1

Growth in elevated CO2 enhances temperature response of photosynthesis in

wheat

Aitor Alonso, Pilar Pérez and Rafael Martínez-Carrasco*

Institute of Natural Resources and Agrobiology of Salamanca, CSIC, Apartado 257, E-

37071 Salamanca, Spain

*correspondence: Rafael Martínez-Carrasco. Fax: +34 923 219 609; E-mail:

rafael.mcarrasco@irnasa.csic.es

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Abstract

The temperature dependence of C3 photosynthesis may be altered by the growth

environment. The effects of long-term growth in elevated CO2 on photosynthesis

temperature response have been investigated in wheat (Triticum aestivum L.) grown in

controlled chambers with 370 or 700 µmol mol-1 CO2 from sowing through to anthesis.

Gas exchange was measured in flag leaves at ear emergence, and the parameters of a

biochemical photosynthesis model were determined along with their temperature

responses. Elevated CO2 slightly decreased the CO2 compensation point and increased

the rate of respiration in the light and Rubisco Vcmax, although the latter effect was

reversed at 15 ºC. With elevated CO2, Jmax decreased in the 15 ºC-25 ºC temperature

range and increased at 30 ºC and 35 ºC. The temperature response (activation energy) of

Vcmax and Jmax increased with growth in elevated CO2. CO2 enrichment decreased the

RuBP-limited photosynthesis rates at lower temperatures and increased Rubisco- and

RuBP-limited rates at higher temperatures. The results show that the photosynthesis

temperature response is enhanced by growth in elevated CO2. We conclude that if

temperature acclimation and factors such as nutrients or water availability do not

modify or negate this enhancement, the effects of future increases in air CO2 on

photosynthetic electron transport and Rubisco kinetics may improve the photosynthetic

response of wheat to global warming.

Abbreviations: A, net rate of CO2 uptake; Ac, Rubisco limited rate of photosynthesis; Aj

RuBP limited rate of photosynthesis; Cc chloroplastic CO2 concentration; Ci,

intercellular CO2 concentration; Γ*, CO2 compensation point in the absence of dark

respiration; H, activation energy; J, electron transport rate; Jmax, potential rate of

electron transport; Kc, Rubisco Michaelis constant for CO2; Ko, Rubisco Michaelis

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constant for O2; Q, photosynthetically active photon flux density; Q2, photosynthetically

active photon flux density absorbed by Photosystem II; θ, convexity parameter; R,

molar gas constant; Rd, mitochondrial respiration rate in the light; Tk, leaf absolute

temperature (K); τ, Rubisco specificity factor; Vcmax, maximum Rubisco rate of

carboxylation.

4

Introduction

Photosynthesis in C3 plants is affected by both short- and long-term changes in

atmospheric temperature and CO2 concentration. In C3 plants, carbon assimilation

increases with a rise in CO2 concentration, first, because the Km of the enzyme for CO2

is close to the current atmospheric concentration, so higher CO2 concentration increases

the velocity of carboxylation; and second, because CO2 competitively inhibits the

oxygenation reaction thus decreasing photorespiratory CO2 loss and increasing the

efficiency of net carbon uptake (Long et al. 2004). In the long term, the increase in

atmospheric CO2 predicted by climate change decreases Rubisco activity and amount,

often associated with decreased transcripts for the enzyme small subunit (Drake et al.

1997, Moore et al. 1999), although other studies show it is due to a lower nitrogen

status (Riviere-Rolland et al. 1996, Nakano et al. 1997, Farage et al. 1998, Geiger et al.

1999, Pérez et al. 2005). We have found additional effects of prolonged growth in

elevated CO2, such as a decrease in Rubisco specific activity with a high nitrogen

supply, which is probably due to increased levels of day-time Rubisco inhibitors (Pérez

et al. 2005). Few studies have addressed the changes in temperature dependence of the

maximum rate of carboxylation by Rubisco (Vcmax) and the potential rate of electron

transport (Jmax) under elevated CO2 concentrations. Regarding rice, there were increases

in both the absolute value of, and the temperature optimum for, the light-saturated

photosynthetic rate at growth CO2 caused by elevated CO2 in FACE conditions;

however, the temperature dependence (activation energy) of Vcmax was not affected by

growth CO2 (Borjigidai et al. 2006). In contrast, growth in field chambers under

elevated CO2 increased the temperature response of Rubisco-limited photosynthesis and

of both in vitro and in vivo Vcmax (Alonso et al. 2008). If a negative relationship exists

between Vcmax and Rubisco specificity for CO2 relative to O2 (τ; Spreitzer and Salvucci

5

2002, Zhu et al. 2004), this would then imply that elevated CO2 leads to a sharper

decrease in τ with temperature (Long 1991). However, further research is required,

since Rubisco might not strictly adhere to this assumed inverse relationship (Zhu et al.

2004).

Photosynthetic electron transport in loblolly pine trees decreases only with elevated

CO2 when low temperatures inhibit translocation (Hymus et al. 1999). There was a

significant decrease (20%) in photosynthetic electron transport (Hymus et al. 2001) in

nitrogen-deficient conditions, at which elevated CO2 induced a 42% decrease in Vcmax.

This decrease compares with an average reduction of 13% with growth in free air CO2

enrichment facilities (Long et al. 2004). In contrast, these authors found increases in

electron transport at elevated CO2 with a high nitrogen supply. Along with Rubisco

activity, elevated CO2 has also been observed to decrease Ribulose-1, 5-bisphosphate

(RuBP) regeneration in forest species (Medlyn et al. 1999). The potential rate of

electron transport (Jmax) increases exponentially with temperature, with deactivation

being observed beyond a point which may vary with growth temperature (Yamasaki et

al. 2002, Bernacchi et al. 2003). Except for one observation in which the activation

energy of Jmax is no different for leaves grown in ambient and elevated CO2 in rice

(Borjigidai et al. 2006), it is not known whether Jmax temperature response is modified

by elevated growth CO2.

The aim of this study was to investigate the effects of growth from germination at

twice the present atmospheric CO2 on photosynthesis temperature response in wheat,

using gas exchange analysis. We intended to gain further evidence that elevated growth

CO2 increases the activation energy of Vcmax (Alonso et al. 2008) and to explore

whether, in association with this, the inverse change occurs in τ. In addition,

modifications with growth CO2 in temperature responses of respiration in the light and

6

Jmax were investigated. Finally, a biochemical photosynthesis model (Farquhar et al.

1980) was used to assess the temperature dependence of Rubisco- and RuBP

regeneration-limited photosynthesis as a function of growth CO2.

7

Materials and Methods

Plant material

Spring wheat (Triticum aestivum L. cv. Alcalá) was sown in 16 l pots (35 seeds per

pot) containing perlite, and placed in a growth chamber with 350 µmol m-2 s-1 photon

flux density (fluorescent and incandescent lamps) with a 14-h photoperiod, 20 ºC/10 ºC

day/night temperatures and 60% relative humidity. The pots were watered twice a week,

being fed once a week with a nutrient solution (Israel et al. 1990) containing 4 mM

CaCl2, 1.5 mM MgSO4, 0.4 mM HNa2PO4, 0.94 mM H2NaPO4, 10.1 mM KNO3, 0.1

mM iron citrate and micronutrients (Hewitt 1966): 0.5 mM Mn SO4, 0.1 mM Cu SO4,

0.1 mM ZnSO4, 5.0 mM H3BO3, 10.0 mM NaCl and 0.05 mM Na2MoO4. The same

growth chamber was used for two successive crops so as to minimize chamber effects.

One of the crops was grown at ambient (370 µmol mol-1) CO2 and the other at elevated

(700 µmol mol-1) CO2 throughout the experiment. Elevated CO2 was obtained by

injecting pure gas mixed with the chamber air by fans. The CO2 concentration was

continuously monitored by an infrared gas analyzer which signal was transferred into a

proportional, integral, derivative controller regulating the opening time of the CO2

solenoid feed valve. As the chambers were in an isolated building with restricted access,

actual CO2 concentrations showed little variation and were very close to set values.

Photosynthetic parameters and temperature dependence functions

Farquhar et al. (1980) proposed that the rate of net photosynthesis is the minimum of

two factors, the Rubisco-limited rate of photosynthesis (Ac), and the RuBP-

regeneration-limited rate of photosynthesis (Aj):

A= min (Ac, Aj)-Rd (1)

where Rd is mitochondrial respiration in the light.

8

Using the CO2 concentration in the chloroplast (Cc) rather than the intercellular CO2

concentration (Ci), the Rubisco-limited photosynthesis is given by:

)KO(1KCCV)C*Γ(1A

occ

ccmaxcc

++−= (2)

where Vcmax is the maximum rate of carboxylation by Rubisco, Γ* is the CO2

compensation point in the absence of dark respiration, Kc and Ko are the Rubisco

Michaelis constants for CO2 and O2, respectively, and O is oxygen concentration. The

term (1-Γ*/Cc) accounts for the CO2 released through photorespiration. In turn, Γ* is

related to the Rubisco specificity for CO2 relative to O2 (τ):

τ=

O5.0*Γ (3)

The RuBP-limited photosynthesis rate is:

*10.5Γ4.5CJC)/C*Γ(1Aj

c

cc

+−= (4)

where J is the rate of electron transport. J is related to the absorbed irradiance, Q2, by:

θJ2-(Q2+Jmax)J+Q2 Jmax=0 (5)

with solution for J:

θ2JQθ4)JQ()JQ(

J max22

max2max2 −+−+= (6)

where θ is the curvature of the light response curve (taken to be 0.88), and Jmax the

maximum rate of electron transport. Q2 is related to incident irradiance Q:

2)f1(QabsQ2

−= (7)

where abs is the absorptance of leaves (assumed to be 0.8) and f (assumed to be 0.25)

corrects for the spectral quality of light (Humphries and Long 1995).

The temperature responses of the parameters Γ*, Rd, Vcmax and Jmax adjusted to a

reference temperature of 25 ºC are described by the Arrhenius function (von Caemmerer

9

2000; Medlyn et al. 2002):

⎟⎠⎞

⎜⎝⎛ −

=k

k25 R298T

298)H(Texp ParameterParameter (8)

where Parameter25 is the parameter value at 25 ºC, H is the activation energy, R the

molar gas constant (8.314 J mol-1 K-1) and Tk the leaf absolute temperature.

Gas exchange measurements and parameter estimations

Gas exchange measurements at differing CO2 concentrations and temperatures were

used to solve the photosynthesis model parameters and their temperature dependencies.

At ear emergence, six flag leaves from each growth CO2 concentration were measured

between 2 and 8 h after the start of photoperiod. Measurements were performed outside

the growth chamber, in a glasshouse under natural light and ambient air. Gas exchange

was recorded with an infrared gas analyzer (CIRAS-2, PP Systems, Hitchin, Herts., UK)

with differential operation in an open system, using a 1.7 cm2-window leaf chamber,

300 ml min-1 air flow rate, and 1500 µmol m-2 s-1 irradiance, except when stated

otherwise. Leaf temperature was controlled by the analyzer’s Peltier system.

Gas exchange between leaf chambers and the surrounding air can lead to erroneous

estimations of A and Ci (Jahnke and Krewitt 2002, Pons and Welschen 2002, Long and

Bernacchi 2003, Flexas et al. 2007b, Rodeghiero et al. 2007). A comparison of

homobaric and heterobaric plant species has shown that leakage between the leaf and

gasket surfaces is much larger than possible leakage through the leaf mesophyll (Flexas

et al. 2007b). Estimations of A and Ci were corrected for chamber leakage by using a

photosynthetically inactive leaf, as described by Flexas et al. (2007b). A leaf was killed

by immersion in boiling water, blotted and then enclosed in the assimilation chamber.

Apparent photosynthesis and transpiration were recorded at all measurement

temperatures following the same protocol used for A/Ci curves (see below).

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Temperature did not modify the recordings obtained. Partially wet dead leaves gave

significant apparent transpiration, as expected (Fig. 1A); however, when the dead leaf

was dry, water leakage (Fig. 1 B) was only small, in contrast with Rodeghiero et al.

(2007), and of no consequence for A and Ci determinations. For example, an apparent

transpiration rate of 0.2 mmol m-2 s-1 at an air CO2 concentration of 115 µmol mol-1

(Fig. 1 B) would cause relative changes of -1.0% in A and 1.6% in Ci; with 0.1 mmol m-

2 s-1 apparent transpiration rate at 12 µmol mol-1 CO2, the respective figures would be

0.1% and -1.5%. Moreover, CO2 response was similar with both wet and dry dead

leaves. Consequently, we made no correction for water diffusion. CO2 diffusion through

the gasket of the leaf chamber was dependent both on air CO2 concentration and the

order in which this was changed. Thus, on decreasing CO2 from 115 to 12 µmol mol-1

CO2, inward diffusion progressively decreased (Fig. 1C, inset), and on increasing air

CO2 concentration from 370 to 1800 µmol mol-1, outward diffusion increased (Fig 1C).

This was probably due to the change in CO2 gradient between the chamber and external

air. Leakage yielded apparent CO2 exchange rates from -0.96 µmol m-2 s-1 (with 115

µmol mol-1 CO2) to 1.8 µmol m-2 s-1 (with 1800 µmol mol-1 CO2). CO2 exchange of

living leaves was corrected by subtraction of the regressions of apparent photosynthesis

of dead leaves over air CO2 concentration. Ci values were then recalculated using the

manufacturer’s formulae (Flexas et al. 2007b).

From the corrected A/Ci curves, Cc (Ci - A/gm) was estimated using the mesophyll

conductance (gm) determined by nonlinear curve fitting of equations 2 and 4,

minimizing the sum of squared model deviations from the data (Sharkey et al. 2007;

http://www.blackwellpublishing.com/plantsci/pcecalculation/). An iterative, least

squares analysis procedure was followed to first determine Cc and gm with Rd and Γ*

values taken from Sharkey et al. (2007), then to convert the Ci-based Γ* values obtained

11

in this study (see below) into Cc-based Γ* (measured Rd is the same with Ci and Cc), and

finally re-calculate Cc with the Rd and Γ* values obtained. This assumes a constant gm

for the entire range of Ci, whereas Flexas et al. (2007a) have shown that gm declines

with increasing Ci. According to these authors, however, the use of a single gm value has

negligible effects on the parameterization of Vcmax and Jmax, except when gm is largely

reduced (e.g., under severe water stress).

At 5 ºC intervals from 15 to 35 ºC for leaf temperature, gas exchange was

successively measured with approximately 115, 90, 60, 24 and 12 µmol mol-1 air CO2

concentration, at two (200 and 1500 µmol m-2 s-1) rather than three light intensities, to

determine Rd and Γ* following the Laisk procedure (1977). Choosing more light

intensities would limit the accuracy of the Rd and Γ* assessment. The Laisk procedure

assumes that while photorespiration closely depends on light intensity, Rd does not

change with irradiance. Hence, the intersection of the A/Cc response lines at the two

light intensities represents Γ* when extrapolated to the Cc axis, and Rd when

extrapolated to the A axis (Laisk 1977, Bernacchi et al. 2001). Atkin et al. (2000) found

that Rd was inhibited by low irradiance at moderate-to-high temperatures (15°C–30°C),

however, higher irradiance had little additional effect on respiration at these

temperatures. In this work, therefore, the Laisk procedure was adopted and Γ* and the

Rd values obtained were used in model calculations. For this and subsequent

measurements, gas exchange parameters were recorded as soon as inlet air CO2

concentration was stable, but not necessarily steady-state (Long and Bernacchi 2003).

The maximum rate of carboxylation by Rubisco, Vcmax, at each measurement

temperature, was obtained from the response of A to Cc at low CO2 concentrations (12

to 115 µmol mol -1 CO2 in external air) and 1500 µmol m-2 s-1 irradiance by substituting

f for (Cc-Γ*)/(Cc +Kc (1+O/Ko)) in equation (2):

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A=Vcmax· f – Rd (9)

and solving Vcmax as the slope of a linear function of f (Long and Bernacchi 2003).

Although it is satisfactory, the non-linear curve fitting of equation (2) with the utility

proposed by Sharkey et al. (2007) offers less precision than fitting a straight line. The

Cc-based Kc and Ko and the parameters describing their dependence on temperature

were taken from Bernacchi et al. (2002):

⎟⎠⎞

⎜⎝⎛

⋅⋅−

⋅=k

kc

298)298(99.80exp38.272K

TRT

(10)

⎟⎠⎞

⎜⎝⎛

⋅⋅−

⋅=k

ko

298)298(72.23exp82.165K

TRT

(11)

At each temperature, air CO2 concentration was subsequently increased from 12

µmol mol-1 to 370, 550, 700, 1100, 1500 and 1800 µmol mol-1 at 1500 µmol m-2 s-1

irradiance. From the A/Cc response curve at each measurement temperature, the light

and CO2 saturated photosynthesis rate, Amax, was estimated with the aid of

Photosynthesis Assistant software (http:// www.scientific.force9.co.uk) by fitting the

rectangular hyperbola:

RACCEACCE

maxc

maxc−

+⋅⋅⋅

=A (12)

where CE is carboxylation efficiency (the initial slope of the A/Cc response curve,

representing the increase in photosynthetic rate achieved per unit increase in CO2) and

R is respiratory processes (dark and light). Amax was used to determine Jmax by

rearranging equation (4):

*ΓC)*5.10)(4.5CR(AJ

c

cdmaxmax

−Γ++

= (13)

With the values for Rd, Γ*, Vcmax and Jmax obtained at each temperature, the

corresponding values at 25 ºC and activation energies were obtained by fitting equation

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(8).

Statistical analyses

Photosynthesis parameters and their temperature dependence functions were

separately calculated for each leaf. The linear regressions for Γ∗, Rd and Vcmax (equation

9) estimations were fitted using an Excel (Microsoft) spreadsheet, and the temperature

dependence functions (equation 8) of the photosynthesis parameters were fitted by

nonlinear regression with GenStat 6.2 statistical software. The data from separate

calculations in each leaf were compared for growth CO2 effects in either a two-way

analysis (temperature and CO2; photosynthesis parameters, see Table 1) or one-way

analysis (CO2; temperature dependence parameters, see Table 2) of variance with no

blocking using GenStat 6.2.

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Results

Figure 2 shows the estimations of Rd and Γ* based on A/Cc responses at two light

intensities and at the two extreme measurement temperatures in a particular leaf. The

figure points to an increase with temperature in both parameters, as presented in Table

1. Growth in elevated CO2 increased respiration in the light (Table 1). The CO2

compensation point (Table 1) was significantly, albeit slightly, lower (3.8% on average)

in elevated as opposed to ambient CO2. Raising temperatures increased Γ* similarly in

plants grown in ambient and elevated CO2. However, the magnitude of CO2 diffusion

leaks seriously affected the accuracy of Rd and Γ* estimates.

Vcmax was increased by warmer temperatures and by elevated CO2 (between 17% and

32%), although the latter effect was reversed at 15 ºC (Table 1). The CO2 x temperature

interaction indicated that growth in elevated CO2 enhanced the Vcmax temperature

response. Jmax and the light- and CO2-saturated photosynthesis rate (Amax) also increased

with temperature (Table 1). Relative to ambient CO2, elevated CO2 tended to decrease

these parameters in the 15 ºC-25 ºC temperature range (between -9% and -1%) and

significantly increased them at 30 ºC and 35 ºC (between 12% and 18%); the increase

with temperature in Jmax and Amax was, therefore, higher in elevated as opposed to

ambient CO2. The Jmax/Vcmax ratio decreased with temperature. Elevated CO2 increased

this ratio at 15 ºC (7%) and decreased it at higher temperatures (between -23% and -

10%); consequently, elevated CO2 enhanced the decrease with temperature in

Jmax/Vcmax.

The temperature responses of photosynthesis parameters in plants grown in ambient

and elevated CO2 were examined with Arrhenius functions yielding the activation

energies and values for each parameter at a reference temperature of 25 ºC (Fig. 3). The

fitted 25 ºC values of respiration in the light were higher, and those of the CO2

15

compensation point were similar, in elevated as opposed to ambient CO2 (Table 2).

Vcmax at 25 ºC was higher in elevated as opposed to ambient CO2, while Jmax and Amax at

25 ºC did not significantly differ between CO2 concentrations. The slopes of the

Arrhenius functions (activation energies, H) for Rd and Γ* showed no significant

differences between CO2 concentrations (Table 2). At variance to this, the activation

energies corresponding to Vcmax, Jmax and Amax where higher in elevated as opposed to

ambient CO2, indicating that CO2 enrichment increased the temperature response of

these parameters.

The calculated photosynthesis parameters were incorporated in the Farquhar et al.

(1980) photosynthesis model to examine the effect of growth CO2 concentration on

photosynthesis responses to chloroplastic CO2 and temperature (Fig. 4). Modeled curves

fitted well to experimental data, except for those values - at certain temperatures -

corresponding to the measurement at 370 µmol mol-1 air CO2. A change in Rubisco

activation state upon the sudden increase in CO2 concentration from 12 to 370 µmol

mol-1 CO2 could account for the divergence between model and measurement. The

transition from Rubisco-limited – at lower CO2 concentrations – to RuBP-limited

photosynthesis occurred at higher intercellular CO2 concentrations (275-375 µmol mol-

1) in plants grown in ambient as opposed to elevated CO2 (200-320 µmol mol-1 CO2)

and increased with temperature. Growth in elevated CO2 changed only slightly the CO2

response of Rubisco-limited photosynthesis rates at 15 ºC and increased it at warmer

temperatures. In turn, elevated CO2 decreased RuBP-limited photosynthesis response to

chloroplastic CO2 concentration in the 15-25 ºC range, but increased it with higher

temperatures.

To model the response of photosynthesis to temperature, measured Cc values were

used. As these varied with temperature and CO2 concentration used for measurement,

16

non-smooth curves were obtained (Fig. 5). Photosynthesis response to temperature was

negative at low air CO2 concentrations (Fig. 5) and became progressively positive as

CO2 increased, as may be expected from the increase in photorespiration with

temperature and the gradual inhibition of this process as CO2 increases (Long, 1991). A

steep increase in photosynthesis from 15 ºC to 30-35 ºC was observed at 1800 µmol

mol-1 air CO2 concentration (Fig. 5). Growth CO2 had little effect on photosynthesis

temperature response in the lower range of CO2 concentrations. At high Cc,

photosynthesis in elevated growth CO2 was lower at 15-25 ºC, and higher at 30-35 ºC,

than in ambient growth CO2, implying an enhanced photosynthesis response to

temperature in plants grown in elevated CO2; moreover, at 1800 µmol mol-1 Cc,

photosynthesis peaked at about 30 ºC in ambient CO2 plants, but at higher temperatures

in their elevated CO2 counterparts.

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Discussion

Measurements of the CO2 dependence of photosynthesis indicate that long-term

exposure to elevated CO2 increased Vcmax, in contrast to many preceding studies

reporting a down-regulation of Rubisco amount and activity (Riviere-Rolland et al.

1996, Nakano et al. 1997, Drake et al. 1997, Farage et al. 1998, Geiger et al. 1999,

Moore et al. 1999, Long et al. 2004, Pérez et al. 2005). The limited stimulation of

photosynthesis at elevated CO2 is nearly always associated with the inability of plants to

acquire sufficient nitrogen, resulting in the commonly observed reduction in leaf

nitrogen content in plants grown under CO2 enrichment (Rogers et al. 2006).

Unfortunately, we did not analyze leaf nitrogen content, although growth in large pots

(Curtis 1996) with frequent and ample nutrient supply (Geiger et al. 1999) may have

prevented the nitrogen and Rubisco decrease in elevated CO2. In contrast to this, Jmax at

25 ºC and lower temperatures tended to decrease with growth under elevated CO2. A

decline in RuBP regeneration capacity in elevated CO2 has been observed in two out of

five C3 species examined (Sage et al. 1989) and in a meta-analytical study of forest

species, in which Vcmax and leaf nitrogen concentration declined (Medlyn et al. 1999).

As the photosynthetic response to CO2 concentrations in the chloroplast (i.e. A-Cc

curves) is significantly less affected by leakage in the IRGA leaf chamber than A-Ci

curves (Flexas et al. 2007), and a correction for these leaks was applied, our

photosynthetic parameterization can be considered reasonably accurate.

Growth in elevated CO2 involved adjustments in both photosynthesis rate and

temperature response. Here we show that exposure to elevated CO2 enhances

photosynthesis temperature response measured at the same CO2 concentrations, which

indicates it is an acclimatory response. In the following this acclimation is discussed

based on changes of the photosynthetic parameters derived from the model by Farquhar

18

et al. (1980). First, growth in elevated CO2 increased net photosynthesis temperature

response (equation 1) despite an increase in respiration in the light, in agreement with

Davey et al. (2004), and in contrast to the inhibition of respiration which has been

reported (for a review, see Drake et al. 1999, Wang and Curtis 2002). This respiration

enhancement in elevated growth CO2 was apparent despite limitations in day respiration

estimates caused by diffusion leaks in the IRGA leaf chamber (Flexas et al. 2007b). An

increase in respiration may be due to the higher leaf soluble carbohydrate content

usually found (Moore et al., 1999, Pérez et al. 2005) under elevated CO2. Indeed, Stitt

et al. (1999) have shown that an increased level of carbohydrate leads to an increased

level of glycolytic enzymes and, thence, to a stimulation of respiration. The increase in

Vcmax in elevated CO2 may be counterbalanced by increased respiration rates, resulting

in limited enhancement of Rubisco-limited photosynthesis.

Second, elevated CO2 altered RuBP-limited photosynthesis temperature response

through an increase in Jmax activation energy. The temperature dependence of electron

transport at the plastoquinone and water-oxidation sites is shown to be influenced by

growth temperature by the temperature at which the leaves developed (Yamasaki et al.

2002). Whether growth in elevated CO2 affects electron transport temperature

dependence at the same sites is worth investigating. Niinemets et al. (1999) have

suggested that a lower Jmax activation energy is associated with decreases in membrane

fluidity due to increased lipid saturation. However, elevated CO2 has been observed to

increase lipid saturation (Huang et al. 1999), and yet it increased the activation energy

for Jmax. Clearly, more work is needed to understand the reasons for this increased

temperature response.

Third, we have found that the known increase with temperature in Vcmax (von

Caemmerer, 2000) is enhanced by growth under air CO2 enrichment. This is consistent

19

with the in vivo and in vitro temperature responses of Rubisco activity in plants grown

in field chambers (Alonso et al. 2008). Deactivation of Rubisco activase may lead to

decreased Rubisco activation at high temperatures, although this is more likely at

temperatures higher than those examined in this study (Crafts-Brandner and Salvucci,

2000) and in plants acclimated to low than to high temperature (Way and Sage 2008).

Increases in Vcmax with temperature have been accounted for by the strong temperature

dependence of the catalytic opening and closure of the Rubisco active site (Zhu et al.

1998). Moreover, it has been suggested that higher temperatures loosen the catalytic site

of Rubisco thus facilitating the release of inhibitors (Schrader et al. 2006). Further

research is required to verify whether growth in elevated CO2 increases Vcmax

temperature response through an altered flexibility of the Rubisco active site’s outer

parts or the easier release of inhibitors.

Fourth, an inverse relationship between Rubisco rate of catalysis and specificity for

CO2 relative to O2 has been suggested (Zhu et al. 2004, Mitchell et al. 2005, Spreitzer et

al. 2005), as well as an association between increased catalysis and decreased affinity

(increased Km) for CO2 (Sage 2002). Contrary to this, and although inaccuracies in Rd

determination arising from chamber leaks (see above) also apply to gas exchange-based

Γ* measurements, a remarkable finding in our study is that the increase in Vcmax and its

temperature response under elevated growth CO2 was associated with slightly decreased

Γ* and thus with increased τ. Significant variability has been found in τ among closely

related C3 species; this variability was related to environmental factors, with a

comparatively high specificity factor being associated with an increase in the activation

energy of the transition state intermediates for oxygenation relative to carboxylation

(Galmés et al. 2005). The relationship between τ and Vcmax (τ = Vcmax Ko/Vomax Kc,

where Vomax is the maximum rate of oxygenation) suggests that the increase in � may

20

simply be a consequence of the increase in Vcmax with temperature, but we do not know

whether growth in elevated CO2 also changed other Rubisco kinetic parameters (Kc, Ko)

that were not estimated in the present study.

The temperature dependence of carboxylation by Rubisco is thought to be stable

among C3 plants (Badger et al. 1982, Brooks & Farquhar 1985). Our study indicates

that, within the same species, Vcmax shows variations in temperature dependence

induced by growth CO2 concentration. Similarly, changes in the temperature

dependence of the electron transport capacity in response to growth temperature have

been reported for a number of species (Yamori et al. 2005) including wheat (Yamasaki

et al. 2002). A conclusion of our work is that atmospheric CO2 can also cause these

changes. Rather than an alteration in the balance between RuBP regeneration and RuBP

carboxylation (Farquhar and von Caemmerer 1982), changes in the temperature

dependencies of Jmax and Vcmax could modify the temperature response of photosynthesis

in elevated growth CO2.

This study shows that growth in elevated CO2 has important additional effects on

electron transport capacity and on Rubisco kinetics that can improve photosynthesis

temperature response. If temperature acclimation and factors such as nutrient or water

availability do not modify or negate these effects, future increases in atmospheric CO2

may therefore positively affect photosynthesis adjustment to global warming in wheat.

Additionally, considerable research has been devoted to improving Rubisco properties

such as specificity (Chen and Spreitzer 1992, Zhu et al. 1998, Schlitter and Wildner

2000). Understanding the effects of elevated growth CO2 on these properties can offer

useful keys for this objective.

21

Acknowledgments

This work has been funded by the Spanish National Research and Development

Programme (Project BFI2003-01277). A. Alonso was the recipient of an I3P pre-

doctoral fellowship from the CSIC-European Social Fund.

22

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Table 1. Effects of growth CO2 and measurement temperature on photosynthesis parameters (± standard error of mean) in flag leaves of wheat. Parameters were derived from photosynthesis-chloroplastic CO2 responses. Units are µmol mol-1 (Γ*) or µmol m-2 s-1 (other parameters). Growth

CO2 Measurement temperature, ºC

Probability

15 20 25 30 35 CO2 Temp. CO2 x Temp.

Rd Ambient 0.3 (0.03) 0.5 (0.2) 0.0 (0.3) 0.8 (0.3) 1.6 (0.1) <.001 <.001 0.08 Elevated 0. 3 (0.1) 0.8 (0.1) 0.3 (0.2) 1.8 (0.1) 2.1 (0.1) Γ* Ambient 23.7 (1.0) 26.8 (0.8) 32.0 (0.6) 37.2 (0.5) 42.3 (0.6) 0.05 <.001 0.84 Elevated 22.3 (1.6) 26.8 (1.3) 31.1 (1.0) 35.3 (1.0) 40.3 (0.9) Vcmax Ambient 44.8 (1.6) 53.8 (2.4) 93.2 (5.3) 125.5 (5.1) 183.0 (7.7) <.001 <.001 0.002 Elevated 40.0 (3.0) 68.3 (1.9) 108.7 (12.1) 159.2 (6.3) 240.8 (15.1) Jmax Ambient 158.8 (2.9) 204.2 (7.0) 258.4 (6.1) 290.6 (7.6) 312.5 (11.0) 0.08 <.001 <.001 Elevated 151.6 (5.1) 199.8 (6.9) 235.0 (11.5) 326.2 (11.1) 368.2 (19.7) Jmax/Vcmax Ambient 3.6 (0.1) 3.8 (0.2) 2.8 (0.2) 2.4 (0.2) 1.7 (0.05) 0.004 <.001 0.01 Elevated 3.9 (0.2) 2.9 (0.1) 2.3 (0.3) 2.1 (0.07) 1.5 (0.08) Amax Ambient 33.9 (0.6) 43.23 (1.6) 53.4 (1.1) 59.9 (1.4) 64.0 (2.1) 0.05 <.001 <.001 Elevated 32.4 (1.1) 42.6 (1.5) 49.0 (2.4) 68.1 (2.2) 74.7 (3.4)

30

Table 2. Arrhenius model estimates (± standard error of mean) for the photosynthesis parameters temperature response in flag leaves of wheat grown in ambient (370 µmol mol-1) and elevated (700 µmol mol-1) CO2. P is the probability in the analysis of variance for effects of growth CO2.

Growth CO2 Value at 25 ºC H (kJ mol-1)

Rd Ambient 0.40 (0.11 ) 108 (23.0) (µmol m-2 s-1) Elevated 0.86 ( 0.13) 76 (11.6) P 0.02 0.25 Γ* Ambient 31.8 (0.54) 22.1 (1.67) (µmol mol-1) Elevated 30.7 (1.08) 21.4 (2.06) P 0.35 0.80 Vcmax Ambient 88.3 (1.08) 55.3 (2.52) (µmol m-2 s-1) Elevated 105.1 (5.30) 63.2 (2.78) P 0.01 0.06 Jmax Ambient 240.4 (5.00) 23.1 (9.02) (µmol m-2 s-1) Elevated 246.0 (1.38) 32.6 (1.18) P 0.60 >0.001 Amax Ambient 50.1 (0.96) 21.7 (1.31) (µmol m-2 s-1) Elevated 51.5 (1.83) 30.6 (0.87) P 0.5 >0.001

31

Figure legends

Fig. 1. The response of diffusion leak of water vapour (A, B) and CO2 (C) to air CO2

concentration (Ca) in a chamber containing a thermally killed leaf. The dead leaf was

partially wet in (A) and completely dry in (B).

Fig. 2. Photosynthesis chloroplastic CO2 response lines at 1500 (closed symbols) and

200 (open symbols) µmol m-2 s-1 irradiance used for calculations of Γ* and Rd at two

temperatures.

Fig 3. Arrhenius plots (Parameter=Parameter25·exp(H(Tk-298)/R·298·Tk)) for the

photosynthesis parameters determined in flag leaves of wheat grown in ambient (open

symbols and broken lines) and elevated (closed symbols and solid lines) CO2. H is the

activation energy (kJ mol-1) and Parameter25 the parameter value at 25 ºC (shown in

Table 2). The 25 ºC normalized x-axis is (Tk-298)/R·298·Tk, where R is the molar gas

constant and Tk the leaf absolute temperature. The parameters are: (A) respiration in the

light; (B) CO2 compensation point in the absence of dark respiration; (C) maximum

Rubisco-limited rate of carboxylation; (D) maximum rate of photosynthetic electron

transport; (E) light and CO2 saturated photosynthesis rate.

Fig. 4. Measured (symbols), modeled Rubisco-limited (thin lines), and modeled RuBP-

limited (thick lines) photosynthesis- chloroplastic CO2 (Cc) responses at several

temperatures in flag leaves of wheat grown in ambient (open symbols, dotted lines) and

elevated CO2 (closed symbols, solid lines). Insets show the initial parts of the A-Cc

responses. Each point is the mean ± standard error of six replicate leaves.

32

Fig. 5. Measured (symbols) and modeled (lines) photosynthesis temperature responses

at air CO2 concentrations corresponding to Rubisco-limited (90 µmol mol-1 CO2) and

RuBP-limited (700 and 1800 µmol mol-1 CO2) photosynthesis in flag leaves of wheat

grown in ambient (open symbols, broken lines) and elevated (closed symbols, solid

lines) CO2. For modelling, measured Cc values at each CO2 concentration and

temperature were used. Each point is the mean of six replicate leaves.

A

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 500 1000 1500 2000Ca µmol mol-1

E, m

mol

m-2

s-1

B

0.0

0.2

0.4

0.6

0.8

0 500 1000 1500 2000Ca µmol mol-1

E, m

mol

m-2

s-1

C

-3

-2

-1

0

1

2

0 500 1000 1500 2000Ca µmol mol-1

A, µ

mol

m-2

s-1

-2.0

-1.5

-1.0

-0.5

0.0

0 50 100 150

Fig. 1

35 ºC

-4

-2

0

2

4

6

8

0 20 40 60 80 100

Cc, µmol mol-1A,

µm

ol m

- 2 s

-1

Γ*

Rd

Rd= -1.90Γ*= 42.3

15 ºC

-4

-2

0

2

4

6

8

0 20 40 60 80 100

Cc, µmol mol-1

A, µ

mol

m -

2 s-1

Γ*

Rd

Rd= -0.71Γ*= 22.4

Fig. 2

0.0

0.5

1.0

1.5

2.0

2.5

-0.02 -0.01 0 0.01 0.02

Tk-298/R*298*Tk

Rd

0

10

20

30

40

-0.02 -0.01 0 0.01 0.02

Tk-298/R*298*Tk

Γ∗

0

100

200

-0.02 -0.01 0 0.01 0.02

Tk-298/R*298*Tk

Vcm

ax

0

100

200

300

400

-0.02 -0.01 0 0.01 0.02

Tk-298/R*298*Tk

Jmax

0

20

40

60

80

-0.02 -0.01 0 0.01 0.02

Tk-298/R*298*Tk

Amax

Fig. 3

15 ºC

-5

5

15

25

35

45

0 500 1000 1500

Ci, µmol mol-1

A, µ

mol

m -2

s-1

-5

5

15

25

0 100 200 300

20 ºC

-5

5

15

25

35

45

0 500 1000 1500

Ci, µmol mol-1

A, µ

mol

m -2

s-1

-5

5

15

25

0 100 200 300

25 ºC

-5

5

15

25

35

45

0 500 1000 1500

Ci, µmol mol-1

A, µ

mol

m -2

s-1

-5

5

15

25

0 100 200 300

30 ºC

-5

5

15

25

35

45

0 500 1000 1500

Ci, µmol mol-1

A, µ

mol

m -2

s-1

-5

5

15

25

0 100 200 300

35 ºC

-5

5

15

25

35

45

0 500 1000 1500

Cc, µmol mol-1

A, µ

mol

m -2

s-1

-5

5

15

25

0 100 200 300

Fig. 4

Ca 90 µmol mol -1

0

4

8

12

10 15 20 25 30 35 40

T ºC

A, µ

mol

m -2 s

-1

Ca 700 µmol mol -1

0

10

20

30

40

50

10 15 20 25 30 35 40

Temperature, ºC

A, µ

mol

m -2 s

-1

Ca 1800 µmol mol -1

0

10

20

30

40

50

10 15 20 25 30 35 40

Temperature, ºC

A, µ

mol

m -2 s

-1

Fig. 5