Plant Physiol. (1984) 76, 776-781 0032-0889/84/76/0776/06/$0 1.

00/0

Inorganic Carbon Source for Photosynthesis in the Seagrass Thalassia hemprichii (Ehrenb.) Aschers
Received for publication April 4, 1984 and in revised form July 16, 1984

KAY M. ABEL*' Botany Department, James Cook University ofNorth Queensland, Townsville, Australia 4811
ABSTRACT
Photosynthetic carbon uptake of the tropical seagrass Thalassia hemprichil (Ehrenb.) Aschers was studied by several methods. Photosynthesis in buffered seawater in media in the range of pH 6 to pH 9 showed an exponentially increasing rate with decreasing pH, thus indicating that free CO2 was a photosynthetic substrate. However, these experiments
were unable to determine whether photosynthesis at alkaline pH also contained some component due to HC03- uptake. This aspect was further investigated by studying photosynthetic rates in a number of media of varying pH (7.8-8.61) and total inorganic carbon (0.75-13.17 millimolar). In these media, photosynthetic rate was correlated with free CO2 concentration and was independent of the HC03- concentration in the medium. Short time-course experiments were conducted during equilibration of free CO2 and HC03- after injectfon of "C labeled solution at acid or alkaline pH. High initial photosynthetic rates were observed when acidic solutions (largely free C02) were used but not with alkaline solutions. The concentration of free CO2 was found to be a limiting factor for photosynthesis in this plant.

forms: free CO2, HCO3-, and CO3-2. Free CO2 and HCO3- have both been suggested as possible sources of inorganic carbon for photosynthesis in submerged plants. In seawater (pH 8.2) where approximately 89% ofthe inorganic carbon is present as HCO3r, the ionic form has been suggested as the most likely source of photosynthetic carbon (6, 27). High 6'3C values of marine plants were initially attributed to HCO3- utilization (21). This interpretation was reinforced by the discoveries that terrestrial plants with the C4 pathway of photosynthesis also have high 6'3C values (24) and that HCO3- is the substrate for PEP2 carboxylase, the primary carboxylating enzyme of the C4 pathway (11). The presence of high 6'3C values in seagrasses (18), therefore, suggested the operation of the C4 pathway and concomitant direct uptake of HCO3 from seawater.

Unlike terrestrial plants, submerged plants are surrounded by a medium in which inorganic carbon is present in three available

Preliminary work by Benedict and Scott (6) indicating the formation of C4 acids as early products of carbon fixation in seagrasses supported the proposal that the C4 pathway was operable in seagrasses. However, results reported by Andrews and Abel (2), Abel (1), Benedict et al. (7), and Beer et al. (4, 5) showed that most seagrasses produce PGA as the first product of
'Present address: Australian Institute of Marine Science, P.M.B. No. 3, Townsville M.S.O. 48 10, Australia. 2Abbreviations: PEP, phosphoenolpyruvate; RuBP, ribulose 1,5-biphosphate; PGA, glyceric acid 3-phosphate. 776

carbon fixation and hence are C3 plants. In C3 pathway plants, inorganic carbon must be in the form of free CO2 at the site of fixation by the primary carboxylating enzyme RuBP carboxylase. The carbon fixation mechanism of seaes is therefore unable to access seawater HCO3- directly. It is evident that the source of inorganic carbon in seagasses required reinvestigation in light of this information concerning the carbon fixation mechanism. This is particularly so since there is an apparent anomaly between the carbon fixation pathway and the observed 8'3C values. Absence of the C4 pathway does not necessarily rule out the possibility that bicarbonate is taken up by the plant and subsequently converted to free CO2 prior to fixation by RuBP carboxylase. Bicarbonate was proposed as a possible substrate for photosynthesis in aquatic plants as early as 1910 (3), almost 60 years prior to the discovery ofthe C4 pathway. It is possible, for instance, for plants to take up bicarbonate, and convert it to the required CO2 by the action of carbonic anhydrase (26) as has been shown to occur in unicellular algae (15). Low cytoplasmic pH would also release free CO2 from previously absorbed bicarbonate (22). Such mechanisms could allow bicarbonate uptake to occur in conjunction with a C3 fixation mechanism. The classical evidence for photosynthetic bicarbonate uptake in the freshwater angiosperm Myriophyllum, was obtained from experiments conducted over a wide range of carbon concentrations at a low pH, where most of the inorganic carbon was present as C02, and at high pH where most of the carbon was present as HCO3 (25). This technique poses some problems of interpretation as there is always the question of the effect of pH when experiments are conducted well outside the range normally encountered by the plant. For example, Van et al. (28), who obtained similar results to Steemann-Nielsen, were unable to discount the possibility of HCO3 usage at high pH. They speculated that a pH dependent difference in the apparent Km of photosynthesis may be responsible for the greater rates at high pH for a given CO2 concentration. Many of the more recent experiments concerning the carbon source for photosynthesis in aquatic plants have relied on the simpler technique of measuring photosynthesis at a single carbon concentration over a range of pH values. Despite its wide application, this technique, using a wide pH span, is inadequate for the study of carbon uptake at high pH. The pH response curve usually shows a decrease in photosynthesis with increasing pH, hence indicating that free CO2 is a photosynthetic substrate. However, difficulties arise in interpreting the data at high pH where the photosynthetic rates are low compared to rates at low pH and where there may also be a contribution from HCO3 . The results obtained from this type of experiment have led some workers to conclude that HCO3 was taken up by the plant in addition to free CO2 at high pH (10 [freshwater plants]; 4, 5, 20 [seagrasses]). However, other workers have interpreted similar results as indicating that bicarbonate was not taken up by the

INORGANIC CARBON FOR PHOTOSYNTHESIS IN A SEAGRASS

777

plant (9, 30 [freshwater plants]; 7 [seagrasses]). In particular, previous work on the nature of the photosynthetic carbon source in marine angiosperms (4, 5, 7, 20) has relied on this pH response technique and has led to conflicting conclusions concerning the role of HCO3 . The purpose of this study was to examine the source of inorganic carbon for photosynthesis in a species of seagrass known to possess the C3 photosynthetic pathway. In view of conflicting and equivocal interpretations in the literature arising from data obtained by classical methods, other techniques were developed for use in this investigation. Time course experiments conducted during the relatively slow reestablishment of C02/ HCO3 equilibrium from a perturbed state were developed by Cooper et al. (1 1) for the study of the carbon substrate in isolated enzymes. The technique has previously been applied to microscopic algae ( 13, 15) and has been extended here to study carbon uptake in a seagrass. Steemann-Nielsen's basic approach was refined to use media which do not differ greatly in pH. This modification enabled the more detailed investigation of carbon uptake at alkaline pH which was required in this study.
MATERIALS AND METHODS Plant Tissue. Leafy shoots (turions) of Thalassia hemprichii (Ehrenb.) Aschers were collected from intertidal flats at Picnic Bay and Cockle Bay, Magnetic Island, Australia (latitude 19'12'S longitude 14650'E). The consolidated substrate around the roots was loosened with a sharp tool during collection to ensure that the shoots remained intact and undamaged. Plant material was transported to the laboratory within 4 h of collection and stored in aerated seawater in the dark until required. Experiments were conducted on this material within 3 d of collection. There was no evidence of tissue deterioration during this time and no significant difference between the carbon uptake rates under identical conditions 18 and 72 h after collection (1). Segments 4 to 5 cm long of the youngest mature T. hemprichii leaves were used in experiments. Some proportion of the photosynthetic activity measured in these tissues could have been due to microscopic bacteria or algae adhering to the leaf blade. However, since the leaves used were young, the mass of photosynthetic tissue in such epiphytes was so small compared to that of the seagrass leaf that their contribution to the total photosynthesis of the leaf-epiphyte system could have been significant only if the photosynthetic rate of the epiphytes were several orders of magnitude greater than that of the leaf itself. Throughout this study, it is assumed, therefore, that the epiphytic contribution to the observed photosynthetic rates was not significant. Incubation Media. Experiments were conducted in Bicinebuffered seawater of known pH and total carbon concentration prepared as previously described (1, 2). The pH of the medium was determined to the second decimal place using a Metrohm pH meter. This level of accuracy in pH determination was necessary to calculate free CO2 and HCO3 concentrations from pH and total inorganic carbon. Radioisotope Procedures. Experiments were conducted in closed vials placed in a glass sided, temperature controlled water bath and irradiated from the side through 5 cm of water. Light was provided by a self-ballasted, 250 w mercury discharge lamp. Segments of T. hemprichii leaf tissue were placed above a plastic mesh grid in vials containing 15 ml of nonradioactive, buffered seawater. The medium was stirred rapidly during experimental procedures. The tissue was pre-equilibrated at irradiance of 120 w. m' and at the pH and temperature appropriate for the experiment. Vials were completely filled with liquid and sealed with a suba-seal. The carbon concentration of the pre-equilibration medium was slightly less than that of the final experimental medium. Exposure to 14C was initiated by injection of a small volume (usually 10 ,gl) of radioactive carbon solution. Tests with

dye injection showed that complete mixing occurred within 3 s. Both the volume and carbon content of the radioactive solution were included in calculation of the final carbon concentration of the experimental medium. For time course experiments during initial equilibration of CO2 and HCO3 , radioactive carbon was injected in 50 MA of either alkaline solution (HCO3 ) or acidic solution (CO2) to a constant total carbon concentration. Measurements of the pH of Bicine-buffered seawater medium before and after addition of the same amount of HCI or NaOH showed no change in the pH of the medium resulting from such additions. Following periods of up to 310 s exposure to 14C in the light, vials were opened and the leaf tissue quickly rinsed in nonradioactive seawater medium and killed in boiling 80% ethanol. The rinsing and transfer was completed within 3 s. The leaf tissue was extracted and Chl and activity of total extracts determined as previously described (1, 2).
RESULTS Photosynthesis at Variable pH and Constant Total Carbon Concentration. The effect of pH on the photosynthetic rate of T. hemprichii at normal seawater concentration of 2.2 mm total carbon is shown in Figure 1 (inset). The photosynthetic rate decreased exponentially with increasing pH over the whole range 6.23 to 9.05. However, at high pH, above that of seawater, the absolute change in photosynthetic rate per unit increase in pH was very small. Measured photosynthetic rate and the calculated free CO2 and HCO3 concentrations are shown (Fig. 1) for the pH range 7.20 to 9.05. The curves have been scaled to intersect at seawater pH of 8.23. Between pH 7.20 and pH 8.23 calculated bicarbonate concentration was almost constant but the measured photosynthetic rate and the calculated free CO2 concentration both decreased throughout that range. Above pH 8.20, the HCO3: concentration also decreased noticeably. Thus, when total carbon is kept constant up to pH 9.05, in well buffered seawater media, changes in photosynthetic rate near seawater pH of 8.23 and above cannot be clearly described as a function of either free CO2 or HCO3: concentrations. It is not possible to determine from the data presented in Figure 1 whether pH has any effect on photosynthetic rate other than the effect on the concentrations of the carbon species. Photosynthesis at Various Total Carbon Concentrations with pH Values Maintained Close to That of Seawater. Free CO2 and HCO3 concentrations in buffered seawater media were calculated from the pH and total carbon concentration using the first and second apparent dissociation constants for carbonic acid in seawater of known temperature and salinity. Since Bicine is a chelator of Ca2+, no CaCO3 solubility term was required in the calculations even for media of higher than seawater pH and total carbon concentration. Hence, the values of free CO2 and HCO3 concentration in the Bicine-buffered seawater differ from those in natural seawater of the same pH and total carbon concentration. Change in total carbon at constant pH has the same proportional effect on both carbon species. Change in pH at constant total carbon has a proportionally significant effect on the concentration of free CO2 and a small effect on that of HCO3 . Incubation media of total carbon concentration 0.75, 2.35, 6.64, and 13.17 mM (natural seawater 2.35 mM) were made up with pH values of 7.80, 8.21, and 8.61 (close to natural seawater). Combinations of pH and total carbon were chosen such that an increment of both pH and total carbon maintained an approximately constant free CO2 concentration. The particular advantage of these media was that very similar free CO2 concentrations were obtained in media of widely differing HC03- concentrations within a small pH range which would not be expected to affect

778

ABEL
0.06

Plant Physiol. Vol. 76, 1984

e-

12
0

_.

E
0-

II E

5 0

0-

O
0

0 0

FIG. 1. Photosynthetic rates of segments of T. hemprichii leaves (O-O) and calculated concentrations of fiee CO2 (---) and HC030.04a ( . . . ) at different pH values. Scales on the Y axis have been chosen so that the curves intere sect at pH 8.23. Inset shows photosynthetic 0 rate of segments of T. hemprichii leaves as a function of pH in the range of pH 6.23 to 9.05. 0.02 0 0 Experiments were conducted in a closed sysL. tem at 2.2 mm inorganic carbon, 25C, and 130wmA
2

0.02

a N

0
pH

the photosynthetic mechanism directly. The photosynthetic rate of T. hemprichii was measured in each of these 12 media. When plotted against the calculated HCO3 concentration (Fig. 2A), the photosynthetic rates were pH dependent, with higher photosynthetic rates occurring at lower pH for a particular HCO3 concentration. However, when the data were plotted with respect to the calculated free C02 conoentration (Fig. 2B), there was no pH dependence and photosynthesis was a function only of free CO2 concentration. Although these results show a slightly higher photosynthetic rate for a given CO2 concentration at higher pH values in some instances, this was not the case in all experiments conducted (data not shown). '4C Uptake by T. hemprchii during Equilibration of '4CO2 and H14CO3. Figure 3 shows results of short term, time course experiments conducted during the re-establishment of equilibrium between '4CO2 and H'4CO3- following the addition of solution predominated by one of these species. Initial fixation rate was rapid when 4C was provided in the form of `4CC2 and subsequently decreased with time. Conversely, when '4C was supplied as H'4C03-, the initial rate of fixation was slow and increased with time. No significant fixation occurred with either carbon species as substrate in the dark (Fig. 3).

Photosynthesis at Various Calulated Free CO2 Conetrations with Constant or Vaiable pH. Figure 4 shows the response of photosynthetic rate to calculated free CO2 concentrations up to 0.23 mm obtained by the two methods. Data for buffered seawater at pH in the range of 6.95 to 9.05 are from experiments presented in Figure 1. Values for varying total carbon concentration were obtained using buffered seawater media at pH 8.2. These experiments were all conducted with maximum possible stirring in small vials where the velocity of the medium was at least as great as that experienced by plants in the field. The results have been normalized to show photosynthetic rate as a multiple of the rate obtained at seawater free CO2 concentration of 0.014 mm in order to remove the variability due to seasonal or other factors in the plant material used. The relationship between photosynthetic rate and CO2 concentration is very similar, whether the CO2 concentration was adjustd by altering the pH or by changing the total inorganic carbon concentration. A constant, K, similar to the Michaelis constant which describes the affinity of an isolated enzyme system for its substrate, may be derived for CO2 uptake by a leaf. The double reciprocal method of calculation was applied to the data from Figures 2 and 4. The estimates of K so obtained were 0.167 and 0.331 mM, respectively.
pH 7.60

x 0 a
0 0

FIG. 2. A, Photosynthetic rate of segments of T.

hemprichii leaves at pH 7.80 (0), pH 8.21 (@), and pH

8.61 (A) as a function of calculated HC03- concentration. B, Photosynthetic rate of segments of T. hemprichii leaves at pH 7.80 (0), pH 8.21 (@), and pH 8.61 (A) as a function of calculated free CO2 concentration. Experiments were conducted in a closed system at 25C and 130 w-m-2.

.c

"C03 concentration (

mM l

Free CO2 concentration

( mM ) X 10

INORGANIC CARBON FOR PHOTOSYNTHESIS IN A SEAGRASS

0.5
0

00

0.4
201

EF
a

0.3

E
0.2

0AI
to1

779 tion has the potential to show uptake of either free CO2 or HCO3-, or both. Results shown here indicate CO2 uptake where the data fall on a single curve when plotted against free CO2 and on pH-dependent curves when plotted against HCO3: concentration. The pH-dependent curves show higher photosynthetic rate at lower pH for similar HCO3 concentration. If HCO3 were the only substrate, similar experiments would be expected to show a single curve of photosynthetic rate versus HCO3 concentration. The photosynthesis versus C02 curves would then be expected to be pH dependent, with higher photosynthesis at higher pH. If, however, both species were utilized to a significant degree, then pH-dependent curves would be expected for both photosynthesis versus HCO3 and photosynthesis versus CO2 plots. Also, the photosynthesis versus CO2 plots would not pass through the origin as they do in Figure 2B.

0.1

n
0

=-

20

40
Time ( sec )

60

FIG. 3. Time course of carbon fixation by segments of T. hemprichii leaves from supplied 4CO2 in the light (0) and in the dark (0) or from supplied H4CO3- in the light (U) and in the dark (0). Experiments were conducted in a closed system at pH 8.2, 2.2 mm inorganic carbon, 12C and 150 w_m-2 or in the dark.

CL z0 r

a0
IL

CL
t5
0

seawater

concentrato

p
0
0.1
Free C02 concentration (mM)

0.2

FIG. 4. Photosynthetic rate of segments of T. hemprichii leaves as a function of free CO2 concentration. Data were obtained from experiments where the CO2 concentration was varied by varying the total inorganic carbon concentration (i) or by varying the pH (0). Photosynthetic rates are expressed in arbitrary units (1 = photosynthetic rate at 2.2 mm inorganic carbon, pH 8.21). All rates were determined at 25C and 130wmn2. DISCUSSION

The question of whether HCO3- as well as free CO2 is taken during photosynthesis at high pH cannot be resolved by the simple technique of examining the pH response curve which was used by Benedict et al. (7) and Beer et al. (4, 5), as evidenced by the conflicting conclusions reached by the two groups ofworkers on the basis of similar results. The techniques used in this study allow a separate observation ofthe effects offree CO2 and HCO3 at high pH and confirm that HCO3 is not a significant photosynthetic substrate for this species at the naturally high pH of
up

seawater.

The method developed here for investigating photosynthesis in a series of media of different pH and total carbon concentra-

The results of the present study indicate that photosynthesis in T. hemprichii is largely dependent on the free CO2 concentration and the contribution of HCO3 , if any, is small, even at high pH. These conclusions support those of Benedict et al. (7) but are based on more extensive evidence than has previously been obtained for seagases. The formation of acid and alkaline regions in media adjacent to photosynthesizing tissue has long been considered to be the result of HCO3 uptake in association with ion transport. In a series of papers (reviewed in 17) Lucas and co-workers have published evidence for the formation of acid and alkaline bands along the internodal cells of the green alga, Chara. Lucas and his co-workers propose that the alkaline bands are caused by OHefflux and that the acid bands are the result of HCO3 uptake. They suggest that the HCO3 uptake requires sulphydral linkages and the presence of calcium ions, although there is sufficient Cai within the tissue to maintain apparent HCO3 uptake for several hours. It could be argued that if a similar mechanism existed in seagasses, the Bicine used in experimental media in this study may remove the possibility of HCO3- uptake by complexing with the calcium in the seawater. However, similar photosynthetic rates were obtained both in the presence and absence of Bicine under standard conditions (K. M. Abel, unpublished data). Photosynthetic rates obtained in this study are also comparable to those obtained by other workers using a number of seagrasses in seawater without added buffering agents or in media buffered by non-calcium chelating agents (4-7, 12). Moreover, leaf tissue was exposed to the buffered medium for a maximum of 90 min during pre-equilibration and experiments in this study, whereas the bicarbonate uptake mechanism in Chara remained unaffected after several hours in a solution containing the strong calcium complexing agent, EDTA (16). It is unlikely that calcium would be entirely removed from T. hemprichii leaf tissue during a 90-min exposure to Bicine. There are some differences between the results of Cooper et al. (1 1) and those obtained here with whole leaf tissue. The rates of 14C fixation as estimated from the tangent to the two carbon uptake curves (passing through the 1 min value) in Figure 3, are not identical as would be expected if the chemical re-equilibration were the only factor involved. This probably reflects the fact that the fixation site in the whole leaf tissue is not in direct contact with the bulk medium as it is in isolated enzyme extracts and the surface available for exchange between cells and medium is much smaller than in microscopic plants. Although the CO2 and HCO3 may be in equilibrium in the medium itself after 1 min, the pool of substrate available to the carbon fixing enzyme within the cell may not have reached isotopic equilibrium at this time. Lack of isotopic equilibrium in the inorganic pool of carbon within the tissue may be responsible for the extended lag in '4C fixation observed when the initial carbon species was H'4CO3-. The data on initial photosynthetic rates obtained during equil-

780

ABEL

Plant Physiol. Vol. 76, 1984

ibration of the carbon species from a perturbed state (Fig. 3) support those obtained from longer term, steady state experiments (Figs. 1, 2). Although Cooper et al. (1 1) also conducted experiments with carbonic anhydrase, their conclusions concerning the species of carbon used by various enzymes do not rely on this data but were drawn, as here, from data obtained in experiments where the C02/HCO3 equilibration was slow. There are many suggestions in the literature that carbonic anhydrase involved in aquatic plant photosynthesis as a means of effectively increasing the inorganic carbon supply. Raven (22) proposed that carbonic anhydrase may be excreted by aquatic plants to the external medium thereby increasing the concentration of whichever carbon species was absorbed. So far, attempts to detect external carbonic anhydrase have failed (19). Carbonic anhydrase has been detected, usually in small amounts, within aquatic plant cells (5, 13, 14). However, no correlation has been observed between carbonic anhydrase activity and photosynthetic rate. In particular, Graham and Smillie (14) measured carbonic anhydrase activity in several marine plants but could not find a pattern relating this activity to other parameters. Raven and Glidewell (23) pointed out that internal carbonic anhydrase would not necessarily increase photosynthetic rate if photosynthesis was limited by the rate of actual uptake of carbon from the medium. The results reported here do not support a role for naturally occurring carbonic anhydrase in overcoming an otherwise limiting carbon supply. The rapid interconversion of CO2 and HCO3 caused by the action of carbonic anhydrase would be manifested as a dependence of photosynthetic rate on total carbon concentration, rather than on free CO2. Rapid equilibration of the carbon species would effectively allow the plant access to the total carbon pool almost instantaneously. This clearly was not the case in the experiments reported here. It is not known whether seagrasses produce a pH differential across their leaves during photosynthesis as do some of their freshwater relatives. However, such a strategy would serve to increase the availability of free CO2 in the vicinity of the leaf. The slight reduction in CO2 available to the more alkaline surface would be more than offset by the increase at the acid surface. Investigation of the pH at the surface of seagrass leaves should provide further information concerning carbon assimilation in seagrasses. Equally, application of techniques developed for this study to the investigation of carbon assimilation in freshwater angiosperms would provide information on the carbon source for these plants. This information would be particularly valuable for plants such as Potamogeton and Elodea which are known to produce a pH differential across their leaves in the light and have therefore been considered to actively assimilate HCO3 . Knowledge of the actual carbon species taken up by such plants would assist in eluidating the presence, or otherwise of an active HCO3 pump. Since most of the inorganic carbon in seawater is in the form of bicarbonate and diffusion of molecules in water is slow compared with diffusion in air, an ability to use HC03- would be an obvious advantage to a marine plant. Some algae, such as Scenedesmus have been shown to supplement CO2 uptake at high pH by the active uptake of HCO3 (13). It has even been suggested that HCO3 concentrating mechanisms exist in some algae (23) and cyanobacteria (19). It would appear that marine angiosperms, in their reliance on free CO2 as a carbon source, resemble their presumed terrestrial ancestors, rather than these algae in this aspect of their physiology. The estimates of K (Fig. 4) are at least an order of magnitude greater than the concentration of free CO2 in seawater, clearly indicating that the supply of carbon is a limiting factor in photosynthesis of T. hemprichii. Carbon is limiting up to several-

fold the concentration of CO2 in seawater and there is no apparent inhibition of photosynthesis at high CO2 concentrations as was observed in the freshwater plant Elodea (29). There are limitations to the supply of CO2 to a submerged leaf because of the slow diffusion of CO2 through the relatively thick liquid boundary layer. This supply may be augmented to some extent by the reservoir of respired CO2 contained within gas lacunae. These physical and anatomical conditions effectively create a somewhat leaky 'closed system.' Restriction of the external carbon supply has the effect of elevating the measured 613C values of plant tissue (8). Andrews and Abel (2) and Benedict et al. (7) have previously suggested that this situation might apply to seagrasses. However, the suggestion was made in the absence of clear evidence for the species of inorganic carbon utilized by seagrasses. There, consequently, was some doubt concerning the possible contribution of HC03- to the observed 6'3C values of seagrasses. Evidence obtained here supports the proposal that high 6'3C values of seagrasses are the result of physical constraints on the supply of free CO2 for photosynthesis.
Acknowledgments-I wish to thank Prof. D. J. Griffiths and Dr. P. F. Brownell for their advice and support and Dr. E. A. Drew and Dr. D. Kinsey for reading the manuscript. I also gratefully acknowledge the support of the Australian Institute of Marine Science which made equipment and facilities available throughout the
study.
LITERATURE CITED

1. ABEL KM 1983 Photosynthesis in two tropical seagrasses with special reference to carbon metabolism. MSc thesis. James Cook University of North Queensland 2. ANDREWS TJ, KM ABEL 1979 Photosynthetic carbon metabolism in seagrasses "C labeling evidence for the C3 pathway. Plant Physiol 63: 650-656 3. ANGELSTEIN V 1910 Uber die Kohlensaureassimilation submerser Wasserpflanzen in Bikarbonat und Karbonatlosungen. Beitr Biol Pflanz 10: 87-117 4. BEER S, A ESCHEL, Y WAISEL 1977 Carbon metabolism in seagrasses I. Utilization of exogenous inorganic carbon species in photosynthesis. J Exp Bot 28: 1180-1189 5. BEER S, A SHOMER-ILAN, Y WAISEL 1980 Carbon metabolism in seagrasses II. Patterns of photosynthetic CO2 incorporation. J Exp Bot 31: 1019-1026 6. BENEDICT CR, JR ScoTT 1976 Photosynthetic carbon metabolism of a marine grass. Plant Physiol 57: 876-880 7. BENEDICT CR, WL WONG, JHH WONG 1980 Fractionation of the stable isotopes of inorganic carbon by seagrasses. Plant Physiol 65: 512-517 8. BERRY JA, JH TROUGHTON 1974 Carbon isotope fractionation by C3 and C4 plants in "closed" and "open" atmospheres. Carnegie Inst Wash Year Book
785-790 9. BROWN JMA, FI DROMGOOLE, MW ToWSEY, J BROWSE 1974 Photosynthesis and photorespiration in aquatic macrophytes. The R Soc N Z Wellington Bull 12: 243-249 10. BROWSE JA, JMA BROWN, FI DROMGOOLE 1979 Photosynthesis in the aquatic macrophyte Egeria densa II. Effects of inorganic carbon conditions on "C fixation. Aust J Plant Physiol 6: 1-9 11. COOPER TG, TT TCHEN, HG WOOD, CR BENEDicr 1968 The carboxylation of phosphoenolpyruvate and pyruvate. The active species of CO2 utilized by phosphoenolpyruvate carboxykinase, carboxytransphosphorylase and pyruvate carboxylase. J Biol Chem 243: 3857-3863 12. DREW EA 1979 Physiological aspects of primary production in seagrasses. Aquat Bot 7: 139-150 13. FINDENEGG GR 1980 Inorganic carbon transport in microalgae II. Uptake of HCO3- ions during photosynthesis of five microalgal species. Plant Sci Lett 18: 289-297 14. GRAHAM D, RM SMILLIE 1976 Carbonate dehydratase in marine organisms of the Great Barrier Reef. Aust J Plant Physiol 3: 113-119 15. IMAMURA M, M TsUZUKI, Y SHIRAIMA, S MIYACHI 1983 Form of inorganic carbon utilized for photosynthesis in Chlamydomonas reinhardtii. Plant Cell Physiol 24: 533-540 16. LuCAS WS 1979 Alkaline band formation in Chara corallina: due to 0Hefflux or H+ influx? Plant Physiol 63: 248-254 17. LUCAS WS 1983 Photosynthetic assimilation of exogenous HCO3 by aquatic plants. Annu Rev Plant Physiol 34: 71-104 18. MCMILLAN C, PC PARKER, B FRY 1980 Carbon-1 3 to carbon- 12 ratios in seagrasses. Aquat Bot 9: 237-250 19. MILLER AG, B COLMAN 1980 Evidence for HCO3 transport by the blue-green

alga (Cyanobacterium) Coccochloris peniocystis. Plant Physiol 65: 397-402

20. OGATA E, T MATSUI 1965 Photosynthesis in several marine plants of Japan in relation to carbon dioxide supply, light and inhibitors. Jpn J Bot 19: 83-98 21. PARK R, S EPSTEIN 1961 Metabolic fractionation of 13C and '2C in plants.

Plant Physiol 36: 133-138

INORGANIC CARBON FOR PHOTOSYNTHESIS IN A SEAGRASS

22. RAVEN JA 1970 Exogenous inorganic carbon sources in plant biosynthesis. 27. Biol Rev 45: 167-221 23. RAVEN JA, SM GLIDEWELL 1978 C4 characteristics of photosynthesis in the C3 28. alga Hydrodictyon africanum. Plant Cell Environ 1: 185-197 g BN, S EPSTEictyon191Toctgreof'3C/"C 29 ratios for 24 SMIHydrodS plants. higher 1971 Two categoes of29. SMITH EPSTEIN 24. Plant Physiol 47: 381f384 25. STEEMANN-NIElsEN E 1947 Photosynthesis of aquatic plants with special 30. reference to the carbon-sources. Dan Bot Ark 12: 1-71 26. STEEMANN-NIELSEN E 1960 Uptake of CO2 by the plant 1. CO2 or bicarbonate

781

ion. Encycl Plant Physiol 5: 70-84 STEEMANN-NIELsEN E 1975 Marine Photosynthesis with Special Emphasis on the Ecological Aspects. Elsevier, Amsterdam, pp 141 VAN TK, WT HALLER, G BowEs 1976 Comparison of the photosynthetic of three submersed aquatic plants. Plant Physiol 58: 761-768 characteristics WEBER JA, JD TENHUNEN, CS YocuM, DM GATES 1979 Variation of photosynthesis in Elodea densa with pH and/or high CO2 concentrations. Photosynthetica 13: 4554-458 WINTER K 1978 Short-term fixation of carbon by the submerged aquatic angiosperm Potamogeton pectinalus. J Exp Bot 29: 1169-1172