CHAPTER 2 BIOCHEMICAL MODEL OF C 3 PHOTOSYNTHESIS APPLIED
2.4 RESULTS
2.4.1 Leaf model prediction of in vivo photosynthesis in wheat
In order to understand the effect of temperature on the calculations of Vcmax , experiments in the cabinet at 21 and 2% O2 were performed, focused on the initial slope of CO2
response curves. Values of Vcmax and J derived from gas exchange measurements and the model fitted to them depend on photosynthetic kinetic constants that have been assumed in the model. The most frequent kinetic constants used to fit the photosynthetic model have been derived from Atriplex glabriuscula and Nicotiana tabacum (Table 2.2.a and 2.2.b). However, using these kinetic constants, the modelled response did not describe the wheat CO2 response curve observed at different temperatures and oxygen concentrations (Figure
44
2.2.a and 2.2.b.). The observed A measured in vivo with gas exchange did not correspond to predictions made using the model (Equation 2.1 and 2.2) with kinetic constants from Table 2.2.a. Using kinetic constants obtained by Bernacchi, 2001 & 2002 derived from tobacco (Table 2.2.b, Figure 2.2.b), improved the fitting but there were still inconsistences, especially at 2% O2. The fitting was substantially improved by deriving new activation
energies (Table 2.2.c, Figure 2.2.c).
Table 2.2 The kinetic constants used to model CO2 assimilation rate in Figure 2.2 a, b & c.
Kinetic constants/Specie (a) N. tabacum2,4
A. glabriuscula1 (b) N. tabacum (c) New constants for wheat Kc Value at 25°C (μbar) 2592 272.384 2724 E (kJ mol-1) 59.41 80.994 93.72±2.56 Ko Value at 25°C (mbar) 1792 165.824 166 E (kJ mol-1) 361 23.724 33.6±5.96 Γ* Value at 25°C (μbar) 37.434 37.434 37.746 E (kJ mol-1) 24.464 24.464 24.426
Vcmax Value at 25°C (μmol
m-2 s-1) 134 113.9 117.5 E (kJ mol-1) 64.81 65.33 635 Vomax E (kJ mol-1) 65.86 32.58+ 27.1±4.86 Rd Value at 25°C (μmol m-2 s-1) 1.37 1.27 1.14 E (kJ mol-1) 46.393 46.393 46.393
References 1(Badger and Collatz, 1977)
2(von Caemmerer et al., 1994)
3(Bernacchi et al., 2001) 4(Bernacchi et al., 2002)
5(Evans, 1986) 6This report, n=6
E: Activation Energy; +Vomax calculated from Bernacchi et al. (2002) constants. Vcmax and Rd values at 25 °C belong to
each plot.
Figure 2.2 CO2 assimilation rate, A, as a function of chloroplastic CO2, Cc, at two different
O2 concentrations (Open symbols, 2% O2; Closed symbols 21% O2) and four temperatures
(square 15 °C, diamond 25 °C, triangle 30 °C, circle 35 °C). Symbols are the observed A
from one leaf of Triticum aestivum cv. Merinda. Lines are the predicted A using each column of kinetic constants from table 2.2. Cc was calculated from Equation 2.3, the
assumed value of mesophyll conductance, gm, from Equation 2.4 and the temperature
dependence of gm,25 from Equation 2.5. Flow rate 500 μmol s-1, irradiance 1800 μmol quanta
45 2.4.1.1 Kinetic constants for respiration
From the literature a similar value of E for Rd has been observed in tobacco and wheat (Evans, 1986; Bernacchi et al., 2001). In order to confirm this, dark respiration (Rdark) in leaves of wheat was measured at 15, 20, 25, 30 & 35 °C and fitted using E= 46.39 kJ mol-1
(Table 2.2). This activation energy derived from Tobacco matches that observed here for wheat dark respiration (Figure 2.3). The temperature response was similar whether obtained with increasing or decreasing temperatures (not shown). We assume the temperature dependency for Rd is the same as that for dark respiration.
Figure 2.3 The temperature response of dark respiration, Rdark, for flag leaves of Triticum
aestivum cv. Espada, Hawkeye, Mace and Merinda. Each point is the mean of three to six leaves. The error bars are the standard error from the leaves measured. Each leaf was measured after a CO2 response curve and 30 minutes of darkness, at five temperatures from
15 °C to 35 °C. The line is the predicted respiration from 10 °C to 40 °C using Equation 2.9,
E=46.39 kJ mol-1, R=8.314 J mol-1 K-1, Rdark(25 ̊C) = 1.2 μmol CO2 m-2 s-1. Flow rate of 500 μmol
s-1 and at 400 μmol CO2 mol-1 for inlet CO2.
2.4.1.2 Kinetic constants for the compensation point
Two important points can be extracted from the initial slope of the CO2 response curve: Γ,
where the curve crosses the x axis (A = 0), and Γ*, the CO2 partial pressure on the curve
when A = -Rd (von Caemmerer, 2000). From the observed CO2 response curves measured
on Merinda, at 15 °C, 25 °C, 30 °C and 35 °C, at 21 and 2 % O2 (Figure 2.2.), a line fitted
to the first two points was used to calculate Γ (Equation 2.7), and then Γ* observed (Γ*obs) using Equation 2.8, assuming that E of Vcmax in wheat is 63 kJ mol-1 (Evans, 1986), E of Rd is 46.39 kJ mol-1 (section 2.4.1.1), and value25 for K
c and Ko from Bernacchi et al. (2001, 2002).
Under 21% O2, the observed CO2 compensation point, Γ, closely matched that observed
for tobacco(Figure 2.4.a).However, under 2% O2 the observed Γ was slightly less than
46
Figure 2.4 Temperature dependence of the CO2 compensation point in 2 and 21% O2.
a) CO2 compensation point when A=0. Circles are the observed Γ derived from six A:Ci
curves from flag leaves of Triticum aestivum cv. Merinda (Equation 2.8). Dashed lines are Γ calculated with kinetic constants from Bernacchi et al. 2002 (Equation 2.7).
b) CO2 photocompensation point when A= -Rd, the solid lines are Γ*new calculated from the
new kinetic constants.
The average value for the CO2 photocompensation point, Γ*new, from six plants of Merinda
measured at 21 % O2 and 25 °C was 35.4±0.41 μbar using an atmospheric partial pressure
for Canberra of 938 mbar. When adjusted to sea level (oxygen partial pressure 210 mbar), this equates to 37.74 μbar. The value of E for Γ* was obtained by summing E for Vomax and
Kc, and subtracting E for Vcmax and Ko after the fitting (24.4 kJ mol-1, Table 2.2.c). This value is similar to that reported for tobacco (Table 2.2.b). Thus, the value and temperature dependence of the CO2 photocompensation point appears to be similar between wheat and
tobacco.
2.4.1.3 Fitting observed values in the model
Minimising the variance between Γ*obs and Γ*new helped to constrain the solution of the fitting which also tried to minimise the variance between A observed and predicted at each temperature and both oxygen concentrations using Equation 2.1. This procedure was repeated for six different plants of Merinda. The average activation energies for Kc and Ko obtained from Merinda (Table 2.2.c) were then used to fit data collected from other genotypes (Mace, Hawkeye and Espada) at different temperatures in the cabinet to assess their general validity.
47 Figure 2.5 CO2 assimilation rate, A, as a function of chloroplastic CO2, Cc a) and
intercellular CO2, Ci b), in 21% oxygen at five different leaf temperatures (Square 15 °C,
cross 20 °C, diamond 25 °C, triangle 30 °C, circle 35 °C). Model curves are A predicted with the new kinetic constants in Table 2.2.c. Symbols are the observed A of one flag leaf from
Triticum aestivum cv. Mace. Flow rate 500 mol s-1, irradiance 1800 μmol quanta m-2 s-1. Cc
was calculated from Equation 2.3.
The ability of the new constants to fit CO2 response curves is illustrated in Figure 2.5. Two
versions are shown. The fundamental data obtained from the LI6400 (A:Ci) are shown in panel b), whereas A:Cc curves are shown in panel a) because it was necessary to include gm
when fitting Equations 2.1, 2.2, 2.3, 2.4 and 2.5. The initial slope of the CO2 response
curves is used to derive Vcmax, which increases at higher temperatures. For example, from
A:Cc curves, Vcmax was 50, 78, 121, 184 and 277 μmol CO2 m-2 s-1 at 15, 20, 25, 30, 35 ºC
respectively (Figure 2.5). As temperatures increase, the affinity of Rubisco for CO2
decreases such that the initial slope changes little. The increase in respiration rate also contributes to the increase in Γ at higher temperatures. At high CO2 partial pressures, the
RuBP regeneration limited rate increased by 30% from 15 to 30 ºC before declining at 35 ºC. The transition from a Rubisco to an RuBP regeneration limitation happens at a lower chloroplastic CO2 partial pressure (230 to 250 μbar) compared to intercellular CO2 partial
pressure (240 to 280 μbar), but did not vary greatly with temperature.
2.4.2 Assessing the new kinetics constants for wheat in the field