Plant Cell Physiol. 49(4): 583–591 (2008)
doi:10.1093/pcp/pcn030, available FREE online at www.pcp.oxfordjournals.org
ß The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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The Role of Electron Transport in Determining the Temperature Dependence of
the Photosynthetic Rate in Spinach Leaves Grown at Contrasting Temperatures
Wataru Yamori
1, 4,
*, Ko Noguchi 2, Yasuhiro Kashino
3
and Ichiro Terashima
2
1
Department of Biology, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka, 560-0043 Japan
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan
3
Department of Life Science, University of Hyogo, 3-2-1 Kamigohri, Ako-Gun, Hyogo, 678-1297 Japan
2
Introduction
In many plants, the temperature response of photosynthesis depends on the growth temperature (Berry and
Björkman 1980, Yamori et al. 2005). The optimum
temperature for photosynthesis increases with increasing
growth temperature. According to the C3 photosynthesis
model (Farquhar et al. 1980), the photosynthetic rate is
limited either by the ribulose bisphosphate (RuBP) carboxylation rate (Pc) at ambient and low CO2, or by the RuBP
regeneration rate (Pr) at high CO2. At temperatures below
the optimum, photosynthetic rates are often limited by
Pr, including the limitation by Pi regeneration (Sharkey
1985, Sage and Sharkey 1987, Labate and Leegood 1988,
Cen and Sage 2005, Sage and Kubien 2007). On the other
hand, the principal limitation of photosynthesis above the
optimum temperature remains unclear. One proposed
mechanism is that the photosynthetic rate is limited by
Pc due to the heat-induced reductions in the activase
activity which regulates the Rubisco activation state (Weis
1981, Kobza and Edwards 1987, Law and Crafts-Brandner
1999, Crafts-Brandner and Salvucci 2000, Salvucci and
Crafts-Brandner 2004, Yamori et al. 2006a). Recently,
Kurek et al. (2007) generated several mutants of Arabidopsis
thaliana which varied in thermostability of Rubisco
activase and showed that the principal limitation of
photosynthesis at high temperature was Rubisco activation
via Rubisco activase. Alternatively, Pr has been proposed as
the limiting step (Schrader et al. 2004, Wise et al. 2004, Cen
and Sage 2005, Makino and Sage 2007). They suggested
that the decrease in Rubisco activation state at high
temperatures is a regulated response resulting from other
limitations including the electron transport rate (ETR). In
all of the studies claiming that the Rubisco activation state
limits the photosynthetic rate at high temperatures, however, analysis of Pr has been omitted. Therefore, we need
to analyze the limitation of photosynthesis by Pr in
plants for which the photosynthetic rate has been reported
Keywords: Electron transport — Photosynthesis — RuBP
carboxylation — RuBP regeneration — Temperature
acclimation — Temperature dependence.
Abbreviations: DBMIB, 2,5-dibromo-3-methyl-6-isopropylp-benzoquinone; DCIP, dichloroindophenol; ETR, electron
transport rate; FBPase, fructose-1,6-bisphosphatase; HT, high
temperature; LT, low temperature; RuBP, ribulose bisphosphate.
4
Present address: Molecular Plant Physiology Group, Research School of Biological Sciences, Building 46, The Australian National
University, Canberra, ACT, 2601 Australia.
*Corresponding author: E-mail, [email protected]; Fax, þ61-2-6125-5075.
583
Editor-in-Chief’s choice
The temperature response of the uncoupled whole-chain
electron transport rate (ETR) in thylakoid membranes differs
depending on the growth temperature. However, the steps that
limit whole-chain ETR are still unclear and the question
of whether the temperature dependence of whole-chain ETR
reflects that of the photosynthetic rate remains unresolved.
Here, we determined the whole-chain, PSI and PSII ETR in
thylakoid membranes isolated from spinach leaves grown at
308C [high temperature (HT)] and 158C [low temperature
(LT)]. We measured temperature dependencies of the lightsaturated photosynthetic rate at 360 kl l–1 CO2 (A360) in HT
and LT leaves. Both of the temperature dependences of wholechain ETR and of A360 were different depending on the
growth temperature. Whole-chain ETR was less than the
rates of PSI ETR and PSII ETR in the broad temperature
range, indicating that the process was limited by diffusion
processes between the PSI and PSII. However, at high
temperatures, whole-chain ETR appeared to be limited by not
only the diffusion processes but also PSII ETR. The C3
photosynthesis model was used to evaluate the limitations of
A360 by whole-chain ETR (Pr) and ribulose bisphosphate
carboxylation (Pc). In HT leaves, A360 was co-limited by Pc
and Pr at low temperatures, whereas at high temperatures,
A360 was limited by Pc. On the other hand, in LT leaves,
A360 was solely limited by Pc over the entire temperature
range. The optimum temperature for A360 was determined by
Pc in both HT and LT leaves. Thus, this study showed that, at
low temperatures, the limiting step of A360 was different
depending on the growth temperature, but was limited by Pc
at high temperatures regardless of the growth temperatures.
584
Temperature acclimation of the electron transport system
to be limited by the Rubisco activation state at high
temperatures.
Pr is generally thought to be determined by the wholechain ETR in thylakoid membranes, since Pr is dependent
on the supply of ATP and NADPH (von Caemmerer 2000).
The CO2-saturated photosynthetic rate at 258C, which
reflects Pr, correlated with the Cyt f content (Price et al.
1995, Price et al. 1998, Ruuska et al. 2000). The temperature
response of the ETR in thylakoid membranes depends on
the growth temperature, with considerable variation
depending on the plant species (Tieszen and Helgager
1968, Armond et al. 1978, Badger et al. 1982, Mitchell and
Barber 1986, Mawson et al. 1986, Mawson and Cummins
1989, Yamasaki et al. 2002). The temperature responses of
the ETR in thylakoid membranes and the CO2-saturated
photosynthetic rate were closely related in Saxifraga cernua
(Mawson et al. 1986, Mawson and Cummins 1989) and
Triticum aestivum (Yamasaki et al. 2002). These studies
indicated that the temperature dependence of the wholechain ETR was limited by that of the PSII ETR (Mawson
and Cummins 1989, Yamasaki et al. 2002). However, it has
not been quantitatively evaluated whether the temperature
dependence of the photosynthetic rate is limited by that of
the ETR.
On the other hand, it has also been reported that the
temperature dependence of the CO2-saturated photosynthetic rate was not correlated with that of the ETR in
Larrea divaricata (Armond et al. 1978, Mooney et al. 1978)
or Nerium oleander (Badger et al. 1982). Therefore, it is
possible that Pr may not necessarily be limited by the ETR.
Thus, it is still unclear whether the temperature dependence
of the ETR limits that of Pr.
Recently, we showed that the temperature dependences
of the photosynthetic rate were different between the leaves
of spinach grown at 158C and those grown at 308C (Yamori
et al. 2005), and that the photosynthetic rate was limited by
Pc across a broad temperature range, irrespective of the
growth temperature (Yamori et al. 2006a, Yamori et al.
2006b). We showed that Pc was partly determined by the
Rubisco activation state at high temperatures (Yamori et al.
2006a). The use of spinach grown at 15 and 308C should
therefore enable us to clarify the limiting step in photosynthesis across a range of temperatures, thereby revealing
the comprehensive mechanisms of temperature acclimation
of photosynthesis.
In the present study, we measured CO2 response curves
of photosynthesis at saturating light intensity in spinach
grown at 158C [low temperature (LT)] and 308C [high
temperature (HT)], and determined the temperature
response of the ETR (whole-chain and PSI and PSII partial
reactions) in isolated thylakoid membranes. We compared the maximum ETRs measured in thylakoid membranes with those estimated from the CO2-saturated
photosynthetic rates in HT and LT leaves. We quantitatively evaluated whether the photosynthetic rate measured
at 360 ml l–1 CO2 was limited by the ETR in HT and LT
leaves. The aims were to determine: (i) whether the
temperature dependence of the whole-chain ETR differed
depending on the growth temperature; (ii) what limited
the temperature dependence of the whole-chain ETR;
(iii) whether the temperature dependence of Pr was limited
by that of the ETR; and (iv) which partial reaction of Pc
and Pr limited the temperature dependence of the photosynthetic rate.
Results
Temperature responses of photosynthetic rate
Temperature responses of the light-saturated photosynthetic rate at 360 ml l–1 CO2 (A360), the initial slope of
the A–Ci curve (IS) and that of the photosynthetic rate at
1,500 ml l–1 CO2 (A1500) all depended on growth temperature (Fig. 1). The optimum temperatures for A360, IS and
A1500 derived from the cubic curves were 30.5, 28.2 and
37.28C, respectively, in HT leaves. In LT leaves, the
optimum temperatures for A360, IS and A1500 were 23.5,
21.6 and 30.08C, respectively. The dark respiration rate (Rd)
of LT leaves was greater than that of HT leaves at any
temperatue (data not shown).
Temperature responses of electron transport rate
Temperature responses of whole-chain, PSI and PSII
ETRs in thylakoid membranes isolated from HT and LT
leaves (HT and LT thylakoids, respectively) are shown
in Fig. 2. The temperature responses of the whole-chain
ETR per unit of Chl differed depending on the growth
temperature (Fig. 2a). The optimum temperature for the
whole-chain ETR was 35 and 308C in HT and LT
thylakoids, respectively (Fig. 2a). At low temperatures, the
whole-chain ETR in LT thylakoids was higher than that in
HT thylakoids, whereas at high temperatures, the wholechain ETR in LT thylakoids was lower than that in HT
thylakoids. The PSII ETR showed a temperature dependence similar to that of the whole-chain ETR (Fig. 2b), and
showed the optimum temperature at 35 and 308C in HT
and LT thylakoids, respectively. On the other hand, the
PSI ETR showed steady increases up to 408C (Fig. 2c).
The PSI ETR at low temperatures was higher in LT
thylakoids than in HT thylakoids, whereas at high
temperatures, it was lower in LT thylakoids than in HT
thylakoids.
ETRs per unit of Chl were converted to a leaf area
basis assuming four electrons per oxygen evolution in wholechain and PSII electron transport, and two electrons per
oxygen evolution in PSI electron transport (Fig. 2d, e, f).
The whole-chain ETR per unit leaf area was greater in LT
Temperature acclimation of the electron transport system
(b) 0.12
15
10
*
5
*
**
HT
LT
0
(c)
A1500 (µmol CO2 m–2 s–1)
20
IS (mol CO2 m–2 s–1)
A360 (µmol CO2 m–2 s–1)
(a)
585
0.10
0.08
0.06
**
**
0.04
**
0.02
40
30
***
*
***
10
0.00
5
***
20
***
**
0
10 15 20 25 30 35 40
5
10 15 20 25 30 35 40
Leaf temperature (°C)
5
Leaf temperature (°C)
10 15 20 25 30 35 40
Leaf temperature (°C)
–1
Fig. 1 Temperature dependences of the net photosynthetic rate at 360 ml l CO2 (A360; a), the initial slope of the A–Ci curve (IS; b) and
the photosynthetic rate at 1,500 ml l–1 CO2 (A1500; c). HT and LT denote the leaves grown at day/night air temperature of 30/25 and
15/108C, respectively (HT, open circle and solid line; LT, filled square and broken line). The data were fitted by cubic curves. Data
represent the means SE, n ¼ 3. The data were analyzed by Student’s t-test. P50.05, P50.01, P50.001.
(b)
500
300
*
200
***
100
**
0
**
10
20
30
40
600
*
400
200
0
Measurement temperature (°C)
200
***
***
100
0
0
***
***
*
*
10
20
30
40
50
Measurement temperature (°C)
*
900
*
600
300
0
**
10
20
30
40
50
Measurement temperature (°C)
500
400
300
** **
200
*** **
***
100
PS I (µmol electron m−2 s−1)
300
1200
(f)
500
400
1500
0
10
20
30
40
50
Measurement temperature (°C)
(e)
500
PS II (µmol electron m−2 s−1)
whole-chain (µmol electron m
−2 −1
s )
(d)
***
*
0
50
1800
PS I (µmol O2 (µmol Chl)−1 h−1)
HT
LT
400
0
(c)
800
PS II (µmol O2 (µmol Chl)−1 h−1)
whole-chain (µmol O2 (µmol Chl)−1 h−1)
(a)
400
300
**
200
***
***
***
***
100
***
0
0
10
20
30
40
50
Measurement temperature (°C)
0
0
**
***
10
20
30
40
50
Measurement temperature (°C)
Fig. 2 Temperature dependences of the electron transport rate per unit Chl of the whole chain (a), PSII (b) and PSI (c), and
electron transport rates per unit leaf area of the whole chain (d), PSII (e) and PSI (f). The Chl contents per unit leaf area were 0.396 and
0.604 mmol m–2 in HT and LT leaves, respectively. HT and LT denote the thylakoid membranes isolated from HT and LT leaves,
respectively (HT, open circle and solid line; LT, filled square and broken line). Data represent the means SE, n ¼ 3. The data were
analyzed by Student’s t-test. P50.05, P50.01, P50.001.
thylakoids than in HT thylakoids, except for at 408C
where it abruptly decreased (Fig. 2d). This temperature
dependence corresponded to that of the PSII ETR per
unit leaf area (Fig. 2e). The PSI ETR per unit leaf area
was always greater in LT than in HT thylakoids (Fig. 2f).
The whole-chain ETR was less than the PSI and PSII
ETRs in both HT and LT thylakoids over the entire
temperature range.
586
Temperature acclimation of the electron transport system
(a)
(b)
150
100
50
Jmax (in vitro)
Jmax (in vivo)
0
10
20
30
40
50
Measurement temperature (°C)
LT
200
200
Jmax (in vivo)
HT
0
(c)
250
Jmax (µmol electron m−2 s−1)
Jmax (µmol electron m−2 s−1)
200
150
100
50
0
Jmax (in vitro)
Jmax (in vivo)
0
10
20
30
40
50
Measurement temperature (°C)
150
100
50
HT
LT
0
0
50
100
150
Jmax (in vitro)
200
Fig. 3 Temperature dependences of the maximum rate of electron transport measured in thylakoid membranes [Jmax (in vitro)] and
estimated from gas exchange measurement [Jmax (in vivo)] in HT (a) and LT leaves (b). Jmax (in vitro) for HT and LT leaves are indicated as
open circles and open squares, respectively. Jmax (in vivo) for HT and LT leaves are indicated as filled circles and filled squares,
respectively. The relationship between Jmax (in vitro) and Jmax (in vivo) in HT and LT leaves (c). The regression lines are: HT (open circle
and solid line), y ¼ 0.66x þ 30.4 (R2 ¼ 0.96); LT (filled square and dashed line), y ¼ 0.73x þ 27.3 (R2 ¼ 0.95). The dotted line indicates
y ¼ x. Data represent means SE, n ¼ 3.
Temperature dependence of Jmax
The in vitro ETR per unit leaf area [Jmax (in vitro)]
was estimated from the thylakoid whole-chain ETR
(Fig. 2d). On the other hand, the in vivo ETR [Jmax
(in vivo)] was estimated from A1500, using Equation 7 (see
Appendix). At low temperatures, Jmax (in vitro) of HT
leaves tended to be less than Jmax (in vivo). At high
temperatures, Jmax (in vitro) exceeded Jmax (in vivo) for
both HT and LT leaves (Fig. 3). However, the temperature
dependences of Jmax (in vitro) were largely similar to that
of Jmax (in vivo) in both HT and LT leaves (Fig. 3a, b). The
optimum temperatures of 35 and 308C in HT and LT leaves,
respectively, were observed for both Jmax (in vitro) and
Jmax (in vivo).
We analyzed relationships between the temperature
dependence of Jmax (in vivo) and that of Jmax (in vitro)
in HT and LT leaves (Fig. 3c). We measured the CO2saturated photosynthetic rate up to 388C, whereas the ETR
in the thylakoid membranes was measured up to 408C.
Therefore, we estimated the ETR at 388C by fitting cubic
curves. Jmax (in vivo) was correlated with Jmax (in vitro) in
both HT (R2 ¼ 0.96) and LT leaves (R2 ¼ 0.95), although
there were deviations between Jmax (in vivo) and Jmax
(in vitro), especially at high temperatures.
Limiting step of A360
We estimated the photosynthetic rate limited by RuBP
carboxylation (Pc) and RuBP regeneration rate (Pr)
(Fig. 4). As described in the Appendix, Pc was calculated from Equation 2, using IS obtained in this study
(Fig. 1b), the Rubisco activation state and Rubisco kinetic
parameters (Yamori et al. 2006a), and internal conductance
(Yamori et al. 2006b). Pr was calculated from Equation 6,
using Jmax (in vitro) and Jmax (in vivo) (Fig. 3). Both Pc
and Pr were estimated at the chloroplastic CO2 concentration. Then, we analyzed the limiting steps of the temperature dependence of A360 by Pc and Pr (Fig. 4).
Pr (in vitro) showed a similar temperature dependence
to Pr (in vivo) in both HT and LT leaves, although Pr
(in vitro) was slightly greater than Pr (in vivo) at high
temperatures (Fig. 4a, b). In HT leaves, both Pc and
Pr generally matched with A360 at temperatures below
25–308C, although Pr was slightly lower than Pc, except for
the data at 108C (Fig. 4a). At temperatures above 25–308C,
A360 reflected Pc rather than Pr. On the other hand, in LT
leaves, A360 reflected Pc over the entire temperature range.
The optimum temperatures for A360, Pc, Pr (in vitro)
and Pr (in vivo) derived from the cubic curves were 30.5,
30.8, 35.1 and 33.08C, respectively in HT leaves (Fig. 4a).
In LT leaves, the optimum temperatures for A360, Pc, Pr
(in vitro) and Pr (in vivo) were 23.5, 25.5, 30.5 and 29.08C.
Growth temperature affected all of the optimum temperatures. For both HT and LT leaves, the optimum temperatures for A360 were very similar to those for Pc.
Discussion
Temperature acclimation of the ETR
The temperature response of the whole-chain ETR in
isolated thylakoid membranes depended on the growth
temperature (Fig. 2a, d). When plants were grown at LT,
the ETR per unit leaf area increased at low temperatures
(Fig. 2d–f), as has been reported (e.g. Mitchell and Barber
1986, Mawson and Cummins 1989, Yamasaki et al. 2002).
The whole-chain ETR showed a temperature dependence
similar to that of the PSII ETR (Fig. 2), as reported in
Temperature acclimation of the electron transport system
(b)
30
HT
25
20
15
10
A360
Pc
Pr (in vitro)
Pr (in vivo)
5
0
5
10 15 20 25 30 35 40 45
Leaf temperature (°C)
Photosynthesis (µmol CO2 m−2 s−1)
Photosynthesis (µmol CO2 m−2 s−1)
(a)
587
30
LT
25
20
15
10
A360
Pc
Pr (in vitro)
Pr (in vivo)
5
0
5
10 15 20 25 30 35 40 45
Leaf temperature (°C)
Fig. 4 Limiting step of the photosynthetic rate by RuBP carboxylation (Pc) and RuBP regeneration rate (Pr) in HT (a) and LT leaves (b). We
estimated Pc (open triangle and solid line) and Pr (square and broken line) at 360 ml l–1 CO2 at saturating light intensities. The measured
photosynthetic rates (A360) were equal to those presented in Fig. 1 (HT, filled circle; LT, filled square). Pc was calculated from Equation 2
in the Appendix, using the initial slope of the A–Ci curve obtained in this study (Fig. 1b), the Rubisco activation state and Rubisco kinetics
parameters (Yamori et al. 2006a), and the internal conductance (Yamori et al. 2006b). On the other hand, Pr (in vitro) and Pr (in vivo) were
calculated from Equation 6 in the Appendix, using Jmax (in vitro) and Jmax (in vivo) obtained in this study (Fig. 3). Both Pc and Pr were
estimated at the chloroplastic CO2 concentration. These data were fitted by cubic curves. Data represent means SE, n ¼ 3.
S. cernua (Mawson and Cummins 1989) and T. aestivum
(Yamasaki et al. 2002). However, the whole-chain ETR was
much lower than the PSI and PSII ETRs in both HT and
LT thylakoids (Fig. 2d–f). This indicates that the wholechain ETR across a broad temperature range was mainly
determined by diffusion processes between the PSI and PSII
reaction centers. This result corresponded to those of
previous studies which suggested that diffusion processes
are one of the crucial factors that regulate the temperature
dependence of the ETR (Barber et al. 1984, Mitchel and
Barber 1986, Ott et al. 1999, Yamasaki et al. 2002).
Plastoquinone and plastocyanin are thought to be the
rate-limiting steps of electron transport between PSI and
PSII, and are closely associated with the lipid layer of
thylakoid membranes. Since the fluidity of the thylakoid
membranes increases with increasing temperature, the ETR
between PSI and PSII would also increase (Nolan and
Smillie 1976, Pike and Berry 1980, Raison and Berry 1979,
Quinn and Williams 1985, Wada and Murata 1990, Nishida
and Murata 1996). However, the PSII ETR started to
decrease at 30–358C, in proportion to the decrease in the
whole-chain ETR (Fig. 2). Therefore, it is thought that,
at high temperatures, the whole-chain ETR would be
determined by the balance of increases in the ETR in the
diffusion processes and decreases in the ETR at PSII.
Limiting step of the RuBP regeneration rate
Jmax (in vivo) was similar to Jmax (in vitro) across
a broad temperature range in both HT and LT leaves
(Fig. 3). Pr (in vivo) and Pr (in vitro) showed similar
temperature dependences, having the same optimum
temperatures in HT and LT leaves, respectively (Fig. 4).
Therefore, it is fair to say the assumption that the
temperature dependence of Pr is determined by that of
the whole-chain ETR is robust (Farquhar et al. 1980,
Farquhar and von Caemmerer 1982, von Caemmerer 2000).
However, Jmax (in vitro) at low temperatures in HT
leaves showed slightly lower values than Jmax (in vivo),
whereas at high temperatures, Jmax (in vitro) showed
higher values than Jmax (in vivo) in both HT and LT
leaves (Fig. 3). In Nerium oleander, stromal fructose-1,6bisphosphatase (FBPase) activity was suggested to be the
limiting step of Pr, because the change in the temperature
dependence of the CO2-saturated photosynthetic rate was
highly correlated with that in stroma FBPase activity
(Badger et al. 1982). By using antisense plants, it has been
suggested that Pr would be limited by the capacities of some
enzymes, such as FBPase (Koßmann et al. 1994, Muschak
et al. 1999, Sahrawy et al. 2004), sedoheptulose 1,7bisphosphatase (SBPase; Harrison et al. 2001, Ölçer et al.
2001, Lefebvre et al. 2005), glyceraldehyde 3-phosphate
dehydrogenase (GAPDH; Price et al. 1995, Ruuska et al.
2000) and phosphoribulokinase (PRKase; Paul et al. 1995,
Banks et al. 1999, Paul et al. 2000). Moreover, it is also
possible that Pr would be limited by the capacity of ATPase
synthase (Farquhar et al. 1980, von Caemmerer 2000).
Therefore, the deviations at low and high temperatures
would imply that the temperature dependence of Pr could
be limited by processes other than the ETR.
Alternatively, it has been reported that alternative
pathways, such as the water–water cycle and cyclic electron
flow around PSI, are stimulated at low and high temperatures (for a review, see Arnon 1995, Asada 2000, Heber
2002, Öquist and Huner 2003, Sharkey 2005). It is also
suggested that the cyclic electron transport would affect the
588
Temperature acclimation of the electron transport system
PSII activity, and thus may limit Pr at high temperature
(Sharkey 2005, Sage and Kubien 2007). Therefore, the
possibility that alternative pathways affect Pr at low and
high temperatures cannot be excluded.
Limiting step of the photosynthetic rate
We clearly showed that the temperature response of
A360 depends on the growth temperature (Fig. 4). In LT
leaves, A360 was solely limited by Pc over the entire
temperature range (Fig. 4b). In contrast, in HT leaves, A360
was co-limited by Pc and Pr at low temperatures, whereas
at high temperatures, A360 was limited exclusively by Pc
(Fig. 4a). It has been reported that, at low temperatures,
photosynthetic rates are often limited by Pr, including the
Pi regeneration capacity (Sharkey 1985, Sage and Sharkey
1987, Labate and Leegood 1988, Cen and Sage 2005, Sage
and Kubien 2007). This study showed that A360 was not
necessarily limited by Pr at low temperatures, as this only
occurred when plants were grown at high temperatures.
The limiting step of the photosynthetic rate at high
temperatures has been debated recently. One proposed
mechanism is the limitation of Pc caused by heat-induced
deactivation of Rubisco (e.g. Law and Crafts-Brandner
1999, Crafts-Brandner and Salvucci 2000, Salvucci and
Crafts-Brandner 2004, Kurek et al. 2007). The other
proposed mechanism of decreases in photosynthetic rate
at high temperatures is the limitation of Pr (e.g. Schrader
et al. 2004, Wise et al. 2004, Cen and Sage 2005, Makino
and Sage 2007). This study clearly shows that at high
temperatures, A360 was limited by Pc rather than by Pr in
both HT and LT leaves. In our previous study (Yamori
et al. 2006a), we indicated that the Rubisco activation state
decreased as the temperature increased above the optimum
temperatures for A360 in HT and LT leaves grown under
conditions identical to those of the present experiment.
Therefore, in spinach, the decrease in A360 at moderately
high temperatures would be partly attributed to the direct
decrease in the Rubisco activation state. This is supported
by the previous study where the optimum temperature
of Rubisco activase in spinach leaves was around 258C
(Salvucci and Crafts-Brandner 2004) and close to the
optimum temperature for A360 in the present study.
Conclusion
We conducted a series of experiments to clarify the
limiting step of the temperature dependence of the photosynthetic rate and, thus, the comprehensive mechanisms of
the temperature acclimation of photosynthesis in spinach
leaves (Yamori et al. 2005, Yamori et al. 2006a, Yamori
et al. 2006b). The present study showed that the temperature dependence of Pr was mainly determined by that of the
ETR (Fig. 3). Our previous studies showed that the
temperature dependence of Pc was determined by that
of Rubisco kinetics (Yamori et al. 2006a), the Rubisco
activation state (Yamori et al. 2006a) and internal
conductance from the intercellular airspace to the chloroplast stroma in spinach (Yamori et al. 2006b). Overall, we
clearly showed that, in LT leaves, A360 was solely limited
by Pc over the entire temperature range, whereas in HT
leaves, A360 was limited by Pc at high temperatures and
co-limited by Pc and Pr at low temperatures (Fig. 4). The
optimum temperature of A360 was determined by the
temperature dependence of Pc in both HT and LT leaves.
Thus, we clearly showed that the limiting step of A360 was
different depending on the growth temperature.
Materials and Methods
Plant growth conditions
Spinach (Spinacia oleracea L. cv. Torai) plants were grown in
vermiculite, as described in Yamori et al. (2005). The day/night
lengths were 8 and 16 h. Photosynthetically active photon flux
density (PPFD) in the daytime was 230 mmol photons m–2 s–1. The
day/night air temperatures were either 30/25 or 15/108C. These
are referred to as the HT and the LT conditions, respectively.
Similarly, the leaves grown at HT and LT are called HT and LT
leaves.
Gas exchange measurements
The light-saturated photosynthetic rate and the dark respiration rate were measured using the most recently fully expanded
leaves (LI-6400; Li-Cor Inc., Lincoln, NE, USA) as described
previously (Yamori et al. 2005). The CO2 dependence of the
photosynthetic rate was examined, based on measurements at CO2
concentrations in the ambient air (Ca) of 50, 100, 150, 200, 360 and
1,500 ml l–1. At each CO2 concentration, net photosynthetic rate (A)
and intercellular CO2 concentration (Ci) were obtained.
Measurements of the electron transport rates in isolated thylakoid
membranes
Chloroplasts were prepared according to the method of
Terashima et al. (1989) with some modifications. Leaves were
homogenized with a blender in a solution containing 0.3 M
sorbitol, 40 mM HEPES-KOH (pH 7.0), 10 mM NaCl, 5 mM
MgCl2 and 0.1% (w/v) bovine serum albumin (BSA). The
homogenate was filtered through a layer of nylon mesh (26 mm)
and the filtrate was centrifuged at 2,000 g at 48C for 1 min. Then,
the chloroplasts were suspended in a solution containing 0.3 M
sorbitol, 40 mM HEPES-KOH (pH 7.0), 10 mM NaCl and 5 mM
MgCl2. Chl contents were determined by the procedure of Porra
et al. (1989).
Immediately before the measurements of the ETR, the
chloroplasts were subjected to osmotic shock in a solution
containing 40 mM HEPES-KOH (pH 7.5), 10 mM NaCl and
5 mM MgCl2 in the cuvette of an oxygen electrode (Hansatech,
King’s Lynn, UK). The ETR in the thylakoid membranes
(5–10 nmol Chl ml–1) was measured with an oxygen electrode,
according to Yamasaki et al. (2002) with some modifications, at
saturating light (3,000 mmol m–2 s–1). Saturating light was supplied
from a slide projector (AF-250, Cabin, Japan) and passed through
an OG 570 long-pass filter (Schott, Mainz, Germany), in order to
prevent the quinone from absorbing the light of short wavelengths.
For measurements of the whole-chain ETR (H2O!methyl
viologen), 10 mM methylamine, 1 mM sodium azide and 1 mM
Temperature acclimation of the electron transport system
methyl viologen were added to the suspension. For measurements
of the PSI ETR [dichloroindophenol (DCIP) and ascorbate!methyl viologen], 10 mM methylamine, 1 mM sodium
azide, 1 mM methyl viologen, 10 mM DCMU, 300 mM DCIP and
2 mM sodium ascorbate were added. For measurements of the PSII
ETR (H2O!phenyl-p-benzoquinone), 10 mM methylamine, 1 mM
phenyl-p-benzoquinone and 1.0 mM 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) were added. The PSII ETR at
408C was much lower than the whole-chain ETR, when 10 mM
methylamine, 1 mM phenyl-p-benzoquinone and 1.0 mM DBMIB
were used for the assay. Therefore, only at 408C, we measured the
PSII ETRs in the buffer containing 1 mM methylamine, 0.1 mM
phenyl-p-benzoquinone and 0.1 mM DBMIB.
Model analysis of the limiting step of the photosynthetic rate
Using the C3 photosynthesis model (Farquhar et al. 1980), the
maximum rate of RuBP carboxylation (Vcmax) and the RuBP
carboxylation rate (Pc) were estimated from the initial slope of the
A vs. Ci curve (IS) obtained with the data measured at a Ca of 50,
100, 150 and 200 ml l–1 (see Appendix). Recently, we showed that
the temperature dependence of Pc was limited by the Rubisco
activation state and Rubisco kinetic parameters (Yamori et al.
2006a), and by internal conductance (Yamori et al. 2006b) in
spinach leaves grown under conditions identical to those of the
present study. Therefore, for calculations of Pc, we took account of
the temperature dependences of the Rubisco activation state,
Rubisco kinetic parameters and internal conductance in HT and
LT leaves, respectively.
The Jmax (in vivo) and Pr (in vivo) were estimated from A at
the highest Ca of 1,500 ml l–1 CO2 (see Appendix). Jmax (in vitro)
and Pr (in vitro) were estimated from the measured whole-chain
ETR in the isolated thylakoid membranes (see Appendix). Since
the ETR from H2O to methyl viologen was measured as wholechain electron transport, the maximum ETR was calculated for
four electron flow per one oxygen evolution. Moreover, Pr was
estimated at the chloroplastic CO2 concentration level as well as
Pc, using the internal conductance (Yamori et al. 2006b).
Funding
Japan Society for the Promotion of Science (to W.Y.).
Acknowledgments
We are grateful to Dr. John Evans (Australian National
University) for valuable comments on the manuscript.
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Appendix
A. C3 photosynthesis model
Based on the C3 photosynthesis model (Farquhar et al.
1980), net leaf photosynthetic rate, A, can be expressed
using the minimum rate of two partial reactions:
A ¼ minfPc ,Pr g Rd ,
ð1Þ
where Pc (mmol m–2 s–1) is the RuBP-saturated rate of
photosynthesis, Pr (mmol m–2 s–1) is the RuBP-limited rate
of photosynthesis and Rd (mmol m–2 s–1) is the day
respiration rate. Pc is expressed as a function of the
chloroplastic CO2 concentration (Cc, ml l–1), according to
Yamori et al. (2006a):
Pc ¼
Vcmax ðCc Þ
R Rd ,
Cc þ Kc ð1 þ O=Kc Þ
ð2Þ
Temperature acclimation of the electron transport system
where Vcmax (mmol m–2 s–1) is the maximum rate of the
RuBP carboxylation, Kc (ml l–1) and Ko (ml l–1) are the
Michaelis constants for CO2 and O2, respectively, O (ml l–1)
is the O2 concentration, (ml l–1) is the CO2 compensation
point in the absence of day respiration, and R is the
Rubisco activation state. Beause the initial slope of the A vs.
Cc curve [IS(Cc)] is identical to dPc(Cc)/dCc at Cc ¼ ,
Vcmax is expressed as:
Vc max ¼
ISðCc Þ þ Kc ð1 þ O=Ko Þ
:
R
ð3Þ
Using the initial slope of the A vs. intercellular CO2
concentration (Ci) curve [IS(Ci)], IS(Cc) was calculated as:
ISðCc Þ ¼
gi ISðCi Þ
,
gi ISðCi Þ
ð4Þ
where gi is the internal conductance, which is the
conductance for CO2 diffusion from the intercellular
airspace to the chloroplast stroma. The temperature
dependence of gi was obtained for spinach leaves grown
under conditions identical to those of the present experiment
(Yamori et al. 2006b). Then, using A, Ci and gi, Cc was
calculated as:
Cc ¼ Ci A=gi :
ð5Þ
On the other hand, Pr is expressed as:
Pr ¼
Jmax ðCc 4Cc þ 8
Þ
Rd ,
ð6Þ
where Jmax (mmol m–2 s–1) is the maximum rate of
electron transport. Using the photosynthetic rate at a high
CO2 concentration of 1,500 ml l–1 (A1500), Jmax is
expressed as:
Jmax ¼
ðA1500 þ Rd Þð4Cc þ 8
Cc Þ
:
ð7Þ
B. Limiting step of the photosynthetic rate
To analyze the limitation of the temperature dependence of the photosynthetic rate at 360 ml l–1 CO2 under
saturating light intensities, we estimated the photosynthetic
591
rate limited by Pc and Pr, respectively. The photosynthetic
rate limited by Pc was evaluated according to Yamori et al.
(2006a, 2006b). To estimate the temperature dependence of
Pc from Equation 2, we assumed that day respiration rates
(Rd) were half the dark respiration rates in HT and LT
leaves. We first calculated the temperature dependence of
Kc(1 þ O/Ko) values, using Equation 3. Temperature dependences of the Rubisco activation state (R), the maximum
rate of RuBP carboxylation (Vcmax) and the CO2 compensation point in the absence of day respiration ( )
were obtained from spinach leaves grown under conditions identical to those of the present experiment
(Yamori et al. 2006a). IS(Cc) was determined from IS(Ci)
measured in this study and gi obtained from spinach leaves
grown under conditions identical to those of the present
experiment (Yamori et al. 2006b), using Equation 4. Cc was
determined from the photosynthetic rate at 360 ml l–1 CO2
and Ci measured in this study, and from gi (Yamori et al.
2006b), using Equation 5. The data for the temperature
dependences of the parameters were obtained by fitting
cubic curves to the actual data. Cc thus estimated at 258C
was 275.9 and 190.0 ml l–1 for HT and LT leaves,
respectively.
Next, we calculated absolute values of Vcmax (mmol
m–2 s–1) to match the Rubisco contents between the
measured photosynthetic rates in this study and the
estimated Pc. Vcmax values on a leaf area basis were
estimated from Equation 2, at each temperature, using the
photosynthetic rates obtained in this study. Then, Vcmax
values estimated for each temperature were averaged for the
HT and LT leaves, respectively. Vcmax values thus
estimated at 258C were 35.7 and 64.1 mmol m–2 s–1 for HT
and LT leaves, respectively.
On the other hand, to estimate temperature dependence of Pr from Equation 6, we used the identical values of
Rd, Cc and , which were obtained in the estimation of Pc,
in HT and LT leaves, respectively. The electron transport
rate in the isolated thylakoid membranes was used for
calculations of Pr (in vitro). Since the electron transport
rate from H2O to methyl viologen was measured as wholechain electron transport, the maximum electron transport
rate was calculated for four electron flow per one oxygen
evolution. Pr (in vivo) was estimated from estimated from
A1500, using Equations 6 and 7.
(Received December 31, 2007; Accepted February 18, 2008)