CHAPTER 2 BIOCHEMICAL MODEL OF C 3 PHOTOSYNTHESIS APPLIED
The estimate of Vcmax from CO2 response curves depends on the values assumed for the
kinetic parameters of Rubisco. As there are few complete sets of values, it is common to assume those determined for tobacco (Bernacchi et al., 2001; Bernacchi et al., 2002). However, it became apparent that using these values from tobacco to analyse wheat measured at different temperatures was problematic. Consequently a detailed
characterisation of CO2 response curves measured under a range of temperatures and two
oxygen concentrations was undertaken.
This chapter highlights the importance of adjusting the biochemical model by using kinetic parameters derived for wheat. The new parameters were compared against repeated
photosynthetic measurements across a range of leaf temperatures measured in the field or growth cabinet. The new parameter values improved the ability to derive an estimate of Vcmax from A:Ci curves measured in the field at leaf temperatures other than 25 °C compared to when activation energies from tobacco were assumed.
2.5.1 Rubisco kinetic constants for wheat
Some kinetic constants from tobacco calculated in vivo helped to fit observed data for wheat in vivo. In vitro value25 for Kc and Ko in wheat differ from those obtained in vivo in tobacco, mainly for Ko. In vitro Ko for wheat is reported as 304 μbar (Makino et al., 1988) and 328 μbar (Galmés et al., 2014) while value25 of Kc for tobacco is half lower than in vitro values (166 μbar). Reports for Kc and Ko measured in vitro for different species, make it difficult to select the set of value25 and E that should be used in the fittings, and an inadequate choice could result in poor predictions (Table 2.2.a). A common way to derive
Vcmax and J is with a published spreadsheet in excel (Sharkey et al., 2007) which uses kinetic constants obtained in vivo from tobacco (Bernacchi et al., 2001; Bernacchi et al., 2002). Previously, value25 had been reported for Kc, Ko, * and kcat based on analysis of CO2
response curves of transgenic tobacco which had reduced Rubisco content (von
Caemmerer et al., 1994). The spreadsheet with that set of kinetic constants has been used in most of the higher plants, assuming that all the plants have the same mechanism for CO2
assimilation. In part it is true, since prediction of A at 25, 30 and 35 ºC were acceptable when the kinetic constants from Bernacchi et al., 2001 & 2002 were used to predict observed data from wheat (Table 2.2.b). However, there were some inconsistencies in the fitting that were fixed for wheat. The new E of Kc and Ko for wheat increased and Vomax decreased in comparison with tobacco. We fixed the value of E for Vcmax for wheat at 63 kJ
mol-1 (Evans, 1986) which is slightly less than that for tobacco, 65.3 kJ mol-1 (Bernacchi et al., 2001). Perhaps these differences are normal since it has been reported that E for kcatc in
vitro varies from 48 to 72 kJ mol-1 across crops (soybean, rice, cotton, tobacco, tomato,
spinach, wheat) and E of kcatc in vitro for wheat is higher than for tobacco (Galmes et al., 2015). As well, E of kcatc in vitro from land plants showed a positive correlation with the optimum growth temperature and Rubisco specificity factor (Sc/o) (Galmes et al., 2015).
Tobacco is from warmer environments than wheat and the temperature required for optimal growth is higher (25 and 20 ºC, respectively), which could relate to the differences in E of Kc and Ko obtained in this experiment.
It is also reported that the specificity factor (Sc/o) in vitro for tobacco is 82 and 107 mol mol- 1 for wheat (Parry et al., 1989; Whitney et al., 2009). Interestingly, the assumed value25 for Kc and Ko helped to solve the rest of the kinetic parameters in wheat where Γ* resulted in almost similar values (Table 2.2). Γ* reflects Sc/o in vivo. The discrepancy of Sc/o in vitro and in vivo need more work to understand the two different answers.
2.5.2 Effect of temperature on estimating Vcmax25
Vcmax calculated with the new constants for wheat varied at increasing temperature (15, 20,
25, 30, 35 ºC, Vcmax of 50, 78, 121, 184, 277 μmol CO2 m-2 s-1 respectively, Figure 2.5) and
during the day temperatures can vary from 23 to 34 ºC (Figure 2.1), for this reason conversion to value25 (Vcmax) is important analysing Rubisco activity for field
measurements. However, measurements at temperature higher than 30 ºC should be reconsidered in wheat because the wheat growth and Rubisco could be affected. The optimum growth temperature in wheat during anthesis and grain filling is 20 ºC and the maximum cardinal temperature (when wheat can recover) is 31 ºC at anthesis and 35.4 ºC during grain filling (Porter and Gawith, 1999). Measuring in the field (experiments Aus3 and Mex) the highest temperatures were during anthesis and grain filling between noon and early afternoon. Short periods of heat may not detrimentally affect wheat plants when grown under good management and with irrigation. Thus it should be possible to obtain
Vcmax25 from Vcmax derived at higher temperatures from plants when they are grown under yield potential conditions. However, when high temperatures are expected in the field, it would be better to restrict gas exchange measurements to the morning.
In the case of Rubisco, activation state can be affected by CO2 concentrations and
temperature. In fitting the model for C3 photosynthesis to calculate Vcmax, a full Rubisco activation state was assumed. However, in sweet potato, it has been shown that the
53 Rubisco activation state varies depending on CO2 concentration. It is higher at low CO2
(Ci=140 μbar) and lower at high CO2 concentrations (Ci=500 μbar). Rubisco activation state also decreased at temperatures above 30 ºC. When measured at ambient CO2 (Ci=250 μbar), activation state decreased from the highest value of 85 % at 30 ºC to 65 % at 40 ºC (Sage and Kubien, 2007). Two ideas can be extracted from this information. First, we may have underestimated Vcmax if Rubisco was not fully activated. Second, plants growing in the field are subjected to variation in temperature. If this altered the activation state of Rubisco, then Vcmax derived from field surveys could be confounded by temperature.
For wheat, gm did not increase greatly at higher temperatures (von Caemmerer and Evans,
2015), but the ability of Rubisco to select CO2 over O2 decreases such that photorespiration
increases from 25 to 35 ºC (Figure 2.3). It was possible to fit CO2 response curves at 35 ºC
to calculate Vcmax25 in wheat and by making measurements under both 2% and 21% O2, we
were able to untangle the changes in carboxylation and oxygenation as temperature varied. Nevertheless, measurements from plants that have been exposed to 30 to 35 ºC for a long time need to be used with caution and one needs to be aware that when deriving Rubisco activity, certain assumptions are required.
2.5.3 Effect of temperature in respiration
Respiration occurs in the mitochondria, and it is the process by which plants consume O2
and release CO2. Respiration increases with increasing temperature. At temperatures
around 5 °C, enzymes have lower maximum catalytic activity and membranes are less flexible, both of which could reduce respiratory flux. At 25°C, enzymatic performance and availability of substrates both increase compared to that at 5°C and membranes are more flexible. These changes help to increase the flux through respiration. However, at higher temperatures, membranes became very fluid, the activity of some enzymes decrease and substrates may become limited, all of which affect the respiration flux (Atkin and Tjoelker, 2003). In Figure 2.3, dark respiration rate per unit leaf area increased with increasing temperature almost doubling from 25 to 35 ºC. Increasing respiration has also been observed for Spinacia oleracea L. cv. Torai grown at 15/10 °C and 30/25 °C (Yamori et al., 2005).