The role of carbonic anhydrase in photosynthesis and the activity of the carbon-concentrating-mechanism in bryophytes of the class Anthocerotae

Transcription

1 RESEARCH New Phytol. (2000), 145, The role of carbonic anhydrase in photosynthesis and the activity of the carbon-concentrating-mechanism in bryophytes of the class Anthocerotae ELIZABETH C. SMITH* AND HOWARD GRIFFITHS Department of Chemical and Life Sciences, University of Northumbria, Newcastle upon Tyne, NE1 8ST, UK Department of Agricultural and Environmental Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne, UK Received 10 August 1998; accepted 9 August 1999 SUMMARY The role of carbonic anhydrase in the carbon-concentrating-mechanism of bryophytes of the class Anthocerotae was investigated by comparing the gas-exchange characteristics of material which had been incubated in the membrane-permeable Carbonic Anhydrase inhibitor ethoxyzolamide, with those of untreated material and material which had been incubated in buffer solution. In Phaeoceros laevis (Anthocerotae), incubation in ethoxyzolamide caused a depression in the rate of gross assimilation and a decrease in CO affinity beyond that which could be attributed to increased diffusion limitation. A range of liverworts and mosses, in which a carbonconcentrating-mechanism is absent, were also investigated. These showed no depression of rates of gross assimilation after incubation in ethoxyzolamide relative to those of untreated material. The CO compensation point and CO uptake characteristics of Phaeoceros laevis were significantly affected by incubation in ethoxyzolamide. Values of CO compensation point for Phaeoceros laevis rose from Pa, after incubation in buffer, to 20 Pa after incubation in ethoxyzolamide. The CO compensation point for the liverworts Pellia epiphylla and Marchantia polymorpha was not significantly affected by incubation in ethoxyzolamide. Measurements of the release of CO at the end of a short (15 min) period of illumination revealed that, after suppression of carbonic anhydrase activity, the rapid release of a CO pool occurred in Phaeoceros laevis but not in the liverworts. There were also significant differences between values for fractionation measured in units per mil (), measured instantaneously, for Phaeoceros laevis incubated in ethoxyzolamide, compared with fractionation values for this species after incubation in buffer. Incubation in ethoxyzolamide caused fractionation values to rise from , indicating that the carbon-concentrating-mechanism of this species had been inactivated. Incubation in ethoxyzolamide had no effect on fractionation values for the liverworts. The convexity of the light saturation curves of liverworts and Phaeoceros laevis was also investigated, but there were no differences between groups before or after the two treatments. The data indicate an important role for carbonic anhydrase in the functioning of the carbon-concentrating-mechanism in the Anthocerotae. Key words: Anthocerotae, bryophyte, carbon-concentrating-mechanism, carbon isotope discrimination, carbonic anhydrase, photosynthesis. Abbreviations: CA, carbonic anhydrase; C I, inorganic carbon; CCM, crassulacean acid metabolism; CCM, carbon-concentrating-mechanism; EZ, ethoxyzolamide;, fractionation measured in units per mil (); δ a, δc of CO in ambient air (8); δ p, δc of plant organic material; Γ, CO compensation point; K., CO concentration at which half maximum rates of assimilation are reached; PDB, Pee Dee Belemite; θ, convexity (curvature of light response curve); φ, initial slope of light response curve *Author for correspondence (tel ; fax ; e.c.smithunn.ac.uk).

2 30 RESEARCH E. C. Smith and H. Griffiths INTRODUCTION Measurements of K (external CO concentration. at which half maximal rates of assimilation are reached) and Γ (CO compensation point) have shown that the Anthocerotae have a higher affinity for CO than other liverworts and mosses studied. These gas-exchange characteristics are similar to those of organisms which utilize C photosynthesis, crassulacean acid metabolism (CAM) or a biophysical carbon-concentrating-mechanism (CCM) (Smith & Griffiths, 1996a,b). This is in contrast to the majority of bryophytes, which are characterized by C photosynthetic responses (Green & Snelgar, 1982; Proctor et al., 1992; Green & Lange 1994; Rice & Giles 1994; Raven et al., 1998) modified by the effects of CO diffusion limitation (Williams & Flanagan, 1996). The Anthocerotae also show reduced discrimination against C during photosynthesis, with fractionation measured in units per mil () ( ) values intermediate between those typical of C and C higher plants (Smith & Griffiths, 1996a,b) and uptake of HCO by the Anthocerotae has been demonstrated (Bain & Proctor, 1980). A number of aquatic organisms including the cyanobacteria and some microalgae employ a CCM to elevate intracellular levels of CO at the site of Rubisco, preventing oxygenase activity and reducing photorespiration (Raven, 1985; Badger & Andrews, 1987; Badger & Price, 1992). The unusual gasexchange characteristics of the Anthocerotae may be attributed to the activity of a CCM rather than to C photosynthesis or CAM, but at present unequivocal evidence for the mechanism of this CCM is lacking. Cyanobacteria have the most effective inorganic carbon (C i )-accumulating mechanisms, generating fold increases in internal C i concentrations depending upon external CO concentration (Badger & Andrews, 1987, Kaplan et al., 1991). The cyanobacterial CCM is based upon the operation of four distinct characteristics: an inorganic carbon delivery system supplying HCO into the cell interior (a pump using CO or HCO, Badger & Price, 1992); the absence of carbonic anhydrase (CA) in the cell interior which prevents interconversion of HCO and possible leakage; a closed, leak-proof compartment containing Rubisco (carboxysome) and the localization of CA in carboxysomes to provide CO for Rubisco. Some microalgal and aquatic macrophytes have a less efficient mechanism which increases internal CO concentrations to times those of external CO (Badger & Price, 1992) and the interplay between the various CCM components is less certain than for cyanobacteria. The role of carbonic anhydrase has been implicated in a number of locations: external; periplasmic; chloroplast or intrathylakoid space (Husic,1990; Palmqvist et al., 1990; Sultemeyer et al., 1991), although CA activity has been found throughout the cell (Eriksson et al., 1996). One recent hypothesis suggests that a range of CCM characteristics might be engendered by the intrathylakoid CA, with the localised enrichment provided by subsequent CO leakage supplying Rubisco (Raven, 1997). Investigations of CA activity associated with microalgae which demonstrate CCM activity indicate that the activity of both periplasmic and intracellular CA increases in low-c i -grown cells and that this increase is associated with increased affinity for the uptake of both CO and HCO (Miyachi et al., 1985: Palmqvist et al., 1994a). However, the precise subcellular localization of isozymes of CA has yet to be achieved and CA isozymes of the β type have been shown to exhibit high intracellular activity in microalgae which have been shown to lack a CCM (Palmqvist et al., 1995) indicating that these enzymes are not exclusively associated with CCM activity. The induction and location (plasmalemma or chloroplast envelope) of the inorganic carbon transport system, are also open to debate (Rotatore & Colman, 1990; Palmqvist et al., 1994a). Additionally, the pyrenoid, a starch-coated structure containing Rubisco (Vaughn et al., 1992) present in the chloroplast of many microalgae and some Anthocerotae, might play a functional role, being directly analogous with the cyanobacterial carboxysome, or providing a means of separating oxygenic PSII from Rubisco. Alternatively, the pyrenoid might provide tight packaging of Rubisco fuelled by CO from the intrathylakoid CA which would enhance the CO :O ratio at Rubisco. Further physiological investigation of members of the Anthocerotae which lack a pyrenoid and in some cases are multiplastidic (Vaughn et al., 1992), is likely to contribute to the elucidation of the precise role of the pyrenoid in the functioning of the CCM. As yet these species have proved difficult to grow in controlled conditions and in this investigation all members of the Anthocerotae studied possessed a pyrenoid. The aim of this investigation was to provide further information about the role of CA in the CCM of the Anthocerotae by combining gasexchange and carbon-isotope discrimination measurements with the use of the membranepermeable CA inhibitor ethoxyzolamide (EZ). MATERIALS AND METHODS Choice of species and acclimation procedure Two species of the Anthocerotae were investigated, Anthoceros punctatus (L.) and Phaeoceros laevis (L.); and the liverworts Plagiochila spp (Dum.) Dum, Pellia epiphylla ((L.) Corda, Metzgeria furcata (L.) Dum., Lunularia cruciata (L.) Dum ex Lindb. and Marchantia polymorpha L and the mosses Philonotis

3 RESEARCH Photosynthesis and the carbon concentrating mechanism in bryophytes 31 fontana (Hedw.) Brid. and Polytrichum commune Hedw. were investigated. The bryophytes were grown under mist on peat in a controlled environment at 17C under a photon fluence rate of 30 µmol m s and a 12-h photoperiod. Comparisons between individuals of each species were made on gametophyte fragments of a similar size after dead or damaged tissue had been removed. Assimilation rates An ADC 225 Mk III (ADC, Hoddesdon, Herts, UK) set in differential mode was connected in an open system to a modified Hansatech leaf disc oxygen electrode (Hansatech, Norwich, UK) which served as a cuvette. The cuvette was maintained at 15C and ambient air was supplied from compressed-air tanks which maintained a constant CO partial pressure of 35 Pa. A chart recorder measured the gas exchange within the cuvette. Gametophyte fragments (approx. 4 cm, 0.5 g f. wt) were cleaned by spraying with distilled water, blotted dry and placed in the cuvette. After a period of dark acclimation, dark respiration rates were recorded, then saturating light was supplied using a red light source (LS-2, Hansatech, Norwich, UK.). The resulting depletion of CO was recorded and converted to a gross assimilation rate expressed per unit chlorophyll. Readings were then taken using gametophyte fragments which had been immersed in 100 mm Hepes buffer (ph 8.2) or the membraneimpermeable CA inhibitor EZ (500 µm) in buffer (ph 8.2) for 45 min (Palmqvist & Badger, 1996). The length of incubation was established by increasing the period of immersion in EZ from 10 min to 1h 30 min and identifying the minimum time which resulted in the suppression of photosynthesis in the Anthocerotae. The experiment was repeated using a range of mosses and liverworts. At least four experimental runs were carried out for each treatment for each species. CO compensation points and CO affinity The modified Hansatech oxygen electrode already described was used in a closed system with the ADC IRGA in absolute mode. A clean piece of gametophyte from a range of bryophytes was placed in the cuvette. Air in the system was purged of CO to a partial pressure of approximately 40 Pa or 10 Pa for measurement of compensation points, by diverting it from the cuvette through a column of Carbasorb. The gametophyte was left to deplete the CO within the system until the compensation point was reached. The procedure was repeated at least five times for each species. Gametophyte fragments of the same species were then treated with buffer and the experiment repeated. Finally, Marchantia polymorpha and Pellia epiphylla were selected for more detailed investigation together with Phaeoceros laevis and the experiment was repeated, incubating the species in EZ (ph, molarities and immersion times as already stated). rates were calculated from the gradient of the trace made as the gametophyte depleted CO (from 40 Pa, n 5) and expressed per unit chlorophyll. CO affinity curves were then plotted. There was no significant difference in values of Γobtained from the traces begun at 40 Pa and 10 Pa (the latter experiments were carried out to ensure that assimilation rates in the longer experiment (40 Pa) were not affected by desiccation), so data were pooled (i.e. n 10 for Γ). Measurement of light dark transients in C i pools after incubation in ethoxyzolamide The apparatus described above (IRGA in differential mode) was used in an open system. After 15 min of illumination, when CO uptake had stabilized, the light was switched off. The chart recorder then traced the return to dark-respiration rates. The experiment was repeated using gametophyte fragments which had been immersed in Hepes buffer (ph 8.2) or EZ (500 µm) in buffer (ph 8.2) for 45 min and at least four experimental runs were carried out for each treatment. Gas exchange and carbon-isotope analysis The detailed experimental protocols for these measurements, using a Walz minicuvette system (Compact Minicuvette System, H. Walz, Effeltrich, Germany), have been described previously (Smith & Griffiths 1996b). Light response curves were constructed for Pellia epiphylla, Marchantia polymorpha and Phaeoceros laevis. Net assimilation was measured at 15C and at incident light intensities of µmol photon m s, beginning with saturating irradiances. Dark-respiration rates were measured at the beginning and end of each light-response curve and used to calculate gross assimilation. The data from four light-response curves were averaged for each species. The light-response curve of photosynthesis is described by (1) (Leverenz & Jarvis, 1979): (θp)((φi)p max )P)(φIP max ) 0 (1) (θ is the convexity of the curve, P is the rate of gross assimilation, I is the incident irradiance, φ is the apparent quantum efficiency of photosynthesis and P max is the maximum rate of gross assimilation.) Mean values from four curves were fitted to (1) to give values for θ. This value has been shown to vary in microalgae depending upon the degree to which the CCM is induced (Palmqvist et al., 1994b). Samples for carbon-isotope discrimination measurements were collected using the system described above which was interfaced to a glass

4 32 RESEARCH E. C. Smith and H. Griffiths Table 1. Gas-exchange characteristics of species used for detailed study Gross photosynthesis (in air) Depression of gross photosynthesis after 45 min incubation (%) Groupspecies nmol CO mg Chl s Hepes (ph 82) EZ (ph 82) Mosses Philonotis fontana 3101 (32) (62) (71) Polytrichum commune 3304 (48) (77) (74) Liverworts Plagiochila spp (44) (65) (62) Pellia epiphylla 5304 (62) (81) (83) Metzgeria furcata 4202 (55) (88) (88) Lunularia cruciata 9802 (46) (60) (63) Marchantia polymorpha (41) (82) (82) Hornworts Anthoceros punctatus 8706 (41) (91) (98) Phaecoceros laevis 7402 (50) (86) (112) Gross photosynthetic rates represent maximum values obtained at optimal gametophyte water contents. Values in parenthesis represent water contents (g H Og DW). Data are mean values (n 4)SE. collection line. During sample collection the plants were mounted on a Perspex plate and maintained at saturating irradiances and 90 98% rh. The average water content of the gametopyhte for each 15-min collection period was obtained by recording the fresh weight of the thallus before and after the gas sample was collected. Gas samples were only collected at maximum rates of net assimilation. Measurements of dark respiration were taken regularly throughout each experimental run and used to calculate the gross assimilation rates which corresponded to each collection. As a result of isotopic discrimination during photosynthesis, the CO leaving the cuvette is enriched in CO in proportion to the extent of discrimination shown by the thallus, with then derived from (2) (Evans et al., 1986): ξ(δ o δ e δ o ξ(δ o δ e ) (2) (δ o δc of air leaving the chamber, i.e. CC of analysis gas, δ e δc of air entering the chamber, i.e. CC of reference gas) and ξ c e c e c o (c e concentration of CO entering the cuvette (µl l ), c o HCO HCO concentration of CO leaving the cuvette µl l ). Collections were taken from samples which had been incubated in buffer and EZ for 45 min. Chlorophyll analysis Chlorophyll analysis was carried out on five replicate samples from each species of bryophyte (Ronen & Galun, 1984). RESULTS rates rates in nonincubated samples and samples incubated in buffer and EZ are shown in Table 1. Before incubation in buffer or the membrane-permeable CA inhibitor EZ, the Marchantialean liverworts had the highest assimilation rates per unit chlorophyll. The gross assimilation rates of the Anthocerotae were higher than those of the remaining bryophytes (both liverworts and mosses). There were differences in the effect of incubation in EZ between the mosses and liverworts and the Anthocerotae. In the majority of the plants studied (with the exception of Metzgeria furcata and Marchantia polymorpha) incubation in EZ caused a depression in the gross rate of assimilation beyond that which could be attributed to diffusion limitation (see data for immersion in buffer). The depression of gross assimilation rates relative to those measured in air and in buffer was greatest in the Anthocerotae (mean value 792 for the Anthocerotae vs and 657 for liverworts and mosses, respectively). CO compensation points and CO affinity CO affinity curves are shown in Fig. 1. Values for Γ and the initial slope of the curve for the three species chosen for more detailed study (M. polymorpha, Pellia epiphylla and Phaeoceros laevis) are given in Table 2. Fig. 1a, b shows CO curves plotted for nonincubated and buffer-incubated samples. In all species, incubation in buffer caused

5 the initial slope of the CO affinity curve to decrease as assimilation rates were reduced owing to infiltration of the gametophyte. As in assimilation rates, Fig. 1. CO -response curves for a range of bryophytes (triangle, base uppermost, Marchantia polymorpha; square, Phaeoceros laevis; triangle, apex uppermost, Pellia epiphylla; circle, Metzgeria furcata; diamond, Polytrichum commune. (a) Traces recorded at optimum rates of net assimilation. (b) Traces recorded after gametophyte fragments had been incubated in Hepes buffer (ph 8.2) for 45min. (c) Traces recorded after gametophyte fragments had been incubated in 500 µm ethoxyzolamide (EZ) buffered at ph 8.2 for 45 min. Means of n 5 with SE. Species 40 CO 2 (Pa) (c) 0 40 CO 2 (Pa) Table 2. Data from light and CO response curves and carbon isotope discrimination values for the hornwort Treatment Convexity of light saturation curve (θ) Initial slope of CO uptake curve (nmol CO mg Pa ) CO compensation point (Pa) 8 (b) CO 2 (Pa) 0 40 Instantaneous carbon isotope discrimination ( ) () Max gross photosyntheses is during sample collection (nmol CO mg Chl s Phaeoceros laevis Hepes (ph 82) (10) EZ (10) Low light (6) Pellia epiphylla Hepes (ph 82) (7) EZ (8) Marchantia polymorpha Hepes (ph 82) (10) EZ (9) Gametophyte fragments were incubated in Hepes buffer (ph 82) and EZ (ph 82) for 45 min prior to collection of data from light and CO saturation curves and on-line (fractionation) measurements. The convexities of light saturation curves for each species were obtained from graphs of mean values of four readings at each photon flux densityse. Data were fitted to equation 1 and the parameters θ (convexity) and φ (initial slope of light-response curve) were estimated. Values for θ are shown. The initial slope of the CO uptake curves and the CO compensation points were calculated for individual CO response curves by linear regression using data points between 0 and 10 Pa. Also shown are values for on-line carbon isotope discrimination and corresponding rates of gross assimilation during 15 min sample collections. Samples were collected from Hepes-incubated gametophye fragments at saturating light and at low light (60 µmol photon m s ). Values in parenthesis represent numbers of replicates for each treatment (a) RESEARCH Photosynthesis and the carbon concentrating mechanism in bryophytes 33

6 34 RESEARCH E. C. Smith and H. Griffiths (a) (b) 1 1 (c) (d) 1 1 (e) (f) 1 1 Fig. 2. Accumulation and release of internal pools of CO during light dark transient measurements for the hornwort Phaeoceros laevis and the liverworts Marchantia polymorpha and Pellia epiphylla. Typical traces are shown for each species after at least four experimental recordings. (a, c, e) Traces from Phaeoceros laevis. (b, d, f) Traces from Marchantia polymorpha (solid line) and Pellia epiphylla (dotted line). (a, b) Traces recorded at optimum rates of net assimilation. (c, d) Traces recorded after gametophyte fragments had been incubated in Hepes buffer (ph 8.2) for 45 min. (e, f) Traces recorded after gametophyte fragments had been incubated in 500 µm ethoxyzolamide (EZ) buffered at ph 8.2 for 45 min. this decrease was most pronounced in M. polymorpha. The compensation points of all species after incubation in buffer were significantly higher than those measured prior to incubation, a possible result of higher rates of respiration in the buffer-incubated samples. Fig. 1c shows curves for EZ-incubated samples. Incubation in EZ increased the CO compensation points of Phaeoceros laevis from 2.2 Pa to 20.3 Pa, decreasing the initial slope of the CO curve from 0.29 to 0.10 nmol CO mg Chl s Pa. There was no significant effect of EZ on the initial slope of the CO uptake curve for M. polymorpha and Pellia epiphylla. There was an increase in the compensation point of the EZ-incubated liverworts but this was not of a magnitude comparable to that seen in the EZ-incubated hornwort. Measurement of light dark transients in C i pools after incubation in ethoxyzolamide The characteristics of the CO traces recorded during light dark transient measurements showed a dependence upon the length of dark acclimation before experimentation. Typical traces representing the results of at least four experimental runs for the selected species are shown in Fig. 2. The charac-

7 RESEARCH Photosynthesis and the carbon concentrating mechanism in bryophytes 35 teristics of the traces were unaffected by incubation in Hepes buffer or EZ in Pellia epiphylla and M. polymorpha (Fig. 2b,d,f); however, after incubation in EZ, the rapid release of a CO pool occurred in the Anthocerotae when illumination ceased (Fig. 2e). Light-response curves There were no significant differences between the convexities of light-response curves for the liverworts and Phaeoceros laevis (Table 2) when incubated in buffer or EZ. Carbon-isotope analysis Instantaneous carbon-isotope discrimination values for Pellia epiphylla, M. polymorpha and Phaeoceros laevis are shown in Table 2. There was no significant difference between values for Pellia epiphylla and M. polymorpha incubated in buffer or EZ. Instantaneous values for Phaeoceros laevis incubated in EZ were significantly higher than those for P. laevis incubated in buffer ( 22.7 and 12.4, respectively). Since incubation in EZ caused a decrease in assimilation rates relative to respiration rates, samples were collected from Phaeoceros laevis under lower light intensities (60 µmol photon m s ). This was to confirm that the change in on-line was a direct result of the treatment effect on the photosynthetic mechanism of Phaeoceros laevis and not the result of a different assimilation: respiration ratio. There was no significant difference between values for samples collected at saturating light and those collected at low light, confirming that the observed changes in were the result of changes in the photosynthetic mechanism operating in Phaeoceros laevis. DISCUSSION The CCMs of the microalgae and cyanobacteria have four main components: (1) a mechanism whereby rapid interconversion between CO and HCO can take place, extracellularly and intracellularly, the latter occurring at typical stromal ph values within the Rubisco-containing compartment or, more effectively, at a low ph in the thylakoid lumen; (2) a C i - transport mechanism at plasma membrane, chloroplast envelope or both; (3) ATP energy to power Ci transport; (4) a diffusion barrier to prevent CO from diffusing away from Rubisco. The first two of these features have been shown to involve CA in a range of eukaryotic algae and cyanobacteria which utilize CCMs and it has recently been suggested that CA might function as a diffusion barrier (Raven, 1997). Our data indicate that inhibition of assimilation occurred in all bryophytes studied when they were treated with EZ. The degree to which this inhibition occurred was greatest in the Anthocerotae. Furthermore, there was a pronounced decline in C i -uptake efficiency (increase in Γ and decrease in initial slope of CO -affinity curve) at low external-co levels when samples of Anthocerotae were incubated in EZ, whereas C i -uptake efficiency in the liverworts was unaffected. In comparison with the amount of C i -accumulation which occurs in cyanobacteria and cyanobiont lichens, the steady-state C i pools in microalgae, phycobiont lichens and the Anthocerotae are much smaller (Palmqvist et al., 1994c, Smith & Griffiths 1996b). The appearance of a release pool on the light dark transient trace, shown by the Anthocerotae after treatment with EZ, is analogous with results gained from similar investigations using phycobiont lichens which have a CCM (Palmqvist et al., 1994c) and might indicate that although active transport is occurring, the C i transported to the stroma is not being utilized by Rubisco when CA activity is suppressed. Alternatively, it has been suggested that the release of any internal C I pool might be obscured by carboxylation of the substrate, RuBP (Badger et al., 1993). Hence the appearance of a C i -release pool might indicate inefficient regeneration of RuBP in EZ-treated Anthocerotae. There were no significant differences between the convexities of the light-saturation curves of Phaeoceros laevis, M. polymorpha and Pellia epiphylla, which would indicate diversion of ATP energy to energize the CCM. Further investigation of the active-transport characteristics of these organisms is needed. At this stage, data indicate that at ph 8.2 carbonic anhydrase does not have a direct role in the light-driven accumulation of CO which occurs in the Anthocerotae. There has also been speculation concerning the nature of the diffusion barrier which prevents the diffusion of CO away from Rubisco. This feature confers lower carbon-isotope discrimination values in organisms with a CCM by producing a closed system in which discrimination by Rubisco is not expressed (Beardall et al., 1982; Sharkey & Berry, 1985). An increase in the CO resistance of the plasma membrane (PM) from low-co cells as the explanation of the existence of a larger pool than in high-co cells has been discounted (Sultemeyer & Rinast, 1996). Plasma-membrane resistance in high- CO cells appeared to be the same as, or slightly higher than that of algae with low C i, regardless of ph. Similarly, studies of starch-free mutants of microalgae (del Pino Plumed et al., 1996) indicate that although the starch sheath which surrounds the pyrenoids of many microalgae might develop in cells acclimated to low C i, CCM activity is not dependent upon this property, and increases in both normal and starch-free mutants on acclimation to low C i. Our results indicate that suppression of carbonic anhydrase activity causes carbon-isotope discrimination values to increase to those characteristic of C

8 36 RESEARCH E. C. Smith and H. Griffiths organisms in which CO diffuses away from Rubisco unimpeded. This implies that, in addition to increasing the rate at which CO is supplied to Rubisco, CA plays an important role in minimizing the leakage of CO from the cell. There are a number of ways in which this might happen. Carbon anhydrase located inside the plasma membrane might act as a leak barrier by restoring the CO :HCO ratio to the equilibrium value, lowering the CO concentration locally and preventing leakage of CO to the apoplast (Raven, 1997). Alternatively, has been suggested that in cyanobacteria the arrangement of CA in zones adjacent to Rubisco prevents the diffusion of CO from Rubisco (Reinhold et al., 1991). Clearly, further investigation of the intracellular location of CA is needed to identify the precise nature of the leak barrier. Recent studies of the carboxysomal Rubisco of cyanobacteria have indicated that packing of Rubisco molecules into the carboxysome, which might be fundamental to the operation of the cyanobacterial CCM might also affect the activity of the Rubisco by imposing a resistance to the diffusion of RuBP and PGA to the enzyme (Satoh et al., 1997). Further work is needed to investigate whether the packing of Rubisco and CA into pyrenoids in microalgae and the Anthocerotae has a functional significance or whether this arrangement imposes inefficiencies upon the activity of the enzyme which might be overcome by CCM activity. Finally, in the absence of information on the amounts of PEP carboxylase in the Anthocerotae, we cannot categorically rule out the possibility that a biochemical CCM analogous with that of C and CAM plants is operating in the Anthocerotae, although preliminary studies indicate that the Anthocerotae do not appear to contain significantly more PEP carboxylase than species of bryophyte without a CCM (E. C. Smith & A. M. Borland, unpublished). In C and CAM plants, primary fixation of carbon as HCO by PEP carboxylase and subsequent decarboxylation of the organic acid product generates high values of p i which maximize the ratio of carboxylase: oxygenase activity of Rubisco. Furthermore, a range of C -C intermediate strategies exists in higher plants (Griffiths 1989; Reiskind et al., 1997) and the possibility cannot be excluded that the Anthocerotae utilize a primitive form of these pathways. In conclusion, it is clear that the CCM of the hornworts has features in common with the CCM of microalgae, relying on CA-dependent interconversions of HCO and CO and active transport. When the CCM is inactivated by EZ, the supply of CO to Rubisco declines, assimilation rates per unit chlorophyll are significantly lower than those of liverworts of a similar structure, and the CO affinity of the Anthocerotae is markedly reduced. The question remains whether the very low efficiency of CO utilization demonstrated by the Anthocerotae when the CCM is inhibited might indicate inherent inefficiencies in the photosynthetic apparatus of these organisms. Alternatively, further investigation of nitrogen allocation might reveal that the CCM enables these organisms to maintain photosynthetic rates without investing as much nitrogen in Rubisco. ACKNOWLEDGEMENTS The authors would like to acknowledge assistance of Dr M. C. F. Proctor, Department of Biology University of Exeter, UK in obtaining samples of Phaeoceros laevis. Dr D. Long, Royal Botanic Garden, Edinburgh, UK and Dr A. J. Richards, Department of Agricultural and Environmental Sciences, University of Newcastle upon Tyne, UK provided assistance with the identification of bryophytes. This work was funded by the Leverhulme Foundation. REFERENCES Badger MR, Andrews TJ Co-evolution of Rubisco and CO concentrating mechanisms. In: Biggins J, ed. Progress in photosynthesis research. Dordrecht, The Netherlands: Martinus Nijhof, Badger MR, Pfanz H, Buedel B, Heber U, Lange OL Evidence for the functioning of photosynthetic carbon dioxide concentrating mechanisms in lichens containing green algal and cyanobacterial photobionts. Planta 191: Badger MR, Price GD The CO -concentrating mechanism in cyanobacteria and microalgae. 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