Plant Cell Physiol. 29(1): 157-165 (1988)
JSPP © 1988
Photosynthetic Characteristics of Spinach Leaves Grown with
Different Nitrogen Treatments
John R. Evans 1 2 and Ichiro Terashima3
3
' Division of Plant Industry, CSIRO, G.P.O. Box 1600 Canberra, A.C.T. 2601, Australia
Plant Environmental Biology Group, Research School of Biological Sciences, The Australian National University,
G.P.O. Box 475, Canberra, A.C.T. 2601, Australia
Spinach plants (Spinacia oleracea L.) were grown hydroponically with different concentrations of nitrate nitrogen, ranging from 0.5 to 12 mM, in a glasshouse under full sunlight. Using
an open gas exchange system, the rate of CO2 assimilation, A, was determined as a function of intercellular partial pressure of CO2, PJ, with a constant amount of absorbed light per unit Chi.
When expressed on a leaf area basis, A measured at high irradiance and at p, = 5O0fjbaT, was proportional to the in vitro rate of uncoupled whole-chain electron transport as well as to Chi content. There was a curvilinear relationship between the mesophyll conductance (the slope of the
A : Pi curve near the CO 2 compensation point) and the in vitro RuBP carboxylase activity. The
curvature did not appear to be due to enzyme inactivation in vivo in leaves with high nitrogen contents. The curvature suggested the presence of a CO 2 transfer resistance between the intercellular
spaces and the site of carboxylation of 2.2 m2 s bar mol" 1 CO2, which is similar to that previously
observed in wheat. This implied that, while nitrogen deficiency increased the ratio of in vitro activity of electron transport to that of RuBP carboxylase, the two activities remained balanced in
vivo.
Irradiance response curves were determined by both net CO2 and O2 exchange. The two
methods gave reasonable agreement at light saturation. The quantum yield measured by O2
evolution was 0.090±0.003 mol0 2 mol~' absorbed quanta, whereas after correcting for pj
= 500//bar, the quantum yield for CO2 assimilation was only 82% of that measured by oxygen
evolution.
Key words: CO 2 assimilation — CO2 transfer resistance — Electron transport — Nitrogen deficiency —RuBP carboxylase — Spinacia oleracea.
While nitrogen deficiency decreases the chlorophyll
content per unit leaf area, the rate of electron transport
and amounts of thylakoid components, each expressed per
unit of chlorophyll, are unaffected (A triplex grown under
high irradiance, Medina 1971, Spinacia, Evans and
Terashima 1987, Terashima and Evans 1988). The relationship between the rate of oxygen evolution per unit of
chlorophyll and absorbed irradiance per unit of chlorophyll was independent of nitrogen treatment (Evans and
Terashima 1987, Terashima and Evans 1988). This suggested
that nitrogen deficiency decreased the amount of thylakoids
2
Present address: Plant Environmental Biology Group, Research School of Biological Sciences, The Australian National
University, G.P.O. Box 475, Canberra, A.C.T. 2601, Australia.
but did not alter their properties. Consequently, under
conditions where electron transport is rate-limiting (such
as at high intercellular p(CO£), there should be a linear
relationship between the rate of CO2 assimilation and
chlorophyll content, both expressed per unit leaf area, for
leaves absorbing the same irradiance per unit of chlorophyll,
There should also be a linear relationship between the rate
of CO 2 assimilation and in vitro electron transport,
The response of the rate of CO2 assimilation, A, to intercellular CO2 partial pressure, Pi, has been successfully
modelled by assuming that at low pit the rate is limited by
the RuBP carboxylase activity, whereas at high pj, the rate
is limited by the rate at which RuBP can be regenerated
(Farquhar and Caemmerer 1982). RuBP regeneration is
generally thought to reflect the rate at which electron
157
158
J. R. Evans and I. Terashima
transport regenerates NADPH and/or photophosphorylation regenerates ATP. However, in some cases RuBP
regeneration can be limited by the availability of inorganic
phosphate in the chloroplasts (Farquhar and Caemmerer
1982, Sharkey 1985, Sharkey et al. 1986, Stitt 1986). The
CO2 response of A when RuBP carboxylase activity is
limiting can be predicted from the kinetic constants for the
enzyme. When the substrate partial pressure of CO2 is
taken as equal to the intercellular ptCOJ, Pi, there should
be a linear relationship between the RuBP carboxylase activity and the mesophyll conductance (the slope of the
A : Pi curve near the CO 2 compensation point). The linear
relationship ignores the possible resistance to CO2 diffusion
from intercellular spaces to the sites of carboxylation
within the chloroplast. For wheat, this resistance was
found to be important (Evans 1983a, Evans et al. 1986).
In contrast to the independence of the ratio of
thyloakoid components to chlorophyll with changing
nitrogen nutrition, the ratio of RuBP carboxylase to
chlorophyll declines with nitrogen deficiency in A triplex
(Medina 1971), Gossypium (Wong 1979), Solarium (Ferrar
and Osmond 1986), Phaseolus (Seemann et al. 1987) and
Spinacia (Evans and Terashima 1987), though not in
Triticum (Evans 1983, 1985a). In spinach, the increase in
the ratio of electron transport to RuBP carboxylase activities with nitrogen deficiency, is therefore expected to
alter the relationship between the rate of CO 2 assimilation
and pi if there is little resistance to CO2 transfer from the intercellular spaces to the sites of carboxylation. The independence of electron transport capacity per unit of
chlorophyll with nitrogen treatment implies that the
different activities per unit leaf area induced by nitrogen
deficiency can be removed by expressing CO 2 assimilation
on a chlorophyll basis. For leaves absorbing the same irradiance per unit of chlorophyll, the rate of CO 2 assimilation
per unit Chi at high p( would be independent of nitrogen
treatment, whereas nitrogen deficient leaves should have a
lower mesophyll conductance. Nitrogen deficiency would
therefore increase the Pi where CO 2 assimilation changes
from an RuBP carboxylase limitation to an electron transport limitation (Evans and Terashima 1987).
This expectation based on the in vitro biochemical
analysis contrasts with observations based on gas exchange
analysis, which showed that potential electron ransport activity approximately matched the RuBP carboxylase activity, irrespective of nitrogen treatment in Phaseolus (Caemmerer and Farquhar 1981), leaf age in Triticum (Evans
1986) and phosphorus treatment in Spinacia (Brooks
1985). The aim of the experiments described here was to
compare the in vivo photosynthetic performance of the leaf
with its underlying biochemistry. We have also compared
net CO2 assimilation with net O 2 evolution as a function of
irradiance in order to facilitate comparisons between
measurements of CO 2 or O2 exchange.
Materials and Methods
Plant material—Spinach plants {Spinacia oleracea L.
cv. Henderson's hybrid 102) were grown hydroponically in
a glasshouse under full sunlight, with a 9 h photoperiod
and 18/13°C day/night temperature. Four nitrate treatments were imposed, 0.5, 1, 4, and 12 mM. Measurements were made on the plants after about 50 days
growth. The chlorophyll content of the leaves was
estimated from leaf discs by the method of Arnon (1949).
The proportion of white light absorbed by the leaf was
measured using an integrating sphere to determine both
leaf transmittance and reflectance. The leaf opposite that
used for gas exchange analysis was used for the leaf disc
oxygen electrode measurements and subsequently for
thylakoid preparations as described previously (Evans and
Terashima 1987). Other samples were taken from the
remaining parts of the leaves for soluble protein/RuBP
carboxylase assays and nitrogen determinations. The
thylakoids were used to assay the whole chain electron
transport activity at light saturation in the presence of an
uncoupler (Terashima and Evans 1987). RuBP carboxylase (EC 4.1.1.39) activity was assayed in crude extracts
following preincubation in the presence of CO2 and Mg2*
as previously described (Evans and Terashima 1987).
Assays were made at 30°C and the activity at 25°C was
taken to be 65% of that at 30°C, using the Q10 of 2.2 (see
Evans 1986). To measure in vivo RuBP carboxylase activity, leaf discs collected in situ under 1,600 //mol quanta
m~ 2 s~' were rapidly frozen in liquid nitrogen. A rapid
extraction was made in CO2-free buffer and the initial activity
compared with that following incubation on ice for 45 min
in the presence of CO2 and Mg 2+ (Brooks 1986).
Gas exchange—The system used is described in detail
elsewhere (Evans 1983a), except that absolute p(CO2) was
calculated from the outputs of two mass flow controllers
(Tylan FC260, Torrance, CA) which mixed CO2-free air of
known humidity with 1 or 10% CO2 in air and the chamber
enclosed the whole leaf. The large leaves produced under
4 and 12 mM nitrate were trimmed to reduce their area to
about 20 cm2 just prior to placing them in the chamber.
The leaf boundary layer conductance in the chamber was
2.5molm~ 2 s~'. Measurements were made with the leaf
temperature at 25 °C and leaf-to-air vapour pressure
difference of 15 mbar. After 45 min equilibration at
340//bar p(CO2) and an irradiance giving 2.3 mmol absorbed quanta mol"' Chi s~' (the irradiance per unit area of leaf
ranging from 800 to 1,500//mol quanta m~2 s"1 to compensate for the different chlorophyll contents which had been
estimated beforehand) rates of CO2 assimilation were
measured with PJ ranging from the compensation point to
about 500//bar. Rate of CO2 assimilation was then
measured as a function of irradiance with p( maintained
near 500//bar. Rates of gas exchange and related bio-
Photosynthetic characteristics of spinach leaves
159
chemical parameters were calculated according to Caemmerer
and Farquhar (1981). The rate of oxygen evolution was
determined as a function of irradiance with a leaf-disc
oxygen electrode (Delieu and Walker 1981: Hansatech,
King's Lynn, U.K.). The leaf temperature was 25°C and
the gas phase was ~ 1 6 % O2 and ~5% CO 2 .
Results
The rate of CO2 assimilation, with Pj = 500/ibar and a
constant absorbed irradiance per unit of chlorophyll was
directly related to the in vitro rate of electron transport
from H2O to methylviologen (Fig. 1). According to the
model of Caemmerer and Farquhar (1981), the CO2 assimilation rate at Pj = 500//bar that can be sustained by the
electron transport rate, J, is A500 = J(pc —r*)/(4.5C
+ 10.5r*) = 0.69J/4, where pc is the ptCOJ at the site of
carboxylation and /"* is the CO2 compensation point in the
absence of non-photorespiratory CO2 evolution in the light
(40/^bar, Brooks and Farquhar 1985). The rate of oxygen
evolution or Hill activity, H = J/4, since there are 4 electrons per oxygen. While the slope of 0.69 is close to that
observed in Fig. 1, the rate of CO2 assimilation was not
measured at light saturation. Allowing for this, the maximum rate of electron transport predicted from the rate of
CO2 assimilation would be 120 mmol O2 mol" 1 Oils" 1 .
The average rate from 24 preparations measured in vitro
was 103 mmol O2 mol" 1 Chi s~'. The in vitro rate of electron transport per unit of chlorophyll was independent of
the nitrogen treatment (Table 1). A strong relationship existed between A500 and chlorophyll: A500 (^molm" 2 s~')
= 82.9xChl(mmolm" 2 ) - 3 . 4 5 , 1^ = 0.845.
Responses of photosynthesis to irradiance, measured
at Pi = 500/ibar (high enough to ensure that the photosyn-
in vitro Rate of electron transport (//mol 0 2 m"2 s~
Fig. 1 Rate of CO2 assimilation versus in vitro rate of electron
transport.
CO2 assimilation was measured at an intercellular
p(CO 2 )=500//bar and an irradiance of 2.3 mmol absorbed
quanta mol~' Chi s"'. Electron transport was measured from
H2O to methylviologen at saturating irradiances. O 12, A 4, D 1, o
0.5 rriM nitrate treatments. Solid points represent the curves in
Fig. 2. y=1.82+0.65x,r 2 =0.85.
thetic rate was limited by electron transport), are shown on
both a unit leaf area basis and a unit chlorophyll basis (Fig.
2). Photosynthetic capacity per unit leaf area, chlorophyll
content and leaf absorptance were all reduced by nitrogen
deficiency (Table 1). The responses of assimilation to irradiance expressed on a chlorophyll basis were independent
of nitrogen treatment, due to the proportionality between
the rate of CO2 assimilation and chlorophyll content (Fig.
2B).
Table 1 The contents of nitrogen and chlorophyll, the electron transport and RuBP carboxylase activities on a
chlorophyll basis and the absorptances of leaves from plants grown with different nitrate concentrations, under full
sunlight
0.5
Nitrate concentration (HIM)
1
4
Nitrogen (mmol m~2)
88 ±6
83
±3
Chlorophyll (mmol m~2)
0 .35 ±0.01
0.27±0.01
Absorptance (400 nm — 700 nm)
0 .85 ±0.02
0.81 ±0.02
Whole chain electron transport H2O -*• MV
99 ±6
98
±8
(mmol O2 mol" 1 Chi s"1)
117
±9
98 ±7
RuBP carboxylase activity
(mmol CO2 mol"' Chi s"')
108 + 3
109
+5
Initial RuBP carboxylase activity {%)
Initial RuBP carboxylase activity expressed as a percentage of fully activated activity (n = 3).
Values are means±S.E.
" Dark control 62 ±2.
114 +
0 .45 ±
0 .87±
97 +
12
155 ± 10
3
0. 58± 0 .02
0 .02
0. 89± 0 .019
0 .016
108 + 8
8
139
+ 12
102
+
199
+ 9
97
+ 12 a
J. R. Evans and I. Terashima
160
"0
1
2
(mol quanta mol"1 Chi s"1)
1000
2000
Irradiance (/tmol quanta m"2 s"')
3
Fig. 2 Rate of CO2 assimilation versus absorbed irradiance.
CO2 assimilation was measured at an intercellular pCCOJ of 500//bar.
Expressed on a leaf area basis (A). Both axes expressed on a chlorophyll basis (B). Symbols as in Fig. 1.
The responses of rate of CO 2 assimilation to p, are
shown in Fig. 3. As expected, for A expressed on a
chlorophyll basis, no effect of nitrogen treatment was evident for pj>300/ibar (the range in which rate of electron
transport should be limiting). The decreasing RuBP carboxylase/chlorophyll ratio with decreasing N-status (Table
1) was expected to result in a smaller mesophyll conductance per unit of chlorophyll for the lower nitrogen
treatments. Contrary to this expectation, there were also
no differences evident in the A : p; curves expressed per
unit of chlorophyll for pj<200//bar. The reason for this
similarity is evident from the relationship between
mesophyll conductace and fully activated in vitro RuBP
carboxylase activity (Fig. 4). The curvature was not due to
enzyme inactivation in the high nitrate treatments (Table
1). Following rapid extraction in the absence of CO2, the initial activities suggested that all the extracted RuBP carboxylase was catalytically active under 1,600//mol quanta
m~2 s~' irrespective of the N-treatments. In the dark, this
declined to ~60%. The curvilinear relationship has been
attributed to a resistance to CO2 transfer from the intercellular spaces to the sites of carboxylation within the
chloroplast. The resistance can be obtained from the double reciprocal plot of mesophyll conductance against RuBP
carboxylase activity i.e. mesophyll resistance against carboxylation resistance. Mesophyll resistance can be regarded as the sum of carboxylation resistance and the CO2
transfer resistance. Therefore, the y-intercept where the
Intercellular p(CO2) (jubar)
Fig. 3 Rate of CO2 assimilation versus intercellular p(CC>2).
CO 2 assimilation was measured at an irradiance of 2.3 mmol absorbed
quanta mol" 1 Chi s~'. (A) is expressed on an area basis, (B) CO 2 assimilation is expressed per unit of chlorophyll with the solid line
predicted from the RuBP carboxylase limited rate. Symbols as in Fig. 1, but represent different leaves from Fig. 2. The irradiances
used were 1,490, 1,350, 1,090 and 900//mol quanta m " 2 s " ' for the 12, 4, 1 and 0.5 mM curves.
Photosynthetic characteristics of spinach leaves
0
40
80
120
0
RuBP carboxylase activity (ju.mol m~2 s"1
0.001
0.002
161
0.003
0.004
1/RuBP carboxylase activity (m 2 s/xmol"')
Fig. 4 Mesophyll conductance versus in vitro RuBP carboxylase activity. (A) The line (i) through the data is the regression from B.
Line (ii) is predited from the kinetic constants for the enzyme used by Evans and Terashima (1987) with the presence of the CO2 transfer
resistance, rw, estimated in B. Mesophyll conductance, gm, is expressed as g m = k / ( l + k x rw), where k=V c (r*+K c (l+O/IC o ))/(Pc +
Kc(l +O/K O )) 2 , r*-40^bar, K c =296^bar, KO=212 mbar, 0 = 2 0 0 mbar and p c =75 ^bar. Dashed line (iii) predicted with r w =0.
Double reciprocal plot of A to obtain the CO 2 transfer resistance (B). rw = 2.24±0.05 m 2 s bar mol"' CO2. For detailed methods for
calculations, see Evans (1986).
carboxylation resistance is zero, equals the CO2 transfer
resistance ( 2 . 2 m 2 s b a r m o n ' CO2, Fig. 4B).
The response of rate of CO2 assimilation per unit of
chlorophyll to P| changed little with nitrogen deficiency
(Fig. 3). The relative capacities for electron transport/
photophosphorylation (this being proportional to the
rate of CO2, assimilation when Pj = 500/ibar, A500, Fig. I)
and RuBP carboxylation are examined in more detail
at a constant irradiance per unit leaf area (Fig. 5). Lines
have been calculated for the means of the ratio of calcula-
0.1
0.2
Mesophyll conductance
2
(mol m" s"' bar"')
ted Hill activity to mesophyll conductance. The means
are 424 and 337mol0 2 /(molC0 2 //bar~') for plants
grown with 0.5 and 12 mM nitrate, respectively. The ratio
defines the transition from RuBP carboxylase to electron
transport limitation, 204 and 158 ^bar, respectively. Incorporation of the CO2 transfer resistance and deriving RuBP
carboxylase kinetic constants from Fig. 4A leads to a curvilinear relationship between A500 and RuBP carboxylase
actvity (Fig. 5B). The data from all treatments fall close
to a single line indicating that the capacity for electron
0
40
80
120
RuBP carboxylase activity
2
(jumol m" s"')
Fig. 5 Rate of CO2 assimilation versus mesophyll conductance.
CO2 assimilation was measured at an intercellular p(CO2) of
500//bar and an absorbed irradiance of l,800/*mol quanta m~2 s"1 (A). The two lines are predicted for transition points at (i) 204 and
(ii) 158 //bar CO2. Solid points represent the curves in Fig. 3. Rate of CO 2 assimilation versus in vitro RuBP carboxylase activity (B).
Line is calculated for a transition at 185 //bar CO2 due to a ratio of Hill activity to mesophyll conductance of 385 mol moP
J. R. Evans and I. Terashima
162
"0
40
80
120
160
RuBP carboxylase activity (//mol m~2 s"1)
Fig. 6 Hill activity versus in vitro RuBP carboxylase activity.
Hill activity measured with a leaf disc oxygen electrode at
2,000^mol quantam~ 2 s~', 1% CO 2 . Plants were grown under
• full sunlight A 30% sunlight and D 15% sunlight. Lines are
calculated for the mean ratios of Hill activity to mesophyll conductance for 100% sunlight (385 mol O 2 //bar mol" 1 CO2) and 30,
15% (273) (data from Terashima and Evans 1988).
transport remains well balanced by the RuBP carboxylase
activity across the nitrate treatment.
A similar analysis is shown for plants grown with
different nitrate treatments and growth irradiances (data
from Terashima and Evans 1988) (Fig. 6). Theoretical lines
are drawn through the mean ratios of Hill activity to
mesophyll conductance (calculated from the in vitro RuBP
carboxylase activity) for the 100%, and for the 30 and 15%
growth irradiances. The mean ratio for full sunlight was
the same as that observed in the independent data set of
Fig. 5B. Also, the mean ratio for the high and low
nitrogen treatments of 337 and 453 mol O2/(moI CO 2
//bar" 1 ) respectively, was similar to that in Fig. 5A.
Growth under reduced irradiance lowered the expected
ratio of Hill activity to mesophyll conductance.
Responses of net CO 2 assimilation and net O2 evolution to changes in irradiance were determined on adjacent
leaves (Fig. 7). All 11 comparisons were consistent with a
trend that was independent of nitrogen treatment. The
light saturated rates obtained with the two methods were in
close agreement. The ratio of the rates calculated from
CO2 assimilation and O2 evolution were 0.90±0.05 (mean
±S.E., n = l l ) which compares with the expected ratio
of 0.92 calculated on the basis that the CO2 assimilation
was measured with pj = 500^bar, while the O2 evolution
was measured at 5% CO 2 which should virtually suppress photorespiration. The average quantum yields were
0.090±0.003 mol O2 mol" 1 absorbed quanta and 0.068 ±
0.003 mol CO2 mol" 1 absorbed quanta. On average, the
quantum yield measured by CO 2 assimilation (allowing for
photorespiration) was only 82% of that measured by O 2
evolution, which results in a curvilinear relationship between the two rates when cmpared at different irradiances
(Fig. 7B).
Discussion
The objectives of these experiments were to compare
the in vivo photosynthetic performance of leaves with vary-
B
50
40
-
30
-
20
-
•
/
A
.0
/f
•
10
0
500
1000
1500
Absorbed irradiance (jUmol quanta m~2 s~')
0
10
1
I
1
20
30
40
1
50
Rate of oxygen evolution (//.mol m" •2 o - n
Fig. 7 Rate of CO 2 assimilation or oxygen evolution versus absorbed irradiance.
Open symbols and dashed lines are the rate of
CO 2 assimilation with P!=500//bar, solid symbols and continuous lines were measured with the leaf disc oxygen electrode with 5% CO 2
O, • 12 mM and o, • 1 niM nitrate. Rate of CO 2 assimilation versus the rate of oxygen evolution (B). • 12 mM, o 1 DIM. The solid line
represents 1 : 1 agreement, corrected by the factor of (1 —FJC), where /i=40//bar and C=500//bar. The dashed line represents the
average relationship between the quanum yields (n = ll).
Photosynthetic characteristics of spinach leaves
ing degrees of nitrogen deficiency with that predicted from
underlying biochemistry. It was expected that nitrogen
deficiency would not alter the electron transport capacity
per unit of chlorophyll but would increase the capacity of
electron transport relative to that of RuBP carboxylation
(Evans and Terashima 1987). According to the model of
Farquhar and Caemmerer (1982), the slope of the curve
relating the rate of CO 2 assimilation to P; near the CO2 compensation point is determined by the RuBP carboxylase activity. At higher Pi (~500^bar), the rate is limited by the
rate of RuBP regeneration which reflects the rate of electron transport/photophosphorylation.
There is considerable evidence demonstrating the relationship between
RuBP carboxylase activity and the mesophyll conductance
(Caemmerer and Farquhar 1981, 1984, Caemmerer and
Edmondson 1986, Evans 1983a, 1986, Evans and Seemann
1984, Brooks 1986), but very limited data concerning the
electron transport limitation (Caemmerer and Farquhar
1981). The measurements made here with spinach leaves
having different electron transport activities confirm the expected limitation by electron transport at high irradiance
and pj = 500/ibar (Fig. 1). However, as in the previous
work (Caemmerer and Farquhar 1981), the electron
transport rate calculated from the gas exhcnage exceeded
the measured in vitro rate by about 20%.
CO2 transfer resistance—The comparison of in vivo
RuBP carboxylase activity (the mesophyll conductance),
with extracted activity, revealed not only a curvilinear relationship but also a greater activity than expected from the
kinetic constants (contrast the data with curves (ii) and (iii)
in Fig. 4A). Given that the initial activity of rapidly extracted enzyme was not improved by incubation with CO2
and Mg 2+ , the curvature in Fig. 4A did not appear to be
due to inactive enzyme in vivo at the higher nitrate levels.
While we cannot rule out the possibility of the presence of
a loosely bound catalytic inhibitor in leaves with high
nitrogen contents, the subsequent discussion will suggest
that the deviation is caused by a CO2 transfer resistance.
Analysis of the curvature yielded an estimate of the CO2
transfer resistance (2.2 m2 s bar mol" 1 CO2 Fig. 4B) similar
to that observed for wheat (Evans 1983a). The double
reciprocal plot analysis assumes that the CO2 transfer resistance is the same for all replicate leaves. While this is
probably untrue, it is difficult to predict how it would differ
with leaf nitrogen content. To the extent that it decreases
with increasing leaf nitrogen, the double reciprocal plot
will underestimate the resistance (Evans 1983b). The existence of the CO2 transfer resistance in wheat has also been
inferred from an independent method which examined
the discrimination against I3CO2 ( 2 . 4 m 2 s b a r m o n ' CO2,
Evans et al. 1986).
A consequence of the resistance is that the in vivo
RuBP carboxylase activity is reduced progressively with increasing amounts per unit leaf area due to the lowering of
163
the p(CC>2) at carboxylation sites. So, for carboxylation to
remain in balance with th electron transport capacity, the
ratio of RuBP carboxylase to electron transport needs to increase with increasing leaf nitrogen content. The comparison of these two capacities estimated in vitro suggested
an excess of electron transport capacity and a dramatic
change in their ratio with leaf nitrogen content (Evans and
Terashima 1987). Incorporation of the CO 2 transfer
resistance and in vivo RuBP carboxylase activity clearly
showed that the capacity for electron transport at 1,800
/rniol quantam~ 2 s~' was closely matched to the RuBP
consumption rate (Fig. 5B). The observed relationship
between A500 and mesophyll conductance in spinach (Fig.
5A) is remarkably similar to that for wheat (Evans 1986).
RuBP carboxylase kinetic constants for modelling—
The rate of CO2 assmilation has been successfully predicted
from the amount of RuBP carboxylase, given the appropriate p ( . However, the kinetic constants used to obtain this agreement vary dramatically among laboratories.
Three examples will be compared at 25°C; the data of
Besford et al. (1985), Makino et al. (1985) and Evans
(1986). In all three, reasonable agreement was claimed between the measured rates of CO2 assimilation and the rates
predicted from the RuBP carboxylase measurements.
This was despite more than a two-fold variation in expected performance of the enzyme. The initial slope of the
curve relating the rate of CO2 assimilation to the p(CO2)
can be described as follows (Farquhar and Caemmerer
1982),
dA/dC = Vcmax/(r* + K,(l + O/Ko))
where Vcmax is the maximum RuBP carboxylase activity, Kc
and Ko are the Michaelis constants for RuBP carboxylase
for CO2 and O2 repectively, O is the p(O2) and r* is the CO 2
compensation point in the absence of non-photorespiratory CO 2 evolution in the light.
The RuBP carboxylase activity in the leaf is sensitive
to the effective A"m(CO2) in the presence of oxygen, and to
the relative activities of the oxygenase and carboxylase reactions. The effective Km(CO2), K c (l+O/K o ), in the presence of 200mbar O2 and the (/"*) for the three sets of
data were 244 (24.1), 399 (27.5) and 575 (37.8) respectively.
At 25°C, A has been estimated to be 43 by an in vitro
technique (Jordan and Ogren 1984) and 40.7 by gas exchange (Brooks and Farquhar 1985). The two lower
values used by Besford et al. (1985) and Makino et al.
(1985) enhanced the carboxylase activity expected in vivo.
By contrast, Evans (1986) used a high turnover rate of
the enzyme (32mol CO 2 mor'enzymes" 1 , Evans and
Seemann 1984) which compares with 16.4 (Makino et al.
1985) and 26.5 mol C O 2 m o r ' enzyme s"1 (Evans and Terashima 1987). To account for the observed CO 2 assimilation rates, its has always been necessary to assume very
164
J. R. Evans and I. Terashima
high affinities for CO 2 and/or high specific activities. Considerable changes to the kinetic constants would be required to account for the data in Fig. 4A.
Balance between electron transport and RuBP carboxylase—The relative capacities for electron transport and
RuBP consumption by RuBP carboxylase define a transition from an RuBP carboxylase limited to an electron
transport limited rate of CO 2 assimilation. Within the
transitional region, both capacities are fully expressed and
therefore utilised with the greatest efficiency. Increasing the
electron transport capacity raises the ps at the transition
and if efficiency were to be maintained, the partial pressure
of intercellular CO 2 would need to be greater. As with leaf
aging in wheat (Evans 1986), nitrogen deficiency in spinach
was associated with a relative increase in the electron
transport capacity (Fig. 5A), although this is not readily apparent from the normalised CO 2 response curves (Fig. 3).
In wheat, the Pj observed under normal external conditions
increased in a manner consistent with p, following the transition point (Evans 1985). A more obvious example of
change in the ratio between electron transport and RuBP
carboxylase capacities is evident in acclimation to different
growth irradiances. Growth under lower irradiance reduces the electron transport capacity for a given RuBP
carboxylase activity (Fig. 6). Therefore, the ratio of Hill
activity to mesophyll conductance, is reduced, as implied
by curves relating the rate of CO2 assimilation to Pj for
Agathis (Langenheim et al. 1984), Pisum (Evans 1987a),
Phaseolus (Seemann et al. 1987) and Piper (Walters and
Field 1987).
O2 versus CO2 measurement—Comparison of the irradiance response curves obtained by measuring CO2 assimilation and O2 evolution showed a consistent difference (Fig.
7). The quantum yields measured by CO 2 assimilation
were unexpectedly lower than those measured by O2 evolution, while the light saturated rates were equal. The irradiance response curve measured by CO 2 assimilation was
obtained after measuring a CO 2 response curve and with
decreasing irradiance, whereas the O2 evolution irradiance
response curve was measured with increasing irradiance
from darkness. No difference has been found when irradiance response curves in the leaf-disc electrode were
measured using increasing or decreasing irradiance (data
not shown). If the irradiance for the CO2 assimilation
measurements had been underestimated, it would result in
both a low quantum yield and the need for a higher irradiance to reach light saturation. In fact, light saturation
was reached at similar irradiances with both methods.
Thus the cause of the curvature in Fig. 7B is uncertain.
Bjorkman and Demmig (1987) also noted that quantum
yields measured by CO 2 assimilation were only 83% of that
measured by the leaf disc oxygen electrode. After correcting for photorespiration, the quantum yields obtained
here by CO 2 assimilation were only 82% of that obtained
by the leaf disc electrode. While the quantum yield of
0.090 mol O2 mol" 1 quanta agrees with the average value in
the literature, no difference was seen between oxygen and
CO2 quantum yields in previous work with pea leaves,
although the measurements were made on different plants
at different locations (Evans 1987b).
This work was carried out during the tenure of a CSIRO postdoctoral fellowship (JRE) with additional support from an ANUCSIRO collaboration grant. H. Ficker's assistance is gratefully
acknowledged. We thank Drs. S. von Caemmerer and G. D. Farquhar for helpful criticism of the manuscript.
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(Received July 29, 1987; Accepted November 2, 1987)