Annals of Botany 88: 131±138, 2001
doi:10.1006/anbo.2001.1440, available online at http://www.idealibrary.com on
Phosphorus De®ciency Impairs Early Nodule Functioning and Enhances Proton Release in
Roots of Medicago truncatula L.
C . TA N G * {{, P. H I N S I N G E R {, J . J . D R E VO N { and B . J A I L L A R D{
{UMR Sol & Environnement, INRA, place Pierre Viala, F-34060 Montpellier, cedex 1, France and {Centre for
Legumes in Mediterranean Agriculture/Soil Science and Plant Nutrition, University of Western Australia, 35 Stirling
Hwy, Crawley 6009, Australia
Received: 17 January 2001
Returned for revision: 15 March 2001 Accepted: 30 March 2001
The e€ect of phosphorus supply (1±15 mM) on proton release and the role of P in symbiotic nitrogen ®xation in medic
(Medicago truncatula L. `Jemalong') was investigated. As P concentration in the nutrient solution increased, shoot
and root growth increased by 19 and 15 %, respectively by day 35, with maximal growth at 4 mM P. A P concentration
of 15 mM appeared to be toxic to plants. Phosphorus supply had no in¯uence on nodule formation by day 12 but
increased nodule number by day 35. Nitrogenase activity was estimated by in situ measurement of acetylene reduction
activity (ARA) in an open-¯ow system. During the assay, a C2H2-induced decline of ARA was observed under all P
concentrations except 4 mM. Speci®c ARA ( per unit nodule weight) doubled when P supply was increased from 1 to
8 mM. This e€ect of P was much greater than the e€ects of P on nodulation and host plant growth. Concentrations of
excess cations in plants decreased with increasing P concentration in the nutrient solution. Phosphorus de®ciency
stimulated uptake of excess cations over anions by the plants and hence enhanced proton release. The results suggest
that P plays a direct role in nodule functioning in medic and that P de®ciency increases acidi®cation which may
# 2001 Annals of Botany Company
facilitate P acquisition.
Key words: Medicago truncatula L. (medic), P de®ciency, C2H2-ID, nitrogenase activity, proton release, cation-anion
balance, open-¯ow system.
I N T RO D U C T I O N
Of all nutrients, shortage of phosphorus (P) has the biggest
impact on legumes which generally rely on N2 ®xation for
nitrogen (N) nutrition. The e€ect of P supply on growth
and N2 ®xation in legumes has been studied extensively, but
the role of P in the symbiotic process remains unclear. The
de®ciency of a nutrient may limit N2 ®xation through its
e€ects of growth and survival of rhizobia, nodule formation, nodule functioning and host plant growth. Robson
et al. (1981) observed that mineral nitrogen (NH4NO3)
increased the growth response of nodulated plants of
subterranean clover to P application, and suggested that P
increased symbiotic N2 ®xation through its e€ect on host
plant growth. In contrast, Israel (1987) demonstrated that
symbiotic N2 ®xation in soybean required more P for
optimal functioning than either host plant growth or N
assimilation in the plant. In other studies, P de®ciency has
been found to decrease nodule number, decrease nodule
mass more than shoot growth, and decrease the speci®c
nitrogenase activity of nodules (Jakobsen, 1985; Singleton
et al., 1985; Israel, 1987; Hart, 1989; Ribet and Drevon,
1995; Vadez et al., 1996; Drevon and Hartwig, 1997).
Annual Medicago species are an important component of
pastures in Mediterranean regions, and are highly responsive to applied P (Bolland, 1985). Medicago truncatula L.
appears to be less tolerant to soil acidity than other annual
* For correspondence. Fax ‡61 8 9380 1050, e-mail cxtang@
cyllene.uwa.edu.au.
0305-7364/01/070131+08 $35.00/00
Medicago species and is therefore generally grown on
neutral to alkaline soils (Ewing, 1991) where P de®ciency is
also common. However, the e€ects of P de®ciency on
symbiotic N2 ®xation in this species are unknown.
Legume plants reliant on N2 ®xation take up more
cations than anions and release H ‡ ions at the root-soil
interface (see Raven et al., 1990; Tang and Rengel, 2001).
Within a species, proton release may increase with the
amount of N2 ®xed. In addition, increased acidi®cation of
the rhizosphere by roots is a widespread response to P
de®ciency (e.g. Imas et al., 1997; Bertrand et al., 1999;
Neumann and RoÈmheld, 1999; Hinsinger, 2001). However,
this has been little studied for legumes reliant on N2
®xation.
The aims of the present study were to assess the role of P
in symbiotic N2 ®xation in M. truncatula and to study how
P de®ciency a€ects cation-anion uptake and hence proton
release.
M AT E R I A L S A N D M E T H O D S
Plants were grown in nutrient solution in a glasshouse with
day/night temperatures of approx. 28/20 8C and a 16 h
photoperiod of natural light supplemented with mercury
vapour lamps. Uniform seeds of Medicago truncatula L.
`Jemalong' were germinated for 2 d at 28 8C in the dark and
then 1 d in the glasshouse on ¯y screens covered with paper
towels above an aerated nutrient solution containing
800 mM CaCl2 and 4 mM H3BO3. Twelve seedlings were
# 2001 Annals of Botany Company
132
Tang et al.ÐE€ect of P on Nodule Function and Proton Release
transplanted (day 0) to 5 l plastic pots containing nutrient
solution of the following composition (mM): K2SO4, 700;
MgSO4, 500; CaCl2, 800; H3BO3, 4; Na2MoO4, 0.1; ZnSO4,
1; MnCl2, 2; CoCl2, 0.2; CuCl2, 1; and FeNaEDTA ( ferric
monosodium salt of ethylenediamine tetraacetic acid), 10.
Phosphorus was applied as KH2PO4 at four levels with a
maximum of 15 mM to minimize possible P toxicity. The P
treatments were replicated three times and were completely
randomized. A dense water suspension of Rhizobium trifolii
2011 was added to the solution at a rate of approx 108 cells
l ÿ1. This solution was unchanged for 6 d and then renewed
with added rhizobia. Five days later, plants were placed in
solutions without added rhizobia; solutions were then
changed every third day. Urea (0.2 mM N) was added for
the ®rst 14 d after transplanting (DAT). Thereafter, the
plants did not receive any external source of N. Solution pH
was adjusted daily with KOH to 6.5 during the ®rst 10 and
then to 6.0. During days 14±35, the calculated depletion of
P in nutrient solutions before renewal ranged from 25±45 %
at 15 mM P to 40±70 % at 1 mM P.
To measure nitrogenase activity, three plants from each
pot were carefully transferred to a 300 ml glass bottle 29
DAT. The bottles were wrapped with aluminium foil to
keep the rooting environment dark. The roots were gently
passed through the hole of a rubber stopper on the bottle
neck, and cotton wool was ®tted at the hypocotyl level. The
bottles contained the same nutrient solution as the
treatment solutions, except that the pH was bu€ered with
0.2 mM MES [2-(N-morpholino)ethane-sulfonic acid].
Nutrient solution in the bottles was renewed daily. To
avoid nodule disturbance, the level of the solution was
lowered to 70 % of the bottle volume 1 d before the assay.
Nitrogenase acetylene reduction activity (ARA) in the
nodules was assayed in situ between 1000 and 1600 h 33±35
DAT, with one replicate each day, in an open-¯ow system as
described by Drevon et al. (1988), using a steady-¯ow gas
mixture of 69 kPa N2, 21 kPa O2 and 10 kPa C2H2. The
C2H4 concentration in the out¯ow was determined 2 to
50 min after C2H2 exposure with a Delsi 30 gas chromatograph (Delsi Instruments, Suresnes, France) equipped with
a ¯ame ionization detector.
The number of nodules was counted on intact plants 12
DAT, and three plants were subsampled from each 5 l pot
14 DAT. The plants in the bottles were harvested after
measuring ARA, and those remaining in the 5 l pots were
harvested 35 DAT. Plants were separated into tops, roots
and nodules. The length of individual fresh nodules was
measured and assigned to one of four classes of mean
length: 4 2.5; 3; 3.5; and 43.5 mm. All plant tissues were
oven-dried at 60 8C prior to chemical analysis. Tissues from
the bottles and 5 l pots of the same treatments were
combined.
Dried plant material was ®nely ground with an iron steel
ball grinder (Retsch, Haan, Germany). A 200 mg subsample of ground material was digested in hot concentrated
HNO3, then HNO3 ÿHClO4 until complete evaporation of
HNO3 according to the A.O.A.C. procedure (A.O.A.C.,
1975). Phosphate was then assayed colourimetrically
(Varian UV-vis photometer CARY, Mulgrave, Australia)
with the vanado-molybdate method (A.O.A.C., 1975).
Sulfur was assayed by inductively coupled plasma emission
spectrometry (Axial ICP Varian VISTA AES, Mulgrave,
Australia) while K, Na, Ca and Mg were assayed by ¯ame
atomic absorption spectrometry (Varian SpectrAA-600,
Mulgrave, Australia). To avoid ionization interference, Cs
was added to the digest for K determination while La was
added to suppress chemical interference in the Ca and Mg
determinations. For N determination, subsamples of 10±
30 mg ground plant material were digested in hot concentrated H2SO4 (McDonald, 1978), with additional salicylic
acid (10 %). Ammonium in the digest was determined
colourimetrically with the phenol hypochlorite method
(Martin et al., 1983). For Cl determination, a water extract
was obtained by shaking 50 mg of ground plant material in
5 ml deionized water for 1 h. After ®ltration through a
0.2 mM nylon ®lter, Cl was assayed by high performance ion
chromatography (Dionex BioLC 2000i, Sunnyvale, California, USA) equipped with a conductimetric detector,
4 mm pre-column and column AS11, ASRS-Ultra 4 mm
suppressor device. The concentration of sulfuric acid in the
suppressor was 13.1 mM (residual conductivity below 5
microS) and the eluent was 11 mM NaOH. Under such
conditions, in the isocratic mode, Cl was detected after
1.55±1.60 min.
Total amounts of H ‡ released by plants were calculated
by summing the amounts of KOH used for pH adjustment
during plant growth and for titrating the used solution after
plant growth to the initial pH. Speci®c H ‡ release was
expressed as the amount of H ‡ released per unit root
biomass. Concentrations of excess cations in the plant were
calculated from individual elements as the sum of charge
concentrations of Ca2‡ , Mg2‡ , K ‡ and Na ‡ minus the sum
ÿ
of H2PO4ÿ , SO2ÿ
4 and Cl (Tang et al., 1998).
Plant dry weights, plant N concentration and data in
Table 2 were subjected to two-way ANOVA (P supply plant parts) using Genstat 5 (Lawes Agricultural Trust,
Rothamsted, UK). Other data were analysed using one-way
ANOVA for di€erences between P treatments.
R E S U LT S
Plant growth
Seventeen DAT, plants grown at 15 mM P displayed
symptoms of toxicity in the form of necrosis on old leaves;
this became more severe with time. By 35 DAT, the dry
weight of shoots and roots had increased by 19 and 15 %,
respectively, as the solution P concentration increased from
1 to 4 mM and then decreased slightly as the P supply
increased further (Fig. 1).
Nodule formation and function
Nodules ®rst appeared 7 DAT; nodule number recorded
12 DAT was not in¯uenced by P supply (Fig. 2). By 35
DAT, the number of nodules tended to increase with
increasing P supply (Fig. 2). Nodules formed at 1 and
15 mM P were, on average, smaller than those formed at 4
and 8 mM P (Fig. 3). For example, over 50 % of nodules in
the 4 and 8 mM P treaments were over 3.5 mm long
Tang et al.ÐE€ect of P on Nodule Function and Proton Release
Nodule number (no. per plant)
Plant dry weight (mg per plant)
5
60
40
20
0
1
4
8
15
P concentration in solution (mM)
F I G . 1. Dry weights of shoot (d) and roots (s) of Medicago truncatula
grown at 1±15 mM P for 35 d. The e€ect of P supply is signi®cant at the
0.05 level. Bars represent s.e.m. of three replicates.
3
2
1
0
2.5
3
3.5
Nodule length (mm)
>3.5
F I G . 3. Distribution of nodule number as a function of mean nodule
length of Medicago truncatula grown at 1±15 mM P for 35 d. Bars
represent s.e.m. of three replicates.
0.8
Total acetylene reduction
(mmol C2H4 h–1 per plant)
Nodule number (no. per plant)
1 mM P
4 mM P
8 mM P
15 mM P
4
12
9
6
3
0.6
0.4
0.2
0.0
0
200
Specific acetylene reduction
(mmol C2H4 h–1 g–1 nodule)
Nodule dry weight (mg per plant)
133
4
150
100
2
50
0
0
1
4
8
15
P concentration in solution (mM)
F I G . 2. The number of nodules at 12 (n) and 35 (s) DAT, and nodule
dry weight at 35 DAT of Medicago truncatula grown at 1±15 mM P.
Bars represent s.e.m. of three replicates.
compared to 25 and 27 % at 1 and 15 mM P, respectively.
The dry weight of nodules increased by a third as the
concentration of P in solution increased from 1 to 4 mM but
did not increase further as P increased to 15 mM (Fig. 2).
Both total (mmol C2H4 h ÿ1 per plant) and speci®c ARA
(mmol C2H4 h ÿ1 g ÿ1 nodule) increased with increasing P
supply, reaching a maximum at 8 mM P (Fig. 4). The
increase in speci®c ARA was more than doubled by P
1
4
8
15
P concentration in solution (mM)
F I G . 4. Total and speci®c acetylene reduction activity (ARA) of
Medicago truncatula grown at 1±15 mM P for 35 d. ARA was the
average ARA measured between 4 and 50 min after C2H2 exposure.
The e€ects of P supply on total and speci®c ARA are signi®cant at the
0.01 and 0.05 levels, respectively. Bars represent s.e.m. of three
replicates.
application, and was much greater than the e€ects of P on
plant growth and nodule formation. A C2H2-induced
decline (C2H2-ID) of ARA was observed in all P treatments
except 4 mM (Fig. 5). When 10 kPa C2H2 acetylene was
introduced into the gas mixture ¯owing through the
134
Tang et al.ÐE€ect of P on Nodule Function and Proton Release
Relative C2H2 reduction activity (% of average between 4–50 min)
140
120
100
80
60
1 mM P
4 mM P
8 mM P
15 mM P
40
140
120
100
80
60
40
0
10
10
10
10
10 0
10
20
Duration of C2H2 exposure (min)
30
40
50
F I G . 5. Time course of acetylene reduction activity (ARA) measured 33±35 DAT in Medicago truncatula grown at 1±15 mM P. ARA is expressed
as a percentage of average ARA between 4 and 50 min after C2H2 exposure. Bars represent s.e.m. of means of three replicates.
nodulated roots, the amounts of C2H4 produced by the
nodule activity increased, reaching a maximum value 4 min
after initial exposure to C2H2, before decreasing. The C2H2ID was sharp and the greatest at 1 mM (34 % of maximum),
followed by 15 and 8 mM P. C2H4 production recovered
after 25 min following the C2H2-ID at 8 mM P. This
recovery of C2H4 production was not observed within
50 min at a P supply of 1 or 15 mM.
Chemical composition
Nitrogen concentration in shoots decreased slightly as P
supply increased from 1 to 4 mM, and then increased with
increasing P supply, reaching a maximum at 15 mM P.
Nitrogen concentration in roots was not a€ected by P
supply. Total amounts of N ®xed by the plants during 14±
35 d after transplanting tended to increase as P supply
increased, with a maximum at 8 mM P (Fig. 6).
Concentrations of P in plant parts increased markedly
with increasing P supply (Table 1). Concentrations of P in
nodules were higher at 1 and 4 mM P and lower when more
P was supplied than those in shoots and roots (Table 1).
The amount of N ®xed per unit P taken up decreased with
increasing P concentration in solution (data not shown).
Increasing the P supply did not signi®cantly a€ect plant
concentrations of Ca, Mg and K. Concentrations of Ca and
Mg were higher in shoots than in roots and nodules,
whereas the opposite was true for K concentrations
(Table 1). Increasing the P supply generally increased
concentrations of S and Cl, except S in shoots and roots,
and Cl in roots at the highest P supply (Table 1). The
concentration of Na in plants was below 0.1 mg g ÿ1 and
was not a€ected by P supply. Concentrations of excess
cations in shoot and root tissues decreased as concentrations of P in the nutrient solution increased, with the
decrease being greater in roots (Table 1). The calculated
amounts of excess cations over anions taken up per plant
(total) and per unit root biomass (speci®c) decreased as P
supply increased (Table 2).
Proton release
Growing plants released protons and decreased the
solution pH. Both total and speci®c proton release tended
to be higher at 1 mM P than at higher levels of P supply. The
amount of protons released per unit root biomass at 1 mM P
was one-third higher than that at 4 and 8 mM P. Similarly,
the molar ratio of protons released to N ®xed was highest at
1 mM P and ranged from 0.54 to 0.82 (Table 2). Total
and speci®c proton releases were generally correlated with
total and speci®c uptake of excess cations, respectively
(Table 2).
N concentration in plant (mg g–1)
Tang et al.ÐE€ect of P on Nodule Function and Proton Release
Phosphorus de®ciency has previously been reported to
decrease nodule mass more than host growth in soybean
(Singleton et al., 1985; Israel, 1987; Ribet and Drevon,
1995; Drevon and Hartwig, 1997), common bean (Pereira
and Bliss, 1989; Vadez et al., 1996), pea (Jakobsen, 1985),
white clover (Hart, 1989) and alfalfa (Drevon and Hartwig,
1997). However, reports on the e€ect of P on nodule
number have been controversial; P de®ciency may increase,
decrease or not a€ect nodule number per unit shoot mass
(Pereira and Bliss, 1989; Gunawardena et al., 1992; Ribet
and Drevon, 1995; Vadez et al., 1996; Drevon and Hartwig,
1997). Di€erences in the responses of nodulation to P
de®ciency appear to be related to legume species, genotype
and experimental conditions.
30
20
10
0
N accumulation in (mg per plant)
2.0
Nodule functioning
1.5
1.0
0.5
0.0
135
1
4
8
15
P concentration in solution (mM)
F I G . 6. Concentration of N in shoots (d) and roots (s) of Medicago
truncatula grown at 1±15 mM P for 35 d and the amount of N
accumulated during 14±35 d after transplanting. Plants received
0.2 mM urea-N during 0±14 d. The e€ect of P supply on N
concentration in the shoot is signi®cant at the 0.01 level. Bars represent
s.e.m. of three replicates.
DISCUSSION
E€ects of P on nodule formation
In the present study, early nodule formation was not
a€ected by increasing P concentrations in the external
solution. Supply of 1 mM P appeared to be sucient for
optimal nodulation when a large number of nodule bacteria
were present. Indeed, the growth and survival of rhizobia
required less than 0.5 mM P (O'Hara et al., 1988). However,
increasing the P supply appeared to increase nodule
formation at a later stage (35 DAT). This increase in
nodule number was independent of host plant growth. As
rhizobia were only added to the nutrient solution in the ®rst
11 d, the rhizobial population was expected to be low in the
nutrient solution after day 11. It is not known whether high
P levels are required for nodule formation when the
rhizobial population is low. Furthermore, the number of
nodules per plant and plant growth rate in this study were
comparable to those reported previously for the same
species (e.g. Ewing, 1991).
Phosphorus had a similar pattern of e€ects on nodule
biomass as on host plant growth, suggesting that P
de®ciency indirectly a€ects subsequent nodule development
by restricting metabolite supply from the host plant.
This study demonstrated that P has a speci®c role in
nodule functioning in. M. truncatula. Nitrogenase activity
per unit nodule mass doubled when the external P
concentration increased from 1 to 8 mM. This e€ect of P
on nodule nitrogenase activity was greater than the e€ect on
any other component of the symbiosis. This increased ARA
by P supply was associated with increases in shoot N
concentration and N accumulation. However, increases in
plant N concentration and accumulation were relatively
small (20±30 %). This inconsistency was probably associated with the presence of urea-N for the ®rst 14 d of the
experiment. Alternatively, acetylene reduction is an instantaneous measure (at an early stage of the symbiosis in the
present study) whereas N concentration and total N
accumulation are cumulative measures over a longer period.
In transfer experiments, an increase in speci®c ARA in
soybean after P addition and a decrease during the onset of
P de®ciency occurred well before any response of host plant
growth and N concentration (Israel, 1993). In soybean, the
decrease in nitrogenase activity under P de®ciency was
suggested to result from inhibition of energy-dependent
reactions in the plant cell fraction of the nodules (Sa and
Israel, 1991). The impaired nitrogenase activity in soybean
nodules was also associated with decreased bacteroid
biomass (Sa and Israel, 1991).
The greater C2H2-ID in nitrogenase activity under P
de®ciency than P suciency in this study is consistent with
previous ®ndings in soybean (Ribet and Drevon, 1995) and
common bean grown hydroponically (Vadez et al., 1997),
and soybean and alfalfa grown in sand (Drevon and
Hartwig, 1997). The C2H2-ID has been suggested to be due
to a shortage of energetic substrates after the initial
transient stimulation. Energy limitation under P de®ciency
might be attributed to an inhibition of the respiratory
cytochrome pathway and/or activation of alternative
respiration (Rychter et al., 1992). Indeed, lower ATP
concentrations (see above, Sa and Israel, 1991), higher
carbon costs of ARA, and lower starch accumulation
(Ribet and Drevon, 1995) have been observed in soybean
nodules in P-de®cient treatments compared with adequate
P controls.
In the present study, the C2H2-ID in nitrogenase activity
also occurred when excess P was supplied (toxic level). The
136
Tang et al.ÐE€ect of P on Nodule Function and Proton Release
T A B L E 1. Concentrations of P, Ca, Mg, K, S, Cl and excess cations in dry plant matter of Medicago truncatula grown at
1±15 mM P for 35 d
P
Ca
Mg
Shoot
1
4
8
15
1.3 + 0.2
6.0 + 0.4
11.9 + 0.7
15.5 + 0.5
23.8 + 1.1
22.8 + 1.0
26.6 + 2.9
27.6 + 2.3
Root
1
4
8
15
2.3 + 0.1
4.2 + 0.1
9.0 + 0.9
15.6 + 1.0
Nodule
1
4
8
15
P supply (mM)
Signi®cance level{
P supply
Plant part
P Plant part
K
S
Cl
Excess cations
(mmol(‡) g ÿ1)
5.5 + 0.8
5.5 + 0.1
6.2 + 0.1
5.8 + 0.3
28.5 + 2.9
29.8 + 0.6
33.1 + 2.0
33.5 + 1.0
5.6 + 1.0
8.3 + 0.5
9.5 + 0.9
8.4 + 0.2
10.4 + 1.1
12.7 + 0.9
14.3 + 0.7
15.0 + 0.5
1.68 + 0.11
1.29 + 0.04
1.20 + 0.20
1.27 + 0.13
4.0 + 0.2
4.1 + 0.1
4.5 + 0.2
4.1 + 0.2
2.2 + 0.1
1.9 + 0.1
2.0 + 0.1
2.3 + 0.1
40.3 + 2.1
40.2 + 0.7
40.2 + 1.2
39.7 + 1.1
6.9 + 0.8
9.2 + 0.6
8.7 + 0.3
6.9 + 0.3
16.3 + 0.8
18.0 + 0.3
18.0 + 0.3
15.7 + 0.7
0.45 + 0.05
0.18 + 0.04
0.08 + 0.04
0.04 + 0.02
5.0 + 0.1
7.4 + 0.2
8.2 + 0.2
9.5 + 0.4
4.1 + 0.1
4.1 + 0.1
3.9 + 0.1
3.9 + 0.1
3.3 + 0.2
3.4 + 0.1
3.2 + 0.1
3.5 + 0.1
35.6 + 1.4
36.8 + 0.9
36.5 + 0.4
36.5 + 0.6
***
**
***
n.s.
***
n.s.
n.s.
***
n.s.
n.s.
***
n.s.
**
n.s.
n.s.
**
***
**
**
***
n.s.
(mg g ÿ1)
{Data were analysed by ANOVA; n.s., not signi®cant; *, ** and ***, signi®cant at the 0.05, 0.01 and 0.001 levels, respectively.
Plants received 0.2 mM urea-N during the ®rst 14 d. Data are the means + s.e. of three replicates.
T A B L E 2. Amounts of excess cations over anions (ECat) taken up and proton (H ‡ ) release, and molar ratio of H ‡ released
to N ®xed (H ‡ /N) by N2-®xing plants of Medicago truncatula grown at 1±15 mM P during 14±35 d after transplanting
P supply (mM)
1
4
8
15
Signi®cance level{
Total ECat uptake
(mmol per plant)
Speci®c ECat uptake
(mmol g ÿ1 dry root)
Total H ‡ release
(mmol per plant)
Speci®c H ‡ release
(mmol g ÿ1 dry root)
H ‡ /N
(molar ratio)
99 + 11
83 + 8
71 + 17
61 + 10
n.s.
3.23 + 0.37
2.35 + 0.21
2.01 + 0.38
1.97 + 0.26
*
80 + 3
73 + 11
68 + 2
72 + 6
n.s.
2.58 + 0.13
2.05 + 0.28
1.97 + 0.12
2.33 + 0.10
n.s.
0.82 + 0.07
0.62 + 0.06
0.54 + 0.05
0.61 + 0.04
*
{Data were analysed by ANOVA; n.s., not signi®cant at P ˆ 0.05; *, signi®cant at the 0.05 level.
Plants received 0.2 mM urea-N during the ®rst 14 d. Date are the means + s.e. of three replicates.
C2H2-ID at high P supply did not correlate with a decrease
in speci®c nitrogenase activity. The C2H2-ID at excess P has
not been reported; the reason for this is unknown. In other
studies, stresses such as salinity decreased the C2H2-ID (see
Ribet and Drevon, 1995 and references therein).
Nodules appeared to be a strong sink for P. Higher P
concentrations were observed in nodules than in roots and
shoots when P supply was limited. This is in agreement with
previous reports in a wide range of legumes (e.g. Robson
et al., 1981; Jakobsen, 1985; Hart, 1989; Israel, 1993; Ribet
and Drevon, 1995; Vadez et al., 1995; Drevon and Hartwig,
1997). The results indicate that nodule function has a high P
requirement. In addition, this study showed that nodules
had lower P concentrations than host plants at high P
supply. The same phenomena have also been observed in
white clover (Hart, 1989) and soybean (Israel, 1987). Thus
nodules appear to have a strategy to regulate P in¯ux,
which minimizes P de®ciency when the supply is low but
avoids excess when the supply is high.
Proton release and P acquisition
The release of protons from roots increased under P
de®ciency; this would facilitate acquisition of P from
rhizosphere soil, especially in soils containing Ca-phosphates such as neutral and calcareous soils, or even acid
soils fertilized with phosphate rocks (Bertrand et al., 1999;
Behi et al., 2000; Hinsinger, 2001). In this study, the
increased acidi®cation under P de®ciency appeared to be
related to the increase in uptake of excess cations over
anions as the patterns of acid release and plant excess cation
uptake were similar. It is not known whether P-de®cient
plants of M. truncatula exude organic acid anions. In some
other species, P de®ciency stimulates organic acid exudation
Tang et al.ÐE€ect of P on Nodule Function and Proton Release
which also contributes to rhizosphere acidi®cation (see
Neumann and RoÈmheld, 1999). There are several previous
reports of increased H ‡ release under P de®ciency e.g. in
tomato (Imas et al., 1997), rape (Bertrand et al., 1999),
chickpea and lupin (Neumann and RoÈmheld, 1999).
However, in the previous studies all the plantsÐincluding
the legumesÐhad been fed with NO3-N or NH4-N
(Hinsinger, 2001). Several of these authors stressed that
decreased NO3ÿ uptake was the major cause of the observed
increase in H ‡ release under P de®ciency (see Hinsinger,
2001). In the present study, P de®ciency decreased the
uptake of S and Cl in the absence of external NO3ÿ , which
might re¯ect a general limitation of anion uptake, an
energy-intensive process, under P-de®cient conditions.
Furthermore, the ability of M. truncatula to accumulate
high P concentrations in the shoot even at low levels of
external P supply may also indicate that P uptake eciency
and/or internal P use eciency is highly expressed in this
species.
Phosphate toxicity has been observed in other legumes
grown in solution culture with P concentrations over 15 mM
(Bell et al., 1990). In the present study, the optimal P
concentration in acid solution was 8 mM which is equivalent
to the level of micro-nutrients. Therefore, a constant, low P
concentration is necessary to avoid both toxicity and
depletion of the nutrient in solution culture; in practice
this is dicult to achieve. However, as stressed by Hinsinger
(2001), P concentrations in the micromolar range ( from 0.1
to a few mM) are most relevant to those found in the soil
solution in many soil environments, especially in strongly
P-®xing soils such as calcareous soils or highly weathered
soils from the tropics.
AC K N OW L E D GE M E N T S
We thank Denis Loisel and HeÂleÁne Payre for maintenance
of the solution experiment and ARA measurements, and
MichaeÈl Clairotte for plant analyses. The senior author also
thanks the Organisation for Economic Co-operation and
Development (OECD) for ®nancial support during his stay
at INRA in 1999 within the OECD Programme `Biological
Resource Management for Sustainable Agricultural
Systems'.
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