Photosynthesis Research 24: 245-252, 1990. (~ 1990 Kluwer Academic Publishers. Printed in the Netherlands
Regular paper
Effect of photon flux density on inorganic carbon accumulation and net CO 2 exchange in a high-CO2-requiring mutant of
Chlamydomonas reinhardtii Martin H. Spalding
Department of Botany, Iowa State University, Ames, Iowa 50011-1020, USA Received 3 October 1989; accepted in revised form 5 January 1990
Key words: inorganic carbon transport, light, Chlamydomonas, CO 2 exchange, photorespiration, photosynthesis Abstract
The effect of photon flux density on inorganic carbon accumulation and photosynthetic CO 2 assimilation was determined by CO 2 exchange studies at three, limiting CO 2 concentrations with a ca-1 mutant of Chlamydomonas reinhardtii. This mutant accumulates a large internal inorganic carbon pool in the light which apparently is unavailable for photosynthetic assimilation. Although steady-state photosynthetic CO 2 assimilation did not respond to the varying photon flux densities because of CO 2 limitation, components of inorganic-carbon accumulation were not clearly light saturated even at 1100/xmol photons m -2 s-1, indicating a substantial energy requirement for inorganic carbon transport and accumulation. Steady-state photosynthetic CO 2 assimilation responded to external CO2 concentrations but not to changing internal inorganic carbon concentrations, confirming that diffusion of CO 2 into the cells supplies most of the CO z for photosynthetic assimilation and that the internal inorganic carbon pool is essentially unavailable for photosynthetic assimilation. The estimated concentration of the internal inorganic carbon pool was found to be relatively insensitive to the external CO 2 concentration over the small range tested, as would be expected if the concentration of this pool is limited by the internal to external inorganic carbon gradient. An attempt to use this CO 2 exchange method to determine whether inorganic carbon accumulation and photosynthetic CO 2 assimilation compete for energy at low photon flux densities proved inconclusive.
Introduction
Chlamydomonas reinhardtii and other unicellular algae use inorganic carbon (Ci) very efficiently for photosynthesis when grown at air levels of CO 2 (air-adapted cells). This results from action of a CO2-concentrating system, which raises the intracellular concentration of CO 2 through active transport of C i (Badger 1987, Spalding 1989). In C. reinhardtii this system appears to involve at least two components; C~ transport to accumulate bicarbonate intracellularly (Badger et al. 1980, Spalding and Ogren 1983, Splading et al.
1983b, Moroney et al. 1987), and an internal carbonic anhydrase (CA) to supply CO 2 for ribulose-l,5-bisphosphate carboxylase/oxygenase (Rubisco) by dehydration of the accumulated bicarbonate (Splading et al. 1983a, Moroney et al. 1985, Moroney et al. 1987). C. reinhardtii mutants with lesions at the ca-1 locus, which require an elevated CO 2 concentration for photoautotrophic growth, are apparently deficient in internal CA (Spalding et al. 1983, Spalding et al. 1985). The mutant cells have very low photosynthetic affinity for external C i but accumulate much more intracellular C i than air-
246 adapted cells of wild type, leading to the conclusion that the accumulated C i is bicarbonate not in chemical equilibrium with CO2. CO 2 exchange studies with ca-1 mutants have demonstrated a transient, exaggerated overaccumulation of C i at the onset of illumination (early-illumination gulp), observed as a high rate of CO 2 uptake followed by a decrease to the steady-state net photosynthetic CO 2 uptake rate (Spalding and Ogren 1985, Suzuki and Spalding 1989b). This transient overaccumulation of C i apparently represents Ci transport uncoupled from photosynthetic CO 2 assimilation. These mutants also exhibit an O2-insensitive post-illumination burst (PIB) of CO 2 efflux which represents release of their large, steady-state intracellular C i pool (Spalding and Ogren 1985, Suzuki and Spalding 1989b). Although the CO2-concentrating system is dependent on photosynthetic electron transport and/or photophosphorylation as the energy source for active C i transport (Badger et al. 1980, Ogawa et al. 1985, S/iltemeyer et al. 1986), little is known about the energy requirements of the system or its interaction with the energy requirements for photosynthetic CO 2 assimilation. Siiltemeyer et al. (1986) reported that 02 uptake in the light, attributed largely to pseudocyclic electron flow, was higher in air-adapted than in CO2-enriched cells of C. reinhardtii and that this 0 2 uptake was not light saturated at 600/zmol photons m -2 s -1. They concluded that the additional pseudocyclic electron flow might result from ATP demand, satisfied by pseudocyclic photophosphorylation, for C i transport in the air-adapted cells. The use of a ca-1 mutant of C. reinhardtii has allowed separation and investigation of the energy requirements for C i accumulation and photosynthetic CO2 assimilation. Using a method similar to that used for determining the action spectrum for C i accumulation in cyanobacteria by Ogawa et al. (1985) the effects of photon flux density on these two dissociated processes in this C. reinhardtii mutant have been examined and are reported here.
Materials and methods
Chlamydomonas reinhardtii high-COz-requiring mutant ca-l-12-1C mt + was grown photoauto-
trophically on a gyratory shaker under constant light (ca. 100/xmol photons m -2 s -1) at 25°C. The cells were grown in a liquid minimal medium consisting of 143 mg/l K2HPO4, 73 mg/l KH2PO4, 400 mg/l NH4NO3, 100 mg/1 MgSO 4 • 7H20, 50mg/l CaC12 . 2 H 2 0 , 1 ml/1 trace elements stock (Surzycki 1971), and 10ml/l of 2.0 M Mops titrated with Tris base to pH 7.1 for air medium and pH 7.6 for CO 2 medium. The pH of both types of cultures was 6.9 to 7.1 during growth. After being grown with aeration by air supplemented with 5% CO2, the cells were adapted to air levels of CO 2 (ca. 0.04% CO2) by resuspension in air medium and further growth without aeration for 24 to 32 h prior to use.
CO2 exchange was monitored in an open system as previously described (Suzuki and Spalding 1989b), except that cells were suspended in 15ml of minimal minimal medium (10-20/zg Chl m1-1) rather than 6 ml as used previously. Fresh cells were used for each measurement, and the 0 2 concentration was 20-21%. Light was provided by a 500 watt slide projector through a red, long-pass filter with a low wavelength cutoff at 620nm. Varying photon flux densities (400-700nm) were obtained using layers of cheesecloth as neutral density filters, and the photon flux density measured with a quantum flux monitor (Li-Cor, model LI-185B). Photosynthetic 0 2 evolution was monitored as described previously (Suzuki and Spalding 1989a), except that cells were used directly from the culture medium (ca. 10/.~g Chl m1-1) without centrifugation and resuspension, but with the addition of 100 units of bovine CA. All 0 2 evolution measurements were made at an 02 concentration equivalent to 21-25% 0 2. Chlorophyll was determined after extraction into 96% (v/v) ethanol (Wintermans and De Mots 1965).
Results and discussion
The n e t C O 2 exchange pattern of C. reinhardtii high-COz-requiring mutant ca-l-12-C was observed through a dark-light-dark sequence at several different photon flux densities (PFD's). The general response of net COg exchange at the higher PFD's (Fig. 1) was similar to that reported previously (Spalding and Ogren 1985,
247
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TIME Fig. 1. Net CO 2 exchange pattern of C. reinhardtii ca-1 mutant during a single dark-light-dark sequence of 5 min25 min-15 min (indicated in figure by light and dark boxes) at ll00 ~mol photons m -2 s -1 and 600 ~1/1 CO 2. Positive exchange rates indicate net CO 2 release from the cells, and negative exchange rates indicate net CO2 uptake by the cells. The dashed line through the CO 2 exchange pattern indicates zero net CO 2 exchange. Components of the pattern are indicated as gross gulp rate, net gulp rate, steady-state net photosynthesis rate (S-S PSN), net post-illumination burst rate (NET PIB) and internal C~ pool size.
Suzuki and Spalding 1989b). This mutant exhibits an early-illumination gulp of CO 2 uptake with a maximum at 2-3 min following onset of illumination. The early-illumination gulp was previously demonstrated to represent a transient, exaggerated overaccumulation into an internal pool of Ci (Spalding and Ogren 1985, Suzuki and Spalding 1989b). The CO 2 gulp is followed by a decline in the net CO 2 uptake rate to a minimum at approximately 10 min, which sometimes represents net CO 2 eflux. Following this CO2-uptake minimum the net CO 2 uptake rate increases for
10-15 min to a steady-state level representing the rate of net photosynthetic CO: assimilation (Spalding and Ogren 1985). Following the lightdark transition, a PIB of CO 2 release is observed before the rate of CO 2 release declines to a steady-state dark respiration rate. It was demonstrated previously that the PIB largely reflects release of the internal pool of C i rather than photorespiratory CO: (Spalding and Ogren 1985, Suzuki and Spalding 1989b). The effect of PFD on various components of the CO 2 exchange response of the ca-1 mutant was investigated. The components studied included the steady-state photosynthetic rate, gross and net CO 2 gulp rates, net PIB rate and internal Ci pool size, as illustrated in Fig. 1. Energy requirements for the high, initial rate of C~ transport should be reflected in the response of the early-illumination gulp to PFD, while the response of the PIB and internal C~ pool size should reflect energy requirements for maintenance of the steady-state, internal C~ pool. At all three CO 2 concentrations examined by CO2 exchange, steady-state photosynthetic rates were apparently CO 2 limited, since the rates did not increase with increases in PFD above 60/zmol photons m -2 s -1 but did increase with increasing CO 2 concentration (Table 1). This CO 2 limitation is consistent with reported K 0 . 5 ( C O 2 ) values for photosynthesis in this mutant (Spalding et al. 1983a, Suzuki and Spalding 1989a). The lowest CO 2 concentration used (130 tzl/l) coincided with the CO 2 compensation concentration for this mutant, consistent with a previous report (Spalding et al. 1983a). Observation of the increase in the rate of photosynthetic 0 2 evolution with increasing PFD at CO 2 saturation (5 mM C~, pH 7) and the lack of increase at a C~ concentration (0.1 mM, pH 7) similar to the highest used in the CO 2 exchange studies (600 ~1/1) confirmed that this mutant was CO 2 limited in the CO2 exchange studies (Table 1). In addition to the CO 2 limitation, a decreased steady-state photosynthetic rate was noted at high PFD (above 350/xmol photons m -2 s -1) in the CO 2 exchange studies but not in the 0 2 evolution studies (Table 1). The decrease may have resulted from photoinhibition, since the CO 2 exchange experiments were measured over a much longer light exposure (20-25 min) than the 0 2 evolution experiments (less than 5 min).
248 Table 1. Effect of photon flux density on steady-state photosynthetic rate of Chlamydomonas ca-1 mutant at different CO 2 concentrations PFD 3
[CO2] '
130 ~1/1
[NaHCO3]2
340/xl/l
600 ~1/1
0.1 mM
5 mM
11 21 21 24 21 20 15 11
20 28 28 29 29 27 29 31
42 58 74 103 121 171 193 196
/xmol CO 2 mg Chl ' h -1 45 60 85 120 200 350 550 1100
0 -1 0 1 0 0 0 -I
8 12 13 11 11 12 7 7
1 Gas phase CO 2 concentrations, corresponding to approximately 4, 10 and 17/zM equilibrium dissolved C O 2 a t 130, 340 and 600/xl/l CO2, respectively. Rates determined during CO 2 exchange studies. 2 Initial inorganic carbon concentrations in soluble phase at pH 7. Equilibrium dissolved CO2 concentrations would be approximately 15 and 727/xM CO 2 at 0.1 and 5 mM NaHCO 3, respectively. Rates determined in 0 2 evolution studies. 3 Photon flux density (620-700nm) in p.mol photons m -2 s-k
In CO 2 exchange studies, exposure of cells to high PFD for 25min resulted in depressed photosynthetic rates in a subsequent 25min measurement with the same sample at low PFD, even following a 20 min dark period (data not shown). Little effect was noted if the first illumination was at low PFD. These observations would be consistent with photoinhibition occurring at low CO 2 and high PFD in these studies, In spite of the lack of increase in steady-state photosynthesis with increasing PFD, the magnitude of both the early-illumination gulp of C O 2 uptake and the PIB increased with increasing PFD at the three C O 2 concentrations investigated (Figs. 2A and 3). These observations indicate that the rate of C i uptake at the onset of illumination and maintenance of the steady-state internal C i pool are essentially uncoupled energetically from CO z assimilation, demonstrating the validity of this method for investigating the energetics of C i transport and C i pool maintenance independent of CO 2 assimilation. Neither the PIB nor the early-illumination gulp was clearly light saturated at the highest PFD (1100/~mol photons m -2 s-l). Although the processes appeared to be light saturated at one CO 2 concentration in some experiments (Figs. 2A and 3), this was not consistently observed. It is possible that the processes were
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PHOTON FLUX DENSITY (~moles photons m "2 s -1)
Fig. 2. Effect o f photon flux density on net PIB rate (A) and estimated internal C i concentration (B) at three CO 2 concentrations: 600/~l/l (11), 340/xl/1 ([3) and 130/xl/l ( 0 ) .
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Regular paper
Effect of photon flux density on inorganic carbon accumulation and net CO 2 exchange in a high-CO2-requiring mutant of
Chlamydomonas reinhardtii Martin H. Spalding
Department of Botany, Iowa State University, Ames, Iowa 50011-1020, USA Received 3 October 1989; accepted in revised form 5 January 1990
Key words: inorganic carbon transport, light, Chlamydomonas, CO 2 exchange, photorespiration, photosynthesis Abstract
The effect of photon flux density on inorganic carbon accumulation and photosynthetic CO 2 assimilation was determined by CO 2 exchange studies at three, limiting CO 2 concentrations with a ca-1 mutant of Chlamydomonas reinhardtii. This mutant accumulates a large internal inorganic carbon pool in the light which apparently is unavailable for photosynthetic assimilation. Although steady-state photosynthetic CO 2 assimilation did not respond to the varying photon flux densities because of CO 2 limitation, components of inorganic-carbon accumulation were not clearly light saturated even at 1100/xmol photons m -2 s-1, indicating a substantial energy requirement for inorganic carbon transport and accumulation. Steady-state photosynthetic CO 2 assimilation responded to external CO2 concentrations but not to changing internal inorganic carbon concentrations, confirming that diffusion of CO 2 into the cells supplies most of the CO z for photosynthetic assimilation and that the internal inorganic carbon pool is essentially unavailable for photosynthetic assimilation. The estimated concentration of the internal inorganic carbon pool was found to be relatively insensitive to the external CO 2 concentration over the small range tested, as would be expected if the concentration of this pool is limited by the internal to external inorganic carbon gradient. An attempt to use this CO 2 exchange method to determine whether inorganic carbon accumulation and photosynthetic CO 2 assimilation compete for energy at low photon flux densities proved inconclusive.
Introduction
Chlamydomonas reinhardtii and other unicellular algae use inorganic carbon (Ci) very efficiently for photosynthesis when grown at air levels of CO 2 (air-adapted cells). This results from action of a CO2-concentrating system, which raises the intracellular concentration of CO 2 through active transport of C i (Badger 1987, Spalding 1989). In C. reinhardtii this system appears to involve at least two components; C~ transport to accumulate bicarbonate intracellularly (Badger et al. 1980, Spalding and Ogren 1983, Splading et al.
1983b, Moroney et al. 1987), and an internal carbonic anhydrase (CA) to supply CO 2 for ribulose-l,5-bisphosphate carboxylase/oxygenase (Rubisco) by dehydration of the accumulated bicarbonate (Splading et al. 1983a, Moroney et al. 1985, Moroney et al. 1987). C. reinhardtii mutants with lesions at the ca-1 locus, which require an elevated CO 2 concentration for photoautotrophic growth, are apparently deficient in internal CA (Spalding et al. 1983, Spalding et al. 1985). The mutant cells have very low photosynthetic affinity for external C i but accumulate much more intracellular C i than air-
246 adapted cells of wild type, leading to the conclusion that the accumulated C i is bicarbonate not in chemical equilibrium with CO2. CO 2 exchange studies with ca-1 mutants have demonstrated a transient, exaggerated overaccumulation of C i at the onset of illumination (early-illumination gulp), observed as a high rate of CO 2 uptake followed by a decrease to the steady-state net photosynthetic CO 2 uptake rate (Spalding and Ogren 1985, Suzuki and Spalding 1989b). This transient overaccumulation of C i apparently represents Ci transport uncoupled from photosynthetic CO 2 assimilation. These mutants also exhibit an O2-insensitive post-illumination burst (PIB) of CO 2 efflux which represents release of their large, steady-state intracellular C i pool (Spalding and Ogren 1985, Suzuki and Spalding 1989b). Although the CO2-concentrating system is dependent on photosynthetic electron transport and/or photophosphorylation as the energy source for active C i transport (Badger et al. 1980, Ogawa et al. 1985, S/iltemeyer et al. 1986), little is known about the energy requirements of the system or its interaction with the energy requirements for photosynthetic CO 2 assimilation. Siiltemeyer et al. (1986) reported that 02 uptake in the light, attributed largely to pseudocyclic electron flow, was higher in air-adapted than in CO2-enriched cells of C. reinhardtii and that this 0 2 uptake was not light saturated at 600/zmol photons m -2 s -1. They concluded that the additional pseudocyclic electron flow might result from ATP demand, satisfied by pseudocyclic photophosphorylation, for C i transport in the air-adapted cells. The use of a ca-1 mutant of C. reinhardtii has allowed separation and investigation of the energy requirements for C i accumulation and photosynthetic CO2 assimilation. Using a method similar to that used for determining the action spectrum for C i accumulation in cyanobacteria by Ogawa et al. (1985) the effects of photon flux density on these two dissociated processes in this C. reinhardtii mutant have been examined and are reported here.
Materials and methods
Chlamydomonas reinhardtii high-COz-requiring mutant ca-l-12-1C mt + was grown photoauto-
trophically on a gyratory shaker under constant light (ca. 100/xmol photons m -2 s -1) at 25°C. The cells were grown in a liquid minimal medium consisting of 143 mg/l K2HPO4, 73 mg/l KH2PO4, 400 mg/l NH4NO3, 100 mg/1 MgSO 4 • 7H20, 50mg/l CaC12 . 2 H 2 0 , 1 ml/1 trace elements stock (Surzycki 1971), and 10ml/l of 2.0 M Mops titrated with Tris base to pH 7.1 for air medium and pH 7.6 for CO 2 medium. The pH of both types of cultures was 6.9 to 7.1 during growth. After being grown with aeration by air supplemented with 5% CO2, the cells were adapted to air levels of CO 2 (ca. 0.04% CO2) by resuspension in air medium and further growth without aeration for 24 to 32 h prior to use.
CO2 exchange was monitored in an open system as previously described (Suzuki and Spalding 1989b), except that cells were suspended in 15ml of minimal minimal medium (10-20/zg Chl m1-1) rather than 6 ml as used previously. Fresh cells were used for each measurement, and the 0 2 concentration was 20-21%. Light was provided by a 500 watt slide projector through a red, long-pass filter with a low wavelength cutoff at 620nm. Varying photon flux densities (400-700nm) were obtained using layers of cheesecloth as neutral density filters, and the photon flux density measured with a quantum flux monitor (Li-Cor, model LI-185B). Photosynthetic 0 2 evolution was monitored as described previously (Suzuki and Spalding 1989a), except that cells were used directly from the culture medium (ca. 10/.~g Chl m1-1) without centrifugation and resuspension, but with the addition of 100 units of bovine CA. All 0 2 evolution measurements were made at an 02 concentration equivalent to 21-25% 0 2. Chlorophyll was determined after extraction into 96% (v/v) ethanol (Wintermans and De Mots 1965).
Results and discussion
The n e t C O 2 exchange pattern of C. reinhardtii high-COz-requiring mutant ca-l-12-C was observed through a dark-light-dark sequence at several different photon flux densities (PFD's). The general response of net COg exchange at the higher PFD's (Fig. 1) was similar to that reported previously (Spalding and Ogren 1985,
247
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10
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S-S PSN
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-10
::L
LU 0 Z -,r" L) X LU ¢q O O I.LU Z
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TIME Fig. 1. Net CO 2 exchange pattern of C. reinhardtii ca-1 mutant during a single dark-light-dark sequence of 5 min25 min-15 min (indicated in figure by light and dark boxes) at ll00 ~mol photons m -2 s -1 and 600 ~1/1 CO 2. Positive exchange rates indicate net CO 2 release from the cells, and negative exchange rates indicate net CO2 uptake by the cells. The dashed line through the CO 2 exchange pattern indicates zero net CO 2 exchange. Components of the pattern are indicated as gross gulp rate, net gulp rate, steady-state net photosynthesis rate (S-S PSN), net post-illumination burst rate (NET PIB) and internal C~ pool size.
Suzuki and Spalding 1989b). This mutant exhibits an early-illumination gulp of CO 2 uptake with a maximum at 2-3 min following onset of illumination. The early-illumination gulp was previously demonstrated to represent a transient, exaggerated overaccumulation into an internal pool of Ci (Spalding and Ogren 1985, Suzuki and Spalding 1989b). The CO 2 gulp is followed by a decline in the net CO 2 uptake rate to a minimum at approximately 10 min, which sometimes represents net CO 2 eflux. Following this CO2-uptake minimum the net CO 2 uptake rate increases for
10-15 min to a steady-state level representing the rate of net photosynthetic CO: assimilation (Spalding and Ogren 1985). Following the lightdark transition, a PIB of CO 2 release is observed before the rate of CO 2 release declines to a steady-state dark respiration rate. It was demonstrated previously that the PIB largely reflects release of the internal pool of C i rather than photorespiratory CO: (Spalding and Ogren 1985, Suzuki and Spalding 1989b). The effect of PFD on various components of the CO 2 exchange response of the ca-1 mutant was investigated. The components studied included the steady-state photosynthetic rate, gross and net CO 2 gulp rates, net PIB rate and internal Ci pool size, as illustrated in Fig. 1. Energy requirements for the high, initial rate of C~ transport should be reflected in the response of the early-illumination gulp to PFD, while the response of the PIB and internal C~ pool size should reflect energy requirements for maintenance of the steady-state, internal C~ pool. At all three CO 2 concentrations examined by CO2 exchange, steady-state photosynthetic rates were apparently CO 2 limited, since the rates did not increase with increases in PFD above 60/zmol photons m -2 s -1 but did increase with increasing CO 2 concentration (Table 1). This CO 2 limitation is consistent with reported K 0 . 5 ( C O 2 ) values for photosynthesis in this mutant (Spalding et al. 1983a, Suzuki and Spalding 1989a). The lowest CO 2 concentration used (130 tzl/l) coincided with the CO 2 compensation concentration for this mutant, consistent with a previous report (Spalding et al. 1983a). Observation of the increase in the rate of photosynthetic 0 2 evolution with increasing PFD at CO 2 saturation (5 mM C~, pH 7) and the lack of increase at a C~ concentration (0.1 mM, pH 7) similar to the highest used in the CO 2 exchange studies (600 ~1/1) confirmed that this mutant was CO 2 limited in the CO2 exchange studies (Table 1). In addition to the CO 2 limitation, a decreased steady-state photosynthetic rate was noted at high PFD (above 350/xmol photons m -2 s -1) in the CO 2 exchange studies but not in the 0 2 evolution studies (Table 1). The decrease may have resulted from photoinhibition, since the CO 2 exchange experiments were measured over a much longer light exposure (20-25 min) than the 0 2 evolution experiments (less than 5 min).
248 Table 1. Effect of photon flux density on steady-state photosynthetic rate of Chlamydomonas ca-1 mutant at different CO 2 concentrations PFD 3
[CO2] '
130 ~1/1
[NaHCO3]2
340/xl/l
600 ~1/1
0.1 mM
5 mM
11 21 21 24 21 20 15 11
20 28 28 29 29 27 29 31
42 58 74 103 121 171 193 196
/xmol CO 2 mg Chl ' h -1 45 60 85 120 200 350 550 1100
0 -1 0 1 0 0 0 -I
8 12 13 11 11 12 7 7
1 Gas phase CO 2 concentrations, corresponding to approximately 4, 10 and 17/zM equilibrium dissolved C O 2 a t 130, 340 and 600/xl/l CO2, respectively. Rates determined during CO 2 exchange studies. 2 Initial inorganic carbon concentrations in soluble phase at pH 7. Equilibrium dissolved CO2 concentrations would be approximately 15 and 727/xM CO 2 at 0.1 and 5 mM NaHCO 3, respectively. Rates determined in 0 2 evolution studies. 3 Photon flux density (620-700nm) in p.mol photons m -2 s-k
In CO 2 exchange studies, exposure of cells to high PFD for 25min resulted in depressed photosynthetic rates in a subsequent 25min measurement with the same sample at low PFD, even following a 20 min dark period (data not shown). Little effect was noted if the first illumination was at low PFD. These observations would be consistent with photoinhibition occurring at low CO 2 and high PFD in these studies, In spite of the lack of increase in steady-state photosynthesis with increasing PFD, the magnitude of both the early-illumination gulp of C O 2 uptake and the PIB increased with increasing PFD at the three C O 2 concentrations investigated (Figs. 2A and 3). These observations indicate that the rate of C i uptake at the onset of illumination and maintenance of the steady-state internal C i pool are essentially uncoupled energetically from CO z assimilation, demonstrating the validity of this method for investigating the energetics of C i transport and C i pool maintenance independent of CO 2 assimilation. Neither the PIB nor the early-illumination gulp was clearly light saturated at the highest PFD (1100/~mol photons m -2 s-l). Although the processes appeared to be light saturated at one CO 2 concentration in some experiments (Figs. 2A and 3), this was not consistently observed. It is possible that the processes were
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Fig. 2. Effect o f photon flux density on net PIB rate (A) and estimated internal C i concentration (B) at three CO 2 concentrations: 600/~l/l (11), 340/xl/1 ([3) and 130/xl/l ( 0 ) .
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