Tree Physiology 28, 11–19
© 2008 Heron Publishing—Victoria, Canada
Does growth temperature affect the temperature responses of
photosynthesis and internal conductance to CO2? A test with
Eucalyptus regnans
C. R. WARREN
School of Biological Sciences, The University of Sydney, NSW 2006, Australia ([email protected])
Received April 26, 2007; accepted May 30, 2007; published online October 15, 2007
Summary Internal conductance to CO2 transfer from intercellular spaces to chloroplasts (gi) poses a major limitation to
photosynthesis, but only three studies have investigated the
temperature dependance of gi. The aim of this study was to determine whether acclimation to 15 versus 30 °C affects the
temperature response of photosynthesis and gi in seedlings of
the evergreen tree species Eucalyptus regnans F. Muell.
Six-month-old seedlings were acclimated to 15 or 30 °C for
6 weeks before gi was estimated by simultaneous measurements of gas exchange and chlorophyll fluorescence (variable
J method). There was little evidence for acclimation of photosynthesis to growth temperature. In seedlings acclimated to either 15 or 30 °C, the maximum rate of net photosynthesis
peaked at around 30 or 35 °C. Such lack of temperature acclimation may be related to the constant day and night temperature acclimation regime, which differed from most other studies in which night temperatures were lower than day temperatures. Internal conductance averaged 0.25 mol m – 2 s – 1 at 25 °C
and increased threefold from 10 to 35 °C. There was some evidence that gi was greater in seedlings acclimated to 15 than to
30 °C, which resulted in seedlings acclimated to 15 °C having,
if anything, a smaller relative limitation due to gi than seedlings
acclimated to 30 °C. Stomatal limitations were also smaller in
seedlings acclimated to 15 °C than in seedlings acclimated to
30 °C. Based on chloroplast CO2 concentration, neither maximum rates of carboxylation nor RuBP-limited rate of electron
transport peaked between 10 and 35 °C. Both were described
well by an Arrhenius function and had similar activation energies (57–70 kJ mol –1). These findings confirm previous studies showing gi to be positively related to measurement temperature.
Keywords: carbon dioxide, diffusion, internal resistance,
mesophyll resistance, photosynthesis, transfer conductance,
transfer resistance.
Introduction
For photosynthesis to occur, CO2 must diffuse from the atmosphere to the site of carboxylation. The CO2 concentration at
the site of carboxylation (Cc) is less than the atmospheric CO2
concentration (Ca ) owing to a series of gas-phase (air) and liquid-phase (mesophyll cells) resistances. In the gaseous phase,
CO2 must diffuse across a boundary layer in the air above the
foliage surface, through stomatal openings, and across intercellular air spaces surrounding mesophyll cells. In the liquid
phase, resistance occurs as CO2 enters the liquid phase at the
surface of mesophyll cells, as CO2 diffuses within the cell to
the chloroplast membrane and from there to the sites of carboxylation (Gaastra 1959, Aalto and Juurola 2002).
The intercellular CO2 concentration (C i) is significantly
higher than the corresponding values of Cc (Evans et al. 1986,
Parkhurst and Mott 1990, Lloyd et al. 1992, Epron et al. 1995).
Internal conductance (gi) determines the difference between Ci
and Cc as a function of photosynthetic rate (A): gi = A/(Ci – Cc).
Internal conductance may limit photosynthesis by 20% or
more, and has a large effect on in vivo estimates of the maximum rate of carboxylation (Vcmax) and of the Michaelis–
Menten constants for carboxylation (Kc) and oxygenation (Ko)
(von Caemmerer et al. 1994, Epron et al. 1995, Bernacchi et al.
2002, Warren et al. 2003a, Ethier and Livingston 2004).
The temperature dependence of gi has received little attention, despite its critical importance, not only in models of photosynthesis, but also for correctly determining Vcmax and for
understanding the major limitations to photosynthesis (Bernacchi et al. 2002). Information about the effect of temperature
on gi may also provide clues as to the factors determining the
value of gi. For example, the temperature response of gi has a
temperature coefficient of about 2 between 10 and 20 °C
(Bernacchi et al. 2002, Warren and Dreyer 2006, Yamori et al.
2006), which is consistent with a role of one or more protein-facilitated processes in the determination of gi. At temperatures greater than 20 °C, the temperature responses of gi differ
among species. In Nicotiana tabacum L., gi increases up to
35 °C and decreases strongly as temperatures approach 40 °C
(Bernacchi et al. 2002); whereas in Spinacia oleracea L. and
Quercus canariensis Willd., gi is temperature independent or
decreases slightly from 20 to 35 °C (Warren and Dreyer 2006,
Yamori et al. 2006).
Variation in the temperature response of gi among species
may reflect genotypic species differences or be a result of acclimation to different growth temperatures. It is well known
12
WARREN
that the temperature dependence of photosynthesis is affected
by growth temperatures such that growth at elevated temperatures increases the photosynthetic temperature optimum
(Berry and Björkman 1980). Yamori et al. (2006) showed that
growth temperature has similar effects on photosynthesis and
gi, such that growth at elevated temperatures increases both the
photosynthetic temperature optimum and the temperature at
which gi is maximal. These effects of acclimation temperature
are subtle, however, compared with effects attributable to species.
So far as I am aware, information on acclimation of gi to
growth temperature is available only for the single species,
S. oleracea (Yamori et al. 2006). However, there are large differences in photosynthetic temperature optimum, even among
closely related species, which raises the possibility that there
are also large differences among species in the acclimation of
gi to temperature (Slatyer 1977, Slatyer and Ferrar 1977, Berry
and Björkman 1980, Ferrar et al. 1989). Thus, I set out to determine the temperature response of gi in the evergreen tree
species Eucalyptus regnans F. Muell. In particular, I investigated the effect of temperature between 10 and 35 °C on both
gi and the photosynthetic temperature optimum in E. regnans
seedlings.
Internal conductance was determined by the variable J method (Di Marco et al. 1990, Loreto et al. 1992) and
the relative limitation posed by stomatal conductance (gs) and
gi was estimated. Calculated Cc was used to estimate the temperature responses of the rates of Rubisco carboxylation
(Vcmax) and RuBP-limited electron transport (Jmax).
Materials and methods
Plant material and growth conditions
Seed of Eucalyptus regnans (CSIRO ATSC Seedlot 15158)
from the Australian Tree Seed Centre, Kingston, ACT, Australia was sown on June 29, 2006 in a 1:1 (v/v) mixture of coarse
sand and soil (from an E. regnans forest) and stratified at 4 °C
for 4 weeks. Germination took place in a fully sunlit greenhouse at the University of Sydney, Camperdown, NSW, Australia (33°53′ S, 151°11′ E, 30 m a.s.l.). About 1 month later,
when the germinated seedlings had one or two pairs of true
leaves, they were transferred to 210-ml plastic tubes (50 mm
square × 125 mm high) filled with the same 1:1 sand:soil mixture. After a further 2 months, seedlings were transferred to
1.6-l plastic pots (140 mm diameter, 140 mm high) and grown
in the greenhouse until the end of January, at which time seedlings were around 25 cm tall. Mean daytime maximum and
minimum temperatures in the greenhouse were 25 and 20 °C,
respectively.
At the end of January, temperature acclimation treatments
were imposed by randomly allocating seedlings to one of two
growth chambers. Both growth chambers had identical 12-h
photoperiods with a photosynthetic photon flux (PPF) at leaf
height of around 500 µmol m – 2 s –1 and differed only in temperature. One chamber was maintained at 15 °C (day and
night), and the other was maintained at 30 °C (day and night).
Measurements were made 7–11 weeks after treatments were
imposed.
Experimental protocol
Gas exchange and chlorophyll fluorescence measurements
were made on one attached leaf from each of six seedlings per
acclimation treatment. For each seedling, measurements were
made at leaf temperatures of 10, 15, 20, 25, 30 and 35 °C. The
same leaf was measured at each temperature to minimize variability in photosynthetic capacity.
Measurements were made in a small, temperature-controlled measurement chamber held within 0.5 °C of the target
leaf temperature. Inside the chamber, seedlings were subjected
to a 16-h photoperiod at a PPF of ~200 µmol m – 2 s – 1 at leaf
height. Two seedlings were transferred from the growth chamber to the measurement chamber about 2 h before measurements were taken. Temperatures were measured in a random
order. Seedlings were allowed 1 h to acclimate to each new
temperature before measurements were taken.
Gas exchange and chlorophyll fluorescence systems
Three main measurements were made: (1) a calibration of the
relationship between linear electron transport (J) estimated
from chlorophyll fluorescence (Jf) and J estimated from gas
exchange under non-photorespiratory conditions (Ja); (2) the
CO2 responses of gas exchange and chlorophyll fluorescence;
and (3) simultaneous estimation of intercellular photocompensation point (Ci*) and the mitochondrial respiration in the
light (Rd ) by the Laisk (1977) method. Simultaneous gas exchange and fluorescence measurements (i.e., 1 and 2) were
made with an LI-6400 gas exchange system equipped with an
integrated fluorescence chamber (LI-6400-40, Li-Cor, Lincoln, NE). Measurements of Ci* and Rd were made with a second LI-6400 equipped with a 2 × 3 cm broadleaf chamber and
an integrated light source (LI-6400-02B, Li-Cor).
Each day, both LI-6400 instruments were calibrated and
re-zeroed with freshly regenerated drierite and new soda lime.
Relative humidity was controlled between 50 and 70% using
the drierite scrubber of the LI-6400 or a dew-point generator.
Measurements were made with leaf temperature controlled to
within ± 1 °C of the target temperature.
Relating chlorophyll fluorescence with rates of electron
transport
The photochemical efficiency of photosystem II (φPSII) was
computed from steady-state fluorescence (F′) and maximal
fluorescence (Fm′) during a light-saturating pulse (Genty et al.
1989):
φ PSII =
Fm ′ − Fm ′
Fm ′
(1)
The rate of J is related to φPSII:
J = 0. 5 φ PSIIα( PPF)
TREE PHYSIOLOGY VOLUME 28, 2008
(2)
TEMPERATURE RESPONSE OF INTERNAL CONDUCTANCE TO CO 2
where α is total leaf absorptance and the factor 0.5 describes
the expected distribution of light between the two photosystems. Fluorescence estimates of linear electron transport
(Jf) are not strictly related to J, because fluorescence measurements primarily indicate the properties of the upper cell layers
and are not representative of the whole leaf. Furthermore, the
distribution of light between photosystems is set to be equal,
but this may not be the case. Because of these uncertainties, no
a priori assumptions were made about relationships between Jf
and J, and an empirical relationship was determined. Such a
“calibration” procedure obviates the need to measure leaf absorptance or to make assumptions about the distribution of
light between photosystems or about how representative fluorescence measurements are of whole-leaf processes.
The relationship between Jf and Ja was determined under
non-photorespiratory conditions (1% O2, 1000 µmol mol – 1
CO2) on one leaf from each of the 12 seedlings at each of the
six measurement temperatures. This method assumes that, under non-photorespiratory conditions, Ja is wholly dependent
on gross photosynthesis (i.e., Ja = 4(A + Rd)). A cylinder delivered a mixture of 1% O2 in N2 (BOC Gas, Australia), which
was scrubbed of CO2 with the soda lime column of the
LI-6400. Carbon dioxide was then added to a concentration of
1000 µmol mol –1 with the LI-6400 CO2 mixer. One leaf per
seedling was placed in the fluorescence chamber (flow rate =
200 µmol s –1) and acclimated to a PPF of 1500 µmol m – 2 s – 1
for at least 30 min before gas exchange was recorded and a saturating light pulse given to determine fluorescence. This procedure was repeated at 1000, 750, 500 and 250 µmol m – 2 s – 1
and then at 50 µmol m – 2 s –1, with a 10-minute wait at each PPF
before gas exchange and chlorophyll fluorescence were measured.
The CO2 responses of gas exchange and chlorophyll
fluorescence
The CO2 responses of gas exchange and chlorophyll fluorescence were measured in six seedlings per treatment at each of
the six measurement temperatures. One leaf per seedling was
placed inside the fluorescence chamber and exposed to a saturating PPF of 1000 µmol m – 2 s –1 and a chamber flow rate of
200 µmol s –1 and CO2 concentration of 400 µmol CO2 mol – 1
(ambient O2 ) for at least 15 min or until gas exchange rates and
fluorescence reached a steady state. Preliminary experiments
established that a PPF of 2000 µmol m – 2 s –1 is saturating at all
leaf temperatures examined. The CO2-response curve was
generated by raising Ca to 2000 µmol mol –1 and then decreasing it in 13 steps to 50 µmol mol –1. At each step, gas exchange
and fluorescence were allowed to stabilize for 5 minutes and
then a saturating light pulse was given and data were recorded.
Intercellular photocompensation point and mitochondrial
respiration in the light
Values of Ci* and Rd were estimated by the Laisk (1977)
method on one leaf from each of six seedlings at each of the six
measurement temperatures. Measurements were made with
the 2 × 3 cm broadleaf chamber at a flow rate of 200 µmol s – 1.
13
One leaf per seedling was placed in the chamber and exposed
to a saturating PPF of 500 µmol m – 2 s – 1 and a chamber flow
rate of 200 µmol s –1 and CO2 concentration of 400 µmol CO2
mol – 1 (ambient O2) for at least 15 min or until gas exchange
rates and fluorescence reached a steady state. Values of Ci*
and Rd were estimated from three partial CO2-response curves
(50–150 µmol CO2 mol – 1) measured at PPFs of 500, 200 and
75 µmol m – 2 s – 1. These irradiances were chosen following
preliminary trials to ensure a large difference in slope of the
three A–Ci curves. Each partial CO2-response curve comprised
at least six measurements, although sometimes only four or
five values were used in regressions, because at the lowest
PPF, the A–Ci relationship above 100 µmol CO2 mol – 1 deviated from linearity. The intersection of the three lines identified Ci* (x-axis) and Rd (y-axis).
Gas exchange parameters
Data for both gas exchange systems were corrected for CO2
diffusion into and out of the leaf chamber according to the
manufacturer’s advice (Anon. 2001). Diffusion leaks are proportional to the difference in CO2 concentration between the
inside and outside of the chamber and the flow rate of air
through the chamber. This is accounted for by a diffusion coefficient that was determined by measuring the diffusion of CO2
into empty chambers (Ca = 0 µmol mol – 1) as a function of the
flow rate of air through the chamber and the gradient of CO2
concentration between the chamber and the ambient air. The
CO2 concentration of the sample cell was measured by the reference IRGA using the match valve, and the diffusion coefficient was used to recalculate the gas exchange data (Anon
2001). As expected, the diffusion coefficient for the 2 × 3 cm
chamber was about one-third that of the smaller fluorescence
chamber. For measurements made under non-photorespiratory
conditions (1% O2), it was necessary to recalculate all gas exchange data because the CO2 and H2O sensitivity of the
LI-6400 is affected by the O2 concentration of the analysis gas.
Internal conductance by the variable J method
Internal conductance was estimated by the variable J method
(Di Marco et al. 1990, Loreto et al. 1992) based on A, Ci and Jf
measured at 400 µmol mol – 1 CO2 and Rd determined by the
Laisk (1977) method. Fluorescence estimates of electron
transport were converted to actual rates of electron transport
(Ja) based on the empirical plant- and temperature-specific relationship determined under non-photorespiratory conditions.
gi =
A
Γ * ( Ja + 8( A + Rd )
Ci −
Ja − 4( A + Rd )
(3)
Internal conductance was calculated using measured Ci* as a
surrogate for the chloroplastic photocompensation point (Γ*)
and the temperature response of Γ* reported by Bernacchi et
al. (2002).
From estimated gi and measured A and Ci, Cc was calculated
as:
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14
WARREN
C c = C i − gA
i
(4)
Maximum rates of carboxylation and electron transport
Values of Vcmax and Jmax were determined on a Cc basis from
CO2-response data and gi determined with measured Ci*. Data
were fitted to the photosynthesis model of Farquhar et al.
(1980), essentially as described by Warren et al. (2003b). Values of Γ*, Kc and Ko and their temperature responses were the
Cc-based in vivo values of Bernacchi et al. (2002). Maximum
quantum yield was assumed to be 0.24 mol (electrons) mol – 1
(photons) (Harley and Tenhunen 1991).
Model temperature responses
Temperature responses to Vcmax, Jmax, Ci* and Rd were fitted by
the equation (Sharpe and Demichele 1977, Leuning 1997):
PT = PTref e
E a  Tref 
1–

RTref 
T 
(5)
where PTref is the parameter value at a reference temperature
Tref (25 °C, 298 K), Ea (J mol –1) is the activation energy, R is
the gas constant (8.3143 J K –1 mol –1), and T (K) is leaf temperature.
Estimating relative limitation imposed by stomatal and
internal conductance
The limitation of A imposed by finite gi and gs was based on estimates of the potential photosynthetic rate assuming these
conductances were either infinite or as measured (Farquhar
and Sharkey 1982). Estimates of A were based on CO2-response curves and mean gi and gs. Rates of net photosynthesis
were estimated assuming gi and gs were as measured (An , the
light-saturated photosynthetic rate at Ca = 360 µmol mol – 1),
assuming gi was infinite and gs as measured (Ail, the light-saturated photosynthetic rate at Cc = Ci), or assuming gi as measured and gs was infinite (Asl, the light-saturated photosynthetic rate at Ci = 360 µmol mol –1). The relative limitations due
to internal (Li) and stomatal (Ls) resistances were estimated as:
Li =
Ail − An
Ail
(6)
Ls =
Asl − An
Asl
(7)
Results
Relationship of fluorescence with electron transport
There was a strong positive relationship between Jf and Ja estimates of J (r 2 > 0.95 for all seedlings at all temperatures). Relationships differed subtly among replicates, and thus, individual relationships were used for each seedling at each temperature (Figure 1).
Intercellular photocompensation point and mitochondrial
respiration in the light
Neither Ci* nor Rd was affected by growth temperature (t-test,
P > 0.05), so data for both acclimation treatments were combined. The temperature response of Ci* was well described by
a simple Arrhenius model (Figure 2, Table 1) but differed
markedly from published data for Ci*. The temperature response of Ci* was similar to that reported for another Eucalyptus species, E. pauciflora Seib. ex Spreng. (Atkin et al. 2000),
but was only about half as sensitive to temperature as several
other species (Brooks and Farquhar 1985, Warren and Dreyer
2006). The rate of Rd was well described by an Arrhenius function (Figure 3), with an activation energy of 55 kJ mol – 1 and a
rate, at 25 °C, of 0.70 µmol m – 2 s – 1.
Stomatal and internal conductance, inter- and intracellular
CO2 concentrations
Stomatal conductance varied between 0.17 and 0.39 mol m – 2
s – 1 and was unaffected by either acclimation or measurement
temperature (data not shown). Internal conductance was also
unaffected by acclimation temperature (t-test, P > 0.05, data
not shown), but generally increased with measurement temperature (Figure 4). Estimates of gi were greater when calculated with measured Ci* than with Γ* from Bernacchi et al.
(2002). Irrespective of how calculated, gi was positively related to temperature and increased threefold from 10 to
35 °C. There were, however, differences in the shape of the
temperature responses. When estimated with measured Ci*, gi
peaked at around 30 or 35°C, whereas when estimated with
Γ*, it increased exponentially.
The CO2 concentration in the sub-stomatal cavities decreased as measurement temperature increased (Figure 5). In
contrast, Cc calculated from gi estimated with Ci*, was generally unaffected by measurement temperature and varied between 194 and 272 µmol mol – 1. Values of Ci and Cc were gen-
Figure 1. Relationship between linear electron transport rate estimated by chlorophyll fluorescence (Jf) and that determined from
gross photosynthesis under non-photorespiratory conditions (Ja ). The
relationship between Jf and Ja was determined at 1% O2 and
1000 µmol CO2 mol – 1 on one leaf from each of six seedlings per treatment at each of six measurement temperatures (10 °C, 䉬; 15 °C, 䉫;
20 °C, 䊏; 25 °C, 䊐; 30 °C, 䉱; and 35 °C, 䉭). Data are representative
of Eucalyptus regnans acclimated to 30 °C. For clarity, regressions are
shown for 10 and 35 °C only (solid and dashed lines, respectively).
TREE PHYSIOLOGY VOLUME 28, 2008
15
TEMPERATURE RESPONSE OF INTERNAL CONDUCTANCE TO CO 2
by acclimation temperature, and thus, a common Arrhenius
function was fitted to all data (Figure 6). Activation energies
were similar for Vcmax (15 °C, 89 kJ mol – 1; 30 °C, 51 kJ mol – 1)
and Jmax (both acclimation treatments, 57 kJ mol – 1).
Relative limitations due to stomatal and internal
conductances
Figure 2. Temperature response of the intercellular photocompensation point (Ci*; solid line, 䉬) in Eucalyptus regnans seedlings compared with published data for Spinacia oleracea (broken line, Brooks
and Farquhar 1985) and Eucalyptus pauciflora (䊐; Atkin et al. 2000).
There was no difference between seedlings acclimated to 15 versus
30 °C, and so data are the means of 12 replicates (error bars = 1 standard deviation). The Ci* data were used to fit an Arrhenius equation:
activation energy = 17 kJ mol –1 and Ci* at 25 °C = 42 µmol mol – 1.
erally greater in seedlings acclimated to 15 °C than in seedlings acclimated to 30 °C.
Light saturated rates of photosynthesis, carboxylation and
electron transport
Eucalyptus regnans had a broad temperature response with the
maximum rate of light-saturated net photosynthesis (Amax ) increasing from around 8 µmol m – 2 s –1 at 10 °C to a maximum
of 12–13 µmol m – 2 s –1 between 30 and 35 °C (Figure 6). Acclimation to 15 °C versus 30 °C did not affect absolute values
of Amax or its temperature response.
The temperature responses of Vcmax and Jmax were similar,
whether gi was determined from Ci* or from Γ* (data not
shown), and for this reason, only the temperature responses of
Vcmax and Jmax determined from Ci* are shown. Seedlings acclimated to 15 °C had a greater Vcmax at 35 °C than seedlings acclimated to 30 °C, whereas at lower temperatures, there were
no differences in Vcmax (Figure 6). Because of this difference,
separate Arrhenius functions for Vcmax were fitted to seedlings
acclimated to 15 and 30 °C. The value of Jmax was unaffected
Relative stomatal and internal limitations were greater in seedlings acclimated to 30 °C compared with seedlings grown at
15 °C (Figure 7). Stomatal and internal limitations generally
increased with temperature, although this relationship was
weak.
Discussion
Temperature response of internal conductance and
photosynthesis
In E. regnans seedlings, there was little evidence that Amax acclimated to growth temperature (e.g., Figure 6). Instead, Amax
peaked between 30 and 35 °C in seedlings acclimated to 15 or
30 °C. It is common for the optimum temperature for Amax to
be greater than the growth temperature by a few degrees
(Slatyer and Ferrar 1977), but it is unusual for there to be no
acclimation of Amax. The extent of temperature acclimation is
thought to be related to the range in seasonal variation in temperature in the species’ native habitat, with large seasonal variations in temperature resulting in large shifts in the optimum
temperature (Badger et al. 1982). In the natural habitat of
E. regnans, mean daily maximum/minimum temperatures
vary from 23 /11 °C in midsummer to 10/4 °C in midwinter.
Although modest, this range is not small enough to explain the
absence of temperature acclimation in E. regnans seedlings,
because temperature acclimation has been shown in other Eucalyptus species from habitats with similar seasonal variation
in temperature (Battaglia et al. 1996). A possible explanation
for the lack of temperature acclimation in this study is that
constant temperature was maintained day and night, whereas
in most studies, night temperature was lower than day temperature (Slatyer 1977, Mooney et al. 1978, Badger et al. 1982). It
Table 1. Arrhenius fits to the temperature response of photosynthetic parameters in Eucalyptus regnans. The intercellular photocompensation
point (Ci*), rate of mitochondrial respiration in the light (Rd ), maximum rate of carboxylation (Vcmax ) and maximum rate of RuBP limited electron
transport (Jmax ) were measured from 10 to 35 °C. Parameters Vcmax and Jmax were determined on a Cc basis using internal conductance calculated
from measured Ci*. To calculate Vcmax and Jmax, the Ci* and Cc-based in vivo values and temperature responses of Kc and Ko were used (Bernacchi
et al. 2002). Quantum yield was assumed to be 0.24. Seedlings were acclimated to 15 or 30 °C, and measurements were made on six seedlings per
treatment. Acclimation temperature did not affect Ci*, Rd or Jmax, so Arrhenius functions were fitted to data for both acclimation treatments combined. There was a significant difference in Vcmax between acclimation temperatures, so separate Arrhenius functions were fitted to seedlings acclimated to 15 and 30 °C. Abbreviations: Cc, chloroplast CO2 concentration; Kc and Ko, Michaelis-Menten constants for carboxylation and
oxygenation, respectively; Ea, activation energy; and P25 °C, mean measured value of the parameter at 25 °C (mean ± SD).
Ci* (µmol mol –1)
Ea (kJ mol –1)
r2
P25 °C
17 ± 3
0.87
42 ± 3
Rd (µmol m –2 s –1)
55 ± 2
0.85
0.7 ± 0.2
Vcmax (µmol m –2 s –1)
15 °C
30 °C
89 ± 5
0.97
53 ± 18
51 ± 5
0.84
66 ± 18
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Jmax (µmol m –2 s –1)
57 ± 7
0.91
121 ± 9
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WARREN
Figure 3. Temperature response of mitochondrial respiration in the
light (Rd ) in Eucalytpus regnans seedlings. There was no difference
between seedlings acclimated to 15 versus 30 °C, so data are the
means of 12 replicates (error bars = 1 standard deviation). The Rd data
were used to fit an Arrhenius equation: activation energy = 55 kJ
mol –1, and Rd at 25 °C = 0.70 µmol m – 2 s –1.
may be that night temperatures are critical for acclimation of
photosynthesis to growth temperature, as has previously been
shown for the development of frost tolerance and cold hardiness (Harwood 1980).
In E. regnans, the shape of the temperature response of gi
(Figure 4) differed depending on whether measured Ci* (Figure 2) was used as a surrogate for Γ* or if the Γ* value for
N. tabacum (Bernacchi et al. 2002) was used. Irrespective of
how gi was calculated, it was positively related to temperature
and increased threefold from 10 to 35 °C. When gi was calculated with the Γ* of N. tabacum, its temperature response was
similar to that observed in N. tabacum (Bernacchi et al. 2002).
In N. tabacum, gi increases with temperature from 10 °C to a
maximum between 35 and 37.5°C, before decreasing at higher
temperatures. Whether there is a decline in gi of E. regnans at
temperatures above 37 °C was not determined. In contrast,
when gi of E. regnans was calculated with measured Ci*, its
temperature response was more like that of Spinacia oleracea
(Yamori et al. 2006) or Quercus canariensis (Warren and
Dreyer 2006) in which gi increased from 10 to 20 °C and was
temperature-independent or declined gradually from 20 to
Figure 4. Temperature response of internal conductance to CO2 (gi ) in
Eucalyptus regnans estimated with measured intercellular photocompensation point (Ci*; 䉬) or published Γ* (䊐). There was no significant effect of acclimation temperature (15 versus 30 °C), so data
for both treatments are combined. Data are means of 12 replicates; error bars are 1 standard deviation.
Figure 5. Temperature responses of the CO2 concentration in the
sub-stomatal cavities (Ci; 䉬, 䉫) and chloroplast (Cc; 䉱, 䉭) in Eucalyptus regnans seedlings acclimated to 15 °C (filled symbols) and
30 °C (open symbols). The Ci and Cc values are given for a PPF of
1000 µmol m – 2 s – 1 at an ambient CO2 concentration of 400 µmol
mol – 1. The Cc was calculated based on measured photosynthetic rates
and internal CO2 conductance calculated with measured Ci* (see Figure 4). Data are the mean of six replicates; error bars are 1 standard deviation.
Figure 6. Temperature responses of the maximum rate of net photosynthesis (Amax ), the maximum rate of carboxylation (Vcmax ) and the
maximum rate of RuBP limited electron transport (Jmax ) in Eucalyptus regnans seedlings acclimated to 15 °C (䉬, solid line) and 30 °C
(䊐, dashed line). The Amax was measured with a light-saturating PPF
of 1000 µmol m – 2 s – 1 at an ambient CO2 concentration of 400 µmol
mol – 1, whereas Vcmax and Jmax were determined from CO2-response
curves on a Cc basis (based on gi determined from measured intercellular photocompensation point, Ci*).The Cc was calculated from
measured rates of photosynthesis and gi (Figure 4). Data are the
means of six replicates; error bars are 1 standard deviation. Arrhenius
functions were fitted to Vcmax and Jmax. Separate Vcmax functions were
fitted to seedlings grown at 15 and 30 °C, whereas a common Jmax
function was fitted to seedlings grown at 15 and 30 °C. Abbreviations:
Cc, chloroplast CO2 concentration; and gi, internal conductance to
CO2.
TREE PHYSIOLOGY VOLUME 28, 2008
TEMPERATURE RESPONSE OF INTERNAL CONDUCTANCE TO CO 2
Figure 7. Temperature responses of relative stomatal limitations (䊏,
䊐) and internal limitations (䉬, 䉫) in Eucalyptus regnans seedlings
acclimated to 15 °C (filled symbols) and 30 °C (open symbols). Data
were calculated from mean photosynthetic parameters.
35°C. Hence, the shape of the temperature response is highly
dependent on which value is used for Γ*, but irrespective of
how gi is calculated, it is positively related to temperature.
Adaptation and acclimation affect the temperature responses of the photosynthetic parameters (Berry and Björkman 1980, Bernacchi et al. 2003). Recently, Yamori et al.
(2006) showed that gi of S. oleracea acclimates to growth temperature; however, acclimation was not observed in E. regnans
in gi or any other measured photosynthetic parameter. Although these discrepancies among species may be a function
of adaptation and acclimation, there are too few data to draw
definite conclusions. However, differences in the shapes of the
temperature responses of gi among species are far larger than
the effect of acclimation on the shape of the temperature responses for other parameters. Acclimation of other photosynthetic parameters generally shifts the optimum but has little
effect on the general shape of the response curve (Slatyer
1977, Mooney et al. 1978, Badger et al. 1982).
The temperature response of gi is consistent with a protein-facilitated process (Bernacchi et al. 2002, Warren and
Dreyer 2006, Yamori et al. 2006). The primary evidence for
this conclusion is that the temperature response of gi is steeper
than would be expected for diffusion of CO2 in water. The
threefold increase in gi from 10 to 35 °C is inconsistent with
the known doubling of rates of CO2 diffusion in water over the
same temperature range (Tamimi et al. 1994). Other support
for a dominant role of proteins comes from earlier studies
showing that gi is related, at least in part, to carbonic anhydrase
activity (Makino et al. 1992, Price et al. 1994) and aquaporin
content (Uehlein et al. 2003, Hanba et al. 2004, Flexas et al.
2006). Nevertheless, arguing that gi is solely a protein-facilitated process ignores the relative constancy of gi from 30 to
35 °C in E. regnans, from 20 to 35°C in Q. canariensis (Warren and Dreyer 2006) and S. oleracea (Yamori et al. 2006) and
from 28 to 38 °C in Eperua grandiflora (Aubl.) Benth. (Pons
and Welschen 2003). It seems likely that gi is determined by
multiple processes with different temperature sensitivities that
result in a complex temperature response.
Internal conductance has complex effects on the comparison between Ci-based and Cc-based Vcmax and Jmax. This is because differences among species in the temperature response
17
of gi (this study, Bernacchi et al. 2002, Warren and Dreyer
2006, Yamori et al. 2006) affect the temperature response of
Cc-based Vcmax and Jmax, and the interpretation of Ci-based
Vcmax and Jmax. In Q. canariensis, Cc-based Vcmax and Jmax increased 10-fold from 10 to 30 °C, which was significantly
greater than the sixfold increase in Ci-based Vcmax and Jmax observed in Q. canariensis (Warren and Dreyer 2006) and many
other species (Dreyer et al. 2001, Medlyn et al. 2002). However, in the present study with E. regnans, Cc-based Vcmax and
Jmax had the “normal” sixfold increase between 10 to 30 °C,
and the activation energies for Vcmax (51–89 kJ mol – 1) and Jmax
(57 kJ mol – 1) were similar to the Ci-based activation energies
for Vcmax and Jmax for a range of tree species (Dreyer et al.
2001). This difference between studies can be attributed to the
differing temperature responses of gi. In Q. canariensis, gi was
relatively constant from 20 to 35 °C, whereas in E. regnans, gi
increased strongly up to 30 °C. Hence, species differences in
the temperature response of gi complicate interpretation of
Ci-based Vcmax and Jmax.
Previous studies with Eucalyptus have shown that acclimation to low temperatures shifts the temperature optimum and
causes a general increase in Amax at all temperatures (Ferrar et
al. 1989, Warren et al. 1998), and the results of the present
study indicate that increased gi may play a role in increased
Amax. It has been speculated that the general increase in Cibased Vcmax and Jmax of Eucalyptus acclimated to low temperatures is a function of increased amounts of enzymes of the Calvin cycle (Ferrar et al. 1989, Warren et al. 1998); however,
another possibility is that increases in Ci-based Vcmax and Jmax
are at least in part a function of increased gi (and thus Cc). This
is supported by the finding that Cc was greater in E. regnans
acclimated to 15 °C (Figure 5).
Methodological considerations
Estimating gi is not easy, and results are always subject to several assumptions. The best approach is to use multiple methods that share few common assumptions (Warren 2006); however, in the present study only the variable J method was used.
The curve fitting method (Ethier et al. 2006) and constant J
method (Bongi and Loreto 1989) were unacceptable because
both assume that gi is unaffected by CO2 concentration—an
assumption now known to be incorrect (Centritto et al. 2003,
Flexas et al. 2007, C. Warren, unpublished data). It would be
useful to support the results obtained here by a similar set of
measurements obtained with the isotope method (Evans et al.
1986). The assumptions underlying the isotope method are
largely independent of those required for the variable J
method, so the methods complement each other. The temperature response of gi has most commonly been measured by the
variable J method (this study, Bernacchi et al. 2002, Warren
and Dreyer 2006), although one study has used the isotopic
method (Yamori et al. 2006) and Warren and Dreyer (2006)
applied the curve-fitting method. There does not seem to be
any general or systematic difference among the three methods,
providing qualified support for the results reported here.
The variable J method is affected by the choice of published
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
18
WARREN
Γ* value versus measured Ci* (Figure 4). It is surprising that
the value of Ci* of E. regnans reported here was so different
from published values of Γ*, because for most species, the two
values are similar (Warren and Dreyer 2006). In an earlier
study with E. pauciflora, it was found that Ci* estimates with
the Laisk method were unreliable at low temperatures (Atkin
et al. 2000). This raises the possibility that Ci* of E. regnans,
determined here by the same method, is also erroneous at low
temperatures. Because of this uncertainty, I also used the Γ*
estimates of Bernacchi et al. (2002) for N. tabacum. This approach is justified by the belief that Γ* is an invariant property
of C3 plants (von Caemmerer 2000), an assumption supported
by experiments showing that Γ* varies little among diverse
species (e.g., Quercus robur L. and herbaceous species; von
Caemmerer et al. 1994, Balaguer et al. 1996). The shape of the
temperature response curve cannot be precisely determined
until Ci* and Γ* of E. regnans are determined by independent
methods.
Neglecting cuticular conductance may have led to a small
overestimate of Ci (Boyer et al. 1997) and an underestimate of
gi (Warren et al. 2004). Unfortunately there are no estimates of
cuticular conductance for E. regnans, but it is likely to be less
than 0.01 mol m – 2 s –1 (Tausz et al. 2005). Inclusion of this
value for cuticular conductance would decrease estimates of Ci
by less than 4 µmol mol –1 and increase gi by less than
0.002 mol m – 2 s –1 (data not shown). Hence, the effect of cuticular conductance on estimates of gi is negligible, not surprisingly, as gs values of between 0.17 and 0.39 mol m – 2 s – 1 were
more than an order of magnitude greater than cuticular conductance.
Acknowledgments
This work was supported by a Discovery Grant and QEII fellowship
from the Australian Research Council and the University of Sydney
major equipment scheme. Prof. Mark Adams (University of New
South Wales) kindly loaned a second LI-6400 gas exchange system.
References
Aalto, T. and E. Juurola. 2002. A three-dimensional model of CO2
transport in airspaces and mesophyll cells of a silver birch leaf.
Plant Cell Environ. 25:1399–1409.
Anon. 2001. Using the LI-6400 portable photosynthesis system.
Li-Cor Biosciences Inc., Lincoln, NE, 946 p.
Atkin, O.K., J.R. Evans, M.C. Ball, H. Lambers and T.L. Pons. 2000.
Leaf respiration of snow gum in the light and dark interactions between temperature and irradiance. Plant Physiol. 122:915–923.
Badger, M.R., O. Bjorkman and P.A. Armond. 1982. An analysis of
photosynthetic response and adaptation to temperature in higherplants: temperature acclimation in the desert evergreen Nerium oleander L. Plant Cell Environ. 5:85–99.
Balaguer, L., D. Afif, P. Dizengremel and E. Dreyer. 1996. Specificity
factor of ribulose bisphosphate carboxylase /oxygenase of Quercus
robur. Plant Physiol Biochem. 34:879–883.
Battaglia, M., C. Beadle and S. Loughhead. 1996. Photosynthetic
temperature responses of Eucalyptus globulus and Eucalyptus
nitens. Tree Physiol. 16:81–89.
Bernacchi, C.J., A.R. Portis, H. Nakano, S. von Caemmerer and
S.P. Long. 2002. Temperature response of mesophyll conductance.
Implications for the determination of Rubisco enzyme kinetics and
for limitations to photosynthesis in vivo. Plant Physiol. 130:
1992–1998.
Bernacchi, C.J., C. Pimentel and S.P. Long. 2003. In vivo temperature
response functions of parameters required to model RuBP-limited
photosynthesis. Plant Cell Environ. 26:1419–1430.
Berry, J. and O. Björkman. 1980. Photosynthetic response and adaptation to temperature in higher plants. Annu. Rev. Plant Physiol.
31:491–543.
Bongi, G. and F. Loreto. 1989. Gas-exchange properties of saltstressed olive (Olea europa L.) leaves. Plant Physiol. 90:
1408–1416.
Boyer, J.S., S.C. Wong and G.D. Farquhar. 1997. CO2 and water vapor exchange across leaf cuticle (epidermis) at various water potentials. Plant Physiol. 114:185–191.
Brooks, A. and G.D. Farquhar. 1985. Effect of temperature on the
CO 2 /O2 specificity of ribulose-1,5-bisphosphate carboxylase oxygenase and the rate of respiration in the light. Planta 165:397–406.
Centritto, M., F. Loreto and K. Chartzoulakis. 2003. The use of low
[CO2] to estimate diffusional and non-diffusional limitations of
photosynthetic capacity of salt-stressed olive saplings. Plant Cell
Environ. 26:585–594.
Di Marco, G., F. Manes, D. Tricoli and E. Vitale. 1990. Fluorescence
parameters measured concurrently with net photosynthesis to investigate chloroplastic CO2 concentration in leaves of
Quercus ilex L. J. Plant Physiol. 136:538–543.
Dreyer, E., X. Le Roux, P. Montpied, F.A. Daudet and F. Masson.
2001. Temperature response of leaf photosynthetic capacity in
seedlings from seven temperate tree species. Tree Physiol. 21:
223–232.
Epron, D., D. Godard, G. Cornic and B. Genty. 1995. Limitation of
net CO2 assimilation rate by internal resistances to CO2 transfer in
the leaves of two tree species (Fagus sylvatica L. and Castanea
sativa Mill.). Plant Cell Environ. 18:43–51.
Ethier, G.J. and N.J. Livingston. 2004. On the need to incorporate sensitivity to CO2 transfer conductance into the Farquhar-von Caemmerer-Berry leaf photosynthesis model. Plant Cell Environ. 27:
137–153.
Ethier, G.J., N.J. Livingston, D.L. Harrison, T.A. Black and
J.A. Moran. 2006. Low stomatal and internal conductance to CO2
versus Rubisco deactivation as determinants of the photosynthetic
decline of ageing evergreen leaves. Plant Cell Environ. 29:
2168–2184.
Evans, J.R., T.D. Sharkey, J.A. Berry and G.D. Farquhar. 1986. Carbon isotope discrimination measured concurrently with gas exchange to investigate CO2 diffusion in leaves of higher plants. Aust.
J. Plant Physiol. 13:281–292.
Farquhar, G.D. and T.D. Sharkey. 1982. Stomatal conductance and
photosynthesis. Annu. Rev. Plant. Physiol. 33:317–345.
Farquhar, G.D., S. von Caemmerer and J.A. Berry. 1980. A biochemical model of photosynthetic CO2 assimilation in leaves of C3 species. Planta 149:78–90.
Ferrar, P.J., R.O. Slatyer and J.A. Vranjic. 1989. Photosynthetic temperature acclimation in Eucalyptus species from diverse habitats,
and a comparison with Nerium oleander. Aust. J. Plant Physiol.
16:199–217.
Flexas, J., M. Ribas-Carbo, D.T. Hanson, J. Bota, B. Otto, J. Cifre,
N. McDowell, H. Medrano and R. Kaldenhoff. 2006. Tobacco
aquaporin NtAQP1 is involved in mesophyll conductance to CO2 in
vivo. Plant J. 48:427–439.
TREE PHYSIOLOGY VOLUME 28, 2008
TEMPERATURE RESPONSE OF INTERNAL CONDUCTANCE TO CO 2
Flexas, J., A. Diaz-Espejo, J. Galmés, R. Kaldenhoff, H. Medrano and
M. Ribas-Carbo. 2007. Rapid variations of mesophyll conductance
in response to changes in CO2 concentration around leaves. Plant
Cell Environ. 30:1284–1298.
Gaastra, P. 1959. Photosynthesis of crop plants as influenced by light,
carbon dioxide, temperature and stomatal diffusion resistance. Meded. Landbouwhogesch. Wageningen, The Netherlands 59:1–68.
Genty, B., J.M. Briantais and N.R. Baker. 1989. The relationship between the quantum yield of photosynthetic electron transport and
quenching of chlorophyll fluorescence. Biochim. Biophys. Acta
990:87–92.
Hanba, Y.T., M. Shibasaka, Y. Hayashi, T. Hayakawa, K. Kasamo,
I. Terashima and M. Katsuhara. 2004. Overexpression of the barley
aquaporin HvPIP2:1 increases internal CO2 conductance and CO2
assimilation in the leaves of transgenic rice plants. Plant Cell
Physiol. 45:521–529.
Harley, P.C. and J.D. Tenhunen. 1991. Modeling the photosynthesis
response of C3 leaves to environmental factors. In Modeling Crop
Photosynthesis—from Biochemistry to Canopy. Vol. 19. American
Society of Agronomy and Crop Science Society of America. Madison, WI, pp 17–39.
Harwood, C.E. 1980. Frost resistance of subalpine Eucalyptus species. 1. Experiments using a radiation frost room. Aust. J. Bot.
28:587–599.
Laisk, A.K. 1977. Kinetics of photosynthesis and photorespiration in
C3 plants. Nauka Publishing, Moscow, 198 p.
Leuning, R. 1997. Scaling to a common temperature improves the
correlation between the photosynthesis parameters Jmax and Vcmax.
J. Exp. Bot. 48:345–347.
Lloyd, J., J.P. Syvertsen, P.E. Kriedemann and G.D. Farquhar. 1992.
Low conductances for CO2 diffusion from stomata to the sites of
carboxylation in leaves of woody species. Plant Cell Environ.
15:873–899.
Loreto, F., P.C. Harley, G. Dimarco and T.D. Sharkey. 1992. Estimation of mesophyll conductance to CO2 flux by three different methods. Plant Physiol. 98:1437–1443.
Makino, A., H. Sakashita, J. Hidema, T. Mae, K. Ojima and B. Osmond. 1992. Distinctive responses of ribulose-1,5-bisphosphate
carboxylase and carbonic anhydrase in wheat leaves to nitrogen nutrition and their possible relationships to CO2 transfer resistance.
Plant Physiol. 100:1737–1743.
Medlyn, B.E., E. Dreyer, D. Ellsworth et al. 2002. Temperature response of parameters of a biochemically based model of photosynthesis. II. A review of experimental data. Plant Cell Environ.
25:1167–1179.
Mooney, H.A., O. Bjorkman and G.J. Collatz. 1978. Photosynthetic
acclimation to temperature in the desert shrub, Larrea divaricata.
1. Carbon dioxide exchange characteristics of intact leaves. Plant
Physiol. 61:406–410.
Parkhurst, D.F. and K.A. Mott. 1990. Intercellular diffusion limits to
CO2 uptake in leaves. Plant Physiol. 94:1024–1032.
Pons, T.L. and R.A.M. Welschen. 2003. Midday depression of net
photosynthesis in the tropical rain forest tree Eperua grandiflora:
contributions of stomatal and internal conductances, respiration
and Rubisco functioning. Tree Physiol. 23:937–947.
19
Price, G.D., S. von Caemmerer, J.R. Evans et al. 1994. Specific reduction of chloroplast carbonic anhydrase activity by antisense RNA
in transgenic tobacco plants has a minor effect on photosynthetic
CO2 assimilation. Planta 193:331–340.
Sharpe, P.J.H. and D.W. Demichele. 1977. Reaction kinetics of poikilotherm development. J. Theor. Biol. 64:649–670.
Slatyer, R.O. 1977. Altitudinal variation in photosynthetic characteristics of snow gum, Eucalyptus pauciflora Sieb. ex Spreng. 3. Temperature response of material grown in contrasting thermal
environments. Aust. J. Plant Physiol. 4:301–312.
Slatyer, R.O. and P.J. Ferrar. 1977. Altitudinal variation in photosynthetic characteristics of snow gum, Eucalyptus pauciflora Sieb.
ex Spreng. 2. Effects of growth temperature under controlled conditions. Aust. J. Plant Physiol. 4:289–299.
Tamimi, A., E.B. Rinker and O.C. Sandall. 1994. Diffusion coefficients for hydrogen sulfide, carbon dioxide and nitrous oxide in
water over the temperature range 293–368 K. J. Chem Engineer.
Data 39:330–332.
Tausz, M., C.R. Warren and M.A. Adams. 2005. Dynamic light use
and protection from excess light in upper canopy and coppice
leaves of Nothofagus cunninghamii in an old growth, cool temperate rain forest in Victoria, Australia. New Phytol. 165:143–155.
Uehlein, N., C. Lovisolo, F. Siefritz and R. Kaldenhoff. 2003. The tobacco aquaporin NtAQP1 is a membrane CO2 pore with physiological functions. Nature 425:734–737.
von Caemmerer, S. 2000. Biochemical models of leaf photosynthesis.
In Techniques in Plant Sciences. CSIRO Publishing, Collingwood,
Australia, 165 p.
von Caemmerer, S., J.R. Evans, G.S. Hudson and T.J. Andrews. 1994.
The kinetics of ribulose-1,5-bisphosphate carboxylase /oxygenase
in vivo inferred from measurements of photosynthesis in leaves of
transgenic tobacco. Planta 195:88–97.
Warren, C.R. 2006. Estimating the internal conductance to CO2
movement. Funct. Plant Biol. 33:431–442.
Warren, C.R. and E. Dreyer. 2006. Temperature response of photosynthesis and internal conductance to CO2: results from two independent approaches. J. Exp. Bot. 57:3057–3067.
Warren, C.R., M.J. Hovenden, N.J. Davidson and C.L. Beadle. 1998.
Cold hardening reduces photoinhibition of Eucalypts nitens and
E. pauciflora at frost temperatures. Oecologia 113:350–359.
Warren, C.R., G.J. Ethier, N.J. Livingston, N.J. Grant, D.H. Turpin,
D.L. Harrison and T.A. Black. 2003a. Transfer conductance in second growth Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco)
canopies. Plant Cell Environ. 26:1215–1227.
Warren, C.R., E. Dreyer and M.A. Adams. 2003b. Photosynthesis–
Rubisco relationships in foliage of Pinus sylvestris in response to
nitrogen supply and the proposed role of Rubisco and amino acids
as nitrogen stores. Trees 17:359–366.
Warren, C.R., N.J. Livingston and D.H. Turpin. 2004. Water stress decreases the transfer conductance of Douglas-fir (Pseudotsuga menziesii) seedlings. Tree Physiol. 24:971–979.
Yamori, W., K. Noguchi, Y.T. Hanba and I. Terashima. 2006. Effects
of internal conductance on the temperature dependence of the
photosynthetic rate in spinach leaves from contrasting growth temperatures. Plant Cell Physiol. 47:1069–1080.
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com