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 with the 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:
Ac 


Vcmax Cc  *
 Rd ,
Cc  K c 1  O / K o 
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:
Ar 

J g Cc   *
4Cc  8*
 R
d
, 2)
where Jg (µmol m-2 s-1) is the rate of electron transport. Temperature dependences of Rubisco kinetics were
obtained in tobacco from Bernacchi et 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:
Ctransition 
K c 1  O / K o J g / 4Vc max  2*
1  J g / 4Vc max
, 3)
(von Caemmerer, 2000).
The Cc measured under Ca = 380 µmol mol-1 was compared to Ctransition. If Cc for A380 is less than
Ctrans, it indicates a limitation by RuBP carboxylation, 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
regeneration exists.
(2) CO2 response of chloroplast 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, Ogren and 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-1 CO2 concentrations is similar to Jf at high CO2 concentration. On
the other hand, if Jf at 380 μmol mol-1 CO2 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-Cc
curve)), since the Ctransition (Jf-Cc curve) indicates the transition point from RuBP carboxylation to RuBP
regeneration limitation as with analyses of CO2 response of CO2 assimilation rate.
Using these two independent methods, it is possible to analyze under what temperatures RuBP
carboxylation or 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. Kalanchoe daigremontiana and K. pinnata plants
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
LMA (g
m-2)
Leaf N (g m-2)
20/10˚C
30/20˚C
163.6±7.6
103.3±2.2
2.29±0.12
1.69±0.04
K. pinnata
20/10˚C
30/20˚C
*
68.3±2.0
53.4±1.6
*
*
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).