bingham
and Stevenson (1993) observed that the number of lateral
wheat root primordia was affected by carbohydrate
supply. Reduction of branching rate and elongation of
laterals occurred just before the decline of primary root
elongation rate, suggesting that the primary root has
priority for carbon allocation. This is consistent with the
findings of Aguirrezabal et al. (1993), who observed with
sunflower that the elongation rate of the taproot was less
reduced at low irradiance than the elongation rate of
first-order laterals.
Consistent with results obtained for other species
(MacLeod, 1990; Pellerin and Tabourel, 1995), these
results suggest that a constant time lag exists between the
differentiation of first-order lateral roots and their emergence (4 d under this study's conditions). The estimated
distance between the root tip and the root segment where
lateral primordia differentiate was 10.6 mm. These data
are in agreement with those of MacLeod and Thompson
(1979), who observed with pea the most apical root
primordium to be located between 11.8 and 18.8 mm
behind the root tip. These findings suggest that the most
apical laterals which arise on roots whose elongation
stopped correspond to the last primordium which differentiated behind the root tip. This observation is consistent
with that of Abadia-Fenoll et al. (1986), who showed
that lateral primordia can differentiate only after root
tissues have reached a certain level of maturity.
Nodules appeared roughly but not strictly acropetally
on the primary roots. A linear relationship was found
between length of the apical part of the root without
nodules and root elongation rate. This strengthens the
assumption of a constant time lag between infection and
appearance of nodules (about 7.7 d under our experimental conditions). The not strictly acropetal sequence
of appearance of nodules may be due either to a not
strictly acropetal sequence of infections or to slight variations in the delay between infections and appearance of
macroscopic nodules.
A relationship was found between the presence/absence
of nodules on a root segment and root elongation rate
during the period between infection and appearance of
nodules on the considered root segment. The proposed
interpretation, which should be tested by further studies,
involves the regulation of both processes by carbohydrate
availability. Calvert et al. (1984) observed that many
infections formed on soybean roots, but relatively few
developed into nodules. Kosslak and Bohlool (1984) have
shown that the number of successful infections may be
affected by photosynthetic capacity of the host plant.
Kasperbauer et al. (1984) and Kasperbauer and Hunt
(1994) showed for soybean and southern pea that greater
photoassimilate allocation to roots was associated with
formation of more nodules. Moreover, Murphy (1986)
showed for pea and other legumes that increasing CO2
atmospheric concentrations from 330 to 1000 [A I"1 was
associated with the formation of more nodules. On
the other hand, root elongation was shown to be dependent on the short-term on carbohydrate availability
(Aguirrezabal^al., 1994; Bingham and Stevenson, 1993).
Under these experimental conditions, variations in primary root elongation rate probably reflected carbon allocation to the root system. Therefore, the link between
elongation rate of the primary root and presence/absence
of nodules may reflect the limitation of both processes by
carbohydrate availability.
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