Effects of growth and measurement light intensities on temperature dependence of CO2 assimilation rate in tobacco leaves

Authors


W. Yamori, Department of Applied Science, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981-8555 Japan. Fax: +81 22 717 8765; e-mail: wataru.yamori@biochem.tohoku.ac.jp

ABSTRACT

Effects of growth light intensity on the temperature dependence of CO2 assimilation rate were studied in tobacco (Nicotiana tabacum) because growth light intensity alters nitrogen allocation between photosynthetic components. Leaf nitrogen, ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) and cytochrome f (cyt f) contents increased with increasing growth light intensity, but the cyt f/Rubisco ratio was unaltered. Mesophyll conductance to CO2 diffusion (gm) measured with carbon isotope discrimination increased with growth light intensity but not with measuring light intensity. The responses of CO2 assimilation rate to chloroplast CO2 concentration (Cc) at different light intensities and temperatures were used to estimate the maximum carboxylation rate of Rubisco (Vcmax) and the chloroplast electron transport rate (J). Maximum electron transport rates were linearly related to cyt f content at any given temperature (e.g. 115 and 179 µmol electrons mol−1 cyt f s−1 at 25 and 40 °C, respectively). The chloroplast CO2 concentration (Ctrans) at which the transition from RuBP carboxylation to RuBP regeneration limitation occurred increased with leaf temperature and was independent of growth light intensity, consistent with the constant ratio of cyt f/Rubisco. In tobacco, CO2 assimilation rate at 380 µmol mol−1 CO2 concentration and high light was limited by RuBP carboxylation above 32 °C and by RuBP regeneration below 32 °C.

INTRODUCTION

Plants in natural habitats are subject to temporal and spatial variations in temperature and light intensity. To predict the temperature dependence of CO2 assimilation rate, the underlying biochemical limitations must be known. According to the C3 photosynthesis model (Farquhar, von Caemmerer & Berry 1980), CO2 assimilation rate is limited either by the capacity of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) to consume RuBP (RuBP carboxylation) or by the capacity of the thylakoid reactions and the Calvin cycle to regenerate RuBP (RuBP regeneration). At the optimum temperature, CO2 assimilation rate at high light under the current CO2 concentration of 380 µmol mol−1 is often limited by RuBP carboxylation (Sage & Kubien 2007). At temperatures below the optimum, CO2 assimilation rates at high light are generally limited by RuBP regeneration, in particular, the component of RuBP regeneration associated with the regeneration of inorganic phosphate during starch and sucrose synthesis (Sharkey 1985; Sage & Sharkey 1987; Labate & Leegood 1988). On the other hand, the principal limitation of CO2 assimilation rate at high light above the optimum temperature remains unclear, as both the capacity of RuBP carboxylation (Crafts-Brandner & Salvucci 2000; Salvucci & Crafts-Brandner 2004; Yamori et al. 2006a,b, 2008) and the capacity of RuBP regeneration (Wise et al. 2004; Cen & Sage 2005; Makino & Sage 2007) have been proposed as the limiting step. The temperature dependence of CO2 assimilation rate also varies with light intensity (Berry & Björkman 1980). CO2 assimilation rate is most responsive to temperature under high light intensity and the temperature response curve of CO2 assimilation rate becomes flatter as light intensity is decreased. Under low light intensity, the rates of light harvesting and electron transport generally limit CO2 assimilation, even at low CO2 concentration.

Many plants acclimate to their growth light environment, and alter the biochemical composition and morphology of leaves and whole plants (Smith 1982; Terashima & Hikosaka 1995; Niinemets, Kull & Tenhunen 1998; Terashima et al. 2005). In general, plants acclimated to low light have lower leaf mass per area (LMA) than plants acclimated to high light. Acclimation to low light enhances nitrogen allocation to the capacity of light acquisition including chlorophyll and light-harvesting complexes, whereas acclimation to high light enhances the capacity of light utilization, through increased synthesis of cytochrome f (cyt f), ATP synthase, Rubisco and other Calvin cycle enzymes (Evans 1987; Terashima & Evans 1988; Hikosaka 1996; Hikosaka & Terashima 1996; Makino et al. 1997). These would have the effect of optimizing the nitrogen partitioning from non-limiting to limiting processes and contribute to efficient use of nitrogen among the photosynthetic apparatus (Evans 1989; Hikosaka & Terashima 1995; Niinemets & Tenhunen 1997; Evans & Poorter 2001; Hikosaka 2004). As a result, the CO2 assimilation rate at low light intensity is usually greater in leaves adapted to low light compared to high light, whereas the CO2 assimilation rate at high light intensity is lower (Björkman 1981). Some studies have reported an increase in the ratio of electron transport capacity to Rubisco activity in plants grown under high versus low light (e.g. Evans & Terashima 1988; Terashima & Evans 1988; Niinemets et al. 1998; Evans & Poorter 2001). Plants grown at high light are also characterized by greater stomatal conductances and mesophyll conductance (the conductance to CO2 diffusion from intercellular airspace to chloroplast) than plants grown at low light (Hanba, Kogami & Terashima 2002; Piel et al. 2002; Niinemets et al. 2006; Warren et al. 2007). In some species, the intercellular or chloroplast CO2 concentration is lower in plants grown at high light than in plants grown at low light (e.g. Evans 1988; Hanba et al. 2002; Oguchi, Hikosaka & Hirose 2005). A change in either Rubisco to electron transport capacity or chloroplast CO2 concentration could alter the underlying limitation of CO2 assimilation at ambient CO2.

The aim of this study was to investigate effects of growth light and the associated differences in photosynthetic nitrogen partitioning on the temperature response of CO2 assimilation at different measurement light intensities. Nicotiana tabacum were selected because the temperature dependencies of Rubisco kinetics, function of electron transport and mesophyll conductance have been reported (Bernacchi et al. 2001, 2002; Bernacchi, Pimentel & Long 2003). Plants were grown under three different light intensities (100, 250, 450 µmol photons m−2 s−1) and various photosynthetic components were quantified. The response of CO2 assimilation rate to CO2 concentration was measured at several light intensities and leaf temperatures. The mesophyll conductance at 25 °C was measured by carbon isotope discrimination with a tunable diode laser absorption spectrometer and applied to the temperature response reported by Bernacchi et al. (2002). Subsequent analysis with the C3 photosynthesis model was performed to identify the limiting step of CO2 assimilation rate. We addressed three questions: (1) If growth light intensity alters the nitrogen allocation between photosynthetic components, does this alter the temperature response of CO2 assimilation rate?; (2) What is the temperature response of the CO2 concentration (Ctrans) at which the transition from RuBP carboxylation to RuBP regeneration limitation occurs?; (3) Does growth light intensity alter the response of Ctrans to temperature and the limiting step of CO2 assimilation rate?

MATERIALS AND METHODS

Plant material

Tobacco (N. tabacum‘W38’) plants were grown in 5 L pots in garden mix containing approximately 2 g L−1 of a slow-release fertilizer (Osmocote; 15:4.8:10.8:1.2 N : P : K : Mg and trace elements, B, Cu, Fe, Mn, Mo, Zn; Scotts Australia) and watered daily. The air temperature was 30 °C during a 12 h day and 25 °C at night, and the relative humidity was 70%. Plants were grown under three different light intensities, 100 (low light, LL), 250 (middle light, ML) and 450 (high light, HL) µmol photons m−2 s−1. A second group of plants grown under identical conditions were used for measurements of chlorophyll a fluorescence and mesophyll conductance to CO2 diffusion (gm).

Gas-exchange and carbon isotope measurements

Gas exchange of leaves was measured with a portable gas exchange system (LI-6400, Li-Cor, Lincoln, NE, USA). The whole portable gas exchange system was enclosed in a temperature-controlled cabinet (Yamori, Noguchi & Terashima 2005; Yamori et al. 2009). The CO2 assimilation rate (A) versus intercellular CO2 concentration (Ci) was measured at several measurement temperatures (15, 20, 25, 30, 35, 40 °C) and light intensities (100, 250, 450 and 1500 µmol photons m−2 s−1). The vapour pressure deficit was kept under 3.0 kPa even at the highest leaf temperature of 40 °C.

The ratio of 13C to 12C of CO2 entering and leaving the leaf chamber (Li-COR 6400) was determined for intact leaves by a tunable diode laser absorption spectrometer (TDL, model TGA100, Campbell Scientific, Inc., Logan, Utah, USA), according to Bowling et al. (2003) and Griffis et al. (2004). Mesophyll conductance (gm) at 25 °C was estimated at 2% O2 and different light intensities as described by Tazoe et al. (2009) and applied to the temperature response reported by Bernacchi et al. (2002). We used the same value of gm for all measuring light intensities and CO2 concentrations (Tazoe et al. 2009).

A response of CO2 assimilation to chloroplast CO2 concentration (Cc) was analysed 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:

image(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 chloroplast CO2 and O2 concentration, respectively, Γ* (µmol mol−1) is the CO2 compensation point in the absence of day respiration and Rday (µmol m−2 s−1) is the day respiration rate. Rday was estimated from ACc curve at Γ* reported by Bernacchi et al. (2002). When CO2 assimilation is limited by the electron transport rate, the CO2 assimilation rate (Ar) is expressed as:

image(2)

where J (µmol m−2 s−1) is the rate of electron transport. The light dependence of J can be described by a non-rectangular hyperbola:

image(3)

where I is the incident light, Φ is the apparent quantum yield, θ represents a curvature factor and Jmax is the maximum rate of electron transport (Ögren & Evans 1993).

An equation for the temperature dependence of the electron transport rate J at 1500 µmol photons m−2 s−1 (J1500) or Jmax was fitted by

image(4)

where TL is the leaf temperature (°C), J(To) is the electron transport rate at the optimum temperature (To) and Ω is the difference in temperature from To at which J falls to e−1 (0.37) of its value at To (June, Evans & Farquhar 2004).

Temperature dependencies of Rubisco kinetic constants of Bernacchi et al. (2002) were used. Fitting was performed with the software Kaleidagraph (Synergy Software, Reading, PA, USA), and Vcmax and J were estimated from A–Cc curves.

Chloroplast CO2 concentration at which the transition from Ac to Ar occurs (Ctrans) was determined as:

image(5)

(von Caemmerer & Farquhar 1981; von Caemmerer 2000).

Chlorophyll fluorescence measurements

Chlorophyll a fluorescence was determined 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), the rate of linear electron transport (Jf) was determined with assumptions of 0.85 for leaf absorptance and 0.5 for the fraction of absorbed light reaching PS II (Genty, Briantais & Baker 1989). CO2 response of Jf was analysed at several leaf temperatures.

Determinations of chlorophyll, Rubisco, cyt f, soluble protein and nitrogen

Immediately after the measurements of gas exchange, leaf discs of 0.5 cm2 were taken and immersed in liquid nitrogen and stored at −80 °C until determinations of chlorophyll, Rubisco, Rubisco activase, cyt f and soluble protein contents. Some leaf discs were dried at 70 °C for 3 d for measurements of dry weight and nitrogen contents.

The frozen leaf sample was ground in liquid nitrogen and homogenized in an extraction buffer containing 50 mm HEPES-KOH buffer (pH 7.8), 5 mm DTT, 10 mm MgCl2, 1 mm EDTA, 1.5% (w/v) polyvinylpyrrolidone, 0.1% (v/v) Triton X-100 and 1.5% (v/v) protease inhibitor cocktail (Sigma, St Louis, Missouri, MO, USA). The total Rubisco catalytic sites were quantified by stoichiometric binding of 14C-carboxy-arabinitol-P2 (CABP-binding technique) according to Ruuska et al. (1998). Rubisco activase content was quantified by immunoblotting with anti-activase antibody using purified activase protein in N. tabacum as a standard (Mate et al. 1996). In the present study, activase content (µmol m−2) was calculated as a monomer with 42 kD. The cyt f content was quantified from a combination of two methods, by immunoblotting with anti-cyt f antibody (Baroli et al. 2008) and by the hydroquinone-reduced, ferricyanide-oxidized difference spectrum of the thylakoid membranes (Bendall, Davenport & Hill 1971). Thylakoid membranes were prepared as described in Ghannoum et al. (2005). For measurements of the hydroquinone-reduced, ferricyanide-oxidized difference spectrum, the difference spectrum was recorded with a dual beam spectrophotometer (model 557, Perkin Elmer, Foster City, CA, USA). The absorbance at 554 nm above a baseline drawn between the absorbances of 540 and 570 nm was measured. The millimolar extinction coefficient of 20 mm−1 cm−1 was used. Chlorophyll was extracted with 80% (v/v) acetone and determined by the procedure of Porra, Thompson & Kriedemann (1989). Soluble protein was quantified by Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA), using bovine serum albumin as a standard.

Leaf nitrogen contents were measured by a C-H-N analyzer (Model EA 1110, Carlo Erba, Milano, Italy).

Statistical analyses

Turkey–Kramer multiple comparison tests were examined with a software, STATVIEW (ver. 4.58, SAS Institute Inc., NC, USA).

RESULTS

Nitrogen partitioning among the photosynthetic apparatus

As growth light intensity increased, LMA, soluble protein, nitrogen (N), Rubisco, cyt f, Rubisco activase (activase) and chlorophyll (Chl) contents increased (Table 1). Chl a/b ratio increased with the increase in growth light intensity, which is indicative of decreased light-harvesting complex. Rubisco/N and cyt f/N were lower in LL leaves than in HL and ML leaves, whereas Chl/N was greater in LL leaves than in ML and HL leaves. Cyt f/Rubisco and activase/Rubisco were similar irrespective of growth light intensities. Both the ratio of chloroplast electron transport rate (J1500) and maximal Rubisco activity (Vcmax) at 1500 µmol photons m−2 s−1 (J1500/Vcmax) and cyt f/Rubisco were independent of growth light intensity (Table 1).

Table 1.  Physiological characteristics of leaves from N. tabacu m grown under three different light intensities (100 (LL leaves), 250 (ML leaves), 450 (HL leaves) µmol photons m−2 s−1)
 LLMLHL
  1. Leaf mass per area (LMA), soluble protein, leaf nitrogen, Rubisco, Cytochrome f, Rubisco activase (activase) and chlorophyll (Chl) were quantified. Activase content (µmol m−2) was calculated as a monomer, assuming a molecular weight of 42 kD. CO2 assimilation rate at 380 µmol mol−1 CO2 concentration (A380) and the chloroplast CO2 concentration (Cc) were analysed at 1500 µmol photons m−2 s−1 (Materials and Methods). The maximum carboxylation rate of Rubisco (Vcmax), the electron transport rate (J1500) and the day respiration rate (Rd) were estimated from CO2 response curves of CO2 assimilation rate at 1500 µmol photons m−2 s−1 at 25 °C (Materials and Methods). Data represent means ± SE, n = 4. Different characters show significant differences (Turkey–Kramer's multiple comparison test, P < 0.05).

LMA (g m−2)14.6 ± 0.6a20.0 ± 1.2b25.4 ± 1.0c
soluble protein (g m−2)4.43 ± 0.07a6.25 ± 0.20b7.44 ± 0.15c
Nitrogen (mmol m−2)80.9 ± 3.0a113.7 ± 5.3b140.9 ± 1.9c
Rubisco (µmol m−2)1.37 ± 0.03a2.57 ± 0.17b3.18 ± 0.09c
Cyt f (µmol m−2)0.66 ± 0.01a1.17 ± 0.06b1.48 ± 0.01c
Activase (µmol m−2)1.97 ± 0.05a3.38 ± 0.16b4.06 ± 0.19c
Chl a + b (mmol m−2)0.446 ± 0.021a0.509 ± 0.024ab0.538 ± 0.013b
Chl a/b3.17 ± 0.04a3.62 ± 0.03b3.77 ± 0.07b
Rubisco/Nitrogen (µmol mol−1)17.0 ± 0.6a22.5 ± 0.7b22.6 ± 0.8b
Cyt f/Nitrogen (µmol mol−1)8.19 ± 0.26a10.3 ± 0.10b10.5 ± 0.15b
Chl/Nitrogen (mmol mol−1)5.54 ± 0.36a4.47 ± 0.06b3.81 ± 0.05c
Cyt f/Rubisco (mol mol−1)0.482 ± 0.008a0.459 ± 0.013a0.466 ± 0.013a
Activase/Rubisco (mol mol−1)1.44 ± 0.04a1.32 ± 0.04a1.28 ± 0.06a
A380 at 25 °C (µmol m−2 s−1)9.10 ± 0.7a17.9 ± 1.0b22.2 ± 0.2c
Cc at 25 °C (µmol mol−1)231 ± 6a250 ± 8a234 ± 8a
Rd at 25 °C (µmol m−2 s−1)1.27 ± 0.09a2.48 ± 0.24b3.91 ± 0.39c
Vcmax at 25 °C (µmol m−2 s−1)56.0 ± 2.4a92.0 ± 6.6b122.9 ± 3.2c
J1500 at 25 °C (µmol m−2 s−1)68.3 ± 1.1a122.4 ± 5.9b159.6 ± 2.4c
J1500/Vcmax at 25 °C1.23 ± 0.05a1.34 ± 0.04a1.30 ± 0.03a

Temperature dependence of CO2 assimilation rate

Greater growth light intensity increased CO2 assimilation rate at 380 µmol mol−1 CO2 concentrations (A380) at 1500 µmol photons m−2 s−1 at all leaf temperatures (Fig. 1a). Temperature dependence of A380 differed depending on the measurement light intensities in HL leaves (Fig. 1b). A380 at 100 µmol photons m−2 s−1 was similar between LL leaves and HL leaves, and A380 at 250 µmol photons m−2 s−1 was similar between ML leaves and HL leaves (data not shown). The optimum temperature increased with increasing measurement light intensity but was not influenced by growth light intensity (Fig. 1c).

Figure 1.

Temperature dependence of CO2 assimilation rate at 380 µmol mol−1 CO2 concentration (A380) at 1500 µmol photons m−2 s−1 for tobacco leaves grown at different light intensities (LL, ML and HL represent 100, 250 and 450 µmol photons m−2 s−1) (a) or measured at several measurement light intensities with HL leaves (b). Data were fitted by cubic curves. Arrow shows the optimum temperature of A380. The optimum temperature as a function of measuring light intensity in leaves grown at different light intensities (c). Data represent means ± SE, n = 4.

Effects of growth and measuring light intensity on mesophyll conductance (gm)

Measurements of mesophyll conductance (gm) were made on a second group of plants at several light intensities. There was no strong dependence of gm on measuring light intensity, but it was slightly lower at the lowest measuring light intensity in ML and HL leaves (Fig. 2a). There was, however, a strong relationship between A380 and gm at 1500 µmol photons m−2 s−1 (R2 = 0.89; Fig. 2b). Greater growth light intensity increased gm. Since A380 were slightly different between first and second group of plants, we used this regression line to estimate gm (the gm at 25 °C was 0.128 ± 0.018, 0.364 ± 0.027 and 0.486 ± 0.017 in LL, ML and HL leaves, respectively). The predicted values of Cc at ambient CO2 concentration of 380 µmol mol−1 and 1500 µmol photons m−2 s−1 were similar, irrespective of growth light intensities (Table 1).

Figure 2.

(a) The dependence of mesophyll conductance (the conductance to CO2 diffusion from intercellular airspace to chloroplast, gm) on light intensitiy in HL (n = 3), ML (n = 2) and LL (n = 3) plants. (b) Relationship between gm and CO2 assimilation rate at 21% O2, 380 µmol mol−1 CO2 concentrations (A380) at 25 °C and 1500 µmol photons m−2 s−1. The regression line is y = 0.027x – 0.1185 (R2 = 0.89).

Determination of the limiting step of CO2 assimilation rate from the analysis of A–Cc curve

Examples of the relationships between A and Cc, and between Jf and Cc at 1500 µmol photons m−2 s−1 are shown for measurements at 25 and 40 °C in Fig. 3. A–Cc responses were well described by the model of Farquhar et al. (1980) and the curves showed a transition from RuBP carboxylation (Ac) to RuBP regeneration (Ar) limitation at all leaf temperatures and light intensities examined in this study (see also Supporting Information Fig. S1 for A–Cc responses at several measurement light intensities in HL leaves). For each leaf temperature and light intensity, 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.

Figure 3.

CO2 assimilation rate (A) versus chloroplast CO2 concentration (Cc) at 1500 µmol photons m−2 s−1 at 25 °C (a) & 40 °C (b), respectively. CO2 assimilation rate at 380 µmol mol−1 CO2 concentration (A380) at 25 °C is shown as a solid symbol in HL leaves (circle), ML leaves (triangle) and LL leaves (square). CO2 assimilation rate limited by RuBP carboxylation (Ac: solid line) was estimated from Eqn 1 in Materials & Methods, whereas CO2 assimilation rate limited by RuBP regeneration (Ar: dotted line) was estimated from Eqn 2 in Materials & Methods. The electron transport rate (Jf) from Chl fluorescence versus Cc at 1500 µmol photons m−2 s−1 at 25 °C (c) and 40 °C (d), respectively. Jf at 380 µmol mol−1 CO2 concentration is shown as a solid symbol.

We analysed ACc curves to determine under what light intensities and temperatures RuBP carboxylation (Ac) or RuBP regeneration (Ar) limited CO2 assimilation rate at 380 µmol mol−1. The Cc measured under Ca = 380 µmol mol−1 was compared with Ctrans, the chloroplast CO2 concentration at which the transition from Ac to Ar limitation occurs (Fig. 4, Eqn 5 in Materials & Methods). Ar limits A when Cc is less than Ctrans, whereas if Cc is greater than Ctrans, Ac is limiting.

Figure 4.

Light dependence of chloroplast CO2 concentration (Ctrans) at which the transition from RuBP carboxylation (Ac) to RuBP regeneration (Ar) limitation occurs (solid circle and line) and Cc for CO2 assimilation rate measured at Ca = 380 µmol mol−1 (open circle and dashed line) at 25 °C (a) and 40 °C (b), respectively, in HL leaves. Ctrans was calculated from Eqn 6 in Materials and Methods. Cc for A380 less than the Ctrans indicates that CO2 assimilation is limited by Ar, whereas Cc for A380 above Ctrans indicates that CO2 assimilation is limited by Ac. Shaded area shows that A380 is limited by Ac, whereas others shows that A380 is limited by Ar. Data represent means ± SE, n = 4.

In HL leaves, A380 at 25 °C was solely limited by Ar regardless of measurement light intensities (Fig. 4a), whereas at 40 °C, A380 below 800 µmol photons m−2 s−1 was limited by Ar but above 800 µmol photons m−2 s−1 by Ac (Fig. 4b). Ctrans increases almost linearly with temperature under high light intensities, whereas the observed values of Cc for A380 was nearly independent of temperature regardless of growth light intensity (Fig. 5). The values for Ctrans were independent of growth light intensity, reflecting the fact that J1500/Vcmax and cyt f/Rubisco were unchanged by growth light intensities. At 1500 µmol photons m−2 s−1, the limiting step changed from Ar to Ac above 30 °C, irrespective of the growth light intensity (Fig. 5).

Figure 5.

Temperature dependence of chloroplast CO2 concentration (Ctrans) 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 HL leaves (circle), ML leaves (triangle) and LL leaves (square). Cc for A380 less than the Ctrans indicates that CO2 assimilation is limited by Ar, whereas Cc for A380 above Ctrans indicates that CO2 assimilation is limited by Ac. Shaded area shows that A380 is limited by Ac, whereas others shows that A380 is limited by Ar. Data represent means ± SE, n = 4.

The relationships between Vcmax and Rubisco, and J1500 and cyt f

Both chloroplast electron transport rate (J1500) and maximal Rubisco activity (Vcmax) at 1500 µmol photons m−2 s−1 at 25 °C increased with growth light intensities in line with increases in Rubisco and cyt f content. Strong linear relationships were found between Vcmax (estimated from ACc curves at 1500 µmol photons m−2 s−1) and Rubisco contents, and between J1500 (estimated from ACc curves at 1500 µmol photons m−2 s−1) and cyt f contents (Fig. 6).

Figure 6.

Relationships between Vcmax at 25 °C (solid line) or at 40 °C (dotted line) and Rubisco contents (a), and between J1500 at 25 °C (solid line) or at 40 °C (dotted line) and cyt f contents (b). Both Vcmax and J1500 were estimated from A–Cc curves at 1500 µmol photons m−2 s−1. The regression lines are shown in each figure; (a) y = 35.3x + 6.6 at 25 °C, y = 98.0x + 28.9 at 40 °C; (b) y = 110.2x – 4.8 at 25 °C, y = 152.1x + 9.4 at 40 °C.

Rubisco catalytic turnover (kcat) estimated from the slope of the relationships between Vcmax and Rubisco contents were 35.3 mol CO2 (mol Rubisco)−1 s−1 at 25 °C and 98.0 mol CO2 (mol Rubisco)−1 s−1 at 40 °C (Fig. 6a). Chloroplast electron transport rate estimated from the slope of the relationships between J1500 and cyt f contents was 110 µmol electrons (mol cyt f)−1 s−1 at 25 °C and 152 µmol electrons (mol cyt f)−1 s−1 at 40 °C (Fig. 6b). The ratios of rates at 40 °C compared to 25 °C were 1.38 and 2.78 for J1500 and kcat, respectively. Rubisco carboxylation becomes limiting at higher temperatures despite electron transport being less temperature responsive, because the affinity of Rubisco for CO2 decreases.

Light and temperature dependences of electron transport rate

Responses of J to measurement light intensities at several leaf temperatures in HL leaves are shown in Supporting Information Fig. S2. The light dependence of J at different leaf temperatures was fitted with Eqn 3 under an assumption of constant apparent quantum yield (ΦJ = 0.386, the observed mean value). Both the curvature factor θJ and Jmax increased with increasing temperature (Table 2).

Table 2.  Parameters for light response curve of electron transport rate, derived from non-rectangular hyperbolic response (Eqn 3)
 15 °C20 °C25 °C30 °C35 °C40 °C
  1. ΦJ, the apparent quantum yield and sets for 0.386 at any temperatures; θJ, a curvature factor; Jmax is the maximum rate of electron transport. Curves are fitted to data shown in supplemental Fig. 2 for HL grown plants.

θJ0.380.750.840.850.840.81
Jmax80.1122.3169.5205.9237.6265.1

The temperature dependence of J1500/cyt f and Jmax/cyt f were analyzed in HL leaves using Eqn 4 (Fig. 7). The difference between Jmax and J1500 increased at higher temperatures. The maximum chloroplast electron transport rate (Jmax) was 114 µmol electrons (mol cyt f)−1 s−1 at 25 °C and 179 µmol electrons (mol cyt f)−1 s−1 at 40 °C. Temperature dependences of Jmax and J1500 were easily fitted with Eqn 4 suggested by June et al. (2004) and the parameters are given in the legend to Fig. 7.

Figure 7.

Temperature dependence of J1500 or Jmax divided by cyt f content in HL leaves. Data were fitted by Eqn 4 in Materials and Methods (June et al. 2004) for Jmax: inline image, for J1500: inline image.

DISCUSSION

Differences in photosynthetic nitrogen partitioning induced by the growth light environment does not alter the balance between RuBP carboxylation and RuBP regeneration capacity

Growth light intensity affected both leaf N contents and N partitioning among the photosynthetic apparatus (Table 1). Plants grown at lower light intensities enhanced the efficiency of light acquisition, through increases in Chl content and decreases in Chl a/b ratio. On the other hand, plants grown at higher light intensities enhanced the capacity of light utilization, through an increase in cyt f and Rubisco contents. Differences in A380 between plants from LL, ML and HL growth light environment were related to differences in leaf N, Rubisco, cyt f and Chl content (Fig. 1, Table 1). These results are in agreement with previous reports that N allocation from non-limiting to limiting processes contributed to efficient use of N among the photosynthetic apparatus (Evans 1989; Hikosaka & Terashima 1995; Hikosaka 2004) and show that N. tabacum is able to acclimate to different light environments.

N. tabacum displayed the classical light acclimation characteristics such as altered LMA, nitrogen content per unit leaf area, chlorophyll per unit nitrogen and chlorophyll a/b ratio. However, the ratios of cyt f/Rubisco and activase/Rubisco did not vary with growth light intensities (Table 1). The ratio of electron transport rate measured at 1500 µmol m−2 s−1 to Rubisco activity (J1500/Vcmax) was also constant across growth light intensities, which meant that Ctrans was also independent of growth light intensity (Fig. 5). The coordinated changes in photosynthetic capacity, stomatal and mesophyll conductances during acclimation to growth light intensity resulted in no differences between Cc for leaves grown at different light intensities measured at 380 µmol mol−1 CO2 concentration. As a result, the limiting step for CO2 assimilation rate at a given measurement light intensity and leaf temperature was independent of growth light intensity (Fig. 5). Below 800 µmol m−2 s−1, A380 was solely limited by Ar regardless of leaf temperature and growth light intensities. At 1500 µmol photons m−2 s−1, A380 was limited by Ar below 32 °C and by Ac above 32 °C, irrespective of the growth light intensities.

It has been reported that the ratio of the electron transport rate to Rubisco activity (Jmax/Vcmax) is constant in 109 plant species (Wullschleger 1993; Leuning 1997), but changes with growth light intensity have been observed in some species (Evans 1996; Poorter & Evans 1998; Evans & Poorter 2001). A constancy of the Jmax/Vcmax ratio irrespective of growth light intensity was observed in Alocasia macrorrhiza and Colocasia esculenta (Sims & Pearcy 1989), Oryza sativa (Makino et al. 1997), Plantago asiatica (Hikosaka 2005) and N. tabacum (this study). On the other hand, the ratio of the electron transport capacity to Rubisco activity was decreased in lower growth light intensity in Phaseolus vulgaris (Seemann et al. 1987), Pisum sativum (Evans 1987, 1988) and Spinacia oleracea (Evans & Terashima 1988; Terashima & Evans 1988). Thus, it is possible that there are interspecific variations in the response of Jmax/Vcmax to the growth light intensity. We conclude that acclimation to growth light intensity generally alters N partitioning between light acquisition and light utilization components of the photosynthetic apparatus, but the response of electron transport capacity and Rubisco activity differs depending on plant species.

Robustness of evaluations of the limiting step of photosynthesis

In the present study, we measured gm in plants grown at different light intensities and used Rubisco kinetics reported in N. tabacum (Bernacchi et al. 2002), and evaluated the limiting step of photosynthesis on the basis of Cc, using two independent methods; (1) CO2 response of CO2 assimilation rate; (2) CO2 response of electron transport rate from Chl fluorescence. All methods consistently showed that A380 at 25 °C was limited by RuBP regeneration, whereas at 40 °C, it was limited by RuBP carboxylation. Therefore, it is fair to say that our conclusion was robust.

Previous studies showed that Jmax/Vcmax declined with increases in leaf temperature (Walcroft et al. 1997; Dreyer et al. 2001; Medlyn et al. 2002; Yamori et al. 2005). It follows from the C3 photosynthesis model (Farquhar et al. 1980; von Caemmerer & Farquhar 1981; von Caemmerer 2000) that Ctrans would increase with increasing leaf temperature, which is consistent with our results. Kirschbaum & Farquhar (1984) analysed the temperature response of CO2 assimilation rate in Eucalyptus pauciflora, highlighting the transition point with respect to Ci and that the transition point increased with leaf temperature until 30–35 °C. We have repeated this analysis based on Cc and not on Ci, using the temperature response of gm reported by Bernacchi et al. (2002).

What limits CO2 assimilation rate at high light and high temperature is currently debated. One proposed mechanism is the limitation of Ac caused by heat-induced deactivation of Rubisco (e.g. Law & Crafts-Brandner 1999; Crafts-Brandner & Salvucci 2000; Salvucci & Crafts-Brandner 2004; Yamori et al. 2006a, 2008). The alternative proposition is that decreases in CO2 assimilation rate at high temperatures are due to limitation by Ar (e.g. Wise et al. 2004; Cen & Sage 2005; Makino & Sage 2007). It is highly possible that limiting step of CO2 assimilation differs depending on plant species and growth temperature (Yamori et al. 2010). Since Ctrans increased steeply with temperature at high light intensity and Cc did not change drastically with leaf temperature, it is obvious that in tobacco, CO2 assimilation rates under high light tended to be limited by Ac at higher leaf temperatures, irrespective of the growth light intensities (Fig. 5).

Light dependences of electron transport rate differed depending on the leaf temperature

Chloroplast electron transport rate (J) varied depending on the measurement light and temperature (Fig. S2). The curvature factor of the light response of J (θJ) and Jmax increased with increasing temperature (Table 2). The difference between Jmax and J1500 increased at high light, especially at high temperature (Fig. 7, Fig. S2, Table 2). Temperature dependences of both J1500 and Jmax were easily fitted by the equation of June et al. (2004). The value observed for Jmax exceeded that predicted by the equation of Bernacchi et al. (2003) at higher temperatures (Fig. S3). The parameters values reported here for N. tabacum fall within the range of values reported by June et al. (2004). Plants in natural habitats are subject to daily variations in temperature and light intensity. For the modelling of carbon uptake in leaf, whole plant and canopies, we need to take account of the difference in the light response of electron transport rate depending on the leaf temperature. We could provide the necessary light functions of J depending on the leaf temperature (Table 2).

Variation in electron transport capacity at a common temperature was closely correlated with cyt f content, irrespective of the growth conditions (Fig. 6), as has been reported in tobacco, pea, rice and spinach (Evans 1987; Terashima & Evans 1988; Makino et al. 1994; Price et al. 1998; Yamori et al. 2005). Our estimate of Jmax at 25 °C per mole of cyt f of 114 (mol cyt f)−1 s−1 was less than the estimates of 156 mol electrons (mol cyt f)−1 s−1 reported by Niinemets & Tenhunen (1997) from analyses of several different species at 25 °C. In previous studies, it has been proposed that the temperature dependence of whole-chain electron transport rate was limited by that of the PSII electron transport rate and diffusion processes between PSI and PSII reaction centres including plastoquinone and plastocyanin (Mawson & Cummins 1989; Yamasaki et al. 2002; Yamori et al. 2008) and also affected by cyclic electron transport (Sharkey 2005; Sage & Kubien 2007). It is not resolved what limits temperature dependence of electron transport rate and there may be a number of co-limiting steps. However, it is fair to say that cyt f complex is one of the key rate-limiting steps determining electron transport capacity over a broad temperature range.

CONCLUSION

Low growth light intensity increased the amount of nitrogen partitioned into light harvesting processes but did not alter the balance between electron transport capacity and Rubisco activity (i.e. Jmax/Vcmax). Moreover, the coordinated changes in photosynthetic capacity, stomatal and mesophyll conductances during acclimation to growth light intensity resulted in the same chloroplast CO2 concentration (Cc) at high light. As a result, the limiting step for CO2 assimilation rate was similar irrespective of the growth light intensity. Ctrans, the chloroplast CO2 concentration at which the transition from RuBP carboxylation to RuBP regeneration occurs, increased with leaf temperature. At high temperature, CO2 assimilation rate was limited by RuBP carboxylation, whereas at low temperature, it was limited by RuBP regeneration. On the other hand, CO2 assimilation rate below 800 µmol photons m−2 s−1 was limited by RuBP regeneration irrespective of growth light intensity and leaf temperature. The rate of electron transport is linearly related to cyt f content at all temperatures.

ACKNOWLEDGMENTS

We would like to thank Stephanie McCaffery, Jessica Janek and Simon Dwyer for their technical assistance. This work was supported by a grant from the Japan Society for the Promotion of Science Postdoctoral Fellowships for Research Abroad (to W.Y.).

Ancillary