Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2004? 2004
277907916
Original Article
Low temperatures and C4photosynthesis
D. S. Kubien & R. F. Sage
Plant, Cell and Environment (2004) 27, 907–916
Low-temperature photosynthetic performance of a C4 grass
and a co-occurring C3 grass native to high latitudes
D. S. KUBIEN* & R. F. SAGE
Department of Botany, University of Toronto, 25 Willcocks St., Toronto, ON, M5S 3B2, Canada
ABSTRACT
The photosynthetic performance of C4 plants is generally
inferior to that of C3 species at low temperatures, but the
reasons for this are unclear. The present study investigated
the hypothesis that the capacity of Rubisco, which largely
reflects Rubisco content, limits C4 photosynthesis at suboptimal temperatures. Photosynthetic gas exchange, chlorophyll a fluorescence, and the in vitro activity of Rubisco
between 5 and 35 ∞C were measured to examine the nature
of the low-temperature photosynthetic performance of the
co-occurring high latitude grasses, Muhlenbergia glomerata (C4) and Calamogrostis canadensis (C3). Plants were
grown under cool (14/10 ∞C) and warm (26/22 ∞C) temperature regimes to examine whether acclimation to cool
temperature alters patterns of photosynthetic limitation.
Low-temperature acclimation reduced photosynthetic rates
in both species. The catalytic site concentration of Rubisco
was approximately 5.0 and 20 mmol m-2 in M. glomerata
and C. canadensis, respectively, regardless of growth temperature. In both species, in vivo electron transport rates
below the thermal optimum exceeded what was necessary
to support photosynthesis. In warm-grown C. canadensis,
the photosynthesis rate below 15 ∞C was unaffected by a
90% reduction in O2 content, indicating photosynthetic
capacity was limited by the capacity of Pi-regeneration. By
contrast, the rate of photosynthesis in C. canadensis plants
grown at the cooler temperatures was stimulated 20–30%
by O2 reduction, indicating the Pi-regeneration limitation
was removed during low-temperature acclimation. In M.
glomerata, in vitro Rubisco activity and gross CO2 assimilation rate were equivalent below 25 ∞C, indicating that the
capacity of the enzyme is a major rate limiting step during
C4 photosynthesis at cool temperatures.
Key-words: acclimation; C3 photosynthesis; C4 photosynthesis; low temperature; oxygen-sensitivity; Rubisco.
INTRODUCTION
C4 plants often dominate warm, dry climates where they
have access to at least moderate light intensities (Sage,
Correspondence: David S. Kubien. Fax: +64 6350 5688; e-mail: [email protected]
*Current address: Institute of Molecular Biosciences, Massey University, Private Bag 11 222, Palmerston North, New Zealand.
© 2004 Blackwell Publishing Ltd
Wedin & Li 1999). However, the frequency of C4 species is
reduced in cool climatic zones (Long 1983; Sage et al. 1999),
as shown by a lower proportion of C4 grasses at higher
latitude (Teeri & Stowe 1976) and elevation (Tieszen et al.
1979). Globally, C4 grasses become rare in regions in which
the minimum temperature of the warmest month of the
growing season is less than 8 ∞C (Teeri & Stowe 1976; Long
1983), and the average growing season temperatures are
below 16 ∞C (Sage et al. 1999).
Numerous hypotheses for the rarity of C4 plants in cool
climates have been proposed. Cold-induced failure of
enzymes in the C4 pathway has frequently been suggested
as the leading cause of the poor performance of C4 photosynthesis at low temperature (Long 1983; Edwards et al.
1985; Long 1999; Naidu et al. 2003). Several enzymes of the
carbon-concentrating mesophyll reactions, notably phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) and
pyruvate orthophosphate dikinase (PPDK, EC 2.7.9.1), can
be cold labile in vitro, with dissociation occurring below
approximately 10 ∞C (Sugiyama 1973; Krall & Edwards
1993). These enzymes are not inherently prone to failure at
low temperatures, however, as there is considerable species
and ecotypic variation in their thermal stability (Sugiyama
& Boku 1976; Krall & Edwards 1993; Simon & Hatch
1994). Furthermore, studies have demonstrated that C4
metabolism is qualitatively unaltered by low-temperature
exposure (Caldwell, Osmond & Nott 1977; Matsuba et al.
1997).
An alternative explanation for the poor performance of
C4 photosynthesis at low temperature is that low Rubisco
capacity limits CO2 assimilation at suboptimal temperatures. As used here, Rubisco ‘capacity’ refers to the capacity
of fully activated Rubisco to consume RuBP under a given
set of environmental conditions. In the context of this definition, Rubisco capacity largely reflects Rubisco content
(Sage, Sharkey & Seemann 1990). In the C4 dicot Atriplex
rosea, the activation energy of photosynthesis in vivo and
Rubisco in vitro are similar (Björkman & Pearcy 1971).
Differences in the photosynthesis rates at low temperatures
in Atriplex lentiformis grown at different temperatures
were correlated with changes in the maximum activity of
Rubisco (Pearcy 1977). Pittermann & Sage (2000, 2001)
observed that the rate of net CO2 assimilation was equivalent to the maximum activity of Rubisco in vitro in the high
elevation species Bouteloua gracilis and Muhlenbergia
montanum at suboptimal temperatures. Recent work with
907
908 D. S. Kubien & R. F. Sage
anti-RbcS Flaveria bidentis has shown that Rubisco capacity, specifically the amount of the enzyme, is an important
controlling step over the rate of carbon gain in C4 plants at
low temperatures (Kubien et al. 2003).
Relatively few studies have examined photosynthetic
acclimation to low growth temperatures in C4 species that
naturally occur in cool climates. C3 species show a variety
of low-temperature acclimation patterns, including
increases in the amount of Rubisco (Badger, Björkman &
Armond 1982; Holaday et al. 1992; Hurry et al. 1995) and
the enzymes of sucrose synthesis (Martindale & Leegood
1997; Savitch, Gray & Huner 1997; Stitt & Hurry 2002). The
temperature acclimation responses of carbon metabolism
in C4 plants are less clear. When grown at low temperature,
Atriplex lentiformis increases its Rubisco content relative
to warm-grown plants (Pearcy 1977). In chilling-sensitive
maize the amount of Rubisco is reported to either decline
or be insensitive to growth temperature (Ward 1987;
Pietrini et al. 1999; Naidu et al. 2003). The chilling-tolerant
C4 grass Miscanthus ¥ giganteus maintains high photosynthetic rates when grown at low temperatures because of the
ability to maintain high levels of Rubisco and PPDK; levels
of these enzymes decline when chilling-intolerant maize is
grown at 14 ∞C relative to plants grown at 25 ∞C (Naidu
et al. 2003). The existence of a few C4 species in cool climates (Schwarz & Redmann 1988; Long 1999; Kubien &
Sage 2003) indicates that C4 plants can adapt to low-temperature environments, but the reasons for their apparent
inability to compete with C3 species in cool-climates remain
uncertain.
In the present study, we examine the nature of the rate
limitations of C3 and C4 photosynthesis at low temperatures. Specifically, we test the hypotheses that Rubisco
capacity limits C4 photosynthesis at low temperatures. To
address this question, the photosynthetic temperature
response of the C4 grass Muhlenbergia glomerata is contrasted with that of the C3 species Calamogrostis canadensis. These species frequently co-occur in boreal Canada and
have wide latitudinal ranges; M. glomerata has been
reported above 60∞N (Schwarz & Redmann 1988). This
approach allows for direct comparisons between C3 and C4
species that occupy the same cool climates. Further, using
M. glomerata as a representative C4 grass mitigates the
effects of general chilling intolerance that are often associated with warm-climate C4 species, such as maize. Warm
(26/22 ∞C) and cool (14/10 ∞C) growth temperature regimes
were used to examine photosynthetic acclimation in these
species. We measured photosynthetic gas exchange and
chlorophyll a fluorescence parameters at temperatures
from 5 to 35 ∞C, and in vitro Rubisco activity from 0 to
35 ∞C.
METHODS AND MATERIALS
Plant growth
Rhizomes of Muhlenbergia glomerata (C4) and Calamogrostis canadensis (C3) were collected from a fen near
Plevna, Ontario (45∞0¢ N 76∞5¢ W). Rhizomes were planted
in 6 L pots containing 66% (v/v) Promix (Plant Products,
Brampton, Canada), 17% sand and 17% plant compost.
Plants were grown in two controlled environment chambers
(GC-20; Enconair, Winnipeg, Canada) and maintained
under a 16 h photoperiod with a maximum photosynthetic
photon flux density (PPFD) of 800 mmol m-2 s-1. Plants
were grown at day/night temperatures and relative humidities of 14/10 ∞C and 50/80% (cool-grown) or 26/22 ∞C and
70/80% (warm-grown), respectively. Plants were grown for
10 weeks prior to the beginning of the experiment, and
growth regimes were rotated between two chambers every
2 to 3 weeks to minimize between-chamber variation.
Plants were watered daily and fertilized weekly with 0.5¥
Hoagland’s solution supplemented with 3 mM NH4NO3.
Gas-exchange measurements
The temperature response of photosynthesis was measured
in an open-type leaf gas-exchange system described elsewhere (Kubien et al. 2003); all gas exchange calculations
follow von Caemmerer & Farquhar (1981). Leaf temperature was measured by placing three fine wire thermocouples in contact with the abaxial surface of the leaves.
Illumination was provided by a cool-light source (KL-2500;
Schott, Mainz, Germany) to minimize interference with the
fluorescence detector. The PPFD in the cuvette was measured using a photodiode (G1738; Hamamatsu, Bridgewater, NJ, USA) calibrated against a quantum sensor (Li-190s:
LiCor Inc., Lincoln, NE, USA). Temperature responses
were measured at a constant PPFD of 1300 mmol m-2 s-1,
which is sufficient to saturate photosynthesis in both species
at temperatures less than 25 ∞C (Kubien 2003). Temperature responses were measured at 200 ± 10 mbar O2 in M.
glomerata and C. canadensis, and also at 20 ± 1 mbar O2 in
the C3 species; CO2 was maintained at 370 ± 3 mbar. The
leaf-to-air vapour pressure deficit (VPD) was maintained
at 14 ± 2 mbar at temperatures greater than 10 ∞C; at cooler
temperatures VPD was reduced.
Photosynthesis was measured simultaneously on two to
four leaves from a single plant, and different plants were
used for each replicate. All temperature response measurements were initiated at 25 ∞C and 200 mbar O2. Leaves
were dark-adapted for 30 min before the measurement of
the respiration rate (R) and Fv/Fm. This R-value was used
to correct the measured net CO2 assimilation rate for respiration, following Bernacchi et al. (2001). After the respiration measurement, the leaves were allowed to equilibrate
at the measurement PPFD and temperature for a minimum
of 45 min before measurement of steady-state photosynthesis and fluorescence. Leaf temperature was subsequently
increased in 5 ∞C steps to 35 ∞C, and then decreased to the
lower temperatures; the decrease from 35 to 20 ∞C was
performed in 5 ∞C steps of approximately 10 min each. At
each temperature, the leaves were allowed to equilibrate
for a minimum of 20 min prior to measurements. For the
C3 species, the partial pressure of O2 was reduced to
20 mbar after the initial measurement at 200 mbar; photo-
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 907–916
Low temperatures and C4 photosynthesis 909
synthesis and fluorescence were measured after a 10 min
adjustment to reduced O2, at all temperatures. After the last
measurement was completed, the leaves were warmed to
about 15 ∞C, leaf area was obtained (Li-3000: LiCor Inc.),
and samples (approx. 3 cm2) were frozen in liquid N2. The
leaf samples were stored at -80 ∞C until the enzyme assays
were conducted.
Chlorophyll a fluorescence was measured using a PAM101 (Walz, Effeltrich, Germany) equipped with a blue-light
emitter detector unit (ED-101BL: Walz: Kubien et al. 2003).
The ratio of variable to maximal fluorescence was measured following a 30 min dark period. Reaction centre closure was achieved by applying a 0.8 s pulse of saturating
light (approximately 4000 mmol m-2 s-1). Once photosynthesis had reached steady state at a given temperature, the
quantum yield of photosystem II (PSII) (FPSII) was measured (Genty, Briantais & Baker 1989); three saturating
pulses were applied at 90 s intervals, and Fm¢ was estimated
as the mean fluorescence peak. The rate of linear electron
transport (J) was determined as J = FPSII(PPFDa)f, where
PPFDa is the absorbed light intensity, and f is the fraction
of absorbed light reaching PSII (Krall & Edwards 1992).
Leaf absorbance was measured in the 370–750 nm waveband, using a dual-channel spectrometer (SD2000; Ocean
Optics, Dunedin, FL, USA) and an integrating sphere
(FOIS-1; Ocean Optics). Following Krall & Edwards (1992)
we assumed a value of 0.5 for f at all temperatures, although
in C4 species the value of f may vary depending on biochemical subtype.
Rubisco assays
Rubisco activity in vitro was assayed on samples harvested
from the leaves used during gas exchange measurements.
Leaf samples were rapidly ground (< 90 s) on ice in a Bicine
extraction buffer (approximately 2 mL cm-2) using a glassin-glass homogenizer (Kubien et al. 2003). Aliquots of the
crude extract were used to quantify the amount of Rubisco
using a 14C-carboxy-arabinitol bisphosphate (CABP) binding assay (Butz & Sharkey 1989). The radioactivity in the
filter-bound Rubisco–14CABP complexes was measured by
liquid scintillation spectroscopy.
A 3.15 mL aliquot of the crude leaf extract was added to
350 mL of a Rubisco activating solution (100 mM Bicine,
pH 8.2). For the C4 samples, the activating solution contained 280 mM MgCl2 and 200 mM NaHCO3, giving final
concentrations of 28 mM MgCl2 and 20 mM NaHCO3,
respectively (Sage & Seemann 1993). For the C3 samples,
the activating solution contained 200 mM MgCl2 and
100 mM NaHCO3, giving final concentrations of 20 mM
MgCl2 and 10 mM NaHCO3, respectively (Butz & Sharkey
1989). The crude extract was incubated in activating solution for 20 min at room temperature to fully carbamylate
Rubisco, and thereafter was kept on ice until being assayed.
Rubisco activity assays follow Kubien et al. (2003).
RESULTS
Figure 1. Temperature response of the rate of net CO2 assimilation in (a) Calamogrostis canadensis (C3), and (b) Muhlenbergia
glomerata (C4). Plants were grown at 26/22 ∞C () or 14 /10 ∞C ()
day/night temperature. Photosynthesis was measured at 370 mbar
CO2 and 200 mbar O2, under a PPFD of 1300 mmol m-2 s-1. Each
point represents the mean (± 1 SE) of measurements on four individual plants. Error bars are obscured when smaller than the
symbol.
In both species, plants grown at warm temperatures (26∞/
22 ∞C) had higher net CO2 assimilation (A) rates from 5 to
35 ∞C than plants that were grown at 14/10 ∞C (Fig. 1).
Growth temperature did not affect the thermal optimum of
the C3 species, with maximum A achieved at 20 ∞C in either
case (Fig. 1a). In air, the maximum photosynthesis rates of
the C3 plants were approximately 13 and 10 mmol m-2 s-1 for
the warm- and cool-grown plants, respectively. In M.
glomerata, plants grown at 26/22 ∞C reached a maximum
photosynthetic rate of about 25 mmol m-2 s-1 at 30 ∞C. The
cool-grown plants attained a maximum A of 16 mmol m-2
s-1, near 25 ∞C (Fig. 1b). When grown at warm temperatures, the C4 plants had considerably higher A than the C3
species above 20 ∞C; below 15 ∞C, tha value of A of the C3
species exceeded that of the C4. When grown under cool
conditions, A in M. glomerata exceeded that of C. canadensis at temperatures greater than 15 ∞C.
Reducing the partial pressure of O2 resulted in a slight
upward shift of the photosynthetic thermal optimum in C.
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 907–916
910 D. S. Kubien & R. F. Sage
below 15 ∞C (Fig. 2b). Under low-O2 conditions, photosynthesis was stimulated 25–30% in the cool-grown C3 plants
when measured from 5 to 35 ∞C. The stimulation by low O2
in the warm-grown C3 plants increased over four-fold across
this temperature range (Fig. 2c). Photosynthesis in M.
glomerata was insensitive to O2 at all temperatures, regardless of growth condition (data not shown).
In air, the in vivo electron transport rate (ETR) of C.
canadensis reached a maximum of about 100 mmol m-2 s-1
near the thermal optimum of 20 ∞C (Fig. 3a); under 20 mbar
O2 the maximum ETR was about 75 mmol m-2 s-1 (data not
Figure 2. Temperature response of the rate of net CO2 assimilation in Calamogrostis canadensis (C3) under high and low oxygen.
Plants were grown at (a) 14/10 ∞C, or (b) 26/22 ∞C day/night temperature. Photosynthesis was measured under 20 mbar O2 (, )
or 200 mbar O2 (, ) (mean ±1 SE., n = 4). PPFD and CO2 were
1300 mmol m-2 s-1 and 370 mbar, respectively. After leaves had
reached a steady-state photosynthetic rate in air, oxygen was
reduced to 20 mbar, and a new photosynthetic rate was noted after
a 10 min period. The O2 sensitivity of photosynthesis (c) was calculated as 1 - A20/A200 (Sage & Sharkey 1987). The modelled sensitivity was calculated assuming that Kc and Ko were 260 mbar and
179 mbar, at 25 ∞C, respectively.
canadensis (Fig. 2). Photosynthesis remained sensitive to
O2 across the measured temperature range in C. canadensis
grown at 14/10 ∞C (Fig. 2a). In the warm-grown C3 plants
reduced O2 did not result in higher A at temperatures
Figure 3. Temperature responses of the rate of electron transport
in (a) Calamogrostis canadensis (C3) and (b) Muhlenbergia glomerata (C4). Plants were grown at 26/22 ∞C () or 14/10 ∞C () day/
night temperature. Electron transport was measured in vivo using
chlorophyll a fluorescence (Krall & Edwards 1992), at 370 mbar
CO2, 200 mbar O2, and a PPFD of 1300 mmol m-2 s-1 (mean ±1 SE,
n = 4, some error bars are obscured where smaller than the symbol). Lines indicate the electron transport rate theoretically
required to support the measured rates of gross assimilation. A
requirement of 3H+/ATP and a functional Q-cycle were assumed.
In this case, the minimum requirement in a C3 plant is (3 + 3.5f)Vc,
where f is the number of oxygenations per carboxylation, and Vc
is the carboxylation rate. For the C4, the requirements for the
mesophyll and bundle sheath reactions are 2Vp and 3Vc, respectively, where Vp is the PEP carboxylation rate. The requirement for
the bundle sheath reactions ignores f, as we assume that the CO2
concentration in the stroma is sufficient to eliminate the oxygenase
function. All equations from von Caemmerer (2000).
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 907–916
Low temperatures and C4 photosynthesis 911
Figure 4. The ratio of the quantum yield of PSII (FPSII) to the
quantum yield of gross assimilation (FCO2*) as a function of temperature in Calamogrostis canadensis (a, b) and Muhlenbergia
glomerata (c). Plants were grown at 26/22 ∞C or 14/10 ∞C day/night
temperature. Chlorophyll a fluorescence was measured concurrently with gas-exchange. Photosynthesis was measured at
370 mbar CO2 and 200 mbar O2, under a PPFD of 1300 mmol m-2
s-1; in C. canadensis photosynthesis was also measured at 20 mbar
O2. Each point represents the mean (± 1 SE) of four independent
measurements.
shown). Growth temperature did not affect the relationship
between ETR and measurement temperature in C.
canadensis. In C. canadensis, ETR exceeded the rate
required to support measured rates of photosynthesis at
temperatures less than 30 ∞C (warm-grown plants) and
35 ∞C (cool-grown plants) (Fig. 3a). The in vivo electron
transport rate of M. glomerata had the same thermal opti-
mum as photosynthesis, and exceeded the theoretically
required minimum at temperatures below the respective
thermal optima (Fig. 3b). Below 25 ∞C the warm- and coolgrown C4 leaves had the same ETR. The maximum ETR in
M. glomerata was 140 and 100 mmol m-2 s-1, for the warmand cool-grown plants, respectively (Fig. 3b).
The instantaneous quantum cost of assimilation can be
approximated by the ratio FPSII/FCO2*, where FPSII is the
quantum yield of PSII, and FCO2* is the quantum yield of
gross CO2 assimilation (A*) (Oberhuber & Edwards 1993).
In cool-grown C. canadensis measured at 200 mbar O2,
FPSII/FCO2* remained relatively constant between 16 and
18 molquanta molCO2-1 from 5 to 35 ∞C (Fig. 4a). This value
was reduced to approximately 12 molquanta molCO2-1 when
measured at 20 mbar O2. In warm-grown C. canadensis
measured at 200 mbar O2, the quantum cost of assimilation
increased from about 10–18 molquanta molCO2-1 as temperature increases from 5 to 35 ∞C (Fig. 4b). At low O2, FPSII/
FCO2* was about 9 molquanta molCO2-1 in the warm-grown C3
plants, and was insensitive to measurement temperature.
Growth temperature had no effect on FPSII/FCO2* in M.
glomerata (Fig. 4c). At or above the thermal optimum, the
quantum cost of assimilation in this C4 species was constant
between 10 and 12 molquanta molCO2-1. At suboptimal temperatures, FPSII/FCO2* increased in this C4 species, regardless of growth temperature.
Calamogrostis canadensis had an approximately fourfold greater Rubisco content than M. glomerata (Table 1).
At 25 ∞C, the in vitro kcat of the C4 species was higher than
in the C3 plant. The activation energy of the C3 Rubisco was
nearly 10% higher than in the C4 species. In M. glomerata,
the activation energy of assimilation from 5 to 20 ∞C was
approximately 72 kJ mol-1, which was similar to the activation energy of Rubisco (approximately 66.5 kJ mol-1). In
contrast, the activation energy of CO2 assimilation (measured at 200 mbar O2) in C. canadensis between 5 and 20 ∞C
was considerably lower than that of Rubisco.
The maximum in vitro activity of Rubisco (Vcmax)
exceeded the rate of gross CO2 assimilation (A*) in C.
canadensis at all temperatures (Fig. 5a & b). At the photosynthetic thermal optima, Vcmax was at least three-fold
greater than A* in this C3 species. At 5 ∞C, Vcmax was only
marginally greater than A*, and they were nearly equivalent when A* was measured at 20 mbar O2. In cool-grown
M. glomerata, A* and Vcmax were equivalent between 5 and
20 ∞C, but Vcmax exceeded A* at warmer temperatures
(Fig. 5c). Similarly, Vcmax exceeded A* at 30 and 35 ∞C in M.
glomerata grown at 26/22 ∞C, but the rates were the same
between 5 and 25 ∞C (Fig. 5d).
DISCUSSION
In this study, we present data supporting the hypothesis
that Rubisco capacity, as determined by Rubisco content,
is the principle limitation on C4 photosynthesis at suboptimal temperatures. Rubisco capacity largely reflects Rubisco
content at low temperatures, as indicated by the similarity
between Vcmax and gross CO2 assimilation. Because Rubisco
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 907–916
912 D. S. Kubien & R. F. Sage
Table 1. Biochemical characteristics of Muhlenbergia glomerata (C4) and Calamogrostis canadensis (C3)
Plant
Growth temp.
(∞C)
Catalytic sites
(mmol m-2)
Rubisco
in vitro kcat
(mol mol-1 s-1)
Ea
(kJ mol-1)x
Photosynthesis
Ea
(kJ mol-1)y
M. glomerata (C4)
M. glomerata (C4)
C. canadensis (C3)
C. canadensis (C3)
14/10
26/22
14/10
26/22
4.3 ± 0.2a
5.7 ± 0.4a
19.5 ± 1.4b
20.7 ± 1.3b
5.21 ± 0.21a
4.57 ± 0.29ab
3.79 ± 0.44b
3.56 ± 0.17b
67.3 ± 0.5ab
65.6 ± 0.4a
73.4 ± 2.3c
71.3 ± 1.7bc
70.0 ± 1.8a
73.3 ± 4.8a
39.1 ± 6.8b
37.6 ± 2.4b
The kcat and activation energy (Ea) data are values at 25 °C. The concentration of Rubisco catalytic sites was determined by the 14CABPlabelling assay described in the Methods, assuming 6.5 binding sites per Rubisco (Butz & Sharkey 1989). The Rubisco kcat values were
determined by the incorporation of 14C into acid-stable products. Activation energies were calculated as described by Berry & Raison (1981).
Each value represents the mean (± SE) of four to six measurements. Values with different superscripts are statistically different from each
other (P < 0.05). Statistical analysis was done by ANOVA using S +(MathSoft 1994), with growth temperature and photosynthetic pathway
as main effects. xMeasured between 0° to 35 °C. yMeasured between 5° to 20 °C.
operates in a high CO2 environment in C4 plants, gross CO2
assimilation should reflect Vcmax, and thus Rubisco content
(Sage 2002). At low temperatures, photosynthesis (A) in
warm-grown C. canadensis appears to be limited by the
ability to regenerate Pi in the chloroplast, but growth at 14/
10 ∞C appears to alleviate this limitation. In both species,
electron transport in vivo exceeds the theoretical requirement at temperatures below the thermal optimum, indicating that photosynthesis is not limited by light-harvesting
processes in the thylakoid at low temperatures. If Rubisco
capacity limits the rate of C4 photosynthesis, then leakage
of CO2 from the bundle sheath should increase, and this is
consistent with the observed increase in FPSII/FCO2* at low
temperatures.
Rate limitations on C4 and C3 photosynthesis at
low temperatures
Between 5 and 20 ∞C, the activity of Rubisco in vitro (Vcmax)
and the rate of gross CO2 assimilation (A*) in vivo are
equivalent in M. glomerata grown at 14/10 ∞C, indicating
that Rubisco capacity limits C4 photosynthesis at low temperatures. The similarity between the activation energy (Ea)
of photosynthesis and Vcmax in M. glomerata is also consistent with a Rubisco limitation of C4 photosynthesis. Similar
results have been reported in the C4 grasses Bouteloua
gracilis and Muhlenbergia montanum, where Vcmax and net
photosynthesis rate are equivalent below 17 to 22 ∞C (Pittermann & Sage 2000, 2001). When grown at 26/22 ∞C, the
Rubisco limitation of photosynthesis in M. glomerata
extended to 25 ∞C, probably due to a rise in the electron
transport capacity during acclimation to the warmer growth
temperature. There was no difference in the amount of
Rubisco in cool- and warm-grown M. glomerata, whereas
the in vivo electron transport rate in warm-grown M. glomerata was 25% greater than that of the cool-grown plants.
A rise in electron transport capacity would increase the
capacity to regenerate RuBP and PEP. Increased capacity
of these potentially rate-limiting steps, arising from growth
at warmer growing conditions, allows the Rubisco control
over photosynthesis to extend to warmer temperatures.
In C. canadensis, there is little evidence that Rubisco
limits A below the thermal optimum in plants grown at 26/
22 ∞C. Instead, the capacity to regenerate orthophosphate
(Pi) appears to be the primary control over A, as indicated
by the low level of O2 sensitivity between 5 and 20 ∞C. A
90% reduction in O2 should enhance A in C3 plants, as the
rate of photorespiration is suppressed. This enhancement
generally declines with temperature, reflecting the inhibition of Rubisco oxygenase activity at cooler temperatures
(Fig. 2c; Sage & Sharkey 1987). When O2-sensitivity is
reduced at constant temperature and gas concentrations,
the capacity to regenerate Pi in the chloroplast generally
exerts high control over A (Sharkey 1985; Stitt & Hurry
2002). Pi-regeneration limitations are common in C3 species
when A is measured below the growth temperature (Sage
& Sharkey 1987; Sage, Sharkey & Seemann 1989). Acclimation to low temperature is known to enhance the Piregeneration capacity and thereby reduce the extent of a
Pi-regeneration limitation in C3 plants grown at low temperature (Stitt & Hurry 2002). Consistent with this, Piregeneration limitations are not apparent in cool-grown C.
canadensis, as shown by the relatively high O2 sensitivity.
The response shown for C. canadensis indicates that this
species is prone to Pi-regeneration-limited A when grown
at warmer temperature, but can overcome this limitation
upon acclimation to cool conditions. The precise mechanism of low-temperature acclimation in C. canadensis is
uncertain, but probably involves increases in the capacity
of enzymes associated with carbon metabolism, such as
sucrose phosphate synthase and cytosolic fructose-1, 6-bisphosphatase. Such changes have been previously noted in
C3 grasses (Hurry et al. 1995) and Arabidopsis thaliana
(Stitt & Hurry 2002).
The low-temperature performance of Rubisco
It is important to identify the potential for Rubisco limitations in M. glomerata and C. canadensis, as this highlights
the fundamental differences between the function of
Rubisco in C4 and C3 plants. To differentiate between the
effects of enzyme capacity versus CO2 availability, we have
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 907–916
Low temperatures and C4 photosynthesis 913
Figure 5. The temperature response of the rates of in vitro Rubisco activity (Vcmax) and gross CO2 assimilation (A*) in Calamogrostis
canadensis (C3) and Muhlenbergia glomerata (C4). Plants were grown at 14/10 ∞C (a, c) or 26 /22 ∞C (b, d). CO2 assimilation was measured
at 200 mbar oxygen in both species, and at 20 mbar oxygen in the C3. The in vitro activity of Rubisco (D) was determined by the incorporation
of 14C into acid stable products, following carbamylation of the enzyme. Enzyme assays were conducted on leaves that had been used for
gas-exchange. Each value represents the mean (± 1 SE, error bars obscured where smaller than the symbol) of measurements on four
individual plants.
modelled the temperature dependencies of the Michaelis–
Menten constant for CO2 (Kc), and the amount of CO2
required for the rate of Rubisco carboxylation (Vc) to be
80% of Vcmax (Fig. 6); above this concentration, the increase
in enzymatic reaction rates with increasing substrate concentration slows considerably, and the amount of the
enzyme becomes the principal limitation. For Rubisco from
a C4 species, Kc ranges from about 135–1500 mbar from 5 to
30 ∞C (Fig. 6a). Using the model of von Caemmerer & Furbank (1999) and assuming a bundle-sheath conductance to
CO2 of 3 mmol m-2 s-1, we estimate that the partial pressure
of CO2 in the bundle sheath of M. glomerata exceeds
4 mbar between 5 and 30 ∞C, which is at least 2.5 times Kc.
As shown in Fig. 6a, Rubisco is effectively CO2 saturated
in the bundle sheath of M. glomerata at temperatures below
at least 20 ∞C, because the CO2 level required for Vc to
achieve 80% of Vcmax is below the predicted 4 mbar substrate concentration in the stroma. These simulations indicate that Vc will be limited by the amount of the enzyme
(Vcmax) at low temperatures, assuming that the stromal concentration of RuBP is saturating for Rubisco.
In C3 plants the situation is different; Kc increases from
about 40–400 mbar between 0 and 30 ∞C (conservatively
assuming a value of 260 mbar at 25 ∞C, Fig. 6b). Assuming
that the partial pressure of O2 in the chloroplast stroma is
close to atmospheric (e.g. Os = 200 mbar), then the partial
pressure of CO2 required for Vc to be 80% of Vcmax is higher
than atmospheric between 0∞ and 30 ∞C (Fig. 6b). In C.
canadensis at 5 ∞C, Vc would be about 70% of Vcmax based
on the measured Ci of 325 mbar (Kubien 2003). If Rubisco
capacity is limiting, the CO2 deficiency is manifested by the
inhibition from the oxygenase activity of Rubisco and by
the deficiency of CO2 as a substrate. If RuBP regeneration
capacity is limiting, the CO2-deficiency is manifest only by
oxygenase inhibition (Sage & Reid 1994). If the oxygenase
function of Rubisco is reduced by lowering Os to 20 mbar,
then atmospheric levels of CO2 could effectively CO2-saturate C3 photosynthesis at temperatures up to 10 ∞C, and the
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 907–916
914 D. S. Kubien & R. F. Sage
Figure 6. Temperature effects on the
amount of CO2 required in order for
Rubisco carboxylation (Vc) to proceed at
80% of Vcmax. (a) A C4 Rubisco, assuming
that Kc is 650 mbar and Ko is 450 mbar, at
25 ∞C (von Caemmerer 2000; p. 100). In
this case the partial pressure of O2 in the
stroma (Os) is modelled at 200 mbar. (b)
A C3 Rubisco, with a Kc of 260 mbar (von
Caemmerer 2000; p. 45) and a Ko of
397 mbar (Jordan & Ogren 1984); this
value was selected to minimize the oxygenation function in this simulation. The
Os is modelled at 200 mbar (—) and
20 mbar (– –). The dashed line in each
panel represents the current atmospheric
CO2. The temperature response of Kc
(··–··–) is shown in each panel, modelled
using a Q10 of 2.24 (von Caemmerer 2000;
p. 45). Q10 values of 2.21 and 1.63 were
assumed for Vcmax and Ko, respectively
(von Caemmerer 2000; p. 45).
nature of the Rubisco limitation would be more like that
seen in C4 plants.
Temperature effects on the leakage of CO2 from
the bundle sheath of C4 leaves
The increase in FPSII/FCO2* at cooler temperatures reflects
increased leakage of CO2 (f) from the bundle sheath cells
in M. glomerata. In the C4 dicot Flaveria bidentis, genotypes
with antisense Rubisco constructs (anti-RbcS) show higher
FPSII/FCO2*, and higher estimates of f, than wild-type plants
at warm and intermediate temperatures (Kubien et al.
2003). Both wild-type and anti-RbcS plants show a rise in
leakiness with reduced temperature, and converge on common leakiness estimates at low temperatures where the
Rubisco flux control coefficient indicates complete limitation by Rubisco capacity (Kubien et al. 2003). Increased
CO2 leakiness reduces FCO2*, but not FPSII; hence FPSII/
FCO2* should increase as Rubisco increasingly limits A. The
values of FPSII/FCO2* in M. glomerata at warm temperatures
(> 20 ∞C) are similar to those reported by Oberhuber &
Edwards 1993) for a range of C4 grasses representing each
biochemical subtype. The stability of FPSII/FCO2* at warm
temperatures reflects a constant stoichiometry between the
mesophyll and bundle sheath reactions (Kubien et al. 2003).
As Rubisco becomes a principal control over the rate of
photosynthesis in M. glomerata at cool temperatures
(< 20 ∞C) this stoichiometry is altered, allowing CO2 to
accumulate in the bundle sheath. The capacity of Rubisco
to consume CO2 is increasingly exceeded by the capacity of
the C4 pump as temperature declines. This results in higher
bundle sheath CO2 levels, increasing the diffusion gradient
between the bundle sheath and intercellular airspace, and
hence increasing f (Henderson, von Caemmerer & Farquhar 1992; von Caemmerer & Furbank 1999). Increased
leakiness cannot arise from a limitation in the mesophyll
reactions (e.g. PPDK or PEPCase), as this would reduce
the delivery of CO2 to the bundle sheath and so should have
the opposite effect.
Consequences for C4 plants in cool climates
The CO2-concentrating functions of the mesophyll give C4
species numerous photosynthetic advantages over their C3
competitors, particularly at warm temperatures. At low
temperatures, the Rubisco content of C4 plants imposes a
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 907–916
Low temperatures and C4 photosynthesis 915
low ceiling on the maximum rate of carbon gain. Whereas
in some C4 species the amount and activity of enzymes such
as PPDK and PEPCase may affect low-temperature performance, the inferior photosynthetic performance at low temperatures of many C4 plants occurs because they either do
not, or cannot, hold as much Rubisco as C3 species (Pittermann & Sage 2000). It is well established that they do not;
C3 plants typically have three to six times as much Rubisco
as C4 species (Ku, Schmitt & Edwards 1979; Long 1999). A
possible low-temperature acclimation strategy would be to
increase the amount of Rubisco, but such a response does
not appear to commonly occur in C4 plants (Pearcy 1977;
Pittermann & Sage 2000, 2001; Naidu et al. 2003). C4 species
may have little capacity to increase Rubisco content. This
could be due to a biochemical constraint, whereby synthesis
of photosynthetic proteins is restricted by a need to maintain specific stoichiometric ratios required for effective coordination of the C3 and C4 cycles. The ability to increase
Rubisco content may be constrained physically, because the
localization of Rubisco to the bundle sheath chloroplasts
places an upper limit on the amount of the protein a leaf
may contain. In C4 grasses bundle sheath tissues occupy less
than 30% of the leaf volume; by contrast, the Rubiscocontaining mesophyll tissues occupy more than 50% of a
C3 leaf (Dengler et al. 1994). Furthermore, unlike C3 plants
in which Rubisco-containing chloroplasts can occur on all
axis of the cell, in C4 plants, Rubisco-containing chloroplasts are usually restricted to an inner or outer pole of the
bundle sheath cells, so the actual leaf volume available to
compartmentalize Rubisco is 40 to 70% less then total bundle sheath volume (Dengler & Nelson 1999). If tissue compartmentalization is a limiting factor, then C4 plants would
have to increase the size and/or amount of bundle sheath
tissue to accommodate additional Rubisco. As an acclimation response this is unlikely, as it would require fundamental changes to the specific vascular patterning and
associated intercellular transport that are required to
ensure efficient co-ordination between the carbon-concentrating mechanism and the bundle sheath reactions (Dengler & Nelson 1999). Acclimation to low temperature via
changes in these relationships would likely be detrimental
at warmer temperatures, where the efficiency of C4 photosynthesis is facilitated by high bundle sheath CO2 concentration. Low Rubisco content does not impair carbon gain
at warm temperatures in C4 plants, and is beneficial for their
nitrogen economy. However, this characteristic is detrimental when low-temperature limits the turnover capacity of
Rubisco.
ACKNOWLEDGMENTS
The authors wish to thank Bruce Hall and Andrew Petrie
for assistance with plant growth, Barbara and Garnett
Sproule for permission to collect M. glomerata and C.
canadensis, and the help of two anonymous reviewers. This
work was funded by an NSERC grant (OGP0154273) to
RFS.
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Received 15 January 2004; received in revised form 19 February
2004; accepted for publication 3 March 2004
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 907–916