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Keywords:

  • Jmax;
  • limiting step;
  • mesophyll conductance (gm);
  • nitrogen;
  • photosynthesis;
  • RuBP carboxylation;
  • RuBP regeneration;
  • stomatal conductance (gs);
  • Vcmax

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES
  10. Supporting Information

Effects of nitrogen (N) supply on the limiting step of CO2 assimilation rate (A) at 380 µmol mol−1 CO2 concentration (A380) at several leaf temperatures were studied in several crops, since N nutrition alters N allocation between photosynthetic components. Contents of leaf N, ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) and cytochrome f (cyt f) increased with increasing N supply, but the cyt f/Rubisco ratio decreased. Large leaf N content was linked to a high stomatal (gs) and mesophyll conductance (gm), but resulted in a lower intercellular (Ci) and chloroplast CO2 concentration (Cc) because the increase in gs and gm was insufficient to compensate for change in A380. The A-Cc response was used to estimate the maximum rate of RuBP carboxylation (Vcmax) and chloroplast electron transport (Jmax). The Jmax/Vcmax ratio decreased with reductions in leaf N content, which was consistent with the results of the cyt f/Rubisco ratio. Analysis using the C3 photosynthesis model indicated that A380 tended to be limited by RuBP carboxylation in plants grown at low N concentration, whereas it was limited by RuBP regeneration in plants grown at high N concentration. We conclude that the limiting step of A380 depends on leaf N content and is mainly determined by N partitioning between Rubisco and electron transport components.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES
  10. Supporting Information

According to the model of Farquhar, von Caemmerer & Berry (1980), CO2 assimilation rate in C3 plants is limited either by the capacity of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) to consume RuBP (RuBP carboxylation) or by the capacity of electron transport and Calvin cycle enzymes to regenerate RuBP (RuBP regeneration). Based on this model, the limiting step of CO2 assimilation rate is altered by the following two factors: (1) the balance of the maximum rate of RuBP regeneration (Jmax) to that of RuBP carboxylation (Vcmax), and (2) CO2 concentration in chloroplast (Hikosaka et al. 2006).

Leaf photosynthetic capacity is related to the leaf nitrogen (N) content in many higher plants, since stromal enzymes and thylakoid proteins account for the majority of leaf N (e.g. Evans & Terashima 1988; Terashima & Evans 1988; Evans 1989; Makino & Osmond 1991). Both Jmax and Vcmax are enhanced by increases in leaf N content, but the in vivo Jmax/Vcmax ratio decreases with increasing leaf N content in many plant species (e.g. Sage, Sharkey & Pearcy 1990; Makino, Nakano & Mae 1994; Nakano, Makino & Mae 1997). Thus, leaf N content may affect the limiting step of CO2 assimilation. Nevertheless, there have been few studies that examined how the limiting step of CO2 assimilation rate depends on the leaf N content. The Jmax/Vcmax ratio is affected by two factors: (1-a) N partitioning between the enzymes related to Jmax and Vcmax, and (1-b) Rubisco activation state. The proportion of N in Rubisco increases with increasing leaf N content in several species (Evans & Terashima 1988; Makino, Mae & Ohira 1988; Terashima & Evans 1988; Evans 1989; Makino, Nakano & Mae 1994), except for wheat (Evans 1983; Makino et al. 1992; Sudo, Makino & Mae 2003). As a result, the ratio of Rubisco content to components of electron transport [e.g. cytochrome f (cyt f) ] increases with increasing leaf N in many C3 species (e.g. Evans & Terashima 1988; Terashima & Evans 1988; Makino et al. 1992). Moreover, it was found that Rubisco activation state decreased with increasing leaf N content in wheat leaves (Mächler et al. 1988) and apple leaves (Cheng & Fuchigami 2000). However, in some plant species, no apparent deactivation of Rubisco was found under high N supply (spinach leaves: Evans & Terashima 1988; rice leaves: Nakano et al. 1997). Therefore, when the effect of N partitioning on the underlying mechanisms of the in vivo Jmax/Vcmax ratio is analysed, the ratio of electron transport components to carbamylated Rubisco should be considered. The response of the Jmax/Vcmax ratio and the mechanisms responsible for changes in the Jmax/Vcmax ratio may vary among C3 species.

It was previously assumed that the chloroplast CO2 concentration (Cc) was equal to the intercellular CO2 concentration (Ci) (Farquhar et al. 1980). However, it is now clear that this assumption is incorrect. Many studies have shown that Cc is significantly lower than Ci due to a finite mesophyll conductance from the intercellular airspace to the chloroplast stroma and that this drawdown in CO2 concentration from Ci to Cc substantially limits photosynthesis (e.g. Evans et al. 1986; Terashima et al. 2006; Flexas et al. 2008; Warren 2008; Evans, Kaldenhoff & Terashima 2009). Cc can also affect the limiting step of CO2 assimilation rate Evans & Terashima (1988) argued that a drop in CO2 concentration at the Rubisco carboxylation sites would be greater in plants grown at high N concentration than in plants grown at low N concentration because of differences in mesophyll conductance. In addition, von Caemmerer & Evans (1991) measured mesophyll conductance in wheat leaves grown at different N regimes using a combination of gas exchange and carbon isotope discrimination measurements. This work showed that chloroplast CO2 concentrations tended to be lower in plants grown at high N concentrations than in plants grown at low N concentrations. On the other hand, recently, Li et al. (2009) indicated that chloroplast CO2 concentration increased with leaf N content in rice. There have been few studies that investigated the effects of leaf N content on mesophyll conductance and chloroplast CO2 concentration. Therefore, it is still not well understood whether mesophyll conductance and chloroplast CO2 concentration alter depending on leaf N content, and it is important to thoroughly examine this hypothesis.

Previous studies on the relation between the intercellular CO2 concentration and the net CO2 assimilation rate in several species, including bean (von Caemmerer & Farquhar 1981, 1984), wheat (Evans 1986) and spinach (Evans & Terashima 1988) suggested that the CO2 assimilation rate under growth conditions was always co-limited by RuBP carboxylation and RuBP regeneration regardless of leaf N content. If leaf N content has an effect on the mesophyll conductance as mentioned above, the limiting step of CO2 assimilation rate under different N conditions should be analysed at the chloroplast CO2 concentration level. In the present study, we examined several plant species grown at low, medium or high N availabilities. We quantified several photosynthetic components and measured CO2 response of CO2 assimilation rate and Chl fluorescence. We analysed the limiting step of CO2 assimilation rate at the level of the chloroplast CO2 concentration based on the C3 photosynthesis model, and addressed three questions: (1) Does the nutrient concentration during growth affect mesophyll conduc-tance and thus chloroplast CO2 concentrations?; (2) Does the balance between RuBP carboxylation and RuBP regeneration respond to a change in leaf N content? If this balance changes with leaf N content, the responsible biochemical differences should be identified; and (3) Does the limiting step of CO2 assimilation rate change with leaf-N content?

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES
  10. Supporting Information

Plant material

Rice (Oryza sativa L. cv. Notohikari) and wheat (Triticum aestivum L. cv. Ias) plants were grown hydroponically in an environmentally controlled growth chamber, while spinach (Spinacia oleracea L. cv. Torai) and tobacco (Nicotiana tabacum L. cv. W38) plants were grown in vermiculite. The chamber for all the plants was operated with a day/night temperature of 27/22 °C, an irradiance of 550 µmol quanta m−2 s−1, a 14 h photoperiod and an ambient CO2 concentration of 380 µmol mol−1. The hydroponic solution used for rice and wheat was previously described by Makino et al. (1988). The rice and wheat plants were grown with three N concentrations (mm): 0.3 (0.15 mm NH4NO3), 2.0 (1.0 mm NH4NO3) and 8.0 (2.0 mm NH4NO3 and 4.0 mm NaNO3). These solutions were renewed once a week and were continuously aerated. On the other hand, in spinach and tobacco, nutrient solution and water were supplied alternately every day. The nutrient solution for spinach and tobacco was previously described by Yamori, Noguchi & Terashima (2005) with three N concentrations (mm): 0.6 (0.2 mm KNO3 and 0.2 mm Ca(NO3)2), 2.0 (0.67 mm KNO3 and 0.67 mm Ca(NO3)2) and 10.5 (3.5 mm KNO3 and 3.5 mm Ca(NO3)2).

Analyses of gas exchange, Chl fluorescence and mesophyll conductance

CO2 gas exchange of leaves was measured with a portable gas exchange system (LI-6400, Li-Cor, Lincoln, NE, USA), according to Yamori et al. (2005, 2009). The CO2 assimilation rate versus intercellular CO2 concentration (Ci) was measured at a light intensity of 1500 µmol photons m−2 s−1 under several measurement temperatures. Chl a fluorescence was also determined simultaneously with gas exchange during the temperature response measurements, using Li-Cor's integrated fluorescence chamber head (LI-6400, LI-6400-40 leaf chamber fluorometer). The electron transport rate (ETR) was determined as ETR = ΦPSII × I × 0.5 × 0.85, where ΦPSII is the quantum yield of photosystem II and I is the incident photon flux density (Genty, Briantais & Baker 1989).

The CO2 assimilation rate is limited either by RuBP carboxylation or by RuBP regeneration, according to the C3 photosynthesis model (Farquhar et al. 1980). The A-Ci curve-fitting procedure described by Ethier & Livingstone (2004) was used:

  • image(1)

where Ac (µmol m−2 s−1) is the CO2 assimilation rate limited by RuBP carboxylation at low CO2 concentration, Vcmax (µmol m−2 s−1) is the maximum rate of RuBP carboxylation, Kc (µmol mol−1) and Ko (mmol mol−1) are the Michaelis constants for CO2 and O2, respectively, and Ci (µmol mol−1) and O (mmol mol−1) is the intercellular CO2 and O2 concentration. The model was then fitted to the data by an iterative procedure estimating gm, Vcmax, -Rd and Γ* (Ethier & Livingstone 2004; Sharkey et al. 2007; see Supporting Information Table S1 for Rd obtained in the present study) with Rubisco kinetic parameters as reported by Bernacchi et al. (2002). Fitting was performed with the software Kaleidagraph (Synergy Software, Reading, PA, USA). The gm was first estimated at 25 °C and its temperature response was analysed with an assumption that temperature response of gm changes in proportion to A at 380 µmol mol−1 CO2 concentrations (A380) (Warren & Dreyer 2006; Yamori et al. 2006a). Then, using the estimated gm, the chloroplast CO2 concentration (Cc) was obtained by the following equation:

  • image(2)

On the other hand, CO2 assimilation rate at high CO2 were used to calculate the RuBP regeneration rate (Ar):

  • image(3)

where Jmax (µmol m−2 s−1) is the maximum rate of electron transport. Chloroplast CO2 concentration at which the transition from Rubisco to RuBP regeneration limitation occurs (Ctransition) was determined as:

  • image(4)

(Yamori & von Caemmerer 2009; Yamori, Evans & von Caemmerer 2010a). The limitation on A380 was analysed using a comparison of Cc measured under Ca = 380 µmol mol−1 and Ctransition.

Determinations of chlorophyll, N, cyt f, Rubisco and Rubisco activation state

Immediately after the measurements of gas exchange, leaf samples were taken, immersed in liquid N and stored at −80 °C. Contents of chlorophyll, N, Rubisco and cyt f were quantified according to Sudo et al. (2003). Cyt f is considered to be a key rate-limiting step of electron transport (Price et al. 1998).

Samples used for the Rubisco activation assay were collected from a leaf equilibrated under steady-state conditions in the gas-exchange chamber. After gas-exchange had reached a steady-state rate at 380 µmol mol−1 CO2 concentration, 1500 µmol photons m−2 s−1 and 25 °C for at least 20 minutes, the leaf in the chamber was taken and immediately frozen in liquid N2. The carboxylation rate of Rubisco before and after activation of Rubisco was measured by coupling 3-phosphoglyceric acid formation with NADH oxidation, according to Yamori et al. (2006b).

Statistical analyses

Four individual plants per N treatment were examined in all the experiments. Tukey–Kramer multiple comparison test and regression analysis was conducted using STATVIEW (ver. 4.58, SAS Institute Inc., Cary, NC, USA).

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES
  10. Supporting Information

N partitioning in the photosynthetic apparatus

As leaf N content increased, Rubisco, cyt f and chlorophyll (Chl) content increased in all plant species (Table 1). However, the increase of Rubisco content was always greater than that of the cyt f and Chl content. Chl a/b ratios were similar irrespective of the leaf N content in wheat, rice and tobacco, whereas it decreased with increasing leaf N in spinach.

Table 1.  Physiological characteristics of leaves from plants grown under three different nutrient concentrations (low, medium and high nitrogen)
 WheatRiceSpinachTobacco
Low NMedium NHigh NLow NMedium NHigh NLow NMedium NHigh NLow NMedium NHigh N
  1. Leaf nitrogen, Rubisco, cytochrome f (cyt f), chlorophyll (Chl) and Chl a/b ratio were quantified. Data represent means ± SE, n = 4. Statistical analysis was examined within plant species. Different characters indicate significant differences (Tukey–Kramer's multiple comparison test, P < 0.05).

Nitrogen (mmol m−2)54.3 ± 2.5a113.3 ± 1.9b153.5 ± 3.3c75.2 ± 5.8a120.3 ± 5.2b162.1 ± 5.0c91.3 ± 8.1a142.0 ± 7.5b168.4 ± 5.4c41.9 ± 7.1a71.0 ± 1.9b110.3 ± 8.7c
Rubisco (µmol m−2)1.55 ± 0.08a3.09 ± 0.08b5.07 ± 0.26c2.44 ± 0.08a4.43 ± 0.09b7.58 ± 0.19c2.96 ± 0.22a4.87 ± 0.34b6.53 ± 0.23c1.35 ± 0.15a2.44 ± 0.06b4.15 ± 0.23c
Cyt f (µmol m−2)0.62 ± 0.02a1.11 ± 0.04b1.39 ± 0.05c0.69 ± 0.04a1.12 ± 0.02b1.49 ± 0.03c0.92 ± 0.04a1.30 ± 0.04b1.38 ± 0.06b0.51 ± 0.02a0.78 ± 0.02b0.83 ± 0.03b
Chl a + b (µmol m−2)273 ± 5a527 ± 7b669 ± 22c300 ± 18a520 ± 9b722 ± 14c322 ± 13a483 ± 16b581 ± 8c247 ± 11a412 ± 13b587 ± 18c
Chl a/b3.13 ± 0.03a3.17 ± 0.05a3.24 ± 0.04a3.91 ± 0.05a3.79 ± 0.02a3.77 ± 0.04a3.24 ± 0.03a3.04 ± 0.09ab2.84 ± 0.08b2.97 ± 0.09a2.99 ± 0.09a2.92 ± 0.02a

Temperature dependence of CO2 assimilation rate

A greater leaf N content correlated with an increased CO2 assimilation rate at a CO2 concentration of 380 µmol mol−1 (A380) at all leaf temperatures in all plant species (Fig. 1). The optimum temperature where A380 was maximal differed depending on the plant species, and was greater in rice and tobacco than in wheat and spinach. However, in all examined species, the optimum temperature was not affected by leaf N content.

image

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 plants grown at different nitrogen concentration (high N: open square, medium N: open triangle, low N: open circle) in wheat (a), rice (b), spinach (c) and tobacco (d). Data were fitted to cubic curves. Data represent means ± SE, n = 4.

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Effects of N concentration on mesophyll conductance (gm) and chloroplast CO2 concentration (Cc)

Stomatal conductance (gs) and mesophyll conductance (gm) were measured to estimate the intercellular (Ci) and chloroplast CO2 concentration (Cc) at ambient CO2 concentration of 380 µmol mol−1. The gm was estimated by a combination of gas-exchange and Chl fluorescence as well as by the A-Ci curve-fitting method (Supporting Information Fig. S1 shows a linear relationship between both methods). Greater leaf N content increased gs and gm in all plant species (Fig. 2). There was a relationship between A380 and gs, and between A380 and gm. The estimated Ci and Cc at ambient CO2 concentration of 380 µmol mol−1 were lower in plants grown at higher N content (Fig. 2). The changes in gs and gm as a result of a difference in leaf N content were smaller but correlated with the changes in A380. Therefore, the Ci/Ca, Cc/Ci and Cc/Ca ratios also decreased with increasing leaf N content in all plant species (Supporting Information Fig. S2).

image

Figure 2. Effects of leaf N content on stomatal conductance (gs: a), mesophyll conductance (gm: c), intercellular CO2 concentration (Ci: b) and chloroplast CO2 concentration (Cc: d) at 380 µmol mol−1 CO2 concentration at 1500 µmol photons m−2 s−1 at 25 °C. The correlation line is shown for each plant species. The coefficient of correlation (r) and significance of correlation (P) are 0.89 and <0.001 in rice, 0.96 and <0.0001 in wheat, 0.85 and <0.01 in spinach, and 0.81 and <0.01 in tobacco for Fig. 2a and 0.96 and <0.0001 in rice, 0.96 and <0.0001 in wheat, 0.77 and <0.01 in spinach, and 0.80 and <0.01 in tobacco for Fig. 2b and 0.91 and <0.0001 in rice, 0.87 and <0.001 in wheat, 0.91 and <0.001 in spinach, and 0.84 and <0.01 in tobacco for Fig. 2c and 0.99 and <0.0001 in rice, 0.97 and <0.0001 in wheat, 0.88 and <0.001 in spinach, and 0.80 and <0.01 in tobacco for Fig. 2d.

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Effects of N concentration on Jmax/Vcmax

The response of A and ETR to CO2 was analysed to determine the limiting step of A380. Figure 3 shows examples of this response measured at 25 and 40 °C in tobacco. A-Cc responses could be 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 examined in this study (see also Supporting Information Fig. S3 for A-Cc responses in other plant species).

image

Figure 3. Chloroplast CO2 concentration (Cc) response of CO2 assimilation rate (A) and the electron transport rate (ETR) from Chl fluorescence at 25 and 40 °C in tobacco grown at different nitrogen concentration (high N: open square, medium N: open triangle, low N: open circle). 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 3 in Materials & Methods. A and ETR at 380 µmol mol−1 CO2 concentration is shown as a solid symbol.

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A greater leaf N content decreased the ratio of the maximum electron transport rate to RuBP carboxylation rate (Jmax/Vcmax) (Fig. 4a) and the ratio of cyt f to Rubisco (cyt f/Rubisco) in all species (Fig. 4b). Although the Rubisco activation state decreased with increasing leaf N content in all species (Fig. 4c), the cyt f/carbamylated Rubisco ratio also decreased with increasing leaf N content (Fig. 4d). There were strong relationships between the cyt f/carbamylated Rubisco and Jmax/Vcmax in all species (Fig. 5), indicating that differences in Jmax/Vcmax could be mainly explained by N partitioning between electron transport components and Rubisco.

image

Figure 4. A Jmax/Vcmax ratio (a), cyt f/Rubisco ratio (b), Rubisco activation state (c) and cyt f/carbamylated-Rubisco ratio (d) versus leaf N content. The correlation line is shown for each plant species. The coefficient of correlation (r) and significance of correlation (P) are 0.92 and <0.0001 in rice, 0.85 and <0.001 in wheat, 0.94 and <0.0001 in spinach, and 0.85 and <0.01 in tobacco for Fig. 4a and 0.95 and <0.0001 in rice, 0.94 and <0.0001 in wheat, 0.93 and <0.0001 in spinach, and 0.96 and <0.0001 in tobacco for Fig. 4b and 0.71 and <0.05 in rice, 0.85 and <0.001 in wheat, 0.83 and <0.01 in spinach, and 0.74 and <0.05 in tobacco for Fig. 4c and 0.93 and <0.0001 in rice, 0.76 and <0.05 in wheat, 0.76 and <0.05 in spinach, and 0.92 and <0.0001 in tobacco for Fig. 4d.

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image

Figure 5. Relationship between Jmax/Vcmax ratio and cyt f/carbamylated-Rubisco ratio. The correlation line is shown for each plant species. The coefficient of correlation (r) and significance of correlation (P) are 0.92 and <0.01 in rice, 0.71 and <0.05 in wheat, 0.75 and <0.05 in spinach, and 0.82 and <0.01 in tobacco.

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Limiting step of CO2 assimilation rate from the analysis of A-Cc curve

The limiting step of A is affected by the following two factors: (1) the Jmax/Vcmax ratio and (2) Cc (Hikosaka et al. 2006). The present study showed that both factors changed with leaf N (Figs 2 & 4). The limiting step of A380 was analysed by comparing Cc at Ca = 380 µmol mol−1 with Ctransition, the chloroplast CO2 concentration at which the transition from Ac-limited A to Ar-limited A occurs (see, Materials and Methods). The Ctransition is mainly determined by the Jmax/Vcmax ratio. Ar limits A380 if Cc is less than Ctransition, whereas if Cc is greater than Ctransition, Ac is limiting (Fig. 6). Figure 7 shows examples of the temperature dependence of Cc and Ctransition in tobacco (see also Supporting Information Fig. S4 for the temperature dependence of Cc and Ctransition in other plant species). Both Cc and Ctransition for A380 decreased with leaf N content under all leaf temperatures. However, the observed reductions in Ctransition were always greater than those in Cc. The temperature dependence of Cc and Ctransition was similar irrespective of the leaf N content. In tobacco grown at low N concentrations, A380 above 25 °C was limited by Ac, whereas below 25 °C, A380 was limited by Ar (Fig. 7a). In tobacco grown at medium N concentration, A380 above 30 °C was co-limited by Ac and Ar, whereas below 30 °C, A380 was limited by Ar (Fig. 7b). In tobacco grown at high N concentration, A380 was solely limited by Ar at all leaf temperatures (Fig. 7c).

image

Figure 6. A scheme illustrating the shift in the limiting step of CO2 assimilation. Based on the C3 photosynthesis model, the CO2 assimilation rate at 380 µmol mol−1 CO2 (A380, closed symbols) is limited by RuBP carboxylation (Ac, solid line) or RuBP regeneration (Ar, broken line). Based on this model, the limiting step of CO2 assimilation rate is altered by the following two factors: (1) the balance of the maximum rate of RuBP regeneration (Jmax) to that of RuBP carboxylation (Vcmax), and (2) CO2 concentration in chloroplast. Shaded area shows that A380 is limited by Ar, whereas the unshaded areas indicate that A380 is limited by Ac. When Jmax/Vcmax is increased, chloroplast CO2 concentration at which the transition from Rubisco to RuBP regeneration limitation occurs (Ctransition) is increased, A380 tends to be limited by Ac (a versus b). When Cc for A380 is decreased, A380 tends to be limited by Ac (a versus c). Thus, increases in Jmax/Vcmax or decreases in Cc lead to a limitation of A380 by Ac (d).

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image

Figure 7. 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 tobacco grown at different nitrogen concentration (high N: square, medium N: triangle, low N: circle). Cc for A380 less than the Ctransition indicates that CO2 assimilation is limited by Ar, whereas Cc for A380 above Ctransition indicates that CO2 assimilation is limited by Ac (Ac limitation: inline image, Ac & Ar limitation: inline image, Ac limitation: inline image). Data represent means ± SE, n = 4.

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In the present study, we used Rubisco kinetic parameters as reported by Bernacchi et al. (2002). However, the possibility that Rubisco kinetic parameters are species specific should be borne in mind. Therefore, we performed a sensitivity analysis of the limiting step of A380 in tobacco leaves grown at medium N (Supporting Information Fig. S5) and analysed the effects of Rubisco kinetic parameters on the limiting step of A380, using three different Rubisco kinetic parameters: (1) Rubisco kinetic parameters obtained from Bernacchi et al. (2002); (2) the Rubisco kinetic parameters multiplied by 1.20; and (3) the Rubisco kinetic parameters multiplied by 0.80. The limiting step of A360 did not change depending on the Rubisco kinetic parameters that were used (Supporting Information Fig. S5). Therefore, we conclude that our analysis of the limiting steps for A380 is robust.

Figure 8 summarizes the differences in the limiting step for A380 depending on the plant species and growth N concentration. It is obvious that A380 tended to be limited by Ac in plants grown at low N concentration, whereas A380 tended to be limited by Ar in plants grown at high N concentration. Lower Cc caused a photosynthetic limitation by Ac, whereas lower Ctransition caused a photosynthetic limitation by Ar. Interestingly, lower leaf N content showed lower values for both Ctransition and Cc at Ca = 380 µmol mol−1 in all the plant species. Nevertheless, a lower leaf N content resulted in a limitation by Ac, indicating that the differences in Ctransition depending on leaf N content were an important factor determining the limiting step of A380. The limiting step of A380 varied with plant species, and A380 was mostly limited by Ar irrespective of leaf N content, especially in rice.

image

Figure 8. Summary of differences in the limiting step of the temperature dependence of A380 by RuBP carboxylation (Ac) or RuBP regeneration (Ar) by plant species and growth temperature. This figure was summarized from Fig. 7 and Supporting Information Fig. S4 (Ac limitation: inline image, Ac & Ar limitation: inline image, Ac limitation: inline image). The Jmax/Vcmax ratio and chloroplast CO2 concentration (Cc) at 25 °C were also summarized. Stars (★) indicate the optimum temperature for A380.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES
  10. Supporting Information

The limiting step of CO2 assimilation depends on N partitioning between photosynthetic components and is affected by leaf N content

The limiting step of CO2 assimilation is affected by the following two factors: (1) the balance of Jmax and Vcmax; and (2) CO2 concentration in chloroplast (Fig. 6; Hikosaka et al. 2006). A higher leaf N content was correlated with a lower Jmax/Vcmax ratio and may therefore lead to a greater limitation by RuBP regeneration. However, a higher leaf N content also resulted in a lower Cc, which limits the carboxylation step (Fig. 8). Thus, both factors affected A380 in a complimentary manner. The present study clearly showed that A380 tended to be limited by RuBP carboxylation in plants grown at low N concentration, whereas A380 tended to be limited by RuBP regeneration in plants grown at high N concentrations (Fig. 8). Therefore, the differences in the Jmax/Vcmax ratio as a result of an altered leaf N content were the most important factors for the determination of the limiting step of A380. Moreover, the present study clearly showed that the differences in the Jmax/Vcmax ratio were correlated with differences in the cyt f/Rubisco ratio (Fig. 5), indicating that N partitioning between components determining Jmax and Vcmax was responsible for limiting A380. This corresponds well to earlier results where the limiting step of A380 was analysed in plants grown at different growth temperatures (Yamori et al. 2010b) or at different growth light intensities (Yamori et al. 2010a). Plants grown at a low growth temperature had a higher Jmax/Vcmax ratio and their CO2 assimilation rate tended to be limited by RuBP carboxylation, whereas plants grown at a high growth temperature had lower Jmax/Vcmax ratio and their CO2 assimilation rate tended to be limited by RuBP regeneration (Yamori et al. 2010b). Growth light intensity did not alter the Jmax/Vcmax ratio and the limiting step of CO2 assimilation rate was independent of growth light intensity (Yamori et al. 2010a). Taken together, these results suggest that under various environmental conditions, the Jmax/Vcmax ratio and, thus, the cyt f/Rubisco ratio are the main determinants of limiting step of CO2 assimilation rate.

Previous studies of the A-Ci response of several species, including bean (von Caemmerer & Farquhar 1981, 1984), wheat (Evans 1986) and spinach (Evans & Terashima 1988), suggested that A380 was always co-limited by RuBP regeneration and carboxylation regardless of leaf N content. In these cases, an optimal distribution of N between RuBP regeneration and carboxylation allows for an efficient use of N in the production of photosynthetic proteins (von Caemmerer & Farquhar 1981; Hikosaka 1997). However, the present study showed that the limiting step of A380 was affected by leaf N content (Fig. 8). This conclusion is supported by experiments with antisense Rubisco mutants of tobacco which showed that the control coefficient of Rubisco increased when tobacco was grown under limiting N conditions (Quick et al. 1992; Fichtner et al. 1993). There have been few studies to quantitatively examine the limiting step of CO2 assimilation rate. As the accuracy of the photosynthesis model as well as the measurement methods have been improved recently (Ethier & Livingstone 2004; Sharkey et al. 2007; Bernacchi et al. 2009; von Caemmerer, Farquhar & Berry 2009), it is now possible to analyse the limiting step of CO2 assimilation rate at various conditions more robustly (e.g. Hikosaka et al. 2006; Yamori et al. 2006a, b, 2008, 2009, 2010a,b; Ishikawa, Onoda & Hikosaka 2007; Makino & Sage 2007; Sage & Kubien 2007; Kubien & Sage 2008). These analyses support the idea that A380 at the leaf level is not always co-limited by RuBP carboxylation and regeneration.

Our results suggest the existence of constraints on the N partitioning in the photosynthetic apparatus and imply that N investment may not always be optimal under natural conditions. The amount of Rubisco apparently does not limit A380 when abundant N is available. In fact, since the Rubisco content is often greater than what is required for photosynthesis under a wide range of conditions, it has been proposed that Rubisco has an important additional role as a storage protein (e.g. Huffaker & Peterson 1974; Staswick 1994; Stitt & Schulze 1994; Warren, Dreyer & Adams 2003; Warren & Adams 2004). The finding that the Rubisco activation state decreases with increasing leaf N content supports the idea that Rubisco can serve as a storage protein (Fig. 4c). An excess allocation of N to Rubisco might be advantageous in terms of N acquisition and reutilization. For example, after changes in the growth environment (e.g. from low to high light), such excess amounts of Rubisco can be immediately used to achieve higher rates of photosynthesis. In addition, it has been reported that N deficiency exacerbates the potential for photoinhibition in high light environments (Osmond 1983; Ferrar & Osmond 1986). This may partially explain why photosystems would easily be over-reduced and thus damaged in plants grown at low N, since the capacity for electron transport is greater than that of RuBP carboxylation. On the other hand, in plants with high leaf N content, photosystems would not be easily damaged, since in this case, the capacity for electron transport is lower than the capacity RuBP carboxylation, and leaf N would be expected to be invested in photoprotection processes. Thus, under abundant N conditions, the N partitioning for plants may be optimized to decrease the risk of damage to plants by various stresses.

The extent of photosynthetic limitation by RuBP regeneration was greater in rice than in other plants, irrespective of leaf N content (Fig. 8). This may be caused by the fact that N partitioning into Rubisco is greater in rice than in others (Table 1; Evans 1989; Makino et al. 1992; Nagai & Makino 2009; Yamori et al. 2009). Makino (2003) pointed out that Rubisco activity from rice is not efficient compared with other plants. In rice, to compensate for the inefficient Rubisco activity, N partitioning into Rubisco would be greater than in other species, leading to strong limitation by RuBP regeneration at the present atmospheric CO2 concentration.

It has been reported that Pi regeneration limits A380 as leaf N increases (Sage et al. 1990; Makino et al. 1994). However, a limitation of A380 by Pi regeneration was not observed at the growth temperatures used here, although the saturating CO2 concentration of the A at 15 °C tended to decrease with leaf N content especially in rice and tobacco (data not shown). Therefore, we expect that Pi regeneration rarely occurs at moderate temperatures, but it is possible that Pi regeneration may play a role in the regulation of CO2 assimilation at lower leaf temperatures (e.g. < 15 °C) especially in leaves grown at high N concentration (Sage et al. 1990; Makino et al. 1994).

Effects of leaf N content on Cc and on the Rubisco activation state

Several studies have implied that Cc and the Cc/Ci ratio vary with changes in leaf N content (Evans & Terashima 1988; Makino et al. 1992, 1994); however, few studies have tested this hypothesis, except in wheat (von Caemmerer & Evans 1991) and rice (Li et al. 2009) and, moreover, those two studies showed contradictory results. The present study showed that Ci, Cc, Cc/Ci ratio as well as Ci/Ca ratio was decreased with increasing leaf N content in all species, since increases in gs and gm with leaf N content were insufficient to compensate for changes in A380 (Fig. 2 & Supporting Information Fig. S2). This result was contrary to those reported by Li et al. (2009) who showed that Cc/Ci was higher in rice leaves with higher leaf N content. Considering that rice was used in both studies, the reason for the discrepancy between our results and the work by Li et al. (2009) is unclear. However, we showed that the Cc/Ci ratio decreases with increasing leaf N content irrespective of plant species using two independent methods, as has been recently recommended (for a review, see Pons et al. 2009).

The Rubisco activation state decreased with increasing leaf N content in all plant species (Fig. 4c). The exact mechanism that causes deactivation of Rubisco in plants with high leaf N content is still unclear. Rubisco activity is regulated by Rubisco activase that removes tight-binding inhibitors (Portis 2003; Parry et al. 2008). If N supply alters the Rubisco/activase ratio, the Rubisco activase content may be the limiting factor for the regulation of Rubisco activation. However, Rubisco activase content is not a limiting step for the regulation of steady-state Rubisco activation under mild temperature conditions (e.g. 25 °C: Mate et al. 1996; Hendrickson et al. 2008; Yamori & von Caemmerer 2009). Rubisco activase activity is regulated by the ATP/ADP ratio and redox state in the chloroplast (for a review, see Portis 2003). Therefore, alteration of Rubisco activation state may occur if N supply affects the ATP/ADP ratio and/or redox potential. The Rubisco activation state decreased with reductions in the Jmax/Vcmax ratio or the cyt f/Rubisco ratio (Supporting Information Fig. S6), indicating that the decline in the Rubisco activation state may be a regulated response to a limitation in electron transport capacity. This is supported by previous reports that transgenic tobacco with lower electron transport capacity showed lower Rubisco activation states (Ruuska et al. 2000; Yamori et al. 2011), and that plants with high leaf N content showed lower non-photochemical quenching (NPO), corresponding to lower ΔpH (Miyake et al. 2005), and a lower ATP/ADP ratio (Mächler et al. 1988) than plants with a low leaf N content.

It has previously been discussed why the relationship between A380 and leaf N is not completely linear (e.g. Evans 1989). At high leaf N concentration, A380 increases more slowly that at low leaf N concentrations. There are at least two factors that would cause such a non-linear relationship. One is that a relatively larger amount of Rubisco was inactivated at high leaf N (Fig. 4C), the other possibility is that the drop in CO2 concentration between the intercellular airspace and the chloroplast stroma (Ci–Cc) is larger at high leaf N, leading to lower Cc and thus lower A (Fig. 2 & Supporting Information Fig. S2). We analysed the effects of Rubisco deactivation and a significant drop in CO2 concentration (Ci–Cc) with increasing leaf N on the A (Supporting Information Fig. S7). Ac at a CO2 concentration of 380 µmol mol−1 at 25 °C was analysed under three assumptions: Ac-1) Rubisco is deactivated and Cc decreases with increasing leaf N; Ac-2) Rubisco is not deactivated with increasing leaf N, but Cc decreases with increasing leaf N; Ac-3) Rubisco is not deactivated with increasing leaf N and Cc remains constant irrespective of leaf N. These analyses indicated that if the deactivation of Rubisco and/or drop in CO2 concentration was not taken into account, Ac increased especially at higher leaf N in all the species, and Ac was more linearly related with leaf N (Supporting Information Fig. S7). Therefore, we conclude that the curvilinear relationship between A380 and leaf N was mainly caused by deactivation of Rubisco and by a drop in CO2 concentration between the intercellular airspace and the chloroplast stroma.

CONCLUSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES
  10. Supporting Information

The limiting step of the CO2 assimilation is affected by the following two factors: (1) the balance of Jmax and Vcmax; and (2) the CO2 concentration in chloroplast. A higher leaf N content leads to an increased Jmax/Vcmax ratio and to a strong limitation by RuBP regeneration. However, a high leaf N content also decreased chloroplast CO2 concentrations, resulting in a limitation of photosynthesis by RuBP carboxylation. Thus, the present study indicated that both factors affected the limiting step of A380 in a complimentary manner. However, our results further showed that the effect of leaf N on the differences in the Jmax/Vcmax ratio was more limiting to photosynthesis than the effect on the chloroplast CO2 concentration. In plants grown at higher N concentrations, A380 tended to be limited by RuBP regeneration. It is concluded that the limiting step of A380 depends on the N concentration and is mainly determined by the N partitioning between Rubisco and electron transport components. To facilitate an increase in photosynthesis and crop yield, genetic manipulation of photosynthesis has become a key target. The present study implies that the impact on photosynthesis by manipulation of one enzyme would differ depending on N nutrition.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES
  10. Supporting Information

We would like to thank Dr D. Tholen and Dr L. Irving for valuable comments on the manuscript. This work was supported by grants from the Japan Society for the Promotion (Science Postdoctoral Fellowships to W.Y and Scientific Research no. 20380041 to A.M.), from the Ministry of Education, Culture, Sports, Science and Technology, Japan (Scientific Research on Innovative Area no. 21114006 to A.M.) and from the Ministry of Agriculture, Forestry, and Fisheries of Japan (Genomics for Agricultural Innovation, GPN 0007).

REFERENCES

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  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES
  10. Supporting Information

Figure S1. Relationship between gm estimated from two independent methods; (1) combination method with gas-exchange and Chl fluorescence and (2) A-Ci curve-fitting method. The gm was estimated by combination analysis of gas-exchange and Chl fluorescence from the following equation (Harley et al., 1992; Loreto et al., 1992):

gm = A380/(Ci − Γ*(ETR380 + 8(A380 + Rd))/(ETR380 − 4(A380 + Rd)))

where A380 (μmol m−2 s−1) is the CO2 assimilation rate at 380 μmol mol−1 CO2, ETR380 (μmol m−2 s−1) is the ETR at 380 μmol mol−1 CO2, estimated from Chl fluorescence, Ci (μmol mol−1) is the intercellular CO2 concentration, 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. Rd was estimated from A-Cc curve at Γ* reported by Bernacchi et al. (2001).

Figure S2. Effects of leaf-N on the ratio between intercellular and ambient CO2 concentration (Ci/Ca), the ratio of chloroplast to intercellular CO2 concentration (Cc/Ci) and the ratio of chloroplast to ambient CO2 concentration (Cc/ Ca) in wheat (A), rice (B), spinach (C) and tobacco (D). The correlation line is shown in each plant species. The coefficient of correlation (r) and significance of correlation (P) are 0.95 and <0.0001 in Ci/Ca, 0.89 and <0.001 in Cc/Ci, and 0.99 and <0.0001 in Cc/Ca in wheat (A); 0.94 and <0.0001 in Ci/Ca, 0.97 and <0.0001 in Cc/Ci, and 0.97 and <0.0001 in Cc/Ca in rice (B); 0.92 and <0.001 in Ci/Ca, 0.89 and <0.001 in Cc/Ci, and 0.96 and <0.0001 in Cc/Ca in spinach (C); 0.94 and <0.0001 in Ci/Ca, 0.75 and <0.05 in Cc/Ci, and 0.95 and <0.0001 in Cc/Ca in tobacco (D).

Figure S3. Chloroplast CO2 concentration (Cc) response of CO2 assimilation rate (A) and the electron transport rate (ETR) from Chl fluorescence at 25 °C & 40 °C in plants grown at different nitrogen concentration (high N: open square, medium N: open triangle, low N: open circle). 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 3 in Materials & Methods. A and ETR at 380 μmol mol−1 CO2 concentration is shown as a solid symbol.

Figure S4. 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 plants grown at different nitrogen concentration (high N: square, medium N: triangle, low N: circle). Cc for A380 less than the Ctransition indicates that CO2 assimilation is limited by Ar, whereas Cc for A380 above Ctransition 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.

Figure S5. Sensitivity analyses of the limiting step of A380 in tobacco leaves grown at medium N. Effects of Rubisco kinetic parameters (Kc and Ko ) on the limiting step of A380 was analysed, using three different Rubisco kinetic parameters; (1) Rubisco kinetic parameters obtained from Bernacchi et al. (2002), (2) the Rubisco kinetic parameters multiplied by 1.20 and (3) the Rubisco kinetic parameters multiplied by 0.80. It was assumed that Γ* was unchanged between these analyses, since it has been reported that Kc and Ko was significantly different between rice and wheat but specificity factor of Rubisco (Sc/o ) was not significantly different (Makino et al. 1988).

Figure S6. Relationships between Jmax/Vcmax ratio and Rubisco activation state (A) and between cyt f/Rubisco ratio and Rubisco activation state (B). The correlation line is shown in each plant species. The coefficient of correlation (r) and significance of correlation (P) are 0.65 and <0.05 in rice, 0.72 and <0.05 in wheat, 0.76 and <0.05 in spinach, and 0.75 and <0.05 in tobacco for Fig. A and 0.77 and <0.01 in rice, 0.96 and <0.001 in wheat, 0.81 and <0.01 in spinach, and 0.87 and <0.01 in tobacco for Fig. B.

Figure S7. A relationship between CO2 assimilation rate and leaf-N. It was analysed why a relationship between CO2 assimilation rate and leaf-N was not always linear. There are at least two factors causing such a curvature relationship. One is that Rubisco was inactive with increasing leaf-N (Fig. 4C), the other is that there was a significant drop in CO2 concentration between the intercellular airspace and the chloroplast stroma (CiCc) with increasing leaf-N (Fig. 2 & Supporting Information Fig. S2). Then, we analysed effects of deactivation of Rubisco and a significant drop in CO2 concentration (CiCc) with increasing leaf-N on RuBP carboxylation rate (Ac) at 380 μmol mol−1 CO2 concentration at 25 °C. Ac is expressed as:

Ac = (Vcmax(Cc − Γ*))/(Cc + Kc(1 + O/Ko)) × R* − Rday,

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, R* is the Rubisco activation state, and Rday (μmol m−2 s−1) is the day respiration rate. Ac was analysed under three assumptions; (1) Rubisco deactivates and Cc decreases with increasing leaf-N as was observed in the present study (Ac-1: blue circle), (2) Rubisco does not deactivate with increasing leaf-N but Cc decreases with increasing leaf-N (Ac-2: purple triangle), (3) Rubisco does not deactivate with increasing leaf-N and Cc was similar to plants grown at low nitrogen concentration irrespective of leaf-N (Ac-3: green square). The correlation line is shown in each plant species.

Table S1. Day respiration (Rd ) estimated from A-Ci curve-fitting procedure at 25 °C. Data represent means ± SE, n = 4. Statistical analysis was examined within plant species. Different characters indicate significant differences (Tukey-Kramer&apos;s multiple comparison test, P < 0.05).

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PCE_2280_sm_FigureS5.pdf68KSupporting info item
PCE_2280_sm_FigureS6.pdf45KSupporting info item
PCE_2280_sm_FigureS7.pdf123KSupporting info item
PCE_2280_sm_TableS1.pdf90KSupporting info item

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