Journal of Experimental Botany, Vol. 56, No. 412, pp. 755–763, February 2005
doi:10.1093/jxb/eri052 Advance Access publication 13 December, 2004
RESEARCH PAPER
Seasonal change in the balance between capacities of
RuBP carboxylation and RuBP regeneration affects CO2
response of photosynthesis in Polygonum cuspidatum
Yusuke Onoda*, Kouki Hikosaka and Tadaki Hirose
Graduate School of Life Sciences, Tohoku University, Aoba, Sendai 980-8578, Japan
Received 26 May 2004; Accepted 18 October 2004
Abstract
The balance between the capacities of RuBP (ribulose1,5-bisphosphate) carboxylation (Vcmax) and RuBP regeneration (expressed as the maximum electron transport rate, Jmax) determines the CO2 dependence of the
photosynthetic rate. As it has been suggested that this
balance changes depending on the growth temperature,
the hypothesis that the seasonal change in air temperature affects the balance and modulates the CO2 response of photosynthesis was tested. Vcmax and Jmax
were determined in summer and autumn for young and
old leaves of Polygonum cuspidatum grown at two CO2
concentrations (370 and 700 lmol mol21). Elevated CO2
concentration tended to reduce both Vcmax and Jmax
without changing the Jmax:Vcmax ratio. The seasonal
environment, on the other hand, altered the ratio such
that the Jmax:Vcmax ratio was higher in autumn leaves
than summer leaves. This alternation made the photosynthetic rate more dependent on CO2 concentration in
autumn. Therefore, when photosynthetic rates were
compared at growth CO2 concentration, the stimulation
in photosynthetic rate was higher in young-autumn than
in young-summer leaves. In old-autumn leaves, the
stimulation of photosynthesis brought by a change in
the Jmax:Vcmax ratio was partly offset by accelerated leaf
senescence under elevated CO2. Across the two seasons and the two CO2 concentrations, Vcmax was
strongly correlated with Rubisco and Jmax with cytochrome f content. These results suggest that seasonal
change in climate affects the relative amounts of photosynthetic proteins, which in turn affect the CO2 response of photosynthesis.
Key words: Allocation of photosynthetic proteins, cytochrome f,
Jmax, Rubisco, seasonality, stimulation of photosynthesis, temperature acclimation, Vcmax.
Introduction
Because CO2 is a substrate for photosynthesis, elevation of
atmospheric CO2 concentration is expected to increase the
carbon assimilation of plants (Mott, 1990). Many studies
have focused on the photosynthetic response to elevated
CO2 concentration with short- and long-term experiments
(for a review see Cure and Acock, 1986; Poorter, 1993;
Gunderson and Wullschleger, 1994; Curtis, 1996; Curtis
and Wang, 1998; Wand et al., 1999). CO2 responses of
photosynthesis are determined by stomatal conductance,
and the capacity of RuBP (ribulose-1,5-bisphosphate)
carboxylation and RuBP regeneration (Farquhar et al.,
1980; Mott, 1990). Stomatal conductance regulates the CO2
concentration in the leaf, while RuBP carboxylation and
RuBP regeneration limit the photosynthetic rate at low and
high CO2 concentrations, respectively. Transition of the
limitation from RuBP carboxylation to RuBP regeneration
usually occurs between an ambient and a twice-ambient
CO2 concentration (Stitt, 1991).
* To whom correspondence should be addressed. Fax: +81 22 217 6699. E-mail: [email protected]
Abbreviations: Ac, photosynthetic rate limited by Rubisco activity; Agrowth, photosynthetic rate at growth CO2 concentration; Aj, photosynthetic rate limited by
RuBP regeneration; ANCOVA, analysis of covariance; ANOVA, analysis of variance; Ca, air CO2 concentration; Ci, intercellular CO2 concentration; Colimiting Ci, Ci at which photosynthetic rate is co-limited by RuBP carboxylation and RuBP regeneration; Jmax, maximum rate of electron transport driving
regeneration of RuBP; Kc, Michaelis–Menten constant of Rubisco for CO2; Ko, Michaelis–Menten constant of Rubisco for O2; PFD, photon flux density; OTC,
open top chamber; Rd, day respiration; RuBP, ribulose-1,5-bisphosphate; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; SDS-PAGE, sodium
dodecyl sulphate–polyacrylamide gel electrophoresis; Vcmax, maximum rate of RuBP carboxylation under saturated RuBP; C*, CO2 compensation point in
the absence of Rd.
Journal of Experimental Botany, Vol. 56, No. 412, ª Society for Experimental Biology 2004; all rights reserved
756 Onoda et al.
Long-term treatment of elevated CO2 modulates the leaf
traits and stimulates photosynthesis to various extents
(Poorter, 1993; Gunderson and Wullschleger, 1994; Curtis,
1996). Plants grown in elevated CO2 tend to have a lower
stomatal conductance (reviewed by Drake et al., 1997;
Medlyn et al., 2001) and a lower photosynthetic rate than
those grown in ambient CO2 when compared at a given
CO2 concentration (for a review see Stitt, 1991; Gunderson
and Wullschleger, 1994; Sage, 1994). The lower rate of
photosynthesis has been ascribed to a reduction in the
capacity of RuBP carboxylation (Vcmax) and/or RuBP
regeneration (expressed as the maximum electron transport
rate, Jmax) (Sage, 1994). Reduction in Vcmax is usually
attributed to decreased amounts of Rubisco (RuBP carboxylase/oxygenase) (Jacob et al., 1995; Nakano et al., 1997;
Tissue et al., 1999), and sometimes to a low activation state
(Sage et al., 1989; Cook et al., 1998). The mechanism of
lowering in Jmax, on the other hand, is less clear, because
the capacity of RuBP regeneration is determined through
many biochemical steps in electron transport (von
Caemmerer and Farquhar, 1981; Evans and Terashima,
1987; Sudo et al., 2003) and the Calvin cycle (Strand et al.,
2000; Sudo et al., 2003).
Changes in the balance between Vcmax and Jmax also
affect the CO2 stimulation of photosynthesis, because the
CO2 dependence of the photosynthetic rate is greater when
the rate is limited by RuBP carboxylation than by RuBP
regeneration (Fig. 2). As Vcmax and Jmax limit the photosynthetic rate at low and high concentrations, respectively,
an increase in the Jmax:Vcmax ratio increases the ratio of the
photosynthetic rate at high CO2 to that at low CO2
concentrations (Webber et al., 1994; Sage, 1994; Medlyn,
1996; Hikosaka and Hirose, 1998). This indicates that the
increase in the Jmax:Vcmax ratio enhances the CO2 stimulation of photosynthesis. Although higher Jmax:Vcmax ratios
were reported in several studies in plants grown with
elevated CO2 (Sage et al., 1989; Lewis et al., 1996), many
studies found no change in the ratio (reviewed by Medlyn
et al., 1999). Hence, the effects of changes in the Jmax:Vcmax
ratio have been largely discounted in studies of plant
response to elevated CO2.
Recent studies, however, have found that growth temperature affects the Jmax:Vcmax ratio. Hikosaka et al. (1999)
demonstrated that evergreen Quercus myrsinaefolia leaves
grown at a low temperature had a higher Jmax:Vcmax ratio
than those at a high temperature, and consequently that
photosynthesis was more sensitive to CO2 in plants
acclimated to a low temperature. A similar trend was
found by Wilson et al. (2000), who reported that autumn
leaves had a higher Jmax:Vcmax ratio than summer leaves in
several deciduous tree species in a temperate forest. These
results suggest that seasonal change in temperature alters
the balance between RuBP carboxylation and RuBP regeneration, and affects the extent of CO2 stimulation of
photosynthesis.
It is proposed that seasonal change in the balance
between RuBP carboxylation and RuBP regeneration is
an important factor in predicting the carbon gain of plants
under elevated CO2 environments. Theoretical analysis of
Rubisco kinetics indicates that CO2 stimulation of photosynthesis increases with temperature (Long, 1991), but
such temperature dependence was not always observed in
plants grown in a seasonal climate (Teskey, 1997; Lewis
et al., 2001). This inconsistency may be explained by
a change in the balance between RuBP carboxylation and
RuBP regeneration. In the present study, Vcmax and Jmax
were analysed in plants grown at two CO2 concentrations in
two seasons (mid-summer and mid-autumn). Rubisco and
cytochrome f contents that are considered to be closely
linked with Vcmax and Jmax, respectively, were also determined. The aim of this study was to test the following
hypotheses: (i) leaves have a higher ratio of Jmax:Vcmax in
autumn than in summer, and consequently (ii) the CO2
stimulation of the photosynthetic rate is higher in autumn;
and (iii) seasonal changes in Vcmax and Jmax are associated
with Rubisco and cytochrome f content, respectively.
Young leaves in summer and young and old leaves in
autumn were used to separate the effect of leaf senescence
from the effect of seasonal change in climate and its
interaction with elevated CO2 (Nie et al., 1995; Osborne
et al., 1998; Cook et al., 1998; Jach and Ceulemans, 2000).
Materials and methods
Polygonum cuspidatum Sieb. et Zucc. (=Reynoutria japonica Houtt.,
Fallopia japonica Houtt.), a perennial herb, is distributed widely
across both latitudes and altitudes in the temperate zone of Asia
(Makino, 1961). This species plays an important role in the early
stage of primary succession (Hirose and Tateno, 1984), and has been
well studied both ecologically (Maruta, 1983; Chiba and Hirose,
1993) and physiologically (Kogami et al., 2001; Onoda et al., 2004).
Effects of elevated CO2 on the growth of this species have been
studied (Ishizaki et al., 2003). This species is of particular interest as it
is a vigorous invading species in Europe, America, and New Zealand
(Beerling et al., 1994). Seeds of P. cuspidatum were collected at
a foot of Mt Fuji (358199 N, 1388489 E) in 1998 and stored in
a refrigerator (4 8C) until the start of the experiments.
The experiments were repeated in two years (2001 and 2002).
Plants were grown from seeds germinated on wet sand in an
incubator (20 8C) on 20 May and 8 August in both years to obtain
leaves of different age at the same measuring time. Seedlings were
planted in 4 l pots filled with washed river sand. The pot was
assumed to be large enough for the plants in this study, as the plant
had a relatively small dry mass at the end of growing season (average
14 g). Fifty-four pots were assigned to either ambient (c. 370 lmol
mol1) or high CO2 concentrations (targeted at 700 lmol mol1)
with six open top chambers (OTC; 23232 m3, three chambers for
each CO2) set in the experimental garden of Tohoku University,
Sendai, Japan (388159 N, 1418529 E). During the experiment, the
CO2 concentration in the chambers was monitored every 30 s. There
was no significant difference in CO2 concentration among chambers
for each CO2 treatment. The CO2 concentration in the chamber was
360–400 lmol mol1 for ambient, and 600-800 lmol mol1 for
elevated CO2. The structure of the OTC was detailed in Nagashima
et al. (2003). Every week, each pot received 90 ml of nutrient
Seasonal change in CO2 response of photosynthesis
solution (7 mM [N], Hyponex NPK=5:10:5; M Scott & Sons Co.,
Marysville, OH, USA).
Leaf phenology was recorded every 10 d in 2001 for four randomly
selected plants germinated in May and in August. To document leaf
ages, coloured bands were attached to the petiole when a new leaf
appeared. Plants germinated in May produced leaves continuously
until late August and retained those leaves until the end of growing
season (November). Leaves of plants germinated in May that
expanded fully in August were used for measurements in August
(young leaves, 20 d after leaf emergence) and in October (old leaves,
100 d after leaf emergence). Plants germinated in August produced
leaves until late October, and fully expanded young leaves were used
for a measurement in October (young-October leaves, 20 d after leaf
emergence). Leaves exposed to full sunlight without mutual shading
were used for measurement.
Air temperature in each OTC was measured with a thermocouple
and light intensity on the top of an OTC was measured with a light
sensor (LI-190SA, Li-Cor, Lincoln, NE, USA), and logged every 60
min during the growing season. There was no significant difference in
temperatures between chambers (ANOVA, P >0.1). Maximum,
minimum, and mean temperatures, and photon flux density (PFD)
across the experimental period are shown in Fig. 1. The mean
temperature of August and October was 23.2 and 13.5 8C in 2001,
and 25.3 and 13.3 8C in 2002, respectively.
CO2 dependence of the net CO2 assimilation rate was determined
on attached, fully expanded leaves with an open gas-exchange system
(LI-6400, Li-Cor) on 1–5 August (young-August leaves) and 20–25
October (young- and old-October leaves). To relate gas-exchange
parameters with protein content, photosynthesis was measured at the
same conditions across seasons: leaf temperature in the chamber was
kept at 25 8C, VPD (vapour pressure deficit) <1 kPa, and PFD at 2000
lmol m2 s1 with an artificial light source (LI-6400–02B, Li-Cor).
Maximum temperature
Minimum temperature
Mean temperature
PFD
50
(a) 2001
40
Photon flux density (mol m-2 d-1)
Temperature (oC) or
30
20
10
0
(b) 2002
40
30
20
10
0
May
Jun
Jul
Aug
Sep
Oct
Fig. 1. Growing conditions of plants in 2001 (a) and 2002 (b). Each point
indicates a mean for 10 d. Arrows indicate the time of photosynthetic
measurement.
757
The measurements were replicated in at least six leaves per leaf group
(young-August, young-October, and old-October) from different
individuals.
The photosynthesis curve plotted against intercellular CO2 concentration (A/Ci curve) was analysed to estimate the in vivo
maximum rate of RuBP carboxylation (Vcmax) and the in vivo
maximum rate of electron transport driving regeneration of RuBP
(Jmax). Vcmax was calculated by fitting the following equation
(Farquhar et al., 1980) to the initial slope of A/Ci curves (Ci <300
lmol mol1) with the least square method with Kaleida Graph
(Synergy Software, PA, USA):
Ac =
Vcmax ðCi C Þ
Rd
Ci + Kc ð1 + O=Ko Þ
ð1Þ
where Ac is the photosynthetic rate limited by Rubisco activity, Ci is
intercellular CO2 concentration, C* is the CO2 compensation point in
the absence of day respiration (Rd), and Kc and Ko are MichaelisMenten constants of Rubisco activity for CO2 and O2, respectively.
C*, Kc, and Ko are assumed to be 42.8 lmol mol1, 405 lmol mol1,
and 278 mmol mol1 according to Bernacchi et al. (2001). Rd was
assumed to be 0.02 of Vcmax (von Caemmerer, 2000). Jmax was
calculated by fitting the following equation (Farquhar et al., 1980) to
a near-plateau of A/Ci curves (Ci >600 lmol mol1):
Aj =
Jmax ðCi C Þ
Rd
4Ci + 8C
ð2Þ
Aj is the photosynthetic rate limited by RuBP regeneration. Ci, is the
CO2 concentration at which photosynthesis was co-limited by RuBP
carboxylation and RuBP regeneration, was calculated from equations
1 and 2 with Ac = Aj.
After the photosynthetic measurements, seven leaf discs, 1 cm in
diameter, were punched out from each leaf. Within 60 s, the leaf discs
and the residual parts were frozen with liquid nitrogen and stored at
80 8C until used in chemical analyses. Two leaf discs were used for
the measurement of Rubisco. The amount of Rubisco was determined
spectrophotometrically after formamide extraction of Coomassie
Brilliant Blue R-250-stained large subunit bands that were separated
by SDS–PAGE (Makino et al., 1986; Onoda et al., 2004). Chlorophyll was extracted with 80% acetone and its concentration was
determined with a spectrophotometer (UV-160A; Shimadzu, Kyoto,
Japan) according to Porra et al. (1989). The residual part, excluding
the main vein, was used for measuring cytochrome f. The amount of
cytochrome f was determined from the difference between the
hydroquinone-reduced and the ferricyanide-oxidized spectra of thylakoid membranes according to Bendall et al. (1971). The increase in
the absorbance at 554 nm above the line drawn between the
absorbances at 543.5 and 570 nm was measured. The extinction
coefficient used was 20 mM1 cm1 (Evans and Terashima, 1987).
Although all samples were used for the cytochrome f measurement,
some leaves were not large enough to allow accurate determination of
cytochrome f and were eliminated from the calculation.
Statistical tests were performed using STAT VIEW version 5.0
(SAS Institute Inc, Cary, IN, USA). Leaf group (young-August,
young-October, and old-October) was assumed to be an independent
factor. Since no difference was observed by the analysis of variance
(ANOVA) between data in 2001 and 2002 (ANOVA, P >0.1 for all
variables), two years’ data were pooled, and then analysed with the
following statistics. Significance levels among slopes and among yintercepts in linear regressions were evaluated by analysis of covariance (ANCOVA). Difference in biochemical data among the
three leaf groups was evaluated by ANOVA. Student’s t-test was
used for the effect of the CO2 treatments.
758 Onoda et al.
Results
Figure 2 shows some examples of A/Ci curves in youngAugust, young-October, and old-October leaves of plants
grown in each CO2 treatment. The model of Farquhar et al.
(1980) was fitted to each A/Ci curve. The photosynthetic
rate at 370 lmol mol1 of air CO2 concentration (Ca) was
always limited by RuBP carboxylation, and that at 700
lmol mol1 Ca was limited by RuBP regeneration in
August leaves, but often limited by RuBP carboxylation in
20
15
10
5
(a) Young-August
Photosynthetic rate (µmol m-2s-1)
0
30
20
10
October leaves. When compared at the same Ci, photosynthetic rates were lower in plants grown at elevated CO2.
Photosynthetic rates measured at growth CO2 concentration
(Agrowth, indicated by arrows in Fig. 2) were higher in elevated CO2 plants in all leaf groups. Agrowth varied significantly among leaf groups, the highest values being observed
in young-October leaves and the lowest in old-October
leaves (Fig. 3; Table 1). Significant interaction between leaf
group and CO2 indicates that stimulation of Agrowth
with elevated CO2 varied among leaf groups (Fig. 3; Table
1). Young-October leaves showed a higher stimulation
(44%) than young-August leaves (36%), and the stimulation
was lowest in October-old leaves (27%, P=0.13).
Stomatal conductance at a growth CO2 concentration did
not significantly differ between CO2 treatments, but differed slightly between leaf groups (Tables 1, 2). The Ci:Ca
ratio at a growth CO2 concentration was independent of
CO2 treatment and leaf group (Tables 1, 2).
Both Vcmax and Jmax were highest in young-October
leaves and lowest in old-October leaves. Elevated CO2
treatment tended to reduce both Vcmax and Jmax within a leaf
group (Fig. 4). The extent of reduction by elevated CO2 was
larger in old-October leaves (24% in Vcmax) than in youngOctober leaves (13% in Vcmax), which was responsible for
the smaller stimulation of Agrowth in old leaves. In youngAugust leaves, elevated CO2 also tended to reduce Vcmax
(10%), although it was not significant.
A strong correlation between Jmax and Vcmax was
observed within a leaf group across CO2 treatments
(Fig. 5). ANCOVA indicates that the effect of CO2 treatments was not significant (P >0.1). Leaf groups significantly affected the regression (P <0.0001), but there was no
significant difference between young- and old-October
(b) Young-October
0
30
+44%****
Agrowth (µmol m-2s-1)
25
20
15
10
+36%***
+27%ns
15
10
5
5
(c) Old-October
0
20
0
200
400
600
800
Intercellular CO2 concentration (µmol
1000
1200
mol-1)
Fig. 2. Photosynthetic rate versus intercellular CO2 concentration of
Polygonum cuspidatum grown either at ambient CO2 (370 lmol mol1;
open symbols) or at elevated CO2 (700 lmol mol1; closed symbols) in
young-August (a), young-October (b), and old-October leaves (c). The
model of Farquhar et al. (1980) was fitted. See Materials and methods for
details. Arrows indicate the photosynthetic rate at growth CO2 concentration.
0
YoungAugust
YoungOctober
OldOctober
Fig. 3. Photosynthetic rate measured at growth CO2 concentrations
(Agrowth) in young-August, young-October, and old-October leaves of
Polygonum cuspidatum grown either at ambient CO2 (370 lmol mol1;
open columns) or at elevated CO2 (700 lmol mol1; closed columns).
Bars represent 61 SE (n >6). The difference between CO2 treatments was
tested with Student’s t-test. Significance levels: ***, P <0.001; ns,
P >0.1. The percentages above a pairs of columns denote changes in
Agrowth with elevated CO2.
Seasonal change in CO2 response of photosynthesis
759
Table 1. Significance levels for the effects of leaf group and CO2 treatment on the photosynthetic rate at growth CO2 concentration
(Agrowth ), RuBP carboxylation capacity ( Vcmax), maximum electron transport rate driving RuBP regeneration (Jmax), ratio of Jmax to
Vcmax (J/V), stomatal conductance at growth CO2 concentration (gs), ratio of intercellular CO2 concentration to air CO2
concentration (Ci/Ca) and the CO2 concentration at which photosynthetic rate was co-limited by RuBP carboxylation and RuBP
regeneration (co-limiting Ci)
See Materials and methods for details. Significance levels: ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; +, P < 0.1; ns, not significant.
Factors
Agrowth
Vcmax
Jmax
J/V
gs
Ci/Ca
Co-limiting Ci
Leaf group
CO2
Leaf group 3 CO2
****
****
*
****
***
ns
****
**
ns
****
+
ns
+
ns
ns
ns
ns
ns
****
ns
ns
Table 2. Stomatal conductance at growth CO2 concentration (gs, mol m2 s1), ratio of intercellular CO2 concentration to air CO2
concentration (Ci / Ca), the CO2 concentration at which photosynthetic rate was co-limited by RuBP carboxylation and RuBP
regeneration (co-limiting Ci, lmol mol1), and the ratio of maximum electron transport rate to maximum RuBP carboxylation rate
(J/V) of Polygonum cuspidatum grown either in ambient CO2 (370 lmol mol1; AC) or elevated CO2 (700 lmol mol1; EC)
See Materials and methods for details. Means 6SE (n >6). Different superscripts within a column indicate statistical difference at 5% level (TukeyKramer test among leaf groups).
Leaf group
CO2
Parameters
gs
Young-August
Young-October
Old-October
AC
EC
AC
EC
AC
EC
Ci/Ca
a
0.32160.071
0.31060.044a
0.30460.014a
0.25260.037a
0.20060.039a
0.19060.054a
leaves. Both young- and old-October leaves had a significantly higher Jmax at a given Vcmax than August leaves. An
increase in the Jmax:Vcmax ratio increased the CO2 concentration at which the photosynthetic rate was co-limited by
carboxylation and regeneration of RuBP (Table 2), which
increased the CO2 sensitivity of the photosynthetic rate in
October leaves.
Vcmax strongly correlated with Rubisco content irrespective of CO2 treatments and leaf groups (Fig. 6a). Similarly,
Jmax correlated with cytochrome f content (Fig. 6b). The
ratio of cytochrome f to Rubisco tended to be higher in
October leaves, especially in old leaves, than in August
leaves (Fig. 7).
Discussion
CO2 enrichment stimulated Agrowth, but the extent of
stimulation was significantly different among leaf groups
(Fig. 3; Table 1). The difference in the stimulation could be
ascribed to the change in RuBP regeneration and RuBP
carboxylation capacities with elevated CO2 (Fig. 4), and to
the seasonal change in the balance between the two capacities (Fig. 5). Although the stomatal conductance changed
among leaf groups, the Ci:Ca ratio was almost constant
(Tables 1, 2), indicating that the stomatal limitation of
photosynthesis was not responsible for the different stimulations (Drake et al., 1997; Rey and Jarvis, 1998).
Co-limiting Ci
a
0.76760.031
0.82460.026a
0.73260.009a
0.73160.031a
0.75560.016a
0.77760.054a
a
466623
516625a,b
569634b
634638b,c
731651c,d
948630d
J/V
1.7260.05a
1.8260.04a,b
1.9160.06b
2.0160.06b,c
2.1460.07c,d
2.1860.04d
The largest reduction of Vcmax and Jmax with elevated
CO2 in old-October leaves was responsible for the lowest
CO2 stimulation of Agrowth. The large down-regulation in
old-October leaves may be explained by acceleration of leaf
senescence with elevated CO2 (Nie et al., 1995; Osborne
et al., 1998; Cook et al., 1998; Jach and Ceulemans, 2000)
due to advanced plant developmental stages (Cook et al.,
1998; Ludewig and Sonnewald, 2000) or to sugar accumulation from source/sink imbalance (Rey and Jarvis, 1998).
The higher ratio of Jmax:Vcmax in October leaves than in
August leaves (Table 2) increased the Ci at which the
photosynthetic rate was co-limited by RuBP carboxylation
and RuBP regeneration (Table 2; Fig. 2), and shifted
the inflection point of the A/Ci curve to higher CO2 concentration in October leaves compared with August leaves
(Fig. 2). This caused the photosynthetic rate to be more
dependent on CO2 concentration. Therefore, larger stimulation of Agrowth with elevated CO2 was observed in youngOctober than young-August leaves (Fig. 3). Old-October
leaves also had a higher co-limitation point, but its effect on
stimulation of Agrowth was offset by a greater downregulation of photosynthesis with elevated CO2 (Fig. 4).
Old-October leaves that were fully expanded in
August had a different relationship of Vcmax and Jmax
from young-August leaves, but had an almost identical
relationship of Vcmax and Jmax with that of young-October
leaves (Fig. 5), suggesting that P. cuspidatum could
760 Onoda et al.
160
100
-13%*
120
-10%ns
60
-24%*
40
20
0
(b)
Jmax (µmol m-2 s-1)
Vcmax (µmol m-2 s-1)
(a)
80
80
40
Jmax (µmol m-2 s-1)
-8%*
150
100
-22%*
-6%ns
0
0
20
40
60
80
100
Vcmax (µmol m-2 s-1)
50
0
YoungAugust
YoungOctober
OldOctober
Fig. 4. The in vivo maximum rate of RuBP carboxylation (Vcmax) and the
in vivo maximum rate of electron transport driving RuBP regeneration
(Jmax) in young-August, young-October, and old-October leaves of
Polygonum cuspidatum grown either at ambient CO2 (370 lmol mol1;
open columns) or at elevated CO2 (700 lmol mol1; closed columns). Bars
represent 61 SE (n >6). The difference between CO2 treatments was tested
with Student’s t-test. Significance levels: *, P <0.05; ns, P >0.1. The
percentages above pairs of columns indicate changes with elevated CO2.
acclimate to seasonal climate after full-expansion of the
leaf. Change in the Jmax:Vcmax ratio (Table 2) may be
brought about by the change in the relative amount of
photosynthetic proteins (Fig. 7). Vcmax was strongly correlated with Rubisco (Fig. 6a) as has been observed in many
studies (von Caemmerer and Farquhar, 1981; Makino et al.,
1994a; Jacob et al., 1995), indicating that Vcmax was
primarily determined by the amount of Rubisco. Similarly,
Jmax was strongly correlated with cytochrome f (Evans,
1987; Terashima and Evans, 1988; Price et al., 1998; Sudo
et al., 2003). Jmax may be correlated with the activities
of other proteins, such as fructose-1,6-bisphosphatase
(Fridlyand et al., 1999; Sudo et al., 2003), sedoheptulose
1,7-bisphosphatase (Harrison et al., 2001), sucrose phosphate synthase (Strand et al., 2000), and ATP synthase
(Evans, 1987; Terashima and Evans, 1988). Given that
there is a stoichiometry among these photosynthetic proteins (Evans, 1987; Evans and Terashima, 1988), they are
likely to co-limit Jmax with the cytochrome b6/f complex.
Vcmax and Jmax were calculated assuming that Ci was equal
to the chloroplast CO2 concentration (von Caemmerer et al.,
1994). However, there is a possibility that internal resistance
for CO2 diffusion affected the Jmax:Vcmax ratio. If the
Fig. 5. The relationship between the in vivo maximum rate of electron
transport driving RuBP regeneration (Jmax) and the in vivo maximum rate
of RuBP carboxylation (Vcmax) in young-August (circles), young-October
(squares), and old-October (triangles) leaves of Polygonum cuspidatum
grown either at ambient CO2 (370 lmol mol1; open symbols) or at
elevated CO2 (700 lmol mol1; closed symbols). Regression lines:
y=1.10x+31.36 (R2=0.70) for August leaves; y=1.71x+16.59 (R2=0.95)
for October leaves.
resistance was high, Vcmax might have been overestimated.
As Jmax was less dependent on resistance, the Jmax:Vcmax
ratio would be increased by higher resistance without any
changes in photosynthetic proteins (von Caemmerer, 2000).
Makino et al. (1994b) suggested that internal resistance
increased in rice leaves grown at low temperatures. However, the strong relationships between Vcmax and Rubisco
across leaf groups (Fig. 6) suggest that the effects of the
internal CO2 diffusion resistance were small in the present
study (Hikosaka et al., 1998; Medlyn et al., 2002).
Increase in the Jmax:Vcmax ratio may be beneficial for
photosynthesis in a season with low temperatures. A
Rubisco-limited rate of photosynthesis (Ac) is less sensitive
to temperature because the decrease in carboxylation at low
temperatures is partly compensated by suppression of
photorespiration (Kirschbaum and Farquhar, 1984). On
the other hand, RuBP regeneration-limited rate of photosynthesis (Aj), which depends much less on oxygenation, is
fairly sensitive to temperature (Kirschbaum and Farquhar,
1984; Hikosaka et al., 1999). At moderately low temperatures, the limitation of photosynthetic rate by RuBP regeneration may be stronger than that by RuBP carboxylation.
Therefore, a relative increase in Jmax at low temperatures
may relieve the limitation of photosynthesis by RuBP
regeneration (Hikosaka, 1997; Hikosaka et al., 1999).
Seasonal change in climate involves not only a change in
temperatures, but also a change in light intensities. The
change in light intensity is a potentially confounding factor
in determining the cause of change in leaf traits. However,
Seasonal change in CO2 response of photosynthesis
0.6
90
Cytchrome f / Rubisco (µmol g-1)
Vcmax (µmol m-2 s-1)
(a)
60
30
0
0
1
2
3
0.5
Leafgroup (LG)
CO2
LG X CO2
0.3
0.2
0.1
0
(b)
Jmax (µmol m-2 s-1)
YoungAugust
YoungOctober
OldOctober
Fig. 7. Ratio of cytochrome f to Rubisco in young-August, youngOctober, and old-October leaves of Polygonum cuspidatum grown either
at ambient CO2 (370 lmol mol1; open columns) or at elevated CO2 (700
lmol mol1; closed columns). Bars represent 61 SE (n=3–6). The
difference was tested with ANOVA.
200
150
100
50
0
P <0.001
P > 0.1
P > 0.1
0.4
Rubisco (g m-2)
0
761
0.2
0.4
0.6
Cytochrome f (µmol m-2)
0.8
Fig. 6. Relationship between the in vivo maximum rate of RuBP
carboxylation (Vcmax) and Rubisco content (a), and between the in vivo
maximum rate of electron transport driving RuBP regeneration (Jmax) and
cytochrome f content (b) in young-August (circles), young-October
(squares), and old-October (triangles) leaves of Polygonum cuspidatum
grown either at ambient CO2 (370 lmol mol1; open symbols) or at
elevated CO2 (700 lmol mol1; closed symbols). Regression lines: (a)
y=25.68x+20.20 (R2=0.83); (b) y=161.2x+44.8 (R2=0.77).
change in the Jmax:Vcmax ratio does not occur across growth
light intensities (von Caemmerer and Farquhar, 1981).
Therefore the change in the Jmax:Vcmax ratio in this study
could be ascribed to seasonal temperature change.
The change in the Jmax:Vcmax ratio demonstrated in the
present study occurred within the range of values in
Wullschleger (1993) who reported a strong correlation
between Vcmax and Jmax across a number of species.
Increased Jmax:Vcmax ratios in leaves acclimated to low
temperature (Fig. 5) were also observed in Quercus
myrsinaefolia grown in temperature-controlled growth
chambers (Hikosaka et al., 1999) and in several deciduous
tree species grown in Tennessee, USA (Wilson et al.,
2000). However, this response may not hold in some plant
species, where systematic change in the Jmax:Vcmax ratio
was not observed with respect to temperature acclimation
(Bunce, 2000; Medlyn et al., 2002). Further research is
needed to elucidate factors involved in inter-specific differences in the response.
It has been believed that an increase in atmospheric CO2
concentration would be of greater benefit to tropical or
warm climate plants with respect to carbon gain than
plants in a cool climate (Long, 1991; Sage et al., 1995;
Kirschbaum, 1994, Hikosaka and Hirose, 1998). This
prediction is based on the kinetics of Rubisco and on the
solubility of CO2 and O2; suppression of photorespiration
with elevated CO2 is stronger at higher temperatures (Long,
1991). It was supported by short-term experiments (seconds
to minutes) (Sage et al., 1995; Bunce, 1998) as well as by
some long-term elevated CO2 studies (Lewis et al., 1996;
Ziska and Bunce, 1997). However, a number of studies
reported that temperature dependence of relative CO2 stimulation of Agrowth was much weaker than theoretical predictions (Greer et al., 1995; Teskey, 1997; Wayne et al., 1998;
Bunce, 1998; Lewis et al., 2001). This might be
partly explained by temperature acclimation. Most models
that predicted the effects of climate change on plants did not
take temperature acclimation into account (Medlyn et al.,
2002). Mechanistic understanding of temperature acclimation of photosynthesis is indispensable to predict accurately
the effect of elevated CO2 on vegetation in the future.
Acknowledgements
We thank Kenichi Sato, Toshihiko Kinugasa, Shimpei Oikawa, Yuko
Yasumura, Riichi Oguchi, Shinjiro Ishizaki, Onno Muller, Teruyuki
Takashima, and Borjigidai Almaz for their technical assistance. This
study was partly supported by grants-in-aid from JSPS for young
research fellows (YO), and from the Japanese Ministry of Education,
Culture, Sports, Science, and Technology to KH and TH.
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