Wataru Yamori, Kouki Hikosaka and Danielle A. Way

Temperature response of photosynthesis in C3, C4 and CAM plants: Temperature acclimation and Temperature adaptation

Wataru Yamori, Kouki Hikosaka and Danielle A. Way

Supplemental Appendix

Analysis of the limiting step of C3 photosynthesis

In C3 plants, analyses of CO2 response of (1) CO2 assimilation rate and (2) chloroplast electron transport rate estimated from chlorophyll fluorescence can determine the limiting step of CO2 assimilation by Rubisco or RuBP regeneration at various measurement conditions (Yamori and von Caemmerer 2009; Yamori et al. 2010a, 2011a). It should be noted that there was an error in the description of how we analyzed the limiting step of photosynthesis in the figure legends in our previous paper (Yamori and von Caemmerer 2009; Yamori et al. 2010a, 2011a).

(1) CO2 response of CO2 assimilation rate

A response of CO2 assimilation to chloroplast CO2 concentration (Cc) was analyzed withthe C3 photosynthesis model (Farquhar et al. 1980). When CO2 assimilation is limited by the capacity of Rubisco to consume RuBP, the CO2 assimilation rate (Ac) is expressed as:

,1)

where Vcmax (µmol m-2 s-1) is the maximum rate of RuBP carboxylation on the leaf area basis, Kc (µmol mol-1) and Ko (mmol mol-1) are the Michaelis constants for CO2 and O2, respectively, Cc (µmol mol-1) and O (mmol mol-1) are chloroplastic CO2 and O2 concentration, respectively, Γ* (µmol mol-1) is the CO2 compensation point in the absence of day respiration, and Rd (µmol m-2 s-1) is the day respiration rate. When CO2 assimilation is limited by the RuBP regeneration rate, the CO2 assimilation rate (Ar) is expressed as:

,2)

whereJg (µmol m-2 s-1) is the rate of electron transport. Temperature dependences of Rubisco kinetics were obtained in tobacco from Bernacchiet al. (2002). Fitting was performed with the software Kaleidagraph (Synergy Software, Reading, PA, USA), and Vcmax and Jg were estimated from CO2 assimilation rate at low CO2 concentration and at high CO2 concentration, respectively.

Chloroplast CO2 concentration at which the transition from RuBP carboxylation to RuBP regeneration limitation occurs (Ctransition) was determined as:

,3)

(von Caemmerer, 2000).

The Cc measured under Ca = 380 µmol mol-1was compared to Ctransition. If Cc for A380 is less than Ctrans, it indicates a limitation by RuBPcarboxylation, whereas if Cc for A380 is greater than Ctransition, it indicates a limitation by RuBP regeneration. At Ctransition, a co-limitation by RuBP carboxylation and RuBP regenerationexists.

(2) CO2 response ofchloroplast electron transport rates

The CO2 response of chlorophyll a fluorescence was determined simultaneously with gas exchange by an integrated fluorescence chamber head (LI-6400, LI-6400-40 leaf chamber fluorometer, LI-COR). After measurements of the quantum yield of photosystem II (ΦPSII) (Genty et al.1989), the rate of linear electron transport (Jf) was determined as Jf= ΦPSII x f x I x αleaf, where f is the fraction of absorbed light reaching PSII (assumed 0.5 for C3 plants, Ogrenand Evans, 1993), I is incident photon flux density, and αleaf is leaf absorptance (assumed 0.85).

For each leaf temperature, Jf was constant at high CO2 concentration. A380 is limited by RuBP regeneration rate if Jf at 380 μmol mol-1CO2 concentrations is similar to Jf at high CO2 concentration. On the other hand, if Jf at 380 μmol mol-1CO2 concentration is lower than the constant Jf at high CO2 concentration, this indicates that A380 is limited by RuBP carboxylation.Jf-Cc curve was also analyzed to obtain the chloroplast CO2 concentration at which electron transport rate (Jf) is constant (Ctransition (Jf-Cccurve)), since the Ctransition (Jf-Cccurve) indicates the transition point from RuBP carboxylationto RuBP regenerationlimitation as with analyses of CO2 response of CO2 assimilation rate.

Using these two independent methods, it is possible to analyze under what temperatures RuBP carboxylationor RuBP regeneration limited CO2 assimilation rate at ambient CO2 concentration of 380 µmol mol-1. A recent detailed description of the C3 photosynthesis model can be also found in von Caemmerer et al. (2009) and Bernacchi et al. (2009).

Supplemental Figure S1

CO2 response of photosynthetic rate and the candidates for the limiting steps of photosynthesis in C3 and C4 plants, respectively. In C3 species, at low CO2 concentrations, the photosynthetic rate is limited by RuBP carboxylation, where CO2 diffusion (stomatal conductance and mesophyll conductance: Yamori et al. 2006a) and Rubisco activity (i.e., Rubisco amount, Rubisco kinetics and Rubisco activation: Yamori et al. 2005, 2006b) would affect photosynthetic rate. At high CO2 concentrations, the photosynthetic rate is limited by RuBP regeneration. RuBP regeneration rate in turn is determined by the chloroplast electron transport capacity to generate NADPH and ATP, or the activity of Calvin cycle enzymes involved in regeneration of RuBP or Pi regeneration capacity. In C4 plants, at low CO2 concentrations, photosynthetic rate would be determined by CO2 diffusion (stomatal conductance and mesophyll conductance), carbonic anhydrase activity and PEPC activity. On the other hand, at high CO2 concentrations, photosynthetic rate would be determined by Rubisco activity (i.e., Rubisco amount, Rubisco kinetics and Rubisco activation), PEP regeneration via PPDK, RuBP regeneration (i.e., chloroplast electron transport rate and activity of Calvin cycle enzymes involved in regeneration of RuBP).

Supplemental Figure S2

CO2 response of net CO2 assimilation rate from gas-exchange (Fig. A) and electron transport rate from chlorophyll fluorescence (Fig. B).Net CO2 assimilation rate and electron transport rate is shown as open circles. Solid line shows Rubisco-limited A (Ac), and dotted line shows RuBP regeneration-limited A (Ar).Temperature dependence of chloroplast CO2 concentration (Ctransition) at which the transition from RuBP carboxylation (Ac) to RuBP regeneration (Ar) limitation occurs (solid symbol and line) and Cc for CO2 assimilation rate measured at Ca = 380 μmol mol-1 and 1500 µmol photons m-2 s-1(open symbol and dashed line) in Fig. C and Fig. D.Shaded area shows that A380 is limited by Ac, whereas others show that A380 is limited by Ar. In the case of Figure C, CO2 assimilation rate at low temperature is limited by Ar, whereas at high temperature by Ac. On the other hand, in the case of Figure D, CO2 assimilation rate at low and high temperature is limited by Ar, whereas at around optimum temperature for photosynthetic rate by Ac.

Supplemental Figure S3

Temperature responses of stomatal conductance (gs) and intercellular CO2 concentration(Ci) at night in two CAM plants grown at two different temperature regimes.Kalanchoedaigremontianaand K. pinnataplants were grown during the summer in a glasshouse under natural light conditions in Canberra, Australia. The day/night air temperatures were either 20°C/10°C or 30°C/20°C. Plants were grown in 3 L pots in garden mix containing approximately 2 g L-1 slow-release fertilizer (Osmocote; Scotts Australia Pty Ltd., Castle Hill, Australia) and watered daily. The relationship between intercellular CO2 concentration (Ci)and CO2 fixation rate (A) at various leaf temperatures is also shown. Data represent means ± SE, n = 4-6.

K. daigremontiana / K. pinnata
20/10˚C / 30/20˚C / 20/10˚C / 30/20˚C
LMA (g m-2) / 163.6±7.6 / 103.3±2.2 / * / 68.3±2.0 / 53.4±1.6 / *
Leaf N (g m-2) / 2.29±0.12 / 1.69±0.04 / * / 1.94±0.07 / 1.34±0.05 / *

Leaf mass per unit area (LMA) and leaf nitrogen content (Leaf N)in two CAM plants grown at two different temperature regimes.Data represent means ± SE, n = 4-6. Statistical analysis of the effect of growth temperature was carried out for each individual species (Student's t-test:*P < 0.05).