Photosynthetic CO2-use efficiency in lichens and their isolated photobionts: the possible role of a CO2-concentrating mechanism

The CO₂ dependence of net CO₂ assimilation was examined in a number of green algal and cyanobacterial lichens with the aim of screening for the algal/cyanobacterial CO₂-concentrating mechanism (CCM) in these symbiotic organisms. For the lichens Peltigera aphthosa (L.) Willd., P. canina (L.) Willd. and P. neopolydactyla (Gyeln.) Gyeln., the photosynthetic performance was also compared between intact thalli and their respective photobionts, the green alga Coccomyxa PA, isolated from Peltigera aphthosa and the cyanobacterium Nostoc PC, isolated from Peltigera canina. More direct evidence for the operation of a CCM was obtained by monitoring the effects of the carbonic-anhydrase inhibitors acetazolamide and ethoxyzolamide on the photosynthetic CO₂use efficiency of the photobionts. The results strongly indicate the operation of a CCM in all cyanobacterial lichens investigated and in cultured cells of Nostoc PC, similar to that described for free-living species of cyanobacteria. The green algal lichens were divided into two groups, one with a low and the other with a higher CO₂-use efficiency, indicative of the absence of a CCM in the former. The absence of a CCM in the low-affinity lichens was related to the photobiont, because free-living cells of Coccomyxa PA also apparently lacked a CCM. As a result of the postulated CCM, cyanobacterial Peltigera lichens have higher rates of net photosynthesis at normal CO₂ compared with Peltigera aphthosa. It is proposed that this increased photosynthetic capacity may result in a higher production potential, provided that photosynthesis is limited by CO₂ under natural conditions.     

Photosynthetic carbon acquisition in the lichen photobionts Coccomyxa and Trebouxia (Chlorophyta)

Processes involved in photosynthetic CO₂ acquisition were characterised for the isolated lichen photobiont Trebouxia erici (Chlorophyta, Trebouxiophyceae) and compared with Coccomyxa (Chlorophyta), a lichen photobiont without a photosynthetic CO₂-concentrating mechanism. Comparisons of ultrastructure and immuno-gold labelling of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco; EC 4.1.1.39) showed that the chloroplast was larger in T. erici and that the majority of Rubisco was located in its centrally located pyrenoid. Coccomyxa had no pyrenoid and Rubisco was evenly distributed in its chloroplast. Both species preferred CO₂ rather than HCO₃^? as an external substrate for photosynthesis, but T. erici was able to use CO₂ concentrations below 10–12 μM more efficiently than Coccomyxa. In T. erici, the lipid-insoluble carbonic anhydrase (CA; EC 4.2.1.1) inhibitor acetazolamide (AZA) inhibited photosynthesis at CO₂ concentrations below 1 μM, while the lipid-soluble CA inhibitor ethoxyzolamide (EZA) inhibited CO₂-dependent O₂ evolution over the whole CO₂ range. EZA inhibited photosynthesis also in Coccomyxa, but to a much lesser extent below 10–12 μM CO₂. The internal CA activity of Trebouxia, per unit chlorophyll (Chl), was ca 10% of that of Coccomyxa. Internal CA activity was also detected in homogenates from T. erici and two Trebouxia-lichens (Lasallia hispanica and Cladina rangiferina). In all three, the predominating CA had α-type characteristics and was significantly inhibited by low concentrations of AZA, having an I₅₀ below 10–20 nM. In Coccomyxa a β-type CA predominates, which is much less sensitive to AZA. Thus, the two photobionts differed in three major characteristics with respect to CO₂ acquisition, the subcellular location of Rubisco, the relative requirement of CA and the biochemical characteristics of their predominating internal CA. These differences may be linked to the ability of Trebouxia to accumulate dissolved inorganic carbon internally, enhancing their CO₂ use efficiency at and below air-equilibrium concentrations (10–12 μM CO₂) in comparison with Coccomyxa.     

Carbonic anhydrase activity in leaves as measured in vivo by18O exchange between carbon dioxide and water

Carbonic anhydrase activity of intactCommelina communis L. leaves was measured using mass spectrometry, by following the¹⁸O-exchange kinetics between¹⁸O-enriched carbon dioxide and water. A gas-diffusion model (Gerster, 1971, Planta97, 155–172) was used to interpret the¹⁸O-exchange kinetics and to determine two constants, one (k) related to the hydration of CO₂ and the other (k_e), related to the diffusion of CO₂. Both constants were determined inCommelina communis L. leaves after stripping the lower epidermis to remove any stomatal influence. The hydration constant (k) was 17200 +2200 ·min^–1 (mean±SD, 12 experiments), i.e., about 8 600 times the uncatalyzed hydration of CO₂ in pure water, and was specifically inhibited by ethoxyzolamide, a powerful inhibitor of carbonic anhydrases, half-inhibition occurring around 10^–5 Methoxyzolamide. The diffusion constant (k_e) was 1.18±0.28·min^–1 (mean±SD, 12 experiments) and was only slightly inhibited (about 20%) by ethoxyzolamide. Carbonic anhydrase activity of stripped leaves was not affected by the leaf water status (up to 50% relative water deficits), was strongly inhibited by monovalent anions such as Cl^– or NO ₃ ^– , and decreased by about 50% when the photon flux density during growth was increased from 100 to 500 mol photons·m^–2·s^–1. By studying the effect of ethoxyzolamide (10^–4 M) on photosynthetic O₂ exchange, measured using¹⁸O₂ and mass spectrometry, we found that inhibition of carbonic anhydrase activity by 92–95% had little effect on the response curves of net O₂ evolution to increased CO₂ concentrations. Ethoxyzolamide had no effect on the photosynthetic electron-transport rate, measured as gross O₂ photosynthesis at high CO₂ concentration (>350 l·^–1), but was found to increase both gross O₂ photosynthesis and O₂ uptake at lower CO₂ levels. The chloroplastic CO₂ concentration calculated from O₂-exchange data was not significantly modified by ethoxyzolamide. We conclude from these results that, under normal conditions of photosynthesis, most of the carbonic anhydrase activity is not involved in CO₂ assimilation. Measurement of carbonic anhydrase activity using¹⁸O-isotope exchange therefore provides a suitable model to study the in-vivo regulation of this chloroplastic enzyme in plants submitted to various environmental conditions.Abbreviations CA carbonic anhydrase - C_cc chloroplastic CO₂ concentration - C_e external CO₂ concentration - EZA ethoxyzolamide - k CO₂ hydration rate constant - k_e CO₂ diffusion rate constan - PPFD photosynthetic photon flux density - Rubisco ribulose-1,5 bisphosphate carboxylase oxygenase - RWD relative water deficit The authors wish to thank P. Carrier for technical assistance with mass-spectrometric experiments and Dr. P. Thibault for helpful suggestions and comments. Dr. A. Vavasseur is gratefully acknowledged for supplyingCommelima communis. cultures. P.C., P.T. and A.V. are all from the CEA, Département de Physiologie Végétale et Ecosystèmes, Cadarache, France.     

Evidence for the functioning of photosynthetic CO2-concentrating mechanisms in lichens containing green algal and cyanobacterial photobionts

The photosynthetic properties of a range of lichens containing both green algal (11 species) and cyanobacterial (6 species) photobionts were examined with the aim of determining if there was clear evidence for the operation of a CO₂-concentrating mechanism (CCM) within the photobionts. Using a CO₂-gas-exchange system, which allowed resolution of fast transients, evidence was obtained for the existence of an inorganic carbon pool which accumulated in the light and was released in the dark. The pool was large (500–1000 nmol · mg Chl) in cyanobacterial lichens and about tenfold smaller in green algal lichens. In Hypogymnia physodes (L.) Nyl., which contains the green alga Trebouxia jamesii, a small inorganic carbon pool was rapidly formed in the light. Carbon dioxide was released from this pool into the gas phase upon darkening within about 20 s when photosynthesis was inhibited by the carbon-reduction-cycle inhibitor glycolaldehyde. In the absence of this inhibitor, release appeared to be obscured by carboxylation of ribulose bisphosphate. The kinetics of CO₂ uptake and release were monophasic. The operation of an active CCM could be distinguished from passive accumulation and release accompanying the reversible light-dependent alkalization of the stroma by the presence of saturation characteristics with respect to external CO₂. In Peltigera canina (L.) Willd., which contains the cyanobacterium Nostoc sp., a larger CO₂ pool was taken up over a longer period in the light and the release of this pool in the dark was slow, lasting 3–5 min. This pool also accumulated in the presence of glycolaldehyde, and under these conditions the CO₂ release was biphasic. In both species, photosynthesis at low CO₂ was inhibited by the carbonic-anhydrase inhibitor ethoxyzolamide (EZ). Inhibition could be reversed fully or to a considerable extent by high CO₂. In Peltigera, EZ decreased both the accumulation of the CO₂ pool by the CCM and the rate of photosynthesis. Free-living cultures of Nostoc sp. showed a similar effect of EZ on photosynthesis, although it was more dramatic than that seen with the lichen thalli. In contrast, in Hypogymnia, EZ actually increased the size of the CO₂ pool, although it inhibited photosynthesis. This effect was also seen when glycolaldehyde was present together with EZ. Surprisingly, EZ did not alter the kinetics of either CO₂ uptake or release. Taken together, the evidence indicates the operation in cyanobacterial lichens of a CCM which is capable of considerable elevation of internal CO₂ and is similar to that reported for free-living cyanobacteria. The CCM of green algal lichens accumulates much less CO₂ and is probably less effective than that which operates in cyanobacterial lichens.     

The occurrence of the chloroplast pyrenoid is correlated with the activity of a CO2-concentrating mechanism and carbon isotope discrimination in lichens and bryophytes

The organic-matter carbon isotope discrimination () of lichens with a wide range of photobiont and/or cyanobiont associations was used to determine the presence or absence of a carbon-concentrating mechanism (CCM). Two groups were identified within the lichens with green algal photobionts. One group was characterised by low, more C₄-like values ( < 15), the other by higher, more C₃-like values ( > 18). Tri-partite lichens (lichens with a green alga as the primary photobiont and cyanobacteria within internal or external cephalodia) occurred in both groups. All lichens with cyanobacterial photobionts had low values ( < 15). The activity of the CCM, organic-matter values, on-line values and gas-exchange characteristics correlated with the presence of a pyrenoid in the algal chloroplast. Consistent with previous findings, lichens with Trebouxia as the primary photobiont possessed an active CCM while those containing Coccomyxa did not. Organic values for lichens with Stichococcus as the photobiont varied between 11 and 28. The lichen genera Endocarpon and Dermatocarpon (Stichococcus + pyrenoid) had C₄-like organic values ( = 11 to 16.5) whereas the genus Chaenotheca (Stichococcus — pyrenoid) was characterised by high C₃-like values ( = 22 to 28), unless it associated with Trebouxia ( = 16). Gas-exchange measurements demonstrated that Dermatocarpon had an affinity for CO₂ comparable to those species which possessed the CCM, with K_0.5 = 200–215 1 · 1^–1, compensation point () = 45–48 l · l^–1, compared with K_0.5 = 195 1 · 1^–1, = 441 · 1^–1 for Trebouxioid lichens. Furthermore, lichens with Stichococcus as their photobiont released a small pool (24.2 ± 1.9 to 34.2 ± 2.5 nmol · mg^–1 Chl) of inorganic carbon similar to that released by Trebouxioid lichens [CCM present, dissolved inorganic carbon (DIC) pool size = 51.0 ± 2.8 nmol · mg^–1 Chl]. Lichens with Trentepohlia as photobiont did not possess an active CCM, with high C₃-like organic values ( = 18 to 23). In particular, Roccella phycopsis had very high on-line values ( = 30 to 33), a low affinity for CO₂ (K_0.5 = 400 1 · 1^–1, = 120 1 · ^–1) and a negligible DIC pool. These responses were comparable to those from lichens with Coccomyxa as the primary photobiont with Nostoc in cephalodia (organic = 17 to 25, on-line = 16 to 21, k_0.5 = 388 1 · 1^–1, = 85 1 · 1^–1, DIC pool size = 8.5 ± 2.4 nmol · mg^–1 Chl). The relative importance of refixation of respiratory CO₂ and variations in source isotope signature were considered to account for any variation between on-line and organic . Organic was also measured for species of Anthocerotae and Hepaticae which contain pyrenoids and/or Nostoc enclosed within the thallus. The results of this screening showed that the pyrenoid is correlated with low, more C₄-like organic values ( = 7 to 12 for members of the Anthocerotae with a pyrenoid compared with = 17 to 28 for the Hepaticae with and without Nostoc in vesicles) and confirms that the pyrenoid plays a fundamental role in the functioning of the CCM in microalgal photobionts and some bryophytes.Abbreviations and Symbols CCM carbon-concentrating mechanism - DIC dissolved inorganic carbon (CO₂ + HCO ₃ ^- + CO ₃ ^2- ) - DW dry weight - K_0.5 external concentration of CO₂ at which half-maximal rates of CO₂ assimilation are reached - photobiont photosynthetic organism present in the lichen - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - carbon isotope discrimination (%) - ¹³C carbon isotope ratio (%) This research was funded by Natural Environment Research Council grant no. GR3/8313. The authors would also like to thank Dr. B. Coppins, Royal Botanic Gardens Edinburgh and Prof. A. Roy Perry, National Museum of Wales, for access to herbarium collections, Dr. T. Booth for confocal microscopy work and Dr. A.J. Richards, University of Newcastle upon Tyne and Dr. O.L. Gilbert, University of Sheffield for identifying bryophytes and lichens respectively. E.S. would particularly like to thank Dr. M. Broadmeadow, The Forestry Authority, Farnham, Surrey, and Cristina Máguas, Universidade de Lisboa, for their advice and expertise at the beginning of the project.     

Photosynthesis and apparent affinity for dissolved inorganic carbon by cells and chloroplasts of Chlamydomonas reinhardtii grown at high and low CO2 concentrations

Chloroplasts with high rates of photosynthetic O₂ evolution (up to 120 mol O₂· (mg Chl)^-1·h^-1 compared with 130 mol O₂· (mg Chl)^-1·h^-1 of whole cells) were isolated from Chlamydomonas reinhardtii cells grown in high and low CO₂ concentrations using autolysine-digitonin treatment. At 25° C and pH=7.8, no O₂ uptake could be observed in the dark by high- and low-CO₂ adapted chloroplasts. Light saturation of photosynthetic net oxygen evolution was reached at 800 mol photons·m^-2·s^-1 for high- and low-CO₂ adapted chloroplasts, a value which was almost identical to that observed for whole cells. Dissolved inorganic carbon (DIC) saturation of photosynthesis was reached between 200–300 M for low-CO₂ adapted chloroplasts, whereas high-CO₂ adapted chloroplasts were not saturated even at 700 M DIC. The concentrations of DIC required to reach half-saturated rates of net O₂ evolution (K_m(DIC)) was 31.1 and 156 M DIC for low- and high-CO₂ adapted chloroplasts, respectively. These results demonstrate that the CO₂ concentration provided during growth influenced the photosynthetic characteristics at the whole cell as well as at the chloroplast level.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon - K_m(DIC) coneentration of dissolved inorganic carbon required for the rate of half maximal net O₂ evolution - PFR photon fluence rate - SPGM silicasol-PVP-gradient medium     

Chlorophyll a Fluorescence Emission,Xanthophyll Cycle Activity,and Net Photosynthetic Rate Responses to Ozone in Some Foliose and Fruticose Lichen Species

The lichens Parmelia quercina, Parmelia sulcata, Evernia prunastri, Hypogymnia physodes, and Anaptychia ciliaris were exposed to ozone (O₃) in controlled environment cuvettes designed to maintain the lichens at optimal physiological activity during exposure. Measurements of gas exchange, modulated chlorophyll (Chl) fluorescence, and pigment analysis were conducted before and after exposure to 300 mm³ (O₃) m^–3, 4 h per d for 14 d. No changes in the efficiency of photosystem 2 (PS2) photochemistry, the reduction state of Q_A, or the electron flow through PS2, measured by Chl fluorescence, were detected in any of the five lichen species studied. Additionally, neither photosynthetic CO₂ assimilation nor xanthophyll cycle activity or photosynthetic pigment concentration were affected by high O₃ concentrations. Thus the studied lichen species have significant capacities to withstand oxidative stresses induced by high concentration of O₃.     

Predicting CO2 gain and photosynthetic light acclimation from fluorescence yield and quenching in cyano-lichens

Modulated chlorophylla fluorescence is useful for eco-physiological studies of lichens as it is sensitive, non-invasive and specific to the photobiont. We assessed the validity of using fluorescence yield to predict CO₂ gain in cyano-lichens, by simultaneous measurements of CO₂ gas exchange and chlorophylla fluorescence in five species withNostoc-photobionts. For comparison, O₂ evolution and fluorescence were measured in isolated cells ofNostoc, derived fromPeltigera canina (Nostoc PC). At irradiances up to the growth light level, predictions from fluorescence yield underestimated true photosynthesis, to various extents depending on species. This reflected the combined effect of a state transition in darkness, which was not fully relaxed until the growth light level was reached, and a phycobilin contribution to the minimum fluorescence yield (F_o). Above the growth light level, the model progressively overestimated assimilation, reflecting increased electron flow to oxygen under excess irradiance. In cyanobacteria, this flow maintains photosystem II centres open even up to photoinhibitory light levels without contributing to CO₂ fixation. Despite this we show that gross CO₂ gain may be predicted from fluorescence yield also in cyanolichens when the analysis is made near the acclimated growth light level. This level can be obtained even when measurements are performed in the field, since it coincides with a minimum in non-photochemical fluorescence quenching (NPQ). However, the absolute relation between fluorescence yield and gross CO₂ gain varies between species. It may therefore be necessary to standardise the fluorescence prediction for each species with CO₂ gas exchange.Abbreviations CCM CO₂-Concentrating mechanism - Chl chlorophyll - C_i inorganic carbon - 0 convexity (curvature of the light response curve) - ETR electron transport rate - F_o minimum fluorescence yield - F_m maximal fluorescence yield - F_s fluorescence yield at steady-state photosynthesis - F_v variable fluorescence yield - F_v/F_m dark ratio of variable to maximal fluorescence yield after dark adaptation - F_vF_mmax ratio of variable to maximal fluorescence yield in the absence of quenching - _CO2 maximum quantum yield of CO₂ assimilation - _PS quantum yield of photosystem II photochemistry - GP gross photosynthesis - I irradiance (mol quanta·m^–2·s^–1) - NPQ non photochemical fluorescence quenching - qp photochemical fluorescence quenching     

UPTAKE,EFFLUX, AND PHOTOSYNTHETIC UTILIZATION OF INORGANIC CARBON BY THE MARINE EUSTIGMATOPHYTE NANNOCHLOROPSIS SP.1

Uptake, efflux and utilization of inorganic carbon were investigated in the marine eustigmatophyte Nannochloropsis sp. grown under an air level of CO₂. Maximal photosynthetic rate was hardly affected by raising the pH porn 5.0 to 9.0. The apparent photosynthetic affinity for dissolved inorganic carbon (DIC) was 35 μM DIC between pH 6.5 to 9.0, but increased approximately threefold at pH 5.0 suggesting that HCO₃- was the main DIC species used from the medium. No external carbonic anhydrase (CA) activity could be detected by the pH drift method. However, application of ethoxyzolamide (an inhibitor of CA) resulted an a significant inhibition of photosynthetic O₂ evolution and carbon utilization, suggesting involvement of internal CA or CA-like activity in DIC utilization. Under high light conditions, the rate of HCO₃^? uptake and its internal conversion to CO₂ apparently exceeded the rate of carbon fixation, resulting in a large leak of CO₂ from the cells to the external medium. When the cells were exposed to low DIC concentrations, the ratio of internal to external DIC concentration was about eight. On the other hand, in the presence of 2 mM DIC, conditions prevailing in the marine environment, the internal concentration of DIC was only 50% higher than the external one.     

Role of carbonic anhydrase in photosynthesis and inorganic-carbon assimilation in the red alga Gracilaria tenuistipitata

The mechanism of inorganic-carbon (C_i) accumulation in the red seaweed Gracilaria tenuistipitata Zhang et Xia has been investigated. Extracellular and intracellular carbonic-anhydrase (CA) activities have been detected. Photosynthetic O₂ evolution in thalli and protoplasts of G. tenuistipitata were higher at pH 6.5 than at pH 8.6, where HCO ₃ ^– is the predominant form of C_i. Dextran-bound sulfonamide (DBS), a specific inhibitor of extracellular CA, reduced photosynthetic O₂ evolution at pH 8.6 and did not have any effect at pH 6.5. After inhibition with DBS, O₂ evolution was similar to the rate that could be supported by CO₂ from spontaneous dehydration of HCO ₃ ^– . The rate of photosynthetic alkalization of the surrounding medium by the algal thallus was dependent on the concentration of C_i and inhibited by DBS. We suggest that the general form of C_i that enters through the plasma membrane of G. tenuistipitata is CO₂. Bicarbonate is utilized mainly by an indirect mechanism after dehydration to CO₂, and this mechanism involves extracellular CA.Abbreviations C_i inorganic carbon (CO₂ + HCO ₃ ^– ) - CA carbonic anhydrase - DIC dissolved inorganic carbon (total) - DBS dextran-bound sulfonamide - EZ ethoxyzolamide - NSW natural seawater - PPFD photosynthetic photon flux density - REA relative enzyme activity - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase This research was supported by the Deutsche Forschungsgemeinschaft (Bonn) as a programme of the Sonderforschungsbereich 251 der Universität Würzburg and by the Fonds der Chemischen Industrie (Frankfurt). Joint work in Würzburg was possible thanks to travel grants from the Chancellor of the University of Würzburg, Professor R. Günther, from the Australian National University under the auspices of its Overseas Studies Programme, and from the New Zealand — Federal Republic of Germany Scientific and Technological Exchange Programme, which are gratefully acknowledged. We thank Dr. A. Meyer and Ms. E. Kilian for untiringly conducting part of the experimental work, Ms. G. Theumer and Ms. D. Faltenbacher-Werner for their valuable assistance, and Mr. H. Walz (Walz Company, Effeltrich, FRG) for his skilled help with the calibration of our gas-exchange system for measurements with helox. The Department of Conservation, New Zealand, is thanked for permission to collect lichens.     

The CO2-concentrating mechanism is absent in the green alga Coccomyxa: a comparative study of photosynthetic CO2 and light responses of Coccomyxa, Chlamydomonas reinhardtii and barley protoplasts

Photosynthesis was characterized for the unicellular green alga Coccomyxa sp., grown at low inorganic carbon (C_i) concentrations, and compared with Chlamydomonas reinhardtii, which had been grown so that the CO₂ concentrating mechanism (CCM) was expressed, and with protoplasts isolated from the C₃ plant barley (Hordeum vulgare). Chlamydomonas had a significantly higher C_i-use efficiency of photosynthesis, with an initial slope of the C_i-response curve of 0.7 mol(gChl)⁻¹ h⁻¹ mmol C_im⁻³)⁻¹, as compared to 0.3 and 0.23 mol(gChl)⁻¹ h⁻¹ (mmol C_im⁻³)⁻¹ for Coccomyxa and barley, respectively. The affinity for C_i was also higher in Chlamydomonas, as the half maximum rate of photosynthesis [K_0.5 (C_i)] was reached at 0.18 mol m⁻³, as compared to 0.30 and 0.45 mol m⁻³ for Coccomyxa and barley, respectively. Ethoxyzolamide (EZ), an inhibitor of the enzyme carbonic anhydrase (CA) and the CCM, caused a 17-fold decrease in the initial slope of the photosynthetic Cj-response curve in Chlamydomonas, but only a 1.5- to two-fold decrease in Coccomyxa and barley. The photosynthetic light-response curve showed further similarities between barley and Coccomyxa. The rate of bending of the curve, described by the convexity parameter, was 0.99 (sharp bending) and 0.81–0.83 (gradual bending) for cells grown under low and high light, respectively. In contrast, the maximum convexity of Chlamydomonas was 0.85. The intrinsically lower convexity of Chlamydomonas is suggested to result from the diversion of electron transport from carbon fixation to the CCM. Taken together, these results suggest that Coccomyxa does not possess a CCM and due to this apparent lack of a CCM, we propose that Coccomyxa is a better cell model system for studying C₃ plant photosynthesis than many algae currently used.     

The roles of malate and aspartate in C4 photosynthetic metabolism of Flaveria bidentis (L.)

In C₄ grasses belonging to the NADP-malic enzyme-type subgroup, malate is considered to be the predominant C₄ acid metabolized during C₄ photosynthesis, and the bundle sheath cell chloroplasts contain very little photosystem-II (PSII) activity. The present studies showed that Flaveria bidentis (L.), an NADP-malic enzyme-type C₄ dicotyledon, had substantial PSII activity in bundle sheath cells and that malate and aspartate apparently contributed about equally to the transfer of CO₂ to bundle sheath cells. Preparations of bundle sheath cells and chloroplasts isolated from these cells evolved O₂ at rates between 1.5 and 2 mol · min^–1 · mg^–1 chlorophyll (Chl) in the light in response to adding either 3-phosphoglycerate plus HCO ₃ ^– or aspartate plus 2-oxoglutarate. Rates of more than 2 mol O₂ · min^–1 · mg^–1 Chl were recorded for cells provided with both sets of these substrates. With bundle sheath cell preparations the maximum rates of light-dependent CO₂ fixation and malate decarboxylation to pyruvate recorded were about 1.7 mol · min^–1 · mg^–1 Chl. Compared with NADP-malic enzyme-type grass species, F. bidentis bundle sheath cells contained much higher activities of NADP-malate dehydrogenase and of aspartate and alanine aminotransferases. Time-course and pulse-chase studies following the kinetics of radiolabelling of the C-4 carboxyl of C₄ acids from ¹⁴CO₂ indicated that the photosynthetically active pool of malate was about twice the size of the aspartate pool. However, there was strong evidence for a rapid flux of carbon through both these pools. Possible routes of aspartate metabolism and the relationship between this metabolism and PSII activity in bundle sheath cells are considered.Abbreviations DHAP dihydroxyacetone phosphate - NADP-ME(-type) NADP-malic enzyme (type) - NADP-MDH NADP-malate dehydrogenase - OAA oxaloacetic acid - 2-OG 2-oxoglutarate - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - Pi orthophosphate - Ru5P ribulose 5-phosphate     

Analysis of Qualitative Contribution of Assimilatory and Non-Assimilatory De-Excitation Processes to Adaptation of Photosynthetic Apparatus of Barley Plants to High Irradiance

The adaptation of barley (Hordeum vulgare L. cv. Akcent) plants to low (LI, 50 µmol m^–2 s^–1) and high (HI, 1000 µmol m^–2 s^–1) growth irradiances was studied using the simultaneous measurements of the photosynthetic oxygen evolution and chlorophyll a (Chl a) fluorescence at room temperature. If measured under ambient CO₂ concentration, neither increase of the oxygen evolution rate (P) nor enhancement of non-radiative dissipation of the absorbed excitation energy within photosystem 2 (PS2) (determined as non-photochemical quenching of Chl a fluorescence, NPQ) were observed for HI plants compared with LI plants. Nevertheless, the HI plants exhibited a significantly higher proportion of Q_A in oxidised state (estimated from photochemical quenching of Chl a fluorescence, q_P), by 49–102 % at irradiances above 200 µmol m^–2 s^–1 and an about 1.5 fold increase of irradiance-saturated PS2 electron transport rate (ETR) as compared to LI plants. At high CO₂ concentration the degree of P stimulation was approximately three times higher for HI than for LI plants, and the irradiance-saturated P values at irradiances of 2 440 and 2 900 µmol m^–2 s^–1 were by 130 and 150 % higher for HI plants than for LI plants. We suggest that non-assimilatory electron transport dominates in the adaptation of the photosynthetic apparatus of barley grown at high irradiances under ambient CO₂ rather than an increased NPQ or an enhancement of irradiance-saturated photosynthesis.     

Historical perspective on microalgal and cyanobacterial acclimation to low- and extremely high-CO2 conditions

Reports in the 1970s from several laboratories revealed that the affinity of photosynthetic machinery for dissolved inorganic carbon (DIC) was greatly increased when unicellular green microalgae were transferred from high to low-CO₂ conditions. This increase was due to the induction of carbonic anhydrase (CA) and the active transport of CO₂ and/or HCO₃ ⁻ which increased the internal DIC concentration. The feature is referred to as the `CO₂-concentrating mechanism (CCM)'. It was revealed that CA facilitates the supply of DIC from outside to inside the algal cells. It was also found that the active species of DIC absorbed by the algal cells and chloroplasts were CO₂ and/or HCO₃ ⁻, depending on the species. In the 1990s, gene technology started to throw light on the molecular aspects of CCM and identified the genes involved. The identification of the active HCO₃ ⁻ transporter, of the molecules functioning for the energization of cyanobacteria and of CAs with different cellular localizations in eukaryotes are examples of such successes. The first X-ray structural analysis of CA in a photosynthetic organism was carried out with a red alga. The results showed that the red alga possessed a homodimeric β-type of CA composed of two internally repeating structures. An increase in the CO₂ concentration to several percent results in the loss of CCM and any further increase is often disadvantageous to cellular growth. It has recently been found that some microalgae and cyanobacteria can grow rapidly even under CO₂ concentrations higher than 40%. Studies on the mechanism underlying the resistance to extremely high CO₂ concentrations have indicated that only algae that can adopt the state transition in favor of PS I could adapt to and survive under such conditions. It was concluded that extra ATP produced by enhanced PS I cyclic electron flow is used as an energy source of H⁺-transport in extremely high-CO₂ conditions. This same state transition has also been observed when high-CO₂ cells were transferred to low CO₂ conditions, indicating that ATP produced by cyclic electron transfer was necessary to accumulate DIC in low-CO₂ conditions. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterisation of carbon dioxide and bicarbonate transport during steady-state photosynthesis in the marine cyanobacterium Synechococcus strain PCC7002

Net O₂ evolution, gross CO₂ uptake and net HCO _inf3 ^su– uptake during steady-state photosynthesis were investigated by a recently developed mass-spectrometric technique for disequilibrium flux analysis with cells of the marine cyanobacterium Synechococcus PCC7002 grown at different CO₂ concentrations. Regardless of the CO₂ concentration during growth, all cells had the capacity to transport both CO₂ and HCO _inf3 ^su– ; however, the activity of HCO _inf3 ^su– transport was more than twofold higher than CO₂ transport even in cyanobacteria grown at high concentration of inorganic carbon (C_i = CO₂ + HCO _inf3 ^su– ). In low-C_i cells, the affinities of CO₂ and HCO _inf3 ^su– transport for their substrates were about 5 (CO₂ uptake) and 10 (HCO _inf3 ^su– uptake) times higher than in high-C_i cells, while air-grown cells formed an intermediate state. For the same cells, the intracellular accumulated C_i pool reached 18, 32 and 55 mM in high-C_i, air-grown and low-C_i cells, respectively, when measured at 1 mM external C_i. Photosynthetic O₂ evolution, maximal CO₂ and HCO _inf3 ^su– transport activities, and consequently their relative contribution to photosynthesis, were largely unaffected by the CO₂ provided during growth. When the cells were adapted to freshwater medium, results similar to those for artificial seawater were obtained for all CO₂ concentrations. Transport studies with high-C_i cells revealed that CO₂ and HCO _inf3 ^su– uptake were equally inhibited when CO₂ fixation was reduced by the addition of glycolaldehyde. In contrast, in low-C_i cells steady-state CO₂ transport was preferably reduced by the same inhibitor. The inhibitor of carbonic anhydrase ethoxyzolamide inhibited both CO₂ and HCO _inf3 ^su– uptake as well as O₂ evolution in both cell types. In high-C_i cells, the degree of inhibition was similar for HCO _inf3 ^su– transport and O₂ evolution with 50% inhibition occurring at around 1 mM ethoxyzolamide. However, the uptake of CO₂ was much more sensitive to the inhibitor than HCO _inf3 ^su– transport, with an apparent I₅₀ value of around 250 M ethoxyzolamide for CO₂ uptake. The implications of our results are discussed with respect to C_i utilisation in the marine Synechococcus strain.Abbreviations Chl chlorophyll - C_i inorganic carbon (CO₂ + HCO _inf3 ^su– ) - CA carbonic anhydrase - CCM CO₂-concentrating mechanism - EZA ethoxyzolamide - GA glycolaldehyde - K_1/2 concentration required for half-maximal response - Rubisco ribulose-1,5,-bisphosphate carboxylase-oxygenase D.S. is a recipient of a research fellowship from the Deutsche Forschungsgemeinschaft (D.F.G.). In addition, we are grateful to Donald A. Bryant, Department of Molecular and Cell Biology and Center of Biomolecular Structure Function, Pennsylvania State University, USA, for sending us the wild-type strain of Synechococcus PCC7002.     

PHOTOSYNTHETIC INORGANIC CARBON ACQUISITION IN AN ACID‐TOLERANT,FREE‐LIVING SPECIES OF COCCOMYXA (CHLOROPHYTA)1

The processes of CO₂ acquisition were characterized for the acid‐tolerant, free‐living chlorophyte alga, CPCC 508. rDNA data indicate an affiliation to the genus Coccomyxa, but distinct from other known members of the genus. The alga grows over a wide range of pH from 3.0 to 9.0. External carbonic anhydrase (CA) was detected in cells grown above pH 5, with the activity increasing marginally from pH 7 to 9, but most of the CA activity was internal. The capacity for HCO₃^? uptake of cells treated with the CA inhibitor acetazolamide (AZA), was investigated by comparing the calculated rate of uncatalyzed CO₂ formation with the rate of photosynthesis. Active bicarbonate transport occurred in cells grown in media above pH 7.0. Monitoring CO₂ uptake and O₂ evolution by membrane‐inlet mass spectrometry demonstrated that air‐grown cells reduced the CO₂ concentration in the medium to an equilibrium concentration of 15 μM, but AZA‐treated cells caused a drop in extracellular CO₂ concentration to a compensation concentration of 27 μM at pH 8.0. CO₂‐pulsing experiments with cells in the light indicated that the cells do not actively take up CO₂. An internal pool of unfixed inorganic carbon was not detected at the CO₂ compensation concentration, probably because of the lack of active CO₂ uptake, but was detectable at times before compensation point was reached. These results indicate that this free‐living Coccomyxa possesses a CO₂‐concentrating mechanism (CCM) due to an active bicarbonate‐uptake system, unlike the Coccomyxa sp. occurring in symbiotic association with lichens.     

A CO2-Flux Mechanism Operating via pH-Polarity in Hydrilla Verticillata Leaves With C3 and C4 Photosynthesis

The aquatic angiosperm Hydrilla verticillata lacks Kranz anatomy, but has an inducible, C₄-based, CO₂ concentrating mechanism (CCM) that concentrates CO₂ in the chloroplasts. Both C₃ and C₄ Hydrilla leaves showed light-dependent pH polarity that was suppressed by high dissolved inorganic carbon (DIC). At low DIC (0.25 mol m⁻³), pH values in the unstirred water layer on the abaxial and adaxial sides of the leaf were 4.2 and10.3, respectively. Abaxial apoplastic acidification served as a CO₂ flux mechanism (CFM), making HCO₃ ⁻ available for photosynthesis by conversion to CO₂. DIC at 10 mol m⁻³ completely suppressed acidification and alkalization. The data, along with previous results, indicated that inhibition was specific to DIC, and not a buffer effect. Acidification and alkalization did not necessarily show 1:1 stoichiometry; their kinetics for the apolar induction phase differed, and alkalization was less inhibited by 2.5 mol m⁻³ DIC. At low irradiance (50 μmol photons m⁻² s⁻¹), where CCM activity in C₄ leaves is minimized, both leaf types had similar DIC inhibition of pH polarity. However, as irradiance increased, DIC inhibition of C₃ leaves decreased. In C₄ leaves the CFM and CCM seemed to compete for photosynthetic ATP and/or reducing power. The CFM may require less, as at low irradiance it still operated maximally, if [DIC] was low. Iodoacetamide (IA), which inhibits CO₂ fixation in Hydrilla, also suppressed acidification and alkalization, especially in C₄ leaves. IA does not inhibit the C₄ CCM, which suggests that the CFM and CCM can operate independently. It has been hypothesized that irradiance and DIC regulate pH polarity by altering the chloroplastic [DIC], which effects the chloroplast redox state and subsequently redox regulation of a plasma-membrane H⁺-ATPase. The results lend partial support to a down-regulatory role for high chloroplastic [DIC], but do not exclude other sites of DIC action. IA inhibition of pH polarity seems inconsistent with the chloroplast NADPH/NADP⁺ ratio being the redox transducer. The possibility that malate and oxaloacetate shuttling plays a role in CFM regulation requires further investigation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Rates of glycolate synthesis and metabolism during photosynthesis of Euglena and microalgae grown on low CO2

The rate of glycolate excretion in Euglena gracilis Z and some microalgae grown at the atmospheric level of CO₂ was determined using amino-oxyacetate (AOA). The extracellular O₂ concentration was kept at 240 M by bubbling the incubation medium with air. Glycolate, the main excretion product, was excreted by Euglena at 6 mol·h^-1·(mg chlorophyll (Chl))^-1. Excretion depended on the presence of AOA, and was saturated at 1 mM AOA. A substituted oxime formed from glyoxylate and AOA was also excreted. Bicarbonate added at 0.1 mM did not prevent the excretion of glycolate. The excretion of glycolate increased with higher O₂ concentrations in the medium, and was competitively inhibited by much higher concentrations of bicarbonate. Aminooxyacetate also caused excretion of glycolate from the green algae, Chlorella pyrenoidosa, Scenedesmus obliquus and Chlamydomonas reinhardtii grown on air, at the rates of 2–7 mol·h^-1·(mg Chl)^-1 in the presence of 0.2–0.6 mM dissolved inorganic carbon, but the cyanobacterium, Anacystis nidulans, grown in the same way did not excrete glycolate. The efficiency of the CO₂-concentrating mechanism to suppress glycolate formation is discussed on the basis of the magnitude of glycolate formation in these low-CO₂-grown cells.Abbreviations AOA aminooxyacetate - Chl chlorophyll - DIC dissolved inorganic carbon - HPLC high-pressure liquid chromatography - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase This is the 16th paper in a series on the metabolism of glycolate in Euglena gracilis. The 15th paper is Yokota et al. (1985c)