Carbon dioxide

We shall first consider the extent to which CO2 availability limits the overall rate. Given that CO2 is a substrate that is used by an enzyme (system), then we may plausibly suppose that the photosynthetic rate at any given light intensity will vary with CO2 concentration approximately in accordance with the well-known Michaelis-Menten equation for enzyme kinetics

Km + s where v is the rate of enzyme reaction at substrate concentration s, V is the maximum rate obtainable at saturating substrate concentrations and Km is the dissociation constant of the enzyme-substrate complex, but is also equivalent to the substrate concentration that gives half the maximum rate. Re-expressing the equation in photosynthetic terms we obtain p=_Pm[C(°2]__(11 2)

Figure 11.2 shows an idealized curve of rate versus substrate concentration for an enzymic reaction, in accordance with the MichaelisMenten equation. Observed curves of photosynthetic rate versus CO2

concentration for aquatic plants are approximately of this type, and so by obtaining the Km values from such curves and comparing them with the in situ CO2 concentrations, we may be able to assess to what extent CO2 availability limits photosynthesis in natural waters.

In the isolated state, Rubisco from aquatic plants has a Km value for CO2 of 30 to 70 mM.1489 The Km(CO2) values for living plants, however, can be higher because of diffusive resistance to entry of carbon dioxide into the plant, or lower due to active uptake of CO2. Some values for Km(CO2) determined for phytoplankton and macrophyte species, at pH values low enough (pH <6) to ensure that essentially all the inorganic carbon (Ci) exists as CO2 (or its hydrated form, H2CO3), are listed in Table 11.1: they range from 4 to 185 mM. Fresh water in equilibrium with the atmosphere (^0.035 vol% CO2) at 15°C contains dissolved CO2 at about 14 mM concentration. Calculations for sea water at 26°C indicate a free CO2 concentration of about 12 mM.136 We might therefore expect it commonly to be the case that aquatic plant photosynthesis in natural waters is undersaturated with respect to CO2 concentration, and should respond to an increase in CO2 concentration with an increase in photo-synthetic rate. This need not apply only at saturating light intensity: the assumption that photosynthetic rate depends on CO2 concentration in accordance with the Michaelis-Menten relation implies that even at low light intensities an increase in CO2 concentration above a typical starting value of 12 to 14 mM would lead to an increase in photosynthetic rate. This means that at subsaturating light intensities, photosynthesis by plants within a water body could be simultaneously limited by availability of both light and CO2. The aquatic moss, Fontinalis antipyretica, and the aquatic higher plant, Cabomba caroliniana, both show marked increase in photosynthetic rate with increasing CO2 concentration in the range 10 to 25 mM at low, subsaturating, as well as at high, light intensities.523,1235 Figure 11.3 shows Smith's (1938) data for Cabomba. These examples are, however, somewhat untypical since most aquatic higher plants and algae, in the particular waters in which they naturally occur, do not show much response to an increase in CO2 supply. The reason is that not only can aquatic plants draw on other sources of inorganic carbon, but most of them also have the ability to carry out active transport of Ci, this being commonly referred to as the carbon-concentrating mechanism, or CCM. This topic is reviewed by Falkowski and Raven (2007), by Kaplan and Reinhold (1999), and in the proceedings of the 4th International Symposium on Inorganic Carbon Utilization by Aquatic Photosynthetic Organisms, 2001 (Functional Plant Biology, 2002, Vol. 29).

Table 11.1 Apparent half-rate constants (Km) for CO2for photosynthesis in certain phytoplankton and macrophyte species.

Plant species

Km(CO2) (mM) Reference

Freshwater phytoplankton


Pediastrum boryanum 40

Cosmarium botrytis 170

Chlamydomonas reinhardtii 29

Scenedesmus obliquus 38 Euglenophyta

Euglena gracilis 25 Cyanophyta

Anabaena cylindrica 60

Aphanizomenon flos-aquae 105

Coccochloris peniocystis 121

Plectonema boryanum 100

Freshwater macrophytes

Nitella flexilis 100

Eurhynchium rusciforme 80

Fontinalis antipyretica 170

Elodea canadensis 30

Potamogeton crispus 20

Hydrilla verticillata 170

Myriophyllum spicatum 150

Ceratophyllum demersum 165

Marine phytoplankton


Stichococcus bacillaris 4 Heterokontophyta

Cylindrotheca fusiformis (diatom) 36

Olisthodiscus luteus (raphidophyte) 59 Rhodophyta

Porphyridium cruentum 22 Cyanophyta

Synechococcus sp. 240 Marine macrophytes

Ulva sp. 30

Ulva lactuca 185

19 19 384 384

19 384 384 384

19 19 19 19 19 1394 1394 1394

384 384

86 321

Sea water and most inland waters contain much more inorganic carbon in the form of bicarbonate ion, HCO3~ than in the form of CO2. The different forms of inorganic carbon are interconverted in accordance with

Chlorella Growth Absorbance Co2
Fig. 11.3 Photosynthetic rate as a function of CO2 concentration at different light intensities in the aquatic higher plant Cabomba caroliniana (after Rabinowitch, 1951, based on data of Smith, 1938).

The higher the pH of the water, the more this equilibrium shifts to the right with HCO3~ constituting more than 50% of the total from pH 6.2 to 9.3, and more than 80% between pH 6.7 and 8.8. Given that CO2 tends, as we have seen, to be present at suboptimal concentration, it would clearly be an advantage in many waters for aquatic plants to be able to use bicarbonate as a carbon source for photosynthesis, and in fact many, but not all, species do. The inorganic carbon species used by the carboxylase enzyme is always CO2. In the plants that can utilize bicarbonate, the HCO3~ ions are transported into the cell where they give rise to CO2 by the reversal of the first two of the above interconversion reactions. The reversible dehydration of H2CO3 to give CO2 is catalyzed by carbonic anhydrase, an enzyme that occurs in the chloroplast, but can also be present within the cytoplasm and on the exterior surface of the cytoplasmic membrane. The liberated CO2 is then used in photosynthesis. In the case of the external enzyme, the liberated CO2 diffuses through the cytoplasmic membrane into the cell. The combination of HCO3~ ions with H+ to give H2CO3 leads to a corresponding accumulation of OH- ions (from H2O! H+ + OH) which are excreted from the cells to balance the uptake of HCO3.

So far as marine ecosystems are concerned, photosynthetic utilization of bicarbonate has been found in most of the seaweed species - green, brown and red - which have been examined, but is not universally present. Maberly (1990) found that out of 35 species of marine macro-algae, six species - all rhodophytes - were unable to use HCO3~ and five of these occurred in relatively low-light habitats, beneath a canopy of larger Phaeophyta. He suggested that most species growing at depths where light is low will be unable to use HCO3. The red macroalga Chondrus crispus, although it does not take up bicarbonate directly, nevertheless gains access to this plentiful inorganic carbon pool by dehydrating HCO3~ to CO2 with an external carbonic anhydrase.1254,1255 Out of 29 macroalgal species (13 green, 5 red, 11 brown) studied by Larsson and Axelsson (1999) all except one (Palmaria palmata, red) utilized HCO3~ by means of an external carbonic anhydrase.

In addition to being able to utilize HCO3~ via an external carbonic anhy-drase, some algae also have the ability to actively transport this anion into the cell. Two common and successful green algal species, Cladophora glomerata and Enteromorpha intestinalis, both have bicarbonate transporters.229,771 Some, but not all, green algal species have an inducible bicarbonate transporter, which operates by anion exchange:770,229 this capacity develops at high pH. It was absent from a number of brown and red algal species tested. The brown alga, Hizikia fusiforme, possesses an external carbonic anhydrase, but does not carry out active transport of HCO3~.1506 Choo et al. (2002) found that in addition to an external carbonic anhydrase and an anion exchange-type bicarbonate transporter, Cladophora glomerata can also form a transporter that uses a proton pump of the ATPase type. Protons are electrogenically pumped out of the cell to create an active proton gradient, which then leads to inward transport of H+ and HCO3. Formation of the proton pump is induced by carbon limitation. An additional mechanism that makes use of the proton gradient is the localized combination of H+ and HCO3~ to give H2CO3 from which CO2 is liberated by means of the external carbonic anhydrase. This appears to be present in the charophyte, Chara corallina,393,1088 and in the brown alga, Laminaria saccharina.41 The CO2 thus produced is present at locally high concentration, leading to inward diffusion across the cell membrane. In Chara, proton secretion takes place in specialized acidic bands. These are accompanied by separate alkaline bands1114 where CaCO3 incrustation takes place: these may correspond to sites where OH secretion (or H+ uptake) takes place to conserve alkalinity.85 Even in those macrophytes, such as Ulva, which can utilize bicarbonate it seems that photosynthesis in bright light can in some cases be limited by the level of inorganic carbon in sea water.794a

Bicarbonate utilization is found in some,87,847,1092a but apparently not all,5 seagrasses. Possession of an external, membrane-bound carbonic anhydrase is common among these marine angiosperms.85 While this facilitates uptake of inorganic carbon it does not on its own constitute a CCM. It appears likely that those seagrasses that can carry out active transport of bicarbonate do so by means of a proton pump creating an H+ gradient. This gradient can be used to bring about bicarbonate transport in two ways. There can be inward cotransport of H+ and HCO3, or there can be localized combination of H+ and HCO3~ to give H2CO3 from which CO2 is liberated by means of the external carbonic anhydrase,393,1088,85 followed by inward diffusion across the cell membrane as described for Chara, above. As with Chara, it seems likely that there are spatially separated acid and alkaline zones.85 This particular mechanism for CCM is not universal among seagrasses: out of eight tropical species studied by Uku et al. (2005), it was present in six and absent in two. In Zostera marina, which does utilize this mechanism, Carr and Axelsson (2008) found that a major part of the ATP used to generate the acid zones was derived from mitochondrial respiration. Seagrasses do not appear to possess an active transport mechanism of the anion exchange type.

On the basis of isotope disequilibrium experiments, carried out on phytoplankton from the eastern Subtropical and Equatorial Pacific Ocean, Tortell and Morel (2002) concluded that HCO3 was the principal form of inorganic carbon taken up by all of the in situ populations sampled. All eukaryotic phytoplankton species contain internal carbonic anhydrase: the presence of an external carbonic anhydrase is common but not universal. Among the marine phytoplankton, HCO3~ utilization has been shown in the coccolithophorid Coccolithus huxleyi;1028 in a number of diatom, haptophyte and eustigmatophyte species;256 and in the unicellular red alga Porphyridium cruentum;255 but the green plankter Stichococ-cus bacillaris has a high affinity for CO2 (Table 11.1) and a low affinity for bicarbonate,966 and a number of common marine diatoms appear to be unable to use bicarbonate.296,1127 Dason et al. (2004) found that two marine dinoflagellates, Amphidinium carterae and Heterocapsa oceanica, are unable to take up bicarbonate, but appear to have an active transport system for CO2 itself. This appears to be also the case for two marine chlorophyte species, Nannochloris atomus and N. maculata.598 Symbiotic dinoflagellates (Symbiodinium sp., zooxanthellae) in giant clams have a CCM, and preferentially transport CO2.792 Burkhardt et al. (2001) present evidence that two marine diatom species, Thalassiosira weissflogii and Phaeodactylum tricornutum, possess active transport systems for both HCO3 and CO2. The rhodophyte, Porphyridium cruentum, also actively transports both forms of Ci,598 as do the freshwater eustigmatophyte, Eustigmatos vischeri,597 and certain marine prymnesiophyte species.108,596 Inhibitor studies suggest that an anion-exchange type of HCO3~ transport system occurs in some eukaryotic marine phytoplankton species but not others.992 Maberly et al. (2009) tested species from 12 chrysophyte families for their ability to take up inorganic carbon. The results indicated that chrysophytes as a group do not have a CCM, and in addition lack the ability to make use of bicarbonate as an alternative source of inorganic carbon.

Marine cyanobacteria, such as Synechococcus, actively transport both HCOr and CO2 into their cells.55,1089,56 Within the cell Ci exists predominantly as HCO3. The bicarbonate ions diffuse into the carboxysome, a semi-crystalline structure consisting mainly of Rubisco, but also containing carbonic anhydrase. The latter enzyme maintains a high localized level of CO2 by continuously generating it from HCO3, and this is sufficient to saturate the carboxylase, and competitively suppress the oxygenase activity of Rubisco.384

In fresh waters, according to Hutchinson's (1975) review, most aquatic higher plants can utilize bicarbonate for photosynthesis. Maberly and Madsen (2002), in their review of carbon uptake mechanisms in freshwater angiosperms, report that use of HCO3~ is present in about half the submerged angiosperm species that have been tested, and is more common in lakes of high alkalinity. The species that cannot utilize bicarbonate typically occur in soft waters in which bicarbonate concentrations are low. Aquatic mosses mainly seem to lack the ability to utilize HCO3. Most charophyte species (other than those from soft waters) can utilize HCO3~ as can benthic filamentous algae such as Cladophora. Among the freshwater phytoplankton, the ability to utilize HCO3~ for photosynthesis varies widely. Chlorella emersonii, Scenedesmus quadricauda, Chlamydomonas reinhardtii (green), Ceratium hirundinella (dinoflagellate), Fragilaria crotonensis (diatom), Microcystis aeruginosa and Anabaena cylindrica (blue-green) can all effectively use HCO3 but Chlorella pyrenoidosa (green), Asterionella formosa and Melosira italica (diatoms) cannot19,839,1109,1338,1377 Certain other freshwater Chlorella species, and the freshwater diatom, Navicula pelliculosa, carry out active transport of both HCO3- and CO2.256

Despite the wide occurrence of ability to utilize bicarbonate in the aquatic plant kingdom, it is generally true that, with the possible exception of phytoplankton species adapted to alkaline waters, free CO2 if available is the preferred, i.e. more effectively used, carbon source. For example, the freshwater macrophyte Myriophyllum spicatum, which can use bicarbonate, gives a much higher photosynthetic rate with free CO2 at its optimum concentration than with HCO3~ at its optimum concentration.1297 Bodner (1994) found that the pondweed, Potamogeton natans, used CO2 but was unable to use HCO3: Ranunculus fluitans preferentially used CO2, but was able to switch to HCO3~ at pH values of 9.0 and above, when the CO2 concentration was close to zero. Steemann Nielsen (1975) suggested that one of the reasons for the less effective use of HCO3~ is that energy must be used for its active transport into the plant, whereas CO2 diffuses in freely. The data of Allen and Spence (1981) for freshwater macrophytes in Table 11.1 show that in all cases, including the known bicarbonate users Elodea canadensis and Potamogeton crispus, the apparent Km for HCO3~ is 50- to 100-fold higher (indicating a much lower affinity) than the Km for CO2. Furthermore, to give the same photosynthetic rate as a certain concentration of CO2 (at pH 5.5), HCO3~ (pH 8.8) at 52 to 132 times the concentration was needed. In the case of the blue-green alga Anabaena cylindrica, on the other hand, occurring typically in alkaline eutrophic waters, the Km values for CO2 and HCO3~ are about the same, and the concentrations of CO2 and HCO3~ that produce a certain photosynthetic rate are also about the same. On the basis of their studies, Allen and Spence conclude that despite the ability of most freshwater macrophytes to utilize bicarbonate, they do not in fact obtain much of their carbon from HCO3~ until the pH of the water exceeds 9.0, and at these high pH values their photosynthetic rates are greatly reduced anyway. Allen and Spence suggest that the natural rates of photosynthesis of freshwater macrophytes and some planktonic algae are functions mainly of CO2 (as opposed to HCO3) concentration. This conclusion may not, however, apply to the more effective bicarbonate users among the macrophytes, such as Myriophyllum spicatum. Adams et al. (1978) found that in a series of rather alkaline (pH values mainly 7.5-8.8) Italian lakes, all well supplied with phosphorus, the rate of photosynthesis of M. spicatum varied

Total dist(}iw«d inoraaoic carbo« (mM| □.(J 1JG I.D 3JO 40

Total di[Hlv«i Inorganic carimn (nig CO] equig^Eenn

Fig. 11.4 Variation of photosynthetic rate of Myriophyllum spicatum with total dissolved inorganic carbon concentration in a series of Italian lakes (plotted from data of Adams et al., 1978). Light intensities were at or near saturation in most cases. Using non-linear regression to the MichaelisMenten equation, Adams et al. calculated a Km (half-saturation constant) value of 1.06 mM total dissolved inorganic carbon (46.5 mg CO2 equivalents l_1) and a Pm value of 7.24 mg C g_l dry mass h_1.

Total di[Hlv«i Inorganic carimn (nig CO] equig^Eenn

Fig. 11.4 Variation of photosynthetic rate of Myriophyllum spicatum with total dissolved inorganic carbon concentration in a series of Italian lakes (plotted from data of Adams et al., 1978). Light intensities were at or near saturation in most cases. Using non-linear regression to the MichaelisMenten equation, Adams et al. calculated a Km (half-saturation constant) value of 1.06 mM total dissolved inorganic carbon (46.5 mg CO2 equivalents l_1) and a Pm value of 7.24 mg C g_l dry mass h_1.

with the total dissolved inorganic carbon concentration in accordance with a Michaelis-Menten type of relation (Fig. 11.4). From calculations of the amounts of the different forms of inorganic carbon present, it appeared that photosynthetic rate was related primarily to the concentration of HCO3~ rather than to the concentration of CO2.

Jones (2005) has pointed out that the formation and operation of an active transport system for HCO3~ must impose an energy cost on a plant. In experiments with Elodea nuttallii, growing in water containing bicarbonate, and bubbled either with normal air or air depleted of CO2 (which would favour the switch to HCO3~ use), he found that after 21 days the bicarbonate-using plants photosynthesized at ^58% higher rate, but had achieved only the same growth as the plants predominantly using CO2, suggesting that some of the photosynthetically captured light energy was being used for constructing, maintaining and running the bicarbonate utilization system. Jones estimated that this would correspond in the field to an irradiance of ~80 mmol photons m~2 s_1, below which HCO3~ use would not be expected in this species. Since this is an irradiance commonly experienced by submerged macrophytes in inland waters, HCO3~ use is likely to be confined to areas where ambient light is above the requisite threshold.

Algae such as Anabaena cylindrica, which photosynthesize optimally at pH values in the region of 9.0, make very efficient use of bicarbonate. For these planktonic species and the more effective bicarbonate users among the macrophytes, the natural rate of photosynthesis (as far as carbon supply is concerned) can be regarded as a function of the concentration of HCO3~ plus CO2. The ability of blue-green algae such as Anabaena, Microcystis and Spirulina to continue photosynthesizing in water of high pH, and effectively zero free CO2 concentration could be one of the main factors contributing to their frequent domination of eutrophic lakes in late summer and their invariable domination of highly alkaline waters such as the African soda lakes. The ability of some cyanobacteria to form surface blooms gives them an additional advantage: by placing a dense biomass close to the water surface, they are able to intercept a large proportion of the incoming CO2 from the atmosphere, as well as the incident light.607

Rattray et al. (1991) found that two rooted freshwater macrophytes, Lagarosiphon major and Myriophyllum triphyllum, grew and photosynthe-sized twice as fast in the water of oligotrophic Lake Taupo (New Zealand) as in the nitrogen- and phosphorus-rich water of eutrophic Lake Rotorua. Rattray et al. attributed this to the two-fold higher level of CO2 found to be present in the oligotrophic, than in the eutrophic, water. Soft-water oligotrophic lakes are often characterized by the presence of benthic macrophytes with the isoetid growth form: small plants with short stems and rosettes of leaves.603 Isoetids have solved the problem presented by the low inorganic carbon concentration in these poorly mineralized waters by extracting the CO2 from the sediments, where it is formed from decaying organic matter, through their roots.142,847,1111,846 This CO2 diffuses from the roots to the leaves via longitudinal air-filled channels, and in some cases provides virtually all the photosynthetically fixed carbon. The emergent macrophyte, Cyperus papyrus, also has large intercellular air cavities (aerenchyma) extending down to its rhizomes in the sediments, and is able to photosynthetically capture CO2 coming up from the rhizomes.808

The free CO2 concentrations of 12 to 14 mM referred to earlier apply only to waters in equilibrium with the atmosphere. Such concentrations might normally be found in the surface layer of the oceans, or of unproductive inland waters, or of most inland waters in the winter. However, any biologically productive water body is likely not to be in equilibrium with the atmosphere so far as CO2 is concerned. The free CO2 concentration at any time is likely to differ from the equilibrium value and will vary with time and depth in accordance with where and how fast the processes of consumption (photosynthesis) and production (respiration, decomposition) are taking place. On the basis of model calculations of rates of CO2 diffusion to the cell, and of laboratory measurements of growth rates as a function of CO2 concentration, Riebesell et al. (1993) concluded that during phytoplankton blooms in the ocean, when CO2 concentration in the surface layer falls drastically, the growth rate of marine diatoms can be limited by the CO2 supply to the cell surface. Vadstrup and Madsen (1995) found in oligotrophic low-alkalinity Lake Hampen (Denmark) that the concentration of free CO2 was about five times atmospheric equilibrium concentration in early summer, but had declined virtually to zero by the end of summer.

Free CO2 can be even more affected by changes in pH caused by photosynthesis. Talling (1976, 1979) presented data for the changes in free CO2 concentration in the eutrophic English lake, Esthwaite Water, from April to July 1971. On 19 April, before thermal stratification, the CO2 concentration was the same from the surface down to 10 m depth. By 3 May when the phytoplankton population had begun to increase, and there was some warming of the surface layer, CO2 concentration had fallen by about 33% in the upper 4 m but had increased greatly between 6 and 10 m. During July, thermal stratification became established, the phytoplankton population above the thermocline increased substantially and free CO2 levels in the epilimnion were reduced virtually to zero, while rising to even higher values than before in the 6 to 10 m layer below the thermocline. The amount of inorganic carbon removed by photosynthesis represented about 50% of the total inorganic carbon in the surface layer. The remainder of the ~ 1000-fold decrease in free CO2 was due to the rise in pH from ^7.0 to above 9.0, resulting from the photosynthetic activity of the dense phytoplankton population. Similar changes are likely to take place in any productive water body. For one of the components of the phytoplankton, the diatom Asterionella formosa (not a bicarbonate user), Talling (1979) calculated that on 12 July its photosynthetic rate was virtually zero in the surface layer due to lack of CO2, that it rose between 2 and 3 m due to an increase in CO2 accompanied by reasonably high irradiance values, and that it declined again below this depth because of the fall in irradiance. Thus with increasing depth, its photosynthesis was limited first by a CO2 supply, then by CO2 and light simultaneously, and then mainly by light availability alone. These changes with depth did not apply to total phytoplankton photosynthesis, however, since when the pH rose and the CO2 concentration fell, Asterionella was largely replaced by the bicarbonate users Ceratium and Microcystis.

The sinking rate of diatom cells increases when CO2 uptake becomes limited: the CO2 depletion associated with summer stratification might lead to an increased sinking rate of diatoms, and this may contribute to the paucity of diatoms frequently observed in productive lakes in the summer.622

Immediately next to the surface of any aquatic plant, there is an unstirred layer of water that CO2 and/or HCO3- ions must diffuse across before they can enter the cells and be used for photosynthesis. The thickness of this layer diminishes if the plant is subjected to turbulence or rapid stirring but the layer never disappears completely. Under well-stirred conditions, the unstirred layer might be only about 5 mm thick around a small cell such as Chlorella but 30 to 150 mm thick around the surface of a macrophyte such as Chara:1109,1236 in still or slowly moving water the unstirred layer will often be much more than 150 mm thick. Raven (1970), and Smith and Walker (1980), who have reviewed this topic, conclude that diffusion of CO2 (and presumably HCO3) across this unstirred layer can be an important rate-limiting step in photosynthesis by aquatic macrophytes. An increase in photosynthetic rate of river plants associated with increased flow velocities in natural waters has in fact been demonstrated by Westlake (1967). Wheeler (1980b) found that the photosynthetic rate of the blade of the great kelp, Macrocystis pyrifera, in saturating light, increased about four-fold when the current speed increased from 0 to 5 cm s-1: in low light, however, the increase was only about 50%. Any morphological adaptation that increases the surface-to-volume ratio will help to overcome the diffusion problem and many aquatic macrophyte species have achieved this by evolving highly dissected leaves.603 The more dissected the leaf form, the less the stimulation of photosynthesis brought about by increased turbulence.451 Whether the existence of an unstirred layer around each cell has any significant limiting effect on photosynthesis by phytoplankton remains uncertain.

While the rate of photosynthesis in submerged freshwater macrophytes tends to increase in response to increasing current velocity up to approximately 0.5 cm s-1, there can be inhibitory effects for water velocities beyond ^0.5 to 3 cm s-1.845,991 Using autoradiography of 14CO2-exposed leaves of Elodea canadensis, Nielsen et al. (2006) found that at high current velocity the CO2 uptake was approximately two-fold higher near the leaf periphery compared with the midrib section, whereas at low current velocity the area of relatively high CO2 uptake expanded from the leaf periphery towards the midrib and basal sections. They propose that slow-moving water creeps around the leaf tips and enters the gaps between the whorls of leaves, delivering CO2 to the basal parts of the leaves near the stem. At high water velocity the water does not enter the gaps, but rather skips across, effectively raising the level of the surface. The extent to which this takes place is likely to be very much a function of the plant morphology.

In the majority of terrestrial higher plant species, those which lack the C4 pathway, photosynthesis is significantly inhibited by oxygen at the normal atmospheric level (21%), primarily as a result of the direct competition between O2 and CO2 at the active site of Rubisco. In the oxyge-nase reaction of Rubisco, the oxygen reacts with ribulose bisphosphate to produce phosphoglycollate and phosphoglycerate, and this is the first step in the metabolic pathway known as photorespiration. Photosynthesis by aquatic higher plants and algae, by contrast, in most cases shows relatively little inhibition by oxygen. This appears to be due to the ability of aquatic plants to increase, by one or other of a range of mechanisms, the concentration of CO2 in the proximity of the Rubisco molecules within the cell, thus increasing the CO2/O2 competitive ratio. There is, as we have seen, evidence for active transport of CO2, and in some cases of HCO3~ as well, achieving a many-fold higher internal than external concentration.

As well as the 'biophysical' strategy there are biochemical solutions to the problem of raising the internal CO2 concentration. No submerged aquatic plant has yet been found which exhibits C4 photosynthesis as it occurs in terrestrial C4 species, i.e. with fixation of CO2 into a C4 acid such as malate in the mesophyll cells, followed by transport of this acid to specialized bundle sheath cells where CO2 is released and then re-fixed into carbohydrate by the normal photosynthetic carbon reduction (PCR) cycle. What might, however, be regarded as a structurally abbreviated version of the C4 pathway has been found by Salvucci and Bowes (1983) in the freshwater macrophyte Hydrilla verticillata (Hydrocharita-ceae), where the whole biochemical sequence appears to take place within the same cell. There is also evidence for this kind of C4 photosynthesis in certain other members of the Hydrocharitaceae, including Hydrilla verti-cillata852 and Egeria densa.212 In both cases, formation of the C4 cycle enzymes is induced when the plants are grown under conditions (high temperature, high light, long photoperiods) that result in limiting CO2 levels in the water. In their review of C4 mechanisms in aquatic angiosperms, Bowes et al. (2002) refer to Hydrilla as a facultative C4 that shifts from C3 to C4 in low [CO2].

It seems likely that most of the initial CO2 fixation is carried out by phosphoenolpyruvate (PEP) carboxylase in the cytoplasm. The oxaloa-cetic acid produced is reduced to malic acid. This is transported into the chloroplasts where it is decarboxylated by NADP malic enzyme, and the CO2 is fixed into carbohydrate by the PCR cycle.149 Evidence for a C4 pathway of this type was found in one seagrass species Cymodocea nodosa, but not in ten others.88 There is also evidence for this C4 pathway, but using PEP carboxykinase as the carboxylating enzyme, in the marine green macro alga Udotea flabellum.1118

Among the phytoplankton, Reinfelder et al. (2000) demonstrated short-term labelling of C4 compounds, and transfer of carbon to sugars, in the marine diatom, Thalassiosira weissflogii, which they interpreted as evidence for C4 photosynthesis in this species. Reinfelder et al. (2004) found that a specific inhibitor of PEP carboxylase caused a more than 90% decrease in photosynthesis in cells of T. weissflogii acclimated to low [CO2], but had little effect on a C3-using marine chlorophyte, Chlamydomonas sp. On the basis of similar inhibition experiments, McGinn and Morel (2008) propose that C4-based CO2-concentrating mechanisms are generally distributed in diatoms, and suggest that their possession of a C4-based CCM may have contributed to the evolutionary success of this phyto-plankton phylum, especially in the low CO2 concentrations that characterize present-day surface waters.

Another biochemical CO2-concentrating mechanism, crassulacean acid metabolism (CAM), occurs in some, but not all, of the isoetid macrophyte species of oligotrophic, soft-water, lakes.142,681,1111 Their photosynthetic physiology has been reviewed by Madsen et al. (2002). These aquatic CAM plants have a rosette of short stiff leaves and large roots that can make up more than 50% of the plant biomass. Through their extensive root system they take up CO2 from the sediments, where it is present in high concentration. Transport of CO2 from roots to leaves takes place by diffusion through a system of internal gas spaces (lacunae). Most of the inorganic carbon used in photosynthesis by isoetids comes from sediment CO2, and it is taken up during the night as well as during the day. Possession of CAM enables these plants to fix CO2 during the night by means of PEP carboxylase, in the form of organic acids such as malate, which accumulate in the vacuole. During the day, when light is available, CO2 is liberated by decarboxylation of the C4 acids, and fixed into carbohydrate by the PCR cycle.

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