TY - JOUR
AU - Terashima, Ichiro
AB - Abstract In C4 photosynthesis, a part of CO2 fixed by phosphoenolpyruvate carboxylase (PEPC) leaks from the bundle-sheath cells. Because the CO2 leak wastes ATP consumed in the C4 cycle, the leak may decrease the efficiency of CO2 assimilation. To examine this possibility, we studied the light dependence of CO2 leakiness (φ), estimated by the concurrent measurements of gas exchange and carbon isotope discrimination, initial activities of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and pyruvate, orthophosphate dikinase (PPDK), the phosphorylation state of PEPC and the CO2 assimilation rate using leaves of Amaranthus cruentus (NAD-malic enzyme subtype, dicot) plants grown in high light (HL) and low light (LL). φ was constant at photon flux densities (PFDs) >200 μmol m−2 s−1 and was around 0.3. At PFDs <150 μmol m−2 s−1, φ increased markedly as PFD decreased. At 40 μmol m−2 s−1, φ was 0.76 in HL and 0.55 in LL leaves, indicating that the efficiency of CO2 assimilation at low PFD was greater in LL leaves. The activities of Rubisco and PPDK, and the phosphorylated state of PEPC all decreased as PFD decreased. Theoretical calculations with a mathematical model clearly showed that the increase in φ with decreasing PFD contributed to the decrease in the CO2 assimilation rate. It was also shown that the ‘conventional’ quantum yield of photosynthesis obtained by fitting the straight line to the light response curve of the CO2 assimilation rate at the low PFD region is seriously overestimated. Ecological implications of the increase in φ in LL are discussed. Introduction C4 plants perform efficient CO2 assimilation in warm and dry environments because they have a CO2-concentrating mechanism, the C4 cycle. In this cycle, CO2 is fixed by phosphoenolpyruvate carboxylase (PEPC) in the mesophyll cells, and the resultant C4 acid diffuses into the bundle-sheath (BS) cells and is subject to decarboxylation. By this mechanism, CO2 is concentrated around ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the BS cells. Not all of the CO2 released by C4 acid decarboxylation in the BS cell is fixed by Rubisco and some leaks back to mesophyll cells or intercellular spaces. Leaked CO2 may be refixed by PEPC. Because pyruvate, orthophosphate dikinase (PPDK) in the C4 cycle consumes two ATP molecules to regenerate one phosphoenolpyruvate (PEP) from pyruvate, some energy is wasted each time CO2 leaks out of the BS cells. Therefore, the excess CO2 leak decreases the efficiency of C4 photosynthesis, although to maintain a high CO2 concentration in BS cells some CO2 leakage may be inevitable. The ratio of the rate of CO2 leakage from BS cells to the rate of CO2 fixation by PEPC is defined as the CO2 leakiness (φ) and has been estimated by measuring the carbon isotope discrimination (Δ) during the CO2 exchange (Henderson et al. 1992, Cousins et al. 2006). Although φ remains constant over a wide range of CO2 concentrations and leaf temperatures, φ showed a tendency to increase when measured at low incident photon flux densities (PFDs) such as 300 (Kubásek et al. 2007), 240 (Henderson et al. 1992) and 150 μmol quanta m−2 s−1 (Cousins et al. 2006). However, φ has not been estimated at much lower PFDs. It is unknown whether φ continues to increase as PFD decrease below 150 μmol quanta m−2 s−1. For evaluation of the efficiency of photosynthesis, the initial slope of the light response curve of leaf photosynthesis, regarded as the maximum quantum yield of CO2 fixation, has been used extensively. Ehleringer and Pearcy (1983) investigated quantum yields of C4 monocots and dicots of various C4 subtypes and showed that the quantum yield was lower in NAD-malic enzyme (ME) subtype dicots than in others. They discussed that the lower quantum yield in NAD-ME dicots was probably due to the greater CO2 leakage. In contrast, Henderson et al. (1992) reported a fairly constant φ of 0.2 across the C4 subtypes. The cause of the difference in the quantum yield between C4 subtypes is not clear. The maximum quantum yield for CO2 fixation was usually obtained by fitting a straight line to the data points expressing relationships between the CO2 assimilation rate and PFD. If φ increases as PFD decreases, then quantum yield would also decrease as PFD decreases. Therefore, the ‘conventional’ quantum yields of C4 leaves themselves could be spurious. Although this possibility was theoretically suggested (Berry and Farquhar 1977, Farquhar 1983, von Caemmerer et al. 1997), it has not been verified. In our previous study with Amaranthus cruentus L. (NAD-ME subtype), we measured the stable carbon isotope ratio of leaf dry matter. The values were more negative in leaves grown in low light (LL) than in those grown in high light (HL), implying greater CO2 leakage at low PFDs. We suggested that the quantum yield in C4 plants may be affected by the increase in CO2 leakage at low PFDs (Tazoe et al. 2006). In this study, we measured the CO2 assimilation rate and φ in leaves of A. cruentus plants grown in HL and LL to understand in detail the effects of φ on the quantum yield in C4 leaves. A. cruentus was used because this species is an NAD-ME dicot, and NAD-ME dicots were suggested to have the lowest quantum yield and the highest CO2 leakage among C4 subtypes (Ehleringer and Pearcy 1983). Also, having large leaves, A. cruentus is suitable for accurate measurements of the CO2 assimilation rate and φ at very low PFDs. The measurements of CO2 assimilation rate and φ were conducted with a laboratory-constructed gas exchange system equipped with a large leaf chamber. Using this gas exchange system, we were able to determine accurately the light dependencies of φ and the CO2 assimilation rate. We also investigated the light dependencies of the activation states of key enzymes, the phosphorylation state of PEPC and the initial activities of Rubisco and PPDK, using HL and LL leaves. Based on these data, we discuss the carboxylation balance between the C3 and C4 cycles, which affects φ. Results CO2 assimilation rate and CO2 leakiness Fig. 1a shows the light responses of the net CO2 assimilation rates at an ambient CO2 partial pressure of 37 Pa. To investigate the CO2 assimilation rate, φ and activities of photosynthetic enzymes at the same PFD using the same leaf, we immediately froze the leaf in liquid nitrogen after the measurement of the CO2 assimilation rate at a given PFD. The leaf was allowed to attain the steady state at a given PFD (1–2 h) before the measurement of gas exchange rates, air sampling and freezing (see Materials and Methods). Three to six HL (or LL) leaves were used for each PFD. Thus, the panels in Fig. 1 are different from conventional figures in which data are obtained for the same leaf by changing the PFD. Also, note the change in scales of the abscissa near 400 μmol quanta m−2 s−1. The CO2 assimilation rate increased as the PFD increased. In HL leaves, CO2 the assimilation rate and stomatal conductance declined above 1,000 μmol quanta m−2 s−1 (Fig. 1a, data not shown). Fig. 1 View largeDownload slide Incident photon flux density (PFD) vs. (a) net CO2 assimilation rate, (b) ratio of intercellular to ambient partial pressure of CO2, pi /pa, (c) carbon isotope discrimination, Δ, and (d) CO2 leakiness, φ, at an ambient CO2 partial pressure of 37 Pa. φ was calculated from Equation 4 using the values of Δ and pi /pa. Open and filled symbols denote Amaranthus cruentus leaves grown in high (HL) and low light (LL), respectively. To detect differences between HL and LL leaves, Student's t-test was used. Asterisks indicate significant differences (P < 0.05) between HL and LL leaves. Data points are means ± SE; n = 3–6. Fig. 1 View largeDownload slide Incident photon flux density (PFD) vs. (a) net CO2 assimilation rate, (b) ratio of intercellular to ambient partial pressure of CO2, pi /pa, (c) carbon isotope discrimination, Δ, and (d) CO2 leakiness, φ, at an ambient CO2 partial pressure of 37 Pa. φ was calculated from Equation 4 using the values of Δ and pi /pa. Open and filled symbols denote Amaranthus cruentus leaves grown in high (HL) and low light (LL), respectively. To detect differences between HL and LL leaves, Student's t-test was used. Asterisks indicate significant differences (P < 0.05) between HL and LL leaves. Data points are means ± SE; n = 3–6. The ratio of the intercellular to ambient partial pressures of CO2 (pi/pa) increased as the PFD decreased below 150 μmol quanta m−2 s−1 in LL leaves (Fig. 1b). In HL leaves, this tendency was not apparent. pi/pa tended to be higher in LL leaves than in HL leaves. The differences between HL and LL leaves at 40 and 500 μmol quanta m−2 s−1 were statistically significant (Student's t-test; P < 0.05). Fig. 1c shows how carbon isotope discrimination (Δ) varied with PFD. Although Δ was constant at PFDs above 200 μmol quanta m−2 s−1, it increased at PFDs below 150 μmol quanta m−2 s−1. The Δ values were particularly high at PFDs <60 μmol quanta m−2 s−1 (Fig. 1c). Differences between HL and LL leaves were statistically significant at 80 and 200 μmol quanta m−2 s−1 (Student's t-test; P < 0.05). φ was calculated using Equation 4 (see Materials and Methods) from concurrent measurements of gas exchange rate and Δ. φ was constant at PFDs >200 μmol quanta m−2 s−1 and was around 0.3 (Fig. 1d). However, φ increased as PFDs decreased below 150 μmol quanta m−2 s−1. At 40 μmol quanta m−2 s−1, φ was 0.76 ± 0.06 (mean ± SE, n = 3) in HL and 0.55 ± 0.01 (n = 5) in LL leaves. At low PFDs, φ tended to be greater in HL leaves than in LL leaves. Differences between HL and LL leaves were statistically significant at 60, 80 and 200 μmol quanta m−2 s−1 (Student's t-test; P < 0.05). Rubisco and PPDK activities The initial activity of Rubisco was almost constant at PFDs above 500 μmol quanta m−2 s−1 (Fig. 2a). Activity decreased at PFDs below 250 μmol quanta m−2 s−1. The light dependence of initial activity was similar for both HL and LL leaves. The light dependence of the Rubisco activation state reflected that of the initial activity. At PFDs >500 μmol quanta m−2 s−1, approximately 90% of Rubisco sites were activated, while only 30% were activated at 40 μmol quanta m−2 s−1 (Fig. 2b). Fig. 2 View largeDownload slide (a) Initial activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) per unit leaf area, (b) Rubisco activation state (initial/total activity) and (c) initial activity of pyruvate, orthophosphate dikinase (PPDK) as a function of incident PFD. The PPDK activity was significantly greater in LL leaves than that in HL leaves at PFDs below 250 μmol quanta m−2 s−1 [two-way analysis of variance (ANOVA), P < 0.01]. The interaction of two-way ANOVA (factors: growth light condition and incident PFD) was statistically insignificant (P = 0.86). Data points are means ± SE; n = 3–10. For symbols, see Fig. 1. Fig. 2 View largeDownload slide (a) Initial activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) per unit leaf area, (b) Rubisco activation state (initial/total activity) and (c) initial activity of pyruvate, orthophosphate dikinase (PPDK) as a function of incident PFD. The PPDK activity was significantly greater in LL leaves than that in HL leaves at PFDs below 250 μmol quanta m−2 s−1 [two-way analysis of variance (ANOVA), P < 0.01]. The interaction of two-way ANOVA (factors: growth light condition and incident PFD) was statistically insignificant (P = 0.86). Data points are means ± SE; n = 3–10. For symbols, see Fig. 1. Light dependencies of the initial activity of PPDK are shown in Fig. 2c. The PPDK activity was almost constant at PFDs above 500 μmol quanta m−2 s−1, and decreased at PFDs below 250 μmol quanta m−2 s−1 (Fig. 2c). The PPDK activities were significantly greater in LL leaves than those in HL leaves at PFDs below 250 μmol quanta m−2 s−1 [two-way analysis of variance (ANOVA), P < 0.01]. The interaction between the growth light condition and the incident PFD was not statistically significant (two-way ANOVA, P = 0.86). Total activities of Rubisco and PPDK were very similar between HL and LL leaves (data not shown). The amounts of these enzymes in A. cruentus probably did not respond to growth light conditions as described in Tazoe et al. (2006). PEPC phosphorylation state Fig. 3 shows the light dependencies of the PEPC phosphorylation state. Western blotting was conducted using leaf samples obtained after illumination for 1–2 h. Each lane contained the same amount of soluble protein and thereby contained almost the same amount of PEPC (Fig. 3a, bottom). Western blotting with the primary antibody of the phosphorylated PEPC clearly showed that PEPC was dephosphorylated at low PFDs (Fig. 3a, top). The phosphorylated PEPC bands were quantified by image analysis software. The mean value of the phosphorylated PEPC at 1,000 μmol quanta m−2 s−1 in HL leaves was arbitrarily set at 100%, and the degree of PEPC phosphorylation is shown in Fig. 3b. At PFDs below 1,000 μmol quanta m−2 s−1, the degree of PEPC phosphorylation decreased with a decrease in PFD. Fig. 3 View largeDownload slide (a) Western blotting of the phosphorylated phosphoenolpyruvate carboxylase (PEPC) (top) and PEPC protein (bottom) in LL leaves obtained after 1–2 h of illumination at various incident PFDs. Each lane contains 4 μg of soluble protein. (b) Degree of PEPC phosphorylation as a function of incident PFD. The phosphorylated PEPC bands (a, top) were quantified by image analysis software, and the mean value at 1,000 μmol quanta m−2 s−1 in HL leaves was arbitrarily set at 100%. Data points are means ± SE; n = 3–5. For symbols, see Fig. 1. Fig. 3 View largeDownload slide (a) Western blotting of the phosphorylated phosphoenolpyruvate carboxylase (PEPC) (top) and PEPC protein (bottom) in LL leaves obtained after 1–2 h of illumination at various incident PFDs. Each lane contains 4 μg of soluble protein. (b) Degree of PEPC phosphorylation as a function of incident PFD. The phosphorylated PEPC bands (a, top) were quantified by image analysis software, and the mean value at 1,000 μmol quanta m−2 s−1 in HL leaves was arbitrarily set at 100%. Data points are means ± SE; n = 3–5. For symbols, see Fig. 1. Chlorophyll content and chlorophyll a/b ratio The Chl content was 0.37 ± 0.01 mmol m−2 (mean ± SE, n = 31) in HL leaves and 0.40 ± 0.01 mmol m−2 (n = 55) in LL leaves (Table 1). The estimated leaf absorptance values were 0.80 and 0.81 in HL and LL leaves, respectively. The Chl a/b ratio was lower in LL leaves than in HL leaves (Table 1). Table 1 Chl content, Chl a/b ratio and estimated leaf absorptance Growth light  Chl (mmol m−2)  Chla/b  Estimated absorptance  HL  0.37 ± 0.01a  4.73 ± 0.05a  0.80  LL  0.40 ± 0.01b  4.48 ± 0.03b  0.81  Growth light  Chl (mmol m−2)  Chla/b  Estimated absorptance  HL  0.37 ± 0.01a  4.73 ± 0.05a  0.80  LL  0.40 ± 0.01b  4.48 ± 0.03b  0.81  Data are means ± SE (Chl content and a/b ratio; n = 31–55). Different superscript letters denote significant differences between plants grown in different light conditions by Student's t-test (P < 0.001). Leaf absorptance was estimated from an empirical equation of a rectangular hyperbola expressing absorptance measured with an integrating sphere as a function of the Chl content per unit leaf area (Tazoe et al. 2006). View Large ‘Conventional’ quantum yield Fig. 4 shows relationships between the net CO2 assimilation rates and absorbed PFD in HL (Fig. 4a) and in LL leaves (Fig. 4b). The absorbed PFD was calculated using leaf absorptance values of 0.80 and 0.81 for HL and LL leaves, respectively (Table 1). Fig. 4 View largeDownload slide Absorbed PFD vs. measured (open and filled circles) and modeled CO2 assimilation rate (Amodel; gray triangles) in HL (a) and LL leaves (b). Amodel was calculated from Equation 10. Error bars of Amodel represent the maximum and minimum values, which were calculated with the variable m of 0 and 1, respectively. Solid and dashed lines represent the regression lines of the measured and modeled CO2 assimilation rate, respectively, which were fitted by the least squares method. Data points are means ± SE; n = 3–7 leaves. Absorbed PFD was calculated using leaf absorptance of 0.80 and 0.81 for HL and LL leaves, respectively, a rectangular hyperbola expressing leaf absorptance as a function of chlorophyll amount per unit leaf area (Tazoe et al. 2006). Fig. 4 View largeDownload slide Absorbed PFD vs. measured (open and filled circles) and modeled CO2 assimilation rate (Amodel; gray triangles) in HL (a) and LL leaves (b). Amodel was calculated from Equation 10. Error bars of Amodel represent the maximum and minimum values, which were calculated with the variable m of 0 and 1, respectively. Solid and dashed lines represent the regression lines of the measured and modeled CO2 assimilation rate, respectively, which were fitted by the least squares method. Data points are means ± SE; n = 3–7 leaves. Absorbed PFD was calculated using leaf absorptance of 0.80 and 0.81 for HL and LL leaves, respectively, a rectangular hyperbola expressing leaf absorptance as a function of chlorophyll amount per unit leaf area (Tazoe et al. 2006). Gray triangles show the modeled CO2 assimilation rate (Amodel), which were calculated by Equation 10 (see Materials and Methods) using φ in Fig. 1d. Vertical bars for the gray triangles represent the ranges between the maximum and minimum Amodel values, which were calculated assuming the maximum and minimum involvements of cyclic electron transport around PSI in ATP production. Amodel was very similar to the actual CO2 assimilation rate except for at 30 and 50 μmol absorbed quanta m−2 s−1 in HL leaves (Fig. 4a). The solid and dashed lines were obtained by the least squares method and their slopes correspond to the ‘conventional’ quantum yield. The values of the ‘conventional’ quantum yields obtained for the actual data were 0.053 and 0.054 mol CO2 mol−1 quanta in HL and LL leaves, respectively (Table 2). φ estimated from these ‘conventional’ quantum yields for the actual gas exchange data using Equation 11 (see Materials and Methods) were 0.44 and 0.42 in HL and LL leaves, respectively (Table 2). The ‘conventional’ quantum yields calculated for Amodel (gray triangles) by the least squares method were higher, and φ estimated from these ‘conventional’ quantum yields were much lower than those calculated with the actual data (Fig. 4, Table 2). Probably reflecting the increase in φ with the decrease in PFD, neither the actual data of net CO2 assimilation rate nor the Amodel calculated by Equation 10 using the measured φ were on straight lines. Both of them showed a somewhat concave dependence on the absorbed PFD. Table 2 Regression equations fitted by least squares method to the data for net CO2 assimilation rate, A, and modeled CO2 assimilation rate, Amodel, as a function of absorbed PFD, and estimated φ Growth light  Regression line  Slope (quantum yield) (mol CO2 mol−1 quanta)  R2  Estimated φ  HL (Fig. 2a)  Amodel (dashed line)  0.064  1.00  0.15    A (solid line)  0.053  0.98  0.44  LL (Fig. 2b)  Amodel (dashed line)  0.061  1.00  0.25    A (solid line)  0.054  1.00  0.42  Growth light  Regression line  Slope (quantum yield) (mol CO2 mol−1 quanta)  R2  Estimated φ  HL (Fig. 2a)  Amodel (dashed line)  0.064  1.00  0.15    A (solid line)  0.053  0.98  0.44  LL (Fig. 2b)  Amodel (dashed line)  0.061  1.00  0.25    A (solid line)  0.054  1.00  0.42  Data are shown in Fig. 4. φ was estimated from Equation 11. View Large Discussion Light responses of CO2 leakiness, CO2 assimilation rate and activities of key enzymes In this study, we clearly showed that φ increased at PFDs below 150 μmol quanta m−2 s−1 (Fig. 1d). The extent of the increase in φ at very low PFDs tended to be greater in HL leaves than in LL leaves. This result indicates that the photosynthetic efficiency at low PFD was greater in LL leaves, although it is generally accepted that C4 plants have less potential to acclimate to low PFD than C3 plants (Sage and McKown 2006). Henderson et al. (1992) suggested a possible mechanism for the increase in φ at low PFD as follows. At low PFD, the CO2 concentration in BS cells would be lower than that at high PFD (Furbank and Hatch 1987). If this were the case, the ratio of ribulose 1,5-bisphosphate (RuBP) oxygenation to carboxylation in the BS cells would increase and extra light energy would be needed for the photorespiratory cycle. Then, the CO2 fixation rate in BS cells would be reduced compared with the CO2 fixation rate in the C4 cycle, and the extra CO2 which is not fixed by Rubisco may leak from the BS cells. Although this hypothesis was supported by a C4 model (Laisk and Edwards 2000), it has not been experimentally tested. In the present study, we examined the light responses of the phosphorylation state of PEPC and the activities of Rubisco and PPDK simultaneously to obtain insight into the carboxylation balance between C3 and C4 cycles. In LL leaves, φ at very low PFD was lower and PPDK activity was higher than in HL leaves (Figs. 1d, 2c). The higher activity of PPDK in LL leaves would infer the greater PEP pool size. In Amaranthus edulis (NAD-ME subtype), the PEP pool size at low PFDs was not small (Leegood and von Caemmerer 1988). If this applies to A. cruentus, the regeneration of PEP by PPDK might not limit the CO2 assimilation at low PFDs. Then, the difference in PPDK activity might not be responsible for the difference in φ at low PFDs between HL and LL leaves. The PPDK activity is known to be affected by the ATP/ADP ratio and light levels (Usuda 1988, Nakamoto and Young 1990). Furthermore, Nakamoto and Edwards (1986) reported that PPDK activity was inhibited by DCMU and antimycin A, which are inhibitors of linear electron transport and cyclic electron transport around PSI, respectively. These reports showed that PPDK activity is very sensitive to photosynthetic electron transport or the energy status. The higher chlorophyll contents and absorptance in LL leaves, which lead to the higher electron transport rate and/or higher energy state, could partly explain why the PPDK activity was greater than that in HL leaves at low PFDs (Table 1). It is also possible that the contents of metabolites such as pyruvate and PEP affect PPDK activity, but the effects of these metabolites on PPDK in vivo activity are still unclear (Roeske and Chollet 1989, Nakamoto and Young 1990, Leegood and Walker 1999). We need further investigations to clarify the mechanisms of regulation of the PPDK activity. The Rubisco activation state decreased linearly at PFDs below 250 μmol quanta m−2 s−1 (Fig. 2b), which was similar to an observation in Amaranthus retroflexus (Sage and Seemann 1993). Sage and Seemann (1993) also showed that the RuBP pool size was constant at PFDs below 500 down to 50 μmol quanta m−2 s−1, and it was high enough to maintain CO2 assimilation at low PFDs. It appears that the activation state of Rubisco is regulated to maintain the RuBP pool size. Therefore, CO2 assimilation at low PFDs may not be limited by the availability of RuBP. In other words, the RuBP pool size per se may not affect φ at low PFDs. The in vivo activity of PEPC is regulated by the reversible phosphorylation of a serine residue near the N-terminus in response to PFD (Izui et al. 2004). At high PFDs, PEPC is phosphorylated in vivo by a protein kinase specific to PEPC (PEPC-PK), and phosphorylated PEPC is more active because it becomes less sensitive to its inhibitors, such as malate and aspartate (Chollet et al. 1996). On the other hand, at low PFDs, PEPC is dephosphorylated and it becomes more sensitive to malate and aspartate. In this study, PEPC was phosphorylated at high PFDs, and it was dephosphorylated with a decrease in PFD (Fig. 3), which was similar to the trend revealed by an observation in Panicum maximum (Bailey et al. 2007). If the concentration of aspartate is extremely high at low PFD as described by Leegood and von Caemmerer (1988), the in vivo activity of the dephosphorylated PEPC would be mostly inhibited by aspartate. Therefore, at low PFDs, CO2 fixation by PEPC would be suppressed and the CO2 concentration in the BS cells would be lowered, leading to the increase in φ at low PFD. However, Furumoto et al. (2007) found that in PEPC-PK antisense transgenic Flaveria bidentis plants, PEPC was dephosphorylated even at high PFDs and the plants showed high rates of CO2 fixation comparable with those of the wild type. Perhaps the phosphorylation state of PEPC may not be essential for the C4 photosynthesis. In summary, φ was constant at PFDs above 200 μmol quanta m−2 s−1, but increased at PFDs below 150 μmol quanta m−2 s−1. As PFD decreased, the Rubisco activation state, PPDK initial activity and PEPC phosphorylation state decreased, which indicated that the carboxylation in C3 and C4 cycles simultaneously decreased. Currently, our working hypothesis to be tested is as follows. At PFDs below 150 μmol quanta m−2 s−1, the reduction of carboxylation in C4 cycle causes the decline of CO2 partial pressure in BS cells (ps), and this leads to the occurrence of RuBP oxygenation in addition to RuBP carboxylation in the BS cells. Since the CO2 fixation rate at low PFD is limited by the electron transport rate, the increase in ATP consumption for photorespiration leads to a substantial reduction in the carboxylation efficiency in BS cells. As a result, the carboxylation imbalance between C3 and C4 cycles causes the increase in φ at low PFDs. If the photosynthetic electron transport rate and/or the energy status in LL leaves is higher than in HL leaves, it will help to sustain the carboxylation efficiency in BS cells, which may contribute to the suppression of φ at low PFDs. Quantum yield The quantum yield of CO2 assimilation has been widely used for evaluating the efficiency of photosynthesis at low PFDs in C3 and C4 plants (Ehleringer and Björkman 1977, Ehleringer and Pearcy 1983, Monson et al. 1982). In C4 leaves, the quantum yield has also been used for estimating φ (Furbank et al. 1990). Our results clearly showed that φ increased at PFDs below 150 μmol quanta m−2 s−1 (Fig. 1d). We also analyzed the effects of φ on the quantum yield of CO2 assimilation with a simple model. The ‘conventional’ quantum yields calculated for the actual data of net CO2 assimilation rates were 0.053 and 0.054 mol CO2 mol−1 quanta in HL and LL leaves, respectively (Fig. 4, Table 2), which were very similar to the quantum yields in NAD-ME dicots (0.053 ± 0.001 mol CO2 mol−1 quanta) reported by Ehleringer and Pearcy (1983). φ estimated from these quantum yields were 0.44 and 0.42 in HL and LL leaves, respectively (Table 2), which were different from the measured φ at low PFDs (Fig.1d). Given that φ clearly decreased as PFD increased, the estimation of φ from the ‘conventional’ quantum yield, giving a unique value of φ, is spurious. The actual CO2 assimilation rate in C4 leaves may be influenced not only by φ, but also by other factors such as photorespiration in BS cells. If photorespiration occurs at low PFDs as suggested by Henderson et al. (1992), the ATP requirement for RuBP regeneration will be >3 ATP molecules (Equation 5), which leads to a further decrease in the efficiency of C4 photosynthesis. To investigate the effect of the increase in φ on the quantum yield separately from those of other effects, we calculated Amodel (gray triangles in Fig. 4) from the measured φ (Fig. 1d) using a mathematical model (Equation 10). The ‘conventional’ quantum yields for the Amodel were 0.064 and 0.061 mol CO2 mol−1 quanta, and φ estimated from these quantum yields were 0.15 and 0.25 in HL and LL leaves, respectively (Table 2). Estimated φ were much lower than the actual φ values (Fig. 1d). Obviously, the decrease in φ with the increase in PFD led to the overestimation of the ‘conventional’ quantum yields. Judging from previous reports indicating the increase in φ at moderately low PFDs (Henderson et al. 1992, Cousins et al. 2006, Kubásek et al. 2007), the increase in φ at low PFDs would generally apply to other C4 plants of all subtypes and the ‘conventional’ quantum yields would be generally overestimated. Therefore, we need to re-examine ‘conventional’ quantum yields in C4 plants. Although Amodel were mostly very similar to the actual CO2 assimilation rates (Fig. 4), those at 30 and 50 μmol absorbed quanta m−2 s−1 were lower than the actual CO2 assimilation rates, in particular for the HL leaves. The lower Amodel values at very low PFDs were due to the large φ. Perhaps φ values at very low PFDs were overestimated because the ratio of the partial pressure of CO2 in mesophyll cells (pm) to ps is not very small. Equation 4 can be used for the estimation of φ only when ps is much greater than pm. However, at low PFDs, the CO2 assimilation rates were lower (Fig. 1a) and pi were higher (Fig. 1b) than those at high PFDs, leading to the increase in pm. In such a case, the effects of pm/(ps – pm) on φ cannot be ignored and ps/(ps – pm) will become much greater than 1 (see Equation 3). When φ is calculated using Equation 3 assuming pi = pm and ps = 211 Pa (≈70 μM, Hatch 1992), φ is lower than the φ estimated from Equation 4 by about 10%. If ps is lower than 211 Pa at low PFDs because of the low CO2 fixation rate by PEPC, φ will be even lower. Therefore, it is probable that, at very low PFDs, φ obtained using Equation 4 was overestimated. Nevertheless, since Δ values calculated from the carbon isotope compositions using Equation 1 were much higher at very low PFDs than those at high PFDs (Fig. 1c), the trend for φ to increase as PFD decreases must be true. It has been thought that CO2 leak would be influenced by anatomical characteristics in C4 leaves, for example suberized lamellae in BS cell walls, location of the BS chloroplasts and mitochondria, and interveinal distance (Ehleringer and Pearcy 1983, Ehleringer et al. 1997, Ogle 2003). Due to the clear difference in the ‘conventional’ quantum yield between NAD-ME dicots and other C4 subtypes and monocots, it has been believed that NAD-ME dicots have more leaky anatomy, e.g. absence of suberized BS cell walls and greater interveinal distance. In contrast, Hatch et al. (1995) estimated the CO2 leak rate of C4 monocots and dicots of various C4 subtypes, and showed that the anatomical characteristics did not relate to the differences in the CO2 leak rate. The influence of anatomical characteristics in C4 leaves on the CO2 leak is still unclear. However, the ‘conventional’ quantum yield cannot be used for estimating φ as mentioned above, because φ varies with PFD. As our model showed, the increase in φ as PFD decreased led to a steeper initial slope of the light response curve, which could influence the ‘conventional’ quantum yield. To investigate the influence of anatomical characteristics on φ, one should compare φ at high PFDs using the carbon isotope method. In the surveys of quantum yields for CO2 assimilation in various C3 and C4 plants, the average quantum yield in C4 plants was much greater than that in C3 plants at 30°C (Monson et al. 1982, Ehleringer and Pearcy 1983). It was concluded that C4 plants have higher efficiency of photosynthesis at low PFDs than C3 plants in warm environments. There are, however, only a few shade-tolerant C4 species, whereas there are many shade-tolerant C3 species in such habitats (Sage and McKown 2006). This discrepancy has been discussed, but it has not been clearly explained. In this study, we found that the efficiency of C4 photosynthesis declined at low PFDs because φ increased. In conclusion, φ increased at PFDs below 150 μmol quanta m−2 s−1, although φ might be somewhat overestimated at very low PFDs. The increase in φ in LL leaves was smaller than that in HL leaves, indicating a new aspect of shade acclimation in C4 photosynthesis. The increase in φ led to the decrease in CO2 assimilation rate, and this led to the overestimation of the ‘conventional’ quantum yield and the underestimation of φ. Further studies using the carbon isotope method will help us to understand the mechanisms of the increase in φ at very low PFDs. At the same time, we also need to estimate pm and ps, and the involvement of photorespiration. Materials and Methods Plant materials and growth conditions Seeds of A. cruentus L. were sown in vermiculite in pots (120 mm diameter and 200 mm in height, one plant per pot). The plants were grown under HL and LL conditions in a greenhouse, as in our previous study (Tazoe et al. 2006). For the LL conditions, a frame box (about 1 mm3), whose top and three lateral sides (except the northern side) were covered with layers of black shade cloth, was used. The midday PFD just above the plants was approximately 1,200 μmol quanta m−2 s−1 in HL, while it was 260 μmol quanta m−2 s−1 in LL. Temperatures in the greenhouse during the plant growth were 27.2 ± 6.4°C (mean ± SD) in the day and 21.2 ± 2.1°C at night. Plants were given 100 ml of a nutrient solution containing 2 mM KNO3, 2 mM Ca(NO3)2, 0.75 mM MgSO4, 0.665 mM NaH2PO4, 25 μM Fe-EDTA, 5 μM ZnSO4, 0.5 μM CuSO4, 25 μM H3BO4, 0.25 μM Na2MoO4, 50 μM NaCl and 0.1 μM CoSO4, which was about half the strength of the Hoagland's solution, every other day. Young fully expanded leaves of 2- to 3-month-old plants were selected for the measurements. Gas exchange and carbon isotope measurements Gas exchange measurements were conducted with a laboratory-constructed system as described by Hanba et al. (1999) with some modifications. A leaf chamber was a 0.3 l, 150 mm × 100 mm × 20 mm (depth), aluminum box with a glass window. The chamber was equipped with two small fans to agitate the air, and one copper–constantan thermocouple to monitor the leaf temperature. The air temperature inside the chamber was regulated by circulating water in a jacket attached to the chamber, and the leaf temperature was kept at 32°C. The air temperature in the laboratory was about 28.5°C. Light was provided by a high power metal halide lamp (PCS-UMX250, NPI, Tokyo, Japan), and PFD was measured using a quantum sensor (LI-190SB, Li-Cor, Lincoln, NE, USA). The flow rate of air entering the chamber was monitored with a mass-flow meter and kept at 500 ml min−1. Concentrations of H2O and partial pressure of CO2 in the gas were measured with an infrared gas analyzer (LI-7000, Li-Cor, Lincoln, NE, USA). CO2 partial pressure in the air leaving the leaf chamber was maintained at 37 Pa. Before the measurements, the plants in pots were kept in the dark for at least 18 h. Subsequently, the gas exchange rates of attached leaves were measured at various PFDs. Gas exchange parameters were calculated according to von Caemmerer and Farquhar (1981). Carbon isotope discriminations (Δ) of the CO2 samples collected from the air leaving the leaf chamber with and without a leaf were measured as described by von Caemmerer and Evans (1991) with some modifications. The carbon isotope ratio of the collected CO2 was analyzed with a dual-inlet mass spectrometer (MAT252, Finnigan MAT, Bremen, Germany). After the leaf photosynthesis reached its steady state (1–2 h), the air leaving the chamber was passed through a vacuum line at a rate of 500 ml min−1 for 6 min to trap the CO2 in a sample tube using cold traps. The carbon isotope ratio of the collected CO2 was little affected by the changes in the flow rate or trapping time. Because the mass of N2O is similar to that of CO2, N2O interferes with the analysis of carbon isotope composition. Therefore, the CO2 sample was enclosed in the sample tube with 0.1 g of reduced copper to prevent the effects of N2O. Calculation of carbon isotope discrimination and CO2 leakiness (Δ) was calculated using Equation 1 (Evans et al. 1986).   (1) where δe and δo are the carbon isotope compositions of the air entering and leaving the leaf chamber, respectively, when a leaf is enclosed. ξ = pe/(pe − po), and pe and po are the CO2 partial pressures of the air entering and leaving the chamber, respectively. The precision of Δ is greatly affected by the ξ value (von Caemmerer and Evans 1991). As recommended by Henderson et al. (1992), the ξ value was kept between 3 and 10 by adjusting leaf sizes to determine φ accurately at low PFDs. For gas exchange measurements at 40 and 60 μmol quanta m−2 s−1, two leaves were enclosed in the leaf chamber. Averages of ξ values at 40 and 60 μmol quanta m−2 s−1 were 9.4 and 7.1, respectively. CO2 leakiness (φ) was estimated using the equation proposed by Farquhar (1983):   (2) where ps, pm, pi and pa are partial pressures of CO2 in BS cells, mesophyll cells, intercellular spaces and ambient air, respectively. a is the fractionation during diffusion of CO2 in air through the stomatal pore (4.4‰), b3 is the fractionation by Rubisco (30‰) and s is the fractionation during CO2 leakage from the BS cells (1.8‰). b4 is the combined fractionation by PEPC (2.2‰), by the equilibrium during dissolution of CO2 into water and by conversion to bicarbonate. Taking account of temperature dependencies of the last two processes, b4 of –5‰ was used for estimation of φ at 32°C (Mook et al. 1974, Henderson et al. 1992). Equation 2 can be solved for φ:   (3) Since CO2 is fixed by PEPC in the mesophyll cells and concentrated in the BS cells at high PFDs, ps will be much higher than pm. In such cases, pm/(ps − pm) will be close to 0 and ps/(ps − pm) will be close to 1, then Equation 3 can be simplified to:   (4) Measurements of enzyme activities After the measurements of photosynthesis, leaf discs, 1 cm in diameter, were rapidly frozen in liquid nitrogen (within 1 min) and kept at −80°C for the analysis of enzyme activities. The frozen leaf disks were homogenized in an extraction buffer [50 mM HEPES-KOH at pH 7.4, 1 mM EDTA-2Na, 5 mM dithiothreitol (DTT), 2 mM iodoacetic acid, 10 mM MgCl2, 20% (v/v) glycerol, 2 mM pyruvate, 1 mM phenylmethylsulfonyl fluoride, the Complete Protease Inhibitor (Roche Applied Science, Mannheim, Germany) and 0.1% Triton X-100]. The homogenate was centrifuged at 16,000×g for 30 s at 4°C. The supernatant was used for the measurement of the amount of soluble protein, Western blotting and the assay of enzyme activities. The Rubisco activity was assayed as described by Sawada et al. (1990) with some modifications. The initial Rubisco activity was assayed at 32°C immediately after the extraction (within 5 min after extraction) by adding 50 μl of the supernatant to 840 μl of a Rubisco assay medium (100 mM Bicine-KOH at pH 8.2, 20 mM MgCl2, 20 mM NaHCO3, 5 mM creatine phosphate and 5 mM ATP-2Na), 35 μl of coupling enzymes (10 U ml−1 of phosphocreatine kinase, 25 U ml−1 of glyceraldehyde 3-phosphate dehydrogenase and 10 U ml−1 of phosphoglycerate kinase), 30 μl of 2 mM NADH and 45 μl of 13 mM RuBP. As a control, changes in the absorptance at 340 nm in the Rubisco assay medium without RuBP were measured. Total Rubisco activity was assayed after activating the supernatant of the extraction in an activation medium (100 mM Bicine-KOH at pH 8.2, 100 mM MgCl2 and 200 mM NaHCO3) for 20 min on ice. The PPDK activity was assayed according to the method of Fukayama et al. (2001). The initial PPDK activity was assayed at 32°C immediately after the extraction by adding 50 μl of the supernatant to 913 μl of a PPDK assay medium (50 mM HEPES-KOH at pH 8.0, 10 mM MgCl2, 10 mM NaHCO3, 5 mM glucose-6-phosphate, 10 mM DTT, 2.5 mM KH2PO4, 2 mM pyruvate, 0.2 mM NADH, 0.1 mM EDTA, 5 mM NH4Cl), 2 μl of 6,000 U ml−1 malate dehydrogenase (from swine heart; Toyobo Enzymes, Osaka, Japan) and 10 μl of 50 U ml−1 PEPC (from maize; Biozyme, Blaenavon, UK), and the reaction was started by adding 25 μl of 50 mM ATP. Determination of chlorophyll and soluble protein Chl was extracted from the leaf discs with N,N-dimethylformamide, and the content of Chl and the Chl a/b ratios were determined according to Porra et al. (1989). Using the Chl contents, leaf absorptance was estimated from the empirical rectangular hyperbola expressing the absorptance measured with an integrating sphere as a function of the Chl contents per unit leaf area (Tazoe et al. 2006). The soluble protein content in the extraction was determined using a protein assay kit based on the Bradford method (Bio-Rad Protein Assay, Bio-Rad, Hercules, CA, USA). Western blotting for PEPC The supernatant was mixed with an equal amount of a double-strength loading buffer [50 mM Tris–HCl at pH 6.8, 10% (w/v) lithium dodecylsulfate, 10% β-mercaptoethanol, 0.2% bromophenol blue and 10% glycerol] and treated at 95°C for 5 min. Gel electrophoresis was performed with a 7.5% polyacrylamide gel according to Laemmli (1970). Amounts of loading samples were adjusted from 4 to 24 μg of soluble proteins to visualize weak bands. After electrophoresis, the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Hybond-P, Amersham Biosciences, Buckinghamshire, UK), and blocked with blocking reagent (Blocking One, Nakarai tesque, Kyoto, Japan). The primary antibody was the anti-phosphorylated PEPC, which was prepared in the same way as described in Ueno et al. (2000) except for the use of a synthetic peptide that mimicked the phosphorylated maize C4-PEPC as an antigen. The antibody can recognize phosphorylated PEPCs prepared from various C4 species, including A. cruentus (T. Furumoto and K, Izui, in preparation). The secondary antibody was the peroxidase-linked species-specific whole antibody (ECL Anti-rabbit IgG, Amersham Biosciences). These antibodies were diluted in the immunoreaction enhancer solution (Can Get Signal, Toyobo Enzymes). The phosphorylated PEPC bands were detected as chemiluminescence with the ECL Plus detection reagents (Amersham Biosciences) using the LAS-1000 (Fuji Film, Tokyo, Japan). The chemiluminescent signals were measured with the image analysis software (Image J, public domain software, developed at US National Institutes of Health, available at http://rsb.info.nih.gov/ij/) to quantify the degree of PEPC phosphorylation. The degree of phosphorylation of PEPC was expressed using the most phosphorylated PEPC as the standard. After the immunodetection of the phosphorylated PEPC, the PVDF membranes were incubated in a stripping buffer (62.5 mM Tris–HCl at pH 6.7, 2% SDS and 100 mM β-mercaptoethanol) for 30 min at 50°C to remove the primary and secondary antibodies from the membranes. The PVDF membranes were reprobed with the anti-PEPC raised against maize C4-specific PEPC (a generous gift from Professor H. Nakamoto). The secondary antibody used was the goat anti-rabbit IgG (H+L) alkaline phosphatase conjugate (Bio-Rad). The PEPC bands were detected with the alkaline phosphatase color development reagents (BCIP/NBT Solution, Bio-Rad). The model to calculate the quantum requirement From the measured φ, the CO2 assimilation rate was estimated using a model of the quantum requirement (Siebke et al. 1997) with some modifications. In C4 photosynthesis, three molecules of ATP and two of NADPH will be required for the production of triose phosphate and regeneration of RuBP in the C3 cycle, if there is no photorespiration. In addition, two molecules of ATP for the PEP regeneration are needed and the ATP requirement increases with the increase in φ. If there is no photorespiration, the ATP requirement for 1 mol of CO2 uptake can then be calculated by the following equation.   (5) Allen (2003) claimed that 14 protons were required for the synthesis of three molecules of ATP used in the C3 cycle. Assuming full operation of the Q cycle, the linear electron transport through PSII and PSI generates a gradient corresponding to 12 protons across the thylakoid membrane with 8 quanta. If the other two protons for the synthesis of three molecules of ATP in the C3 cycle are generated only by the linear electron transport, an additional 0.67 quanta are required in PSII. Similarly, 3.11 quanta are required in PSII for the synthesis of two molecules of ATP used in the C4 cycle. Therefore, the quantum requirement in PSII is the sum of 4 quanta for 2 NADPH and 2.57 ATP syntheses, 0.67 quanta for 0.43 ATP synthesis and 3.11 quanta for the extra 2 ATP in the C4 cycle:   (6) ATP can be also produced by the cyclic electron transport around PSI. m is the ratio of the rate of linear electron transport to the total rate of the linear electron transport and the cyclic electron transport around PSI for the synthesis of 0.43 ATP used in the C3 cycle plus 2/(1 – φ) ATP used in the C4 cycle, and varies between 0 and 1. The cyclic electron transport around PSI generates a gradient corresponding to two protons with one quantum assuming full operation of the Q cycle. If the other two protons required for the synthesis of three molecules of ATP in the C3 cycle are generated only by the cyclic electron transport, one quantum is required in PSI. Similarly, 4.66 quanta are required in PSI for the synthesis of 2 ATP used in the C4 cycle. Therefore, the quantum requirement in PSI is the sum of 4 plus 0.67 m quanta (linear electron transport) plus (1 – m) quanta (cyclic electron transport around PSI) for synthesis of three ATP molecules in the C3 cycle. For an extra two ATP molecules in the C4 cycle, 3.11 m plus 4.66 (1 – m) quanta are required:   (7) Siebke et al. (1997) obtained m of 0.5 from the measurements of quantum requirements of PSII and PSI. This indicates that the cyclic electron transport around PSI and the linear electron transport contributed equally to the production of extra ATP. By combining Equations 6 and 7 and substituting 0.5 into m, the total quantum requirement per CO2 is given by:   (8) Thus, if φ is zero, 14.61 quanta are needed for 1 mol CO2 assimilation in C4 photosynthesis. A total of 14.61 quanta were similar to the value of 14.3 quanta calculated in the mathematical model of C4 photosynthesis (Laisk and Edwards 2000). The quantum yield of CO2 fixation (ΦCO2) can be calculated from Equation 8:   (9) Amodel is calculated from ΦCO2, absorbed PFD and the rate of dark respiration (Rd):   (10) Rd was assumed to be 0.42 μmol CO2 m−2 s−1, which was the average rate of dark respiration in HL and LL leaves. φ has usually been estimated from ΦCO2. Equation 9 can be solved for φ as follows:   (11) Funding The 21st Century Center of Excellence Program; Grants-in-Aid for Scientific Research of the Japan Society for the Promotion of Science (16207002). Acknowledgments We thank the Center for Ecological Research (Kyoto University) for the carbon isotope measurements, Mr. M. Nishiyama (Central Workshop, Osaka University) for manufacturing the leaf chamber, Professor K. Izui (Kinki University) for the generous gifts of the anti-phosphorylated PEPC, Professor H. Nakamoto (Saitama University) for the generous gifts of PEPC antisera, Dr. W. Yamori (Australian National University) for technical advice on the measurements of Rubisco activity, and Dr. H. Fukayama (Kobe University) and Dr. M. Miyao (National Institute of Agrobiological Sciences) for technical advice on the measurements of PPDK activity. We also thank Professor S. von Caemmerer and Dr. J. Evans (Australian National University) for their invaluable comments on the manuscript. 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Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org
TI - Relationships Between Quantum Yield for CO2 Assimilation, Activity of Key Enzymes and CO2 Leakiness in Amaranthus cruentus, a C4 Dicot, Grown in High or Low Light
JF - Plant and Cell Physiology
DO - 10.1093/pcp/pcm160
DA - 2008-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/relationships-between-quantum-yield-for-co2-assimilation-activity-of-5sn8yibLiT
SP - 19
EP - 29
VL - 49
IS - 1
DP - DeepDyve
ER -