PHYSIOL. PLANT, 64: 19ft-l%. Copenhagen 1985
The role of phosphorus hi eltrogen fixation by young pea plants
{Pisum sativum)
Iver Jakobsen
Jakobsen, I. 1985. The role of pbosphorus in nitrogen fixatioti by young pea plants
{Pisum sativum). - Physio). Plant. 64: 190-196.
The influence of P on N, fixation and dry matter production of young pea (Pisum sativum L. cv. Bodil) plants grown in a soil-sand mixture was investigated in growth cabinet experiments. Nodule dry weight,, specific C^H^ reduction and P concentration in
shoots responded to P addition before any growth response could be observed. The P
concentration in nodules responded only slightly to P addition. A supply of P to P-deficient plants increased both the nodule dry weight, specific CjH, reduction and P
concentration iti shoots relatively faster than it increased shoot dry weight and P concentration in nodules. Combined N applied to plants when N2 fixation had commenced, increased shoot dry weight only at the highest P levels. This indicates that the
smaller plant growth at the low P levels did not result from N deficiency. The reduced
nodulation and N,fixationin P-defident plants seem to be caused by impaired shoot
metabolism and not by a direct effect of P deficiency of the nodules.
Additional key words - Acetylene reduction, phosphorus deficiency, Rhizobium leguminosarum.
Iver Jakobsen. Agricultural Research Dept, Ris0 National Laboratory, DK-4000 Roskitde, Denmark.
Introduction
Nitrogen fixation in legumes is affected by a range of
mineral nutrients, and it is important to establisti
whether a specific mitrient acts directly upon the nodules or indirectly via the host plant (Sprent and Minehin
1983). It is well established that the requirement of
nodule ftinetion for molybdenum and cobalt and probabiy also for calcium and copper exceeds requiretnents
elsewhere in the plant (Robson 1983), whereas the role
of phosphorus (P) is not fully understood.
In general, P deficiency reduces nodule biomass and
Njfixationrelatively more than plant growth. From this
it was inferred that the effeet of P on N2 fixation was a
direct one upon the nodules (Gates 1974, Mosse et al.
1976, Bonetti et al. 1984). Similar conclusions were
drawn from the observation that shoot N concentrations
often increase with increasing P applications (Andrew
and Robins 1969, Gates and Wilson 1974). However,,
Munns (1977) and Graham and Rosas (1979) suggested
that P deficiency limits Nj fixation mainly by reducing
the growth of the host plant. This view is supported by
observations of the early responses to P addition on
shoot growth, nodulation and Nj fixation in subterranean clover and of the nature of the interaction between
P and combined N on plant growth (Robson et al.
1981).
Nodule formation by pea plants responds strongly to
P addition (Diener 1950). The present work reports on
the role of P in nodule biomass production and Nj fixation in young pea plants. It is based on studies of
changes during recovery from P deficiency and of early
responses to P addition.
Abbreviation — ARA, acetylene reduction activity.
Received 13 November, 1984; revised 7 February, 1985
190
PhysioL Plant. (A, 1985
Materials and methods
General
The growth tnedium used was a 1:4 mixture (w/w) of
sieved (3 mm) clay soil and sand. The soil contained 15
mg P kg"' extractable with 0.5 M NaHCO, (Olsen et al.
1954). The pH of 5.5 (lO^^ M CaCy was adjusted to 7.1
by adding CaCOj. Samples with different P levels were
obtained by spraying a solution of NaH^PO^ on the surface of the soil-sand mixture that was spread in thin
layers. In addition to P, the solution contained nutrients
which were added to all treatments at the following
rates [mg (kg growth medium)"^]: KjSO4 (350),
MgSO, • 7HjO (500), MnSO^ • H,O (20), CuSO^ • 5HjO
(25), ZnSO4-7H2O (10), CoCl,-6H,O (5), H3BO3 (5)
and Na2MoO4-2H2O (5). After nutrient addition the
growth medium was allowed to dry, and was carefully
mixed. Irradiation (800 fcrad, 10 MeV electron beam)
eliminated fungal propagules forming unwanted vesieular-arbuscular mycorrhiza (Jakobsen and Andersen
1982). Either 500 g (Exp. 1) or 1000 g (Exps 2 and 3) of
the growth medium were put into 10 cm pots. Uniforrnly-sized seeds of Pisum sativum L. cv. Bodil were
surface sterilized in 70% ethanol and pre-germioated in
autoclaved vermiculite for 2-3 days. When the radicles
were 1-2.5 cm long, either 2 (Exps 1 and 3) or 3 (Exp.
2) seeds were planted 2 em below the surface of the
growth medium and eaeh seed was inoculated with 5 ml
of a three-day-old yeast mannitoi broth suspension culture of Rhizobium leguminosarum Ris0 strain la (about
10' cells ml"'). The pots were placed in a randomised
block design on trays in a growth eabinet with a 16/8 h
light/dark cycle at 20/14°C. A mixture of white fluorescent and incandescent lamps provided a photosynthetic photon flux density of approximately 450 |i.mol
m~' s~' at platit emergence level. Seedlings emerged
three days after planting and the pots were watered
daily as needed from below (Exp. 1) or from above
(Exps 2 ,and 3).
Experimental designs
Experiment 1. This experiment was designed to examine
the effect of various soil-P levels on dry matter production and N; fixation in young pea plants, and included
five levels of P additions: 0, 15, 30, 60 and 120 mg P (kg
growth medium)^'. Experiment 1 included ten replicate
pots of each P treatment, five of which were harvested
19 days, and the rest 24 days, after the seedlings emerged..
Experiment 2. This experitnent was designed to elucidate time course changes in dry matter production and
N, fixation during a period of recovery from P deficiency. Three levels of P addition were used: 0, 15 and
60 mg P (kg growth medium)"'; 54 pots were prepared
for each of the two lowest P levels and 30 for the highest
level. Six extra pots with both the low and the high P
PhysloL Plam. M, t985
level were included to monitor acetylene reduction activity (ARA) during early growth. Phosphorus deieiency was defined as a markedly lower ARA 15 days
after seedling emergence in plants given no P than in
plants witb the high P supply. At this time six replicate
pots were harvested from each of the three original P
levels, and half of the remaining pots from each of the
two lowest P levels then received the amoutit of
NaH2PO4-solution needed to reach a final addition of 60
mg P (kg growth medium)"'. The resulting five treatments are given in Tab. 3. Six replicates of all treatments were harvested 1, 2, 3 and 7 days later.
Experiment 3. The interaction between P and combined
N on the growth of nodulated peas was investigated in
this experiment. Five levels of P addition [0, 10, 20, 40
and 80 mg, P (kg growth medium)"'] were eombined
with two levels of combined N (0 and 96 mg N per pot as
NHjNOj). Each treatment had 5 replicates. The combined N was added in portions of 24 mg per pot nine
days after seedling emergence, when approximately 30
pink nodules were present on the main root, followed
by 12 mg per pot 3, 6, 8, 9,, 10 and 12 days later. Platits
were harvested 22 days after seedling emergence.
Measurements
At harvest the shoots were cut just above the cotyledons. The roots were gently washed from the soil, rapidly blotted dry with "Kleenex" tissues and incubated
in 500 ml jars where 10% of the air was replaced by
QH,. Nitrogen fixation was measured as ARA. Gas
samples (0.5 ml) for analysis were taken 10 and 20 min
(Exps 1 and 2) or 26 and 52 tain (Exp. 3) after the start
of incubation. The OR, content of the samples was
measured using a HP5730A gas chromatograph (Hewlett Packard), equipped with aflameionization detector
and stainless steel column containing Foropak T (8ft100 mesh). Nitrogen carrier gas flow was 40 ml min"'.
After ARA analysis, roots were stored frozen, and later
all red and pink nodules were removed. Dry weights of
shoots, roots and nodules were recorded after drying at
80°C for 24 h. In the last harvest of Exp. 2 and in Exp. 3,
nodules and roots were not separated. Phosphorus concentrations were measured in all plant parts after wet
digestion using the molybdate-blue method (Murphy
and Riley 1962), and N concentrations were assayed by
a Kjeldahi method. In Exp. 3, inorganic N in the growth
medium was determined in 2 M KCl extracts (Keeney
and Nelson 1982) of samples taken at the start of the experiment and at harvest. Means were compared by LSD
{P = 0.05) when the F test showed significant treatment
effects.
191
Tab. 1. Dry weights (DW) of 19- and 24-day-old pea plants grown at different levels of P addition, ns, non-significant; d, days
(Exp. 1).
DW (g pot - )
P addition
(mg kg"')
Nodales
Shoots
0
15
30
60
120
t^D (0.05)
Roots
19 d
24 d
19 d
24 d
19 d
24 d
0.80
0.79
0.79
0.83
0.78
1.14
1.31
1.31
1.48
1.33
0.03
0.04
0.05
0.06
0.05
0.03
0.05
0.05
0.08
0.07
0.51
0.52
0.46
0.43
0.44
0.67
0.62
0.62
0.61
0.51
ns
0.20
0.01
0.01
ns
0.08
Results
400 r
Expertment 1
The shoot dry weights did not respond to increasing P
additions until 24 days after seedling emergence,
whereas nodule dry weights showed marked responses
at both harvests (Tab. 1). In contrast, the root dry
weights at the second harvest were significantly smaller
at the highest level of P addition (Tab. 1). Total dry
weights were therefore unaffected.
Acetylene reduction activity responded significantly
to P addition at both harvests (Fig. la) in accordance
with the increased nodule dry weights.. The specific nodule activity also responded to P addition (Fig. lb). Nitrogen concentrations in shoots were significantly lower
when P was deficient than at higher P levels, and similar
trends were found in roots and tiodules (Tab. 2). Nodule
P concentrations at the first harvest varied from 4.89 to
6.83 mg P (g dry weigh!)"' at the lowest and highest P
level, respeetively, whereas the corresponding shoot-P
concentrations varied three fold [1.66 to 4,93 mg P (g
dry weight)"', data not presented]. Similar results were
obtained at the second harvest.
At the time when extra P was added, ARA was much
lower when P was deficient than at the highest level
(Fig. 2, day 0), whereas shoot dry weights were not significantly affected by the initial P level (Tab. 3). Acetylene reduction activity was increased as early as one day
after the supply of additional P. This increase was signifieant only in the treatment without initial P addition,
but after one additional day, ARA had increased to the
levels observed with the initially high P addition (Fig.
2). In contrast, significant shoot growth responses were
not observed until two days after additional P was supplied (Tab. 3). Growth ol plants receiving 15 mg P kg"'
initially and supplied with additional P was similar to
that observed for plants receiving 60 mg P kg"' from the
start. Plants without initial P addition, on the other
192
0 15 30
50
120
P ADDITION
mg !kg growth mediumr'
Fig. 1. ARA (a) and specific ARA (b) in 19-day ( • ) ,an_d 24day-old ( • ) pea plants grown at different levels of P addition.
Bars indicate LSD (0.05) (Exp. 1).
Physio!. Planl. 64, 1985
Tab. 2. N concentrations in dry weights of shoots, roots and nodules of 19- and 24-day-old pea piants grown at different levels of P
addition. Nodules from replicate pots were pooled before analysis, n.d., not determined; ns, non-significant; d, days (Exp. 1).
P addition
(mg kg^')
N concentration (%)
Shoots
Roots
Nodules
19 d
24 d
19 d
24 d
19 d
24 d
60'
120
3.11
3.58
3.77
3.96
4.07
3.30
3.73
4.39
4.56
4.'63
2.53
2.56
2.63
2.66
2.79
2.42
2.59
2.64
2.77
2.92
6.94
7.40
7.26
7.26
7,.32
LSD (0.05)
0.46
0.27
0.22
n.d.
7.32
7.40
7.83
7.92
8.05
n.d.
0
15
30
500 r
hand, remained stunted during the period of measurement (Tab. 3). This eontrasts with the high A R A in the
latter treattnent 7 days after supplying additional P (Fig.
2). Nodule dry weights and specific A R A were also increased after the supply of additional P to the P deficient treatments; however, these increases were not
significant until the second day (Fig. 3, Tab. 4).
Phosphorus concentrations in shoots were markedly
lower in P-defieient plants than in those initially supplied with sufficient P (Fig. 4, day 0). Only one day after
the addition of P, P concentrations in shoots had nearly
doubled.. By the third day the P 'Concentrations were
higher than in plants witli an initially sufficient P supply
(Fig. 4). Root P concentrations responded in a similar
manner but with a slower rate of change (data not presented). In contrast, the P concentrations in nodules responded more slowly to the P treatments and were unaffected until two days after the supply of additional P
(Tab. 5).
500
•E 400
r 300
E
S 200
100
0 L
0
1
2
3
7
DAYS AFTER ADDITIONAL P SUPPLY
Fig. 2. ARA in pea plants at various times after snpply of additional P to pots with the two lower of three initial P levels. Bars
indicate LSD (0.05) at each harvest. O, 0 mg P kg^'; • , 0 + 60
kg-(Exp! 2).'
Tab. 3. Shoot dry weights (DW) of pea piants at various times
after supply of additional P to pots with the two lower of three
initial P levels (Exp. 2). ns, non-significant.
P addition
(mg kg-')
Shoot DW (10 mg pot^')
Days after additional P supply
Initial
0
15
60
After
15 days
0
60
0
45
0
LSD (0.05)
Physiol. PJaiil . 54, 1985
0
.1
2
3
7
78
81
82
86
89
88
ns
87
100'
93
103
99
8
93
106
95
113
111
11
136
154
148
184
186
16
8S
86
ns
0
1
2
3
DAYS AFTER ADDITIONAL P SUPPLY
Fig. 3. Nodnle dry weights at various times after supply of additional P to pots with the two lower of three initial P levels.
Bars indicate LSD (0.05) at each harvest. O, 0 mg P kg"'; • , 0
+ 60 mg F kg-; V, 15 mgP kg"'; • , 15 + 45 mgP kg-; Q, 60'
mg P k g - (Exp. 2).
'.
193
Tab. 4. Specific ARA in pea plants at various times after supply of additional P to pots with tiae two lower of three initial P
levels (Exp. 2). ns, non-significaBt.
P addition
(mgkg-')
300
-
Speeific ARA, nmot
(mg nodule DW)-' min-'
Days after additional P supply
15 days
0
1
2
3
4.9
5.4
5.1
5.4
4.7
ns
4.0
8.1
3.6
6.7
5.5
1.3
4.2
5.8
4.5
6.4
5.2
0.9
n
^
0
60
3.9
IS
"
45
0
5.0
60
LSD (0.05)
6.5
1.5
Experiment 3
As in Exp. 2, ARA was markedly lower under P deficiency and addition of combined N reduced ARA by
60-70% at all P levels (Fig. 5a). Initially there were only
6 mg of inorganic N (kg of growth medium)^' without an
0 10 20
iO
80
P ADDITION, mg (kg growth mediuml"'
Fig. 5. ARA (a), shoot dry weight (b) and shoot N concentration (c) in 22~day-old pea plants grown without (O) or
with ( • ) combined N at five levels of P addition. Bars indicate
LSD (0.05) (Exp. 3).
7
DAYS AFTER ADDITIONAL P SUPPLY
Fig. 4. P concentrations in shoots of pea plants at various times
after supply of additional P to pots with the two lower of the
three iaitial P ieveis. Bars indicate LSD (0.05) at each harvest.
O,0mgPltg-';», 0 + 60mgPkg-'; V, lSmgPkg''; ^ , 1 5 +
45 mg P kg-'; D, 60 mg P kg-" (Exp. 2).
Tab. 5. P concentrations in root nodules at various times after
suppiy of additional P to pots with the two lower of three initial
P levels. Nodules from replicate pots were pooled before analysis (Exp. 2).
Nodule-P concentration, mg (g DW) '
P addition
(mgkg-')
Days after additional P suppiy
Initial
0
15
60
194
After •
15 days
0
60
0
45
0
0
1
2
3
6.42
6.35
6.10
6.93
6.03
7.23
6.78
7.14
7.79
6.15
8.18
6.97
7.94
7.88
6.73
•7.25
6.26
7.95
additional N supply. At harvest this was reduced to
about 3 mg N kg'', in contrast to about 40' mg N kg'' in
the N-amended treatments. Although some mineralisation might have occurred during the growth period,
inorganic N from the growth medium would thus have
contributed only slightly to the finai N content in the
plants.
The results from this experiment indicate a positive
Tab. 6. Analysis of variance for ARA, shoot dry weights (DW)
and sboot-N coneentrations in Exp. 3. *** P<0.00]; **
POOI
Souree of
variation
Phosphorus
Combined N
PxN
Error
Mean squares
df
ARA
4
1
3
35
47500"*
202100**'
18040**
3550
Shoot DW Shoot-N cone.
0.944"*
0.063
0.026
0,018
1.518*"
1.911***
0.528**'
0.034
Phwiot,. Planl. 64. t985
although noe-signi£icant interaction between the addition of P and combined N on shoot dry matter production, which was unaffected by combined N at the low P
levels but increased at the high levels (Fig. 5b, Tab. 6).
Shoot N concentrations were significantly increased
by addition of both P and combined N (Fig. 5c, Tab. 6).
However,, the effect of P was small in plants receiving
combined N and this resulted in a significant negative
interaction between N and P on shoot N concentration.
Discussion
The present work with pea indicates that P deficiency
impairs N, fixation in young pea plants indirectly by impairing the metabolism of the shoots and not by direct
action on nodule formation or function.
Phosphorous concentrations in nodules were largely
unaffected by the P supply (Tab. 5). Initially, P concentrations in shoots were much lower than concentrations in nodules at all levels of P supply (Fig, 4). Phosphorus concentrations in shoots increased rapidly after
iP was supplied to P starved plants, but were still similar
to, or slightly less than, concentrations in nodules. An
"overshoot" observed after three days (Fig. 4) was also
obtained in transfer experiments with barley and tomato (Ciarkson and Scattergood 1982). Nutrient concentrations in an organ depend on its sink strength for a
giveo nutrient. Sink strength of nodules for P is high,
probably because nitrogenase has a very high demand
for ATP and because the P concentration in raicrobial
tissue is higher than in plant cells. Thus, P concentration
in nodules is always higher than in other plant organs,
especially when P availability is low.
Before the supply of additional P,, ARA was markedly lower when P was deficient than at the highest P
level (Fig. 2). During the first two days after P supply,
ARA increased rapidly and it appears that N, fixation
was somehow affected by the shoot P concentrations.
Nitrogen fixation has a high sink strength for reduced C,
whose supply is limited by the availability of N and P to
the chloroplasts. The three nutrients together thus form
an autocatalytic cycle, each enhancing productivity mutually: the more P to the chloroplast (shoot in this
study), the more C to the nodule; the more N to the
chloroplast, the more C to the nodule, etc. The supply
of reduced C is one of the principal factors limiting Nj
fixation (Mahon 1983). Photosynthesis, the source of
reduced C, is clearly influenced by the P supply: The
onset of P deficiency in the tropical legume Macroptilium atropurpureum increased the stomatal resistance
and decreased the activity of ribulose-bisphosphate carboxylase (Clarkson et al. 1983), which is rate limiting
for CO2 fixation. Moreover, in a P depletion-recovery
experiment with subterranean clover, even moderate
levels of P deficiency depressed net photosynthesis,
which increased to normal levels within 2-3 days after
the supply of P (Bouma 1967).
Although Exp. 3 showed a highly significant negative
Physiol. Piaol. 64, WS5
interaction between P and combined N on shoot N concentration (Fig. 5c, Tab. 6), shoot growth at the low P
levels was unaffected by combined N and hence not limited by N deficiency. If the requirement of Nj fixation
for P had exceeded the requiremeots elsewhere in the
plant, the interaction between P and combined N on
shoot growth would have been negative, as was the case
between combined N and molybdenum (Anderson and
Spencer 1950) and cobait (Delwiche et al. 1961).
It now appears iikeiy that P deficiency impairs N; fixation as the consequence of shoot P deficiency, but the
results may also be interpreted in other ways. The observation of decreased ARA in P-defident plants prior
to shoot-growth changes (Tabs 1 and 3,, Figs la and 2)
may indicate a specific involvement of P in N,fixationas
suggested by Robson (1983). However, reduced growth
may be regarded as a delayed phenotypic expression of
complex metabolic changes, whereas ARA is a direct
measure of enzymatic activity. Therefore, a decreased
pool of photosynthates caused by P deficiency would
probably limit Nj fixation and, consequently, ARA before shoot-growth changes could be detected. To decide
this question, photosynthetic rates should be measured
simultaneously with ARA.
Shoot N concentrations in plants dependent on Nj
fixation increased with increasing levels of P in the
growth medium (Tab. 2, Fig. 5c)., The absence of this relationship in plants supplied with combined N (Fig. 5c)
suggests that it was neither caused by effects on N metabolism within the plant (Robson et al. 1981) nor by
varying proportions of N-poor to N-rich structural tissues (Munns 1977). Probably the relationship simply resulted from a relatively larger increase in specific nodule
activity than in shoot dry weights with increasing P supply. Increasing the application of P above that required
for maximum dry matter production increased the N
concentrations slightly in plants from Exp. 1 (Tabs 1 and
2). Similar results obtained with tropical legumes by
Andrew and Robins (1969) and Zaroug and Munns
(1979) have been explained as indicating that nodule
function requires more P than plant growth in general
(Smith 1982). However, the evidence is rather weak, as
over-optimal P leveis often result io slightly reduced
shoot dry weights (e,.g. Exp. !, Tab. ,1). Consequently,
the shoot-N content was increased neither in Exp. 1 nor
in the work by Zaroug and Munns (1979),, and in the
work by Andrew and Robins (1969) it was increased
only in three of 11 species investigated.
Acknowledgements -1 thank A. Olsen and A. Sillesen for their
able technical assistance.
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