Tree Physiology 19, 707--715
© 1999 Heron Publishing----Victoria, Canada
Photosynthetic responses to phosphorus nutrition in two-year-old
maritime pine seedlings
DENIS LOUSTAU,1 MOHAMED BEN BRAHIM,1 JEAN-PIERRE GAUDILLÈRE2 and ERWIN
DREYER3
1
INRA, Centre de Bordeaux, Station de Recherches Forestières, BP 45, 33611 Gazinet, France
2
INRA, Centre de Bordeaux, Station d’Agronomie, BP 81, 33883 Villenave d’Ornon, France
3
INRA, Centre de Nancy, Unité d’Ecophysiologie Forestière, BP 35, 54230 Champenoux, France
Received August 5, 1998
Summary We analyzed processes limiting photosynthesis in
two-year-old, container-grown Pinus pinaster Ait. seedlings
subjected to phosphorus (P) deficiency. After withholding P for
3 months, seedlings were supplied P at four relative addition
rates (0, 0.005, 0.01 and 0.02 day −1) in a nutrient recycling
system. At Weeks 12 and 22, responses of photosynthesis to
CO2 and irradiance were measured and the following parameters derived: maximal velocity of carboxylation by Rubisco,
Vm; apparent quantum efficiency of electron transport, α; maximal electron transport rate, Jm; stomatal conductance and relative stomatal limitation of photosynthesis. At Week 22, these
measurements were combined with concurrent measurements
of chlorophyll fluorescence to determine the quantum yield of
PSII, and a theoretical partitioning of total light-driven linear
electron flow between fractions used to regenerate carboxylated and oxygenated RuBP. After 12 weeks of treatment,
needle P concentrations ranged from 0.04 to 0.15 × 10 −2 g
gDW−1, and then remained constant until Week 22. Values of Jm,
α and Vm increased with increasing needle P concentration
(from 30 to 133 µmol m −2 s −1, 0.02 to 0.25 mol mol −1 and 13
to 78 µmol CO2 m −2 s −1 at the lowest and highest needle P
concentrations, respectively). Under ambient conditions, net
assimilation rates in P-deficient seedlings were limited by Vm
under saturating irradiance, and by Jm under limiting irradiance, but not by triose-P regeneration. There was no detectable
change in the partitioning of total light-driven linear electron
flow between the fractions used for carboxylation and oxygenation. Predawn photochemical efficiency of PSII was significantly reduced in seedlings with low P concentrations.
Although stomatal conductance tended to decrease with decreasing needle P concentration, relative stomatal limitation
was not significantly affected. At Week 22, there was an attenuation of the effects of P nutrition on Vm and an increase in α
and Jm that was probably related to cessation of growth and the
seasonal decline in natural irradiance.
Keywords: carboxylation velocity, chlorophyll fluorescence,
electron transport rate, phosphate, photosynthesis, Pinus pinaster, quantum efficiency, stomatal conductance.
Introduction
In higher plants, phosphorus (P) deficiency has been reported
to affect photosynthesis through reduced thylakoidal (Conroy
et al. 1986, Lauer et al. 1989) and stromal processes (Brooks
1986, Sivak and Walker 1986). Short-term modifications of
foliar phosphate concentration through Pi sequestering agents
or by feeding isolated leaves with varying amounts of Pi
generally result in dramatic effects on photosynthesis. In particular, foliar Pi deficiency results in O2-insensitive photosynthesis and the occurrence of high transient rates of
carboxylation when abrupt changes in O2 concentration are
imposed (Leegood and Furbank 1986, Sharkey et al. 1986), in
thylakoid energization (Heineke et al. 1989), and in rapid
changes in Calvin cycle metabolite pools. The extent to which
photosynthesis is limited by stomatal, thylakoid or stromal
effects during longer term P deficiency (weeks to months),
remains a matter of debate. For instance, the apparent quantum
yield of CO2 assimilation was affected by P deficiency in Pinus
radiata D. Don (Conroy et al. 1986) and spinach (Brooks
1986), whereas it remained unchanged in sugar beet (Abadia
et al. 1987). Similarly, maximal carboxylation velocity in
response to P deficiency decreased systematically in C3 plants
such as spinach, wheat and sunflower (Parry et al. 1985,
Brooks 1986, Jacob and Lawlor 1991) but not in C4 plants,
such as sugar cane or maize (Rao and Terry 1989, Jacob and
Lawlor 1992). In addition, a limitation of photosynthesis at
saturating light by the triose-phosphate utilization rate was
demonstrated in experiments in a CO2-enriched atmosphere,
but its occurrence after long-term acclimation to P deficiency
at ambient CO2 concentration is unlikely (Harley et al. 1992,
Lewis et al. 1994).
Little is known about long-term effects of differential P
supply on photosynthesis or possible acclimation to limiting P
supply. Because photosynthesis is an integrated process, some
coupling between the various limitations induced by P restriction should occur, and some degree of acclimation to phosphate availability can be expected. This question is particularly
relevant for long-lived forest trees that have experienced rela-
708
LOUSTAU, BEN BRAHIM, GAUDILLÈRE AND DREYER
tively stable nutrient availability until now, but may be exposed
to changes in atmospheric conditions in the near future.
We investigated the effects of several relative addition rates
of P (0, 0.005, 0.01 and 0.02 day−1) on gas exchange and
chlorophyll a fluorescence of 2-year-old seedlings of maritime
pine grown under ambient conditions in a greenhouse. Net
carbon uptake per seedling was more severely affected by P
deficiency than expected solely from the reduction in photosynthetic surface area. We used the model introduced by Farquhar et al. (1980) to analyze the effects of P supply on
photosynthesis.
Materials and methods
Plant material
In March 1995, 56 fifteen-month-old Pinus pinaster Ait. seedlings originating from the northern part of the species’ natural
range were transplanted to 4 dm3 pots containing perlite. The
seedlings had been raised from seed in 0.5 dm3 pots containing
a mixture of sand and peat and fertilized with slow-release
complete nutrient mixture under standard INRA nursery conditions. The transplanted seedlings were grown in a greenhouse equipped with a cooling system to maintain the
temperature close to ambient. Seedlings were illuminated by
natural light and continuously irrigated with nutrient solution
that was collected at the base of the pots and recirculated by an
immersed pump. Fourteen seedlings were assigned to each of
four independent circulating units. Each unit contained 12 dm3
of nutrient solution maintained at a constant volume by an
automated watering system. The locations of the circulating
units were periodically changed to avoid edge and location
effects.
During the first three months following transplanting
(March to June), seedlings were irrigated with a complete
nutrient solution without phosphate (Nylund and Wallander
1989). In June, carbon and phosphorus concentrations of the
seedlings were determined by destructive analysis of a subsample of 12 randomly selected individuals. During the second
part of the growth period, from July (Week 0) to October
(Week 16), the same nutrient solution was used with phosphorus added to provide relative addition rates (RAR) of 0.0,
0.005, 0.01 and 0.02 day −1. The RAR regimes were applied as
described by Ingestad and Lund (1986). Each RAR was applied to a single circulating unit. Relative growth rate was
assumed to equal the relative addition rate and to remain
constant throughout the growing period. After Week 16,
growth ceased and seedlings were irrigated with a P-free nutrient solution until the end of the experiment.
Gas exchange measurements
During Weeks 11--13 (September) and 21--23 (December),
three seedlings from each treatment were randomly chosen for
gas-exchange and chlorophyll a fluorescence measurements.
After overnight acclimation at room temperature, one CO2 and
one irradiance response curve of net CO2 assimilation rate and
stomatal conductance were made on a sample of five to ten
pairs of fully expanded needles per seedling. The needles were
arranged horizontally in a Plexiglas cuvette covered with a
glass lid connected to an open gas-exchange system with
environmental control (Compact Minicuvette System, Walz,
Effeltrich, Germany). A differential H2O and CO2 gas analyzer
measured the H2O and CO2 concentration difference between
the analysis and reference circuits. Needles were illuminated
unilaterally by an optic fiber illuminator (Fiber illuminator
FL-400, Walz) composed of 200 parallel optic fibers applied to
the glass lid of the cuvette, orthogonal to the needle surface.
Gas exchange was calculated on an irradiated area basis assuming a semi-cylindrical needle shape. The plane face of the
half-cylinders was exposed to illumination. Net CO2 assimilation rate (A), transpiration (E), stomatal conductance for water
vapor (gw), and for CO2 (gc) and substomatal CO2 mole fraction
(ci) were calculated according to von Caemmerer and Farquhar
(1981).
During both measurement periods, CO2 response curves
were determined in saturating light (Q = 1500 µmol m −2 s −1 at
the needle surface), at constant temperature and relative humidity (T = 21 °C, RH = 70%) during the first measurement
period and 25 °C during the second measurement period. The
Q response curves were determined under the same environmental conditions at a CO2 concentration of 360 µmol mol −1.
At Week 12, one irradiance response curve per seedling was
also made under non-photorespiratory conditions (O2 = 0.02
mol mol −1). In addition, at Week 12, the maximal rate of
photosynthesis (Amax ) was measured at saturating incident
light (Q = 1500 µmol m −2 s −1) and CO2 = 1500 µmol mol −1
with 0.21 mol mol −1 O2.
Fluorescence measurements
During Week 22, chlorophyll a fluorescence was measured
with a modulated fluorometer (PAM-2000, Walz). Predawn
photochemical efficiency of PSII (the ratio of variable to maximal fluorescence, Fv /Fm) was measured on fully expanded
dark-acclimated needles of the entire population of seedlings
(n = 56, three measurements per plant). Chlorophyll fluorescence was also recorded concurrently with gas exchange. The
fluorometer fiber optics was inserted in the chamber through a
hole 2.5 cm above the needles, taking care to avoid shading the
needles when the lighting unit was switched on. The Fv /Fm
ratio was recorded before the start of illumination. For each
plant, a light response curve was first made under non-photorespiratory conditions (900 µmol mol −1 CO2 and 0.01 mol
mol −1 O2) to calibrate the individual relationship between
photochemical efficiency of PSII, ΦII (computed as ∆F/Fm′,
Genty et al. 1989), and apparent quantum yield of linear light
driven electron flux (computed as ΦCO 2 = (A + Rd)/Q). The
linear relationship was adjusted as (Valentini et al. 1995, Roupsard et al. 1996):
ΦII = kΦCO2 + b.
(1)
The value of b differed from the expected value of 0 because
of needle geometry (see Discussion).
In a second step, an A--ci response curve was determined
under photorespiratory conditions (0.21 mol mol −1 O2) and the
TREE PHYSIOLOGY VOLUME 19, 1999
PHOSPHORUS EFFECTS ON MARITIME PINE PHOTOSYNTHESIS
calibration coefficient obtained for each plant was used to
calculate total light-driven electron flow (JT) as:
JT = ((ΦII − b)/k)Q .
(2)
Parameter JT was further split into fractions devoted to regeneration of carboxylated (Jc) and oxygenated (Jo) RuBP, as
described in Peterson (1989) and Valentini et al. (1995):
Jc = 1/3[JT + 8(A + Rd)]
(3)
Jo = 2/3[JT − 4(A + Rd )].
(4)
These values were used to estimate the CO2 concentration in
the chloroplast (cc) (Laing et al. 1974):
Jc /Jo = Vc /Vo = Scc /oc,
Maximal velocity of carboxylation (Vm), observed maximal
electron transport rate (Jm), dark respiration (Rd) and apparent
quantum efficiency of electron transport (α) were determined
from the response curves for each seedling. As a first approximation, we assumed that chloroplastic (cc) and substomatal
(ci) CO2 mole fractions were equal within the needle. Based on
the formulation of photosynthesis proposed by Farquhar et al.
(1980) and subsequent authors (Harley et al. 1992, Lewis et al.
1994), Vm and Rd were estimated by nonlinear regressions from
the data of the A--ci curve for 0 < ci < 200 µmol mol −1 according
to the model:
Vm
−R ,
[O2 ]  d

c i + K c 1 +
Ko 

(6)
where Γ* is the photorespiratory CO2 compensation point
representing:
Γ∗ =
K c νo max [O2 ]
,
Ko ν cmax 2
Jm =
(Amax + Rd)4(ci + 2Γ∗ )
.
(c i − Γ ∗ )
(7)
where Kc (332 µmol mol−1) and Ko (271 mmol mol −1) are the
Michaelis constants of Rubisco for carboxylation and oxygenation, respectively, derived from Harley et al. (1992) and
corrected for temperature dependency according to Farquhar
et al. (1980) and Harley et al. (1992). The value of the ratio of
oxygenase to carboxylase maximal velocities, νomax /νcmax , was
fixed at 0.21 (Harley et al. 1985). This led to a Γ* value of 27
(8)
The apparent quantum efficiency of electron transport, α, was
estimated by regression from the initial part of each A--Q curve
(Q < 500 µmol m −2 s −1) by least squares adjustment to the
nonlinear equation:
A = (ci − Γ∗ )
Calculation of photosynthesis parameters
A = ci − Γ∗


µmol mol −1 at 21 °C, and a gas phase specificity coefficient of
Rubisco of around 3890 and 3320 at 21 and 25 °C, respectively
(i.e., 132 and 118 when expressed with reference to the liquid
phase). These values, although frequently used by modelers,
are slightly above the recently published range of values for C3
plants (close to 80--90 at 25 °C in the liquid phase; Kane et al.
1994, Balaguer et al. 1996).
We estimated Jm from Amax as:
(5)
where S is the specificity factor of Rubisco (taken as 2970 at
25 °C; gas phase value is equivalent to 105 in the liquid phase
of the chloroplast stroma at 25 °C); Vc and Vo are rates of RuBP
carboxylation and oxygenation by Rubisco, respectively; cc
and oc are gas phase balance values of CO2 and O2 concentrations in the chloroplast stroma, respectively, and oc is assumed
to equal 0.21 mol mol −1.
709
J
− Rd,
4(ci + 2Γ∗)
(9)
where J, the electron transport rate, was expressed according
to Farquhar and Wong (1984) as the smaller root of:
θ J2 − (Jm + αQ )J + αQJm = 0.
(10)
The convexity factor, θ, was fixed at 0.65. The procedure was
applied separately to the curves obtained at ambient and low
O2 concentrations. Because similar values were obtained at
both O2 concentrations, only those computed under low O2
concentrations are presented.
Relative stomatal limitation of net assimilation in ambient
CO2 (RSL) was calculated from the A--ci curve as (Farquhar
and Sharkey 1982):
RSL =
A360 − Aci
A360
.
(11)
Needle analysis
Immediately after the gas exchange measurements, specific
leaf area (SLA), and N, P, and total chlorophyll concentrations
of the measured needles were determined as described by Ben
Brahim et al. (1996).
Statistical analysis
Effects of the four relative addition rates of P were analyzed by
standard ANOVA and Student Newman Keuls for means comparison, with a first-order risk of 5%. Each seedling was
treated as a replicate. Because all replicates of each RAR were
in the same circulating unit, the unit effect could not be estimated. Some data were analyzed by covariance analysis with
phosphorus concentration as the independent continuous variable and time of measurement (Week 12 or 22) as a discrete
variable. The model used for each independent variable was
derived from:
Y = intercept + (a + bweek )P + cweek + e,
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(12)
710
LOUSTAU, BEN BRAHIM, GAUDILLÈRE AND DREYER
where Y is the independent variable, a is the mean linear effect
of P, bweek is the interaction term, cweek is the time effect and e
is the residual error. All analyses and least squares adjustments
were made with the SAS.7X system software package for Unix
(SAS Inc., Cary, NC).
Results
Needle P concentration differed significantly among the four
RAR regimes, ranging from 0.036 × 10 −2 to 0.160 × 10 −2 g g −1
(Table 1). The RAR regimes had no effects on nitrogen and
chlorophyll concentrations or specific leaf area (SLA). Nitrogen concentration, and, to a lesser extent, phosphorus concentration increased and SLA decreased between Weeks 12 and 22
as growth rate declined.
The response of net assimilation rate (A) to substomatal CO2
mole fraction (ci) at saturating irradiance was strongly affected
by the four RAR regimes (Figure 1). The light response of A
was similarly affected by the RAR treatments (Figure 2). At all
RARs, photosynthesis measured in ambient CO2 remained
oxygen sensitive.
The effects of needle P concentration on several photosynthetic parameters are presented in Figure 3, and were further
analyzed by Equation 12. The model explained 75, 77 and 88%
of the variance of maximal carboxylation velocity (Vm ), maximal electron transport rate (Jm ) and apparent quantum efficiency of electron transport (α), respectively (Table 2). Values
of α were significantly higher at Week 22 than at Week 12.
There was no interaction of time on the phosphorus effects
(regression slopes in Figure 3) on α and Jm; however, the effect
of phosphorus on Vm was significantly attenuated at Week 22
as indicated by the change in the Vm versus P regression slope
between Weeks 11 and 22. There was no significant effect of
RAR of P on stomatal conductance or relative stomatal limitation of photosynthesis at either measurement time (Table 3).
Values of Amax increased with increasing RAR of P (Table 3). Values of Amax were much higher than A measured at
ambient CO2 and saturating light, and were closely correlated
with needle P concentration (r 2 = 0.83, regression not shown).
Figure 1. Responses of net assimilation (A) to substomatal CO2 mole
fraction (ci ) measured in Pinus pinaster seedlings after 12 weeks at
four relative addition rates (RAR) of P (n = 4 × 3 seedlings). Each point
is a discrete measurement. (n: RAR = 0.02, h: RAR = 0.01, e: RAR
= 0.005, s: RAR = 0 day − 1). Lines indicate the average of the curves
fitted according to Equation 6 for each treatment, between 0 and 400
µmol mol − 1 internal CO2 concentration. All measurements were made
at 21 °C, an O2 concentration of 0.21 mol mol − 1, and Q = 1500 µmol
m − 2 s − 1.
Maximal photochemical efficiency of PSII (estimated from
Fv /Fm, measured at predawn on dark adapted needles at Week
22) was reduced in seedlings in the RAR = 0 treatment and
close to optimum in the other RAR treatments (Table 4). Base
fluorescence (Fo) was higher and light-saturated fluorescence
(Fm ) lower in seedlings in the RAR = 0 regime than in seedlings in the other regimes. Values of Fv /Fm approached the
expected maximal value of 0.85 (Björkman and DemmigAdams 1994) when measured on the entire population in the
three higher RAR regimes, and slightly lower when measured
in the 12-needle samples used for gas exchange measurements.
In light-adaptated needles, ΦII decreased with increasing Q
(Figure 4). The decrease was steeper and lower values of Φ
were reached at low P-addition rates than at higher P-addition
rates, indicating lower light-driven electron flows at similar
irradiance.
Table 1. Effects of P relative addition rate on mean values (± SE, n = 3) of needle nitrogen, phosphorus and chlorophyll (Chl) concentrations and
specific leaf area (SLA) of Pinus pinaster seedlings used for gas exchange measurements. Values annotated with different letters are significantly
different.
Week of
Parameter
P Relative addition rate (day −1)
measurement
0.0
0.005
0.01
0.02
12
N (% DW)
P (g m −2)
P (% DW)
Chl (µg gFW− 1)
SLA (m2 kg −1)
2.03 ± 0.05
0.034 ± 0.003 a
0.035 ± 0.001 a
928 ± 68
11.9 ± 0.41
1.73 ± 0.13
0.041 ± 0.007 a
0.05 ± 0.007 b
1065 ± 60
12.5 ± 0.227
2.09 ± 0.17
0.062 ± 0.014 b
0.077 ± 0.008 c
808 ± 45
12.2 ± 0.782
2.05 ± 0.145
0.089 ± 0.002 c
0.12 ± 0.006 c
1014 ± 60
13.1 ± 0.83
22
N (% DW)
P (g m −2)
P (% DW)
Chl (µg gFW− 1)
SLA (m2 kg −1)
2.53 ± 0.29
0.038 ± 0.001 a
0.047 ± 0.003 a
786 ± 52
9.9 ± 0.22
2.77 ± 0.5
0.059 ± 0.003 a
0.06 ± 0.002 a
1606 ± 163
10.9 ± 0.2
2.62 ± 0.35
0.103 ± 0.009 b
0.11 ± 0.004 bc
1098 ± 99
10.7 ± 0.58
2.42
0 .12
0.13
1316
10.8
TREE PHYSIOLOGY VOLUME 19, 1999
± 0.19
± 0.014 b
± 0.013 c
± 137
± 0.16
PHOSPHORUS EFFECTS ON MARITIME PINE PHOTOSYNTHESIS
Figure 2. Responses of net assimilation (A) to irradiance (Q) at 0.21
mol mol − 1 (open symbols, dotted lines) or 0.02 mol mol − 1 O2 (closed
symbols, full lines) measured in Pinus pinaster seedlings after 12
weeks at four relative addition rates (RAR) of P (n = 4 × 3 seedlings).
Each point is an individual measurement and curves indicate the
average of the curves fitted according to Equation 9 for each treatment
at two O2 concentrations. All measurements were made at 21 °C, and
a CO2 concentration of 360 µmol mol −1 (n: RAR = 0.02, h: RAR =
0.01, e: RAR = 0.005, s: RAR = 0 day −1).
The relationship between ΦII and apparent quantum yield of
CO2 fixation measured under non-photorespiratory conditions
(ΦCO 2) was almost linear (Figure 5) but differed slightly among
treatments, diverging from linearity at the highest (low irradiance) and lowest (high irradiance) efficiencies. After discarding these points, we obtained the intercepts and slopes of the
linear regressions for each of the treatments (n = 3) (Table 5).
The y-intercept was close to 0. The calibration curve for each
seedling was used to compute total linear electron flows (JT)
and chloroplast concentrations (cc) (Figure 6).
A comparison of the A--cc curves with the corresponding
A--ci curves (Figure 6) indicated that the computed values of cc
were much lower than those of ci and remained below 500
µmol mol −1 even at the highest atmospheric CO2 concentrations. As a result, data were less scattered and higher correlation coefficients were detected. The RAR treatments caused
different responses of A to cc. Calculation of approximate
maximal carboxylation efficiencies from the initial slope of the
A--cc relationship yielded values close to 0.70, 0.60, 0.50 and
0.27 µmol m −2 s −1 Pa −1 at RARs of P of 0.02, 0.01, 0.005 and
0 day −1, respectively.
Discussion
The estimated optimal P concentration for the growth of twoyear-old maritime pine seedlings is about 0.2 g gDW−1 (authors’
unpublished results). In our experiment, the application of four
RARs of P led to lower P concentrations than those previously
obtained with a flowing nutrient solution system (Ben Brahim
711
Figure 3. Relationship between apparent quantum efficiency of electron transfer, α, maximal observed electron transport rate, Jm, maximal velocity of carboxylation, Vm, and needle P concentration in Pinus
pinaster seedlings subjected to four RARs of P for 12 or 22 weeks (n:
RAR = 0.02, h: RAR = 0.01, e: RAR = 0.005, s: RAR = 0 day −1).
Regression lines are shown on each graph.
et al. 1996). Thus, our RAR = 0 treatment corresponds to
severe P deficiency, whereas the highest RAR of P (RAR =
0.02) is above the optimal concentration for P nutrition.
Under ambient conditions, P nutrition affected photosynthesis mainly through biochemical limitations. Although stomatal
conductance tended to be lower in seedlings in the low RAR
of P treatments, we observed no increase in relative stomatal
limitation of photosynthesis. We suggest that the low stomatal
conductance values reflected a feedback adjustment to photosynthesis rather than a direct effect of P deficiency. Contradictory results have been reported on this point. Thus, stomatal
limitations are usually affected by P nutrition in long-term
experiments (Kirschbaum and Tompkins 1990, Jacob and
Lawlor 1991, Lewis et al. 1994) and only slightly or not at all
in short-term experiments (Freeden et al. 1990). The main
effects of P deficiency that we observed after 12 weeks of
treatment included decreases in: (i) maximal carboxylation
velocity (Vm ); (ii) maximal electron transport rate (Jm ); and
(iii) apparent quantum yield of linear electron transport (α).
The magnitude of the decreases was related to the extent of P
deficiency in the needles.
According to Harley and Sharkey (1991), P-induced reduction of light-saturated photosynthesis may be attributed to
reductions in triose-P utilization rate or maximal carboxylation velocity or maximal capacity of electron transport rate. In
our experiment, Amax was typically 100% higher than the
carboxylation limited rate at ambient CO2 indicating that lightsaturated photosynthesis was not limited by electron transport
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712
LOUSTAU, BEN BRAHIM, GAUDILLÈRE AND DREYER
Table 2. Values of the F statistics (Type III) of the effects of time of measurement (Week), needle phosphorus (P) concentration and their interaction
(Week × P) on photosynthetic parameters. Significance: * = significant with first-order risk = 0.05; ** = significant with first-order risk = 0.01.
Effects
df
α
Jm
Vm
1
1
1
11.9**
61.8**
0.5
0.55
41.3**
2.43
2.1
52.8**
9.4**
24
53.8**
22.2**
18.4**
0.88
0.78
0.75
Week
P
Interaction Week × P
Total
Parameters
R2
Table 3. Mean values (± SE; n = 3) of stomatal conductance, gs, relative stomatal limitation of photosynthesis, RSL, and maximal net assimilation
rates, Amax (µmol m −2 s − 1), (Q = 1500 µmol quanta m −2 s − 1, ambient CO2 = 1500 µmol mol −1, air temperature = 21 °C, O2 = 0.21 mol mol −1) in
seedlings of Pinus pinaster subjected to four relative addition rates of P for 12 or 22 weeks. Measurements of gs were made at 21 °C, 70% RH, a
CO2 concentration of 360 µmol mol −1 CO2 and a Q of 800 µmol m −2 s −1. Values annotated with different letters are significantly different.
Week of
P Relative addition rate (day −1)
Parameter
Measurement
12
22
0.0
0.005
0.01
0.02
gs (mmol H2O m s )
RSL (%)
Amax (µmol CO2 m −2 s −1)
28 ± 7
24
7.5 ± 0.40 a
27 ± 8
50
11.9 ± 1.9 b
46 ± 1
41
15.9 ± 1.2 c
61 ± 17
40
20.6 ± 0.8 d
gs (mmol H2O m −2 s −1)
RSL (%)
27 ± 6
34
27 ± 10
50
34 ± 8
49
46 ± 2
43
−2 −1
Table 4. Maximal photochemical efficiency of PSII (Fv /Fm) measured at predawn on dark-adapted needles of P. pinaster seedlings subjected to
four relative addition rates of P for 22 weeks (n = 14 seedlings per treatment). Values annotated with the same letter are not significantly different.
P Relative addition rate (day −1)
Fv /Fm
0.0
0.005
0.01
0.02
0.74 ± 0.027 a
0.83 ± 0.007 b
0.81 ± 0.010 b
0.83 ± 0.0024 b
Figure 4. Relationship between photochemical efficiency of PSII
(Φ II) and photosynthetic quanta flux density, Q, in seedlings of Pinus
pinaster subjected to four RARs of P for 22 weeks. Measurements
were made at 25 °C at CO2 and O2 concentrations of 900 µmol mol − 1
and 0.01 mol mol−1, respectively (n: RAR = 0.02, h: RAR = 0.01, e:
RAR = 0.005, s: RAR = 0 day −1).
rate under growing conditions. Additionally, the oxygen sensitivity of photosynthesis at ambient CO2 showed that the trioseP utilization rate was not limiting. Thus, P nutrition controlled
the light-saturated photosynthetic rate only through a decrease
in maximal velocity of carboxylation, Vm. Similar results have
been observed in sunflower and maize by Jacob and Lawlor
(1993a) and in Pinus taeda L. by Lewis et al. (1994). Different
results have been reported in response to short-term P deficiencies. A loss of oxygen sensitivity at ambient CO2 concentration
is frequently observed in in vitro fed-leaf experiments (Leegood and Furbank 1986, Sharkey et al. 1986, Sivak and Walker
1986) and in intact plants at increased CO2 concentrations
(Harley et al. 1992, Lewis et al. 1994). Our experiment supports the hypothesis that triose-P utilization limitations occur
mainly during transient stages under natural conditions (Sivak
and Walker 1986). Acclimation of photosynthesis must occur
under long-term P deficiency in maritime pine, possibly as the
result of a classical co-limitation by carboxylation velocity at
saturating irradiance and to RuBP regeneration at low irradiance (Farquhar et al. 1980).
TREE PHYSIOLOGY VOLUME 19, 1999
PHOSPHORUS EFFECTS ON MARITIME PINE PHOTOSYNTHESIS
Figure 5. Relationship between apparent quantum yield of net CO2
assimilation (Φ CO 2), and photochemical efficiency of PSII (Φ II) measured at CO2 = 900 µmol mol −1, O2 = 0.02 mol mol −1, 25 °C and 70%
RH, on 12 Pinus pinaster seedlings. The regressions for each RAR
were derived from Equation 1, and parameter values are given in
Table 5 (n: RAR = 0.02, u: RAR = 0.01, e: RAR = 0.005, s: RAR =
0 day − 1).
In C3 plants, a change in the apparent carboxylation velocity
has been ascribed to a change in amount (Brooks 1986, Jacob
and Lawlor 1992) and activation state of Rubisco (Parry et al.
1985, Lauer et al. 1989, Sawada et al. 1990, Jacob and Lawlor
1992). Alternatively, a reduction in the internal conductance to
CO2 may create a CO2 concentration gradient between the
mesophyll and chloroplast and thereby introduce a bias in the
determination of Vm from gas exchange measurements.
Our computed cc values were generally significantly lower
than the ci values, indicating the occurrence of significant
internal resistance to CO2 diffusion in the needles. Because of
the large scatter of the data, we avoided systematic calculations
of internal conductance to CO2. Consequently, no conclusion
can be drawn about potential P-deficiency related differences
in internal resistance. The occurrence of large differences
among treatment in the response to cc and in carboxylation
efficiency confirms that the major impact of P deficiency is a
reduction in Vm, even when chloroplastic CO2 is taken into
account instead of ci. Hence, a major photosynthetic response
to P deficiency in maritime pine needles may be attributed to
713
a change in carboxylation rate. Although we cannot exclude
other causes that could contribute to the effect of P deficiency
on the A--ci curves (e.g., a change in mesophyll conductance or
Rubisco specificity; Jacob and Lawlor (1993a)), the theoretical or experimental support for these effects is limited.
At low light, P deficiency affected photosynthesis through a
reduction in apparent quantum yield, which displayed a linear
relationship with needle P concentration. Such decreases have
previously been observed over a similar range of needle P
concentrations in Pinus radiata by Conroy et al. (1986), and in
annual species by Brooks (1986), Lauer et al. (1989) and Jacob
and Lawlor (1991). Comparisons with maximal quantum yield
of PSII photochemistry only partly confirmed this result at
Week 22. At the lowest needle P concentration (RAR = 0), the
reduction in maximal quantum efficiency of PSII showed that
the primary processes of light capture and electron transport
were affected by P deficiency (Sharkey 1985, Heineke et al.
1989). The reduction in apparent quantum efficiency of CO2
assimilation at RARs > 0 (Figure 3), as reported, for example,
by Brooks (1986) for spinach, suggests that P deficiency affected the electron transport chain at a later step. A feedback
limitation on electron transport induced by the lack of a final
electron acceptor (Heineke et al. 1989) or a reduction in ATP
synthesis resulting from a decrease in stromal Pi concentration
are possible explanations (Furbank et al. 1987, Robinson and
Giersch 1987).
Although the effects of P nutrition were less pronounced at
Week 22 than at Week 12, needle P concentration increased in
all RAR treatments with time (Table 1). The 20% decrease in
specific needle area and concurrent increases in P and N
concentrations could be attributed to a reduction in growth and
carbon sink activity and to an accumulation of these elements
in the needles. We hypothesize that the attenuation of treatment
effects on Vm at Week 22 (Table 2) was induced by a feedback
limitation associated with growth cessation (Chapin and Wardlaw 1988, Sawada et al. 1990) that had stronger effects on
seedlings with high growth rates than on seedlings with low
growth rates. A similar attenuation of the effects of P deficiency on photosynthesis was reported in Pinus radiata after
20 weeks by Conroy et al. (1986). At Week 22, it is noteworthy
that the increase in α, which was concurrent with a decrease in
Vm, reduced the light-saturation threshold at which photosynthesis is limited by Rubisco activity. This shift coincided with
the decrease in natural irradiance (Q) from a mean daily value
Table 5. Mean values (± SD; n = 3) of the calibration coefficients between the apparent quantum yield of carboxylation (measured under
non-photorespiratory conditions) and the photochemical efficiency of PSII, in seedlings of Pinus pinaster subjected to four RARs of P for 22
weeks. Efficiency changes were created by changing irradiance. Results were compared by ANOVA and a Fisher PLSD. Abbreviations: k = slope
of the relationship; b = y-intercept; and R2 = determination coefficient of the linear model. Values annotated with the same letter are not significantly
different.
P Relative addition rate (day − 1)
k
b
R2
0
0.005
0.01
0.02
12.46 ± 0.6 a
0.09 ± 0.02 a
0.97
14.50 ± 1.0 a
0.06 ± 0.03 a
0.93
12.28 ± 0.76 a
0.21 ± 0.02 b
0.95
12.91 ± 1.2 a
0.14 ± 0.04 a
0.81
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714
LOUSTAU, BEN BRAHIM, GAUDILLÈRE AND DREYER
Figure 6. Relationship between net CO2
assimilation rate and (i) substomatal (ci)
or (ii) chloroplastic (cc) CO2 mole fraction in Pinus pinaster seedlings subjected to four RARs of P for 22 weeks.
All measurements were made at Q = 570
µmol m − 2 s − 1, T = 25 °C, and an O2 concentration of 0.21 mol mol − 1. Each
curve (log regression) represents measurements on three twigs per treatment.
Correlation coefficients (r 2) are displayed for each treatment.
of 44 mol day −1 in July to 10 mol day −1 in November. This
decline is consistent with our hypothesis that the photosynthetic apparatus acclimates to the ambient light regime.
We conclude that photosynthesis of maritime pine needles
in ambient CO2 adapted to phosphorus availability primarily
through a change in maximal velocity of carboxylation and, for
severely P-deficient needles, through a change in quantum
efficiency of electron transport. Attenuation of these effects
was observed at the end of the growing season, and was
attributed to feedback effects linked to the cessation of growth
and a decrease in natural irradiance. We found no evidence of
a phosphorus effect on stomatal limitation or that the triose-P
utilization rate limited photosynthesis at ambient CO2 concentration.
Acknowledgments
C. Lambrot, M. Guèdon, P. Rossetto, M. Sartore and J.L. Grange
afforded invaluable technical assistance during this experiment. The
work was a part of the program Fonctionnement et Protection des
Ecosystèmes Sableux funded by the Région Aquitaine. During his
Ph.D. studies, M. Ben Brahim was supported by la Division de la
Recherche et de l’Expérimentation Forestière du Maroc and the Ministère Français de la Coopération.
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