The effect of pH on the inorganic carbon source for photosynthesis in the seagrass Zostera muelleri irmisch ex aschers

Aquatic Botany, 24 (1986) 199--209

199

Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

THE EFFECT OF pH ON THE INORGANIC CARBON SOURCE FOR PHOTOSYNTHESIS IN THE SEAGRASS ZOSTE_RA MUELLERI IRMISCH EX ASCHERS.

JEN NY MILLHOUSE and S T A N L E Y STROTHER

Division of Biological and Health Sciences, Deakin University, Victoria, 3217 (Australia) (Accepted for publication 9 January 1986)

ABSTRACT Millhouse, J. and Strother, S., 1986. The effect of p H on the inorganic carbon source for photosynthesis in the seagrass Zostera muelleri Irmisch ex Aschers. Aquat. Bot., 24 : 199w209.

The ability of the seagrass Zostera mueUeri Irmisch ex Aschers. to use HCO; a s well as CO 2 for photosynthesis was investigated by measuring photosynthetic 02 e v o l u t i o n over a range of pH values. It was found that the apparent K m CO 2 fell from 0 . 1 2 8 mM at pH 7.9 to 0.016 mM at pH 9.1 indicating that HCO; as well as CO 2 may a c t as a substrate for photosynthesis. The true K m CO s could not be determined due to inhibition of photosynthesis a t pHs less than 7.8. K m CO~ must be at least 0.128 raM, the apparent K m at pH 7.9, a n d is probably of the order of 0.200 mM CO2, the same as that reported for other m a r i n e plants. K m HCO; is about 20 mM when CO2-dependent photosynthesis is m i n i m a l . Such a high Km HCO; resembles values reported for freshwater, rather than m a r i n e plants. Photosynthetic O~ evolution is not saturated with respect to total inorganic c a r b o n in natural seawater (pH 8.2). It is suggested that the distinctive shoulder from pI-t 8.1 to 8.5 in the pH profile of photosynthetic O= evolution at a constant c o n c e n t r a t i o n of inorganic carbon is caused by an effect of pH on HCO~ uptake. The effect of p H on HCO; uptake was determined by constructing a pH profile of photosynthesis a t constant HCO; concentration, and subtracting the estimated contribution of COs t o photosynthesis from this rate. The resultant curve has a m ax i m u m at pH 8.4 and d e c l i n e s sharply at pHs less than 8.

INTRODUCTION

Inorganic carbon in natural seawater is largely present as the bicarbonate ion (HCO~) rather than as hydrated carbon dioxide (CO2). At pH 8.2, 90% of the inorganic carbon is HCO; and only 0.6% is CO2. For this reason, a variety of marine plants has b e e n investigated for their ability t o use HCO; as well as CO2 for photosynthesis. A number o f seagrasses have been investigated for HCO; use: Halophila stipulacea (Forsk.) Aschers., Thalassodendron ciliatum (Forsk.) den Haxtog,

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Halodule uninervis (Forsk.) Aschers., Syringodium isoetifolium (Aschers.) Dandy (Beer et al., 1977), Cymodocea nodosa (Ucria) Aschers., Halophila ovalis (R.Br.) Hook. f. (Beer and Waisel, 1979) and Zostera marina L. (Sand-Jensen and Gordon, 1984). All have been reported to be able to use both HCO~ and CO2 as their carbon source for photosynthesis in natural seawater. CO2 is the carbon source for Thalassia testudinum Banks ex KSnig (Benedict et al., 1980) and T. hemprichii (Ehrenb.) Aschers. (Abel, 1984). Most methods used to investigate HCOa use by plants are based on a comparison of the photosynthetic rate at two or more pH values (Raven, 1970). Dissolved CO2 hydrates to form carbonic acid which dissociates to form HCO~ and ultimately carbonate ions (COl-). The actual amount of each of these forms of inorganic carbon depends on the pH of the medium. By altering the pH of seawater, the proportion of CO2 to HCO~ can be altered. This enables the contributions of CO2 and HCO~ as substrates for photosynthesis to be separated by a comparison of carbon use at two or more pHs. The method most commonly employed to demonstrate HCO~ use for photosynthesis in seagrasses is an analysis of a pH profile of photosynthesis at a constant concentration of inorganic carbon (Beer et al., 1977; Beer and Waisel, 1979; Benedict et al., 1980; Sand-Jensen and Gordon, 1984). If photosynthesis is due only to CO2, then the rate of photosynthesis is expected to decrease in a CO2~lependent manner. Deviations from this curve, especially around pH 8.0--8.5, are considered to indicate HCO~ use. This method, however, is considered inadequate to determine HCO~ use at high pH where the photosynthetic rates are low (Abel, 1984). In order to more reliably determine whether HCO~ is a carbon source for photosynthesis in Zostera muelleri Irmisch ex Aschers., we investigated the effect of pH on photosynthetic oxygen evolution over a range of inorganic carbon concentrations. Results of the influence of pH on the apparent K m for CO2 and HCO~ are presented and compared with those obtained previously (Beer et al., 1977; Beer and Waisel, 1979; Abel, 1984; Sand-Jensen and Gordon, 1984). The influence of pH on the rate of photosynthetic oxygen evolution at constant total inorganic carbon, CO2 and HCO~ is discussed in terms of evidence for a specific HCO~ transporting system. METHODS Intact plants of the seagrass Zostera muelleri Irmisch ex Aschers. were collected from the intertidal mud flats of Swan Bay, a shallow bay off Port Phillip Bay near Queenscliff, Victoria, Australia (38°16'S, 144°39'E). Material used for constant inorganic carbon pH profiles and for apparent K m and Vmax calculations was collected in June (winter) whilst that used

201 in experiments at constant CO2 or HCO; concentration was collected in December (summer). Plants were collected with the roots and surrounding sediment undisturbed, and placed in plastic containers for storage in an aerated seawater aquarium at 15°C, with an irradiance at the plant canopy of 22 /~Em-2 s -1 PAR supplied by "Grolux" fluorescent lights on a 12 h day--night cycle. Plants stored in this manner remained healthy for several months, but were generally used within 2 weeks of collection. Leaves (0.05--0.10 g, 50--150 pg chlorophyll) free of epiphytes were chopped into approximately 2 mm slices with a razor blade and placed in a 5 ml reaction vessel containing 3 ml reaction medium (either natural seawater or artificial seawater containing 540 mM chloride, 454 mM sodium, 55 mM magnesium, 27 mM sulphate, 10 mM calcium and 10 mM potassium ions). The photosynthetic rate was measured polarographically with a Rank Bros. O2 electrode at 20°C. D.O.C. varied from 240--300 #M. Inorganic carbon was supplied as freshly prepared sodium bicarbonate solution, adjusted to the pH of the assay. Light was supplied by a quartz-iodide projector lamp giving a light intensity of 800 ~Em -2 s -1 PAR. The kinetic parameters K m and Vrnax were calculated using the Hofstee equation (a plot of rate versus rate/substrate concentration) {Dixon and Webb, 1964), a linear rearrangement of the Michaelis--Menten equation. Carbonate alkalinity of natural seawater was measured titrimetrically using the method of Strickland and Parsons (1972), and the total a m o u n t of inorganic carbon calculated using the equations of Park (1969). The concentrations of CO2 and HCO; in media enriched with inorganic carbon were calculated using the dissociation constants from H o m e (1969) and equations from Park (1969). The maximum rate of supply of CO2 b y dehydration of HCO~ and COl- was calculated using the equations of Lucas {1975) and dissociation constants from H o m e (1969). Calcium carbonate solubility was determined by adding sodium bicarbonate (adjusted to the pH of the assay) to media containing varying quantities of calcium, magnesium and sodium salts, in an illuminated oxygen electrode reaction vessel. Calcium carbonate precipitation was detected visually and compared with the predicted level of precipitation using the solubility product for calcium carbonate in distilled water or seawater (Home, 1969). Chlorophyll was extracted from leaves ground in acetone and sand. The homogenate was centrifuged and the supernatant was measured spectroscopically using a modified method of Arnon (1949). The concentrations of chlorophylls a and b were calculated using the revised equations of Jeffrey and Humphrey (1975). RESULTS Table I shows the results of a typical experiment to estimate the apparent K m of CO2 and HCO~ for photosynthesis over the pH range 7.9--

202 TABLE I Apparent K m values for CO2 and HCO; as a function of pH' pH

Km CO2 (raM)

Km HCO; (raM)

Vmax (umol 02 rag- ~ chlorophyll min -~)

7.9 8.2 8.4 8.5 8.8 9.1

0.128 0.095 0.061 0.044 0.040 0.016

10.2 13.4 15.8 14.1 21.9 18.2

0.65 0.90 0.90 0.86 0.92 0.58

IPhotosynthetic O2 evolution was measured in natural seawater adjusted to the appropriate pH. Additional inorganic carbon (up to 21 m M at p H 7.9, increasing to 100 m M at p H 9.1) was added as sodium bicarbonate. Apparent K m C O 2 and K m H C O ; were calculated from the apparent K m total inorganic carbon, based on the concentration of CO2 and H C O ; in a closed vessel at each pH.

9.1. If the apparent K m CO2 is unchanged over a range of pH values, then only CO2 is used as a substrate, b u t if the apparent K m CO2 decreases with increasing pH (as the relative proportion of HCO3 to CO2 rises), then HCO; is implicated as a photosynthetic substrate (Raven, 1970). These results therefore support HCO~ use. The converse should be true for the Km HCO;, in that an increase in the apparent K m HCO; indicates CO2 uptake. This occurred up to pH 8.8, after which there was no further rise in the apparent K m H C O ; , which suggests that CO2 was n o t significantly contributing to photosynthesis at high pH. This indicates that the K m-HCO; is around 20 mM at high pH. It is n o t possible to calculate the K m CO2, as the apparent K m CO2 was still increasing at pH 7.9, indicating that b o t h HCO; and CO2 were substrates for photosynthesis at this pH. Vma x remained fairly constant over the pH range investigated, except at pH 7.9 and 9.1 where Vma x was reduced. The lower rate at pH 9.1 m a y be due to calcium carbonate precipitation, which occurs at high pH with high inorganic carbon levels, or to inhibitory effects of high pH on the seagrass itself. Vma x at pH 7.9 is probably artificially low, as inhibition o f photosynthetic oxygen evolution was observed at pHs less than 7.8 or 8.0 (see below). At pH 8.2, the natural pH of seawater, the apparent K m for total inorganic carbon was 14.9 mM, more than six times higher than the inorganic carbon content o f seawater (2.2 mM), indicating that photosynthesis is nowhere near saturated with respect to inorganic carbon at high light intensities under natural conditions. A pH profile of photosynthesis at constant inorganic carbon concentration is shown in Fig. 1. There is a marked deviation from the CO2
203

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Fig. I. Rates of photosynthesis (.... ) and calculated CO2 concentration ( ) as a function of p H in a closed system containing 2.2 or 6.5 m M (inset) total inorganic carbon in natural seawater adjusted to each p H prior to the addition of leaf slices. Vertical bars represent ± S.E.M.

A comparison o f the rate of 02 evolution in the reaction vessel w i t h the m a x i m u m rate of CO2 supply from HCO~ and CO~- indicated t h a t t h e rate of CO2 production is n o t fast enough to a c c o u n t for observed rates o f photosynthesis above pH 8.4 w i t h o u t use o f HCO~ as a p h o t o s y n t h e t i c substrate (data n o t shown). Photosynthetic oxygen evolution declined with decreasing pH b e l o w pH 7.8 (Fig. 1 inset). This inhibition could be due either to a direct p H effect, or to inhibition by high substrate (CO2) concentrations. An analysis of the relationship between photosynthesis and hydrogen ion concentration gives a good correlation (r = 0.94). The correlation b e t w e e n p h o t o synthesis and high CO2 concentrations (Dixon and Webb, 1964) is slightly better (r = 0.97) with a relative inhibition constant for CO2 of 1.1 raM. Inhibition at high CO2 concentrations was also n o t e d at pH 8.4, 8.5 and 8.8 for the inorganic carbon response curves in Table I. Due to this s t r o n g inhibition at low pH, measurements were n o t made below pH 7.4, w h e r e inhibition was already obvious. The K m CO2 could n o t be d e t e r m i n e d as it is usually measured at pH 5, where HCO~ is less than 10% of the t o t a l inorganic carbon, b u t where, in this case, inhibition was strong. The data from both Table I and Fig. 1 indicate that HCO~ as w e l l as

204

CO2 m a y be used for photosynthesis. Further information on the relative contributions of CO2 and H C O ; to net photosynthesis m a y be obtained by investigating the effect of p H on photosynthesis at constant CO2 and HCO;. Fig. 2a shows the results of an experiment in which the concentration of H C O ; is kept constant whilst the concentration of CO2 fallslogarithmically with increasing pH. If Z. muelleri were incapable of using HCO;, then the photosynthetic rate would be expected to fall in proportion to the decrease in CO2 concentration. This would also occur if H C O ; use was equally as efficient over the p H range investigated. O n the other hand if CO2 use was insignificant, the photosynthetic rate would reflect the p H dependence of H C O ; use. It has already been established that H C O ; is a substrate for photosynthesis between p H 8 and 9. The apparent K m CO2 and H C O ; also indicate that CO2 contributes to photosynthesis, at least at the lower pHs. The observed photosynthetic rate in Fig. 2a appears to be the result of constant CO2-dependent photosynthesis and variable H C O ; photosynthesis. The same arguments should hold if the concentration of CO: is kept constant and the concentration of H C O ; varied (Fig. 2b). If it is assumed 1.0

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Fig. 2a. Rates of photosynthesis (.... ) and calculated CO2 concentations ( ) as a function of p H in a closed system containing 8.3 m M H C O ; (variable total inorganic carbon) in artificial seawater. ( ....) represents the estimated rate due to HCO;-dependent photosynthesis if COs-dependent photosynthesis is estimated to follow the [ C O 2 ] curve. Vertical bars represent ± S.E.M.

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that CO2 use is independent of pH, then if HCO; use were also i n d e p e n d e n t of pH, photosynthesis should be proportional to the HCO; concentration. As this was n o t so, the discrepancy may be accounted for in t e r m s of the influence o f pH on HCO; uptake. Despite the relatively high concentrations of COl- at pH 9.1 in Fig. 2b, no precipitation of calcium carbonate was observed. It was found t h a t as little as 1 mM equivalent calcium carbonate precipitation caused detectable cloudiness of the incubation medium. This w o u l d mean that at p H 9.1, less than 1% of the total inorganic carbon was lost due to calcium carbonate precipitation. The results from Fig. 2 are n o t directly comparable with those o f Fig. 1 and Table I as the latter were obtained from winter-grown seagrasses w h i c h tend to have lower photosynthetic rates. 1.1

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Fig. 2b. Rates of photosynthesis ( . . . . ) and concentration of HCO; ( ) as a f u n c t i o n of pH in a closed system containing 43 ~M CO2 (variable total inorganic c a r b o n ) in artificial seawater. Vertical bars represent ± S.E.M.

206

DISCUSSION Zostera muelleri, like many other seagrasses (Beer et al., 1977; Beer and Waisel, 1979; Sand~lensen and Gordon, 1984) is able to use HCO; as well as CO2 for photosynthesis. The apparent K m CO2 could not be measured directly because photosynthesis was inhibited at pHs less than about 7.8--8.0. This phenomenon is widespread, and has been reported for freshwater HCOs- users such as Eiodea canadensis Michx. (Allen and Spence, 1981), and Egeria densa Planch. (sub nomine Elodea densa (Planch.) Casp.) (Weber et al., 1979), in the seagrass, Zostera japonica Aschers. and Graebn. (sub nomine Z. nana Roth) (Ogata and Matsui, 1965), and also in terrestrial plants. This inhibition has been found to be caused by high CO2 concentrations in the HCO~ using freshwater aquatic angiosperm Egeria densa (Weber et al., 1979) rather than to pH per se, although low pH exacerbates this effect. Woo and Wong (1983) showed that inhibition of photosynthesis of high CO2 in cotton leaves appeared to involve CO2 inhibition of the carbon fixing enzyme, ribulose 1,5-bisphosphate carboxylase, with some effect on electron transport activity as well. This is a possible explanation for Z. muelleri. The evidence for HCO; use in Z. rnuelleri raises the question of the mode of uptake. There are four main ways in which HCO; use could occur; by uncatalyzed uptake (diffusion), or by various catalyzed alternatives, namely facilitated diffusion, active transport or external conversion of HCO; to CO2 which is dependent on H ÷ extrusion into the cell wall and surrounding unstirred layer (Walker et al., 1980). The last three methods are virtually indistinguishable in this type of work. HCO; uptake by diffusion is not likely, firstly because the negative charge would make it difficult to cross the hydrophobic plasmalemma. Leaf cells also have a negative potential difference (Jagels, 1973) and thus a large driving force against uptake of negatively charged ions by diffusion. Secondly, the results presented in Fig. 2 indicate that at ambient pHs, HCO; use does not correlate directly with HCO~ concentration. If diffusion were the main mechanism of HCO; uptake, then the presence of HCO; using plants would be expected to be far more widespread. Some form of catalyzed transport of HCO; could explain the results in Fig. 1. Beer et al. (1977) assumed that CO2 use is insignificant above pH 8.2, and showed that HCO; use could then occur by the operation of a transport system which saturates at 2 mM HCO; (the HCO; concentration at pH 8.2). Similar results would be obtained for Z. muelleri if CO2 use is insignificant at high pH. This would mean that at higher levels of total inorganic carbon, the shoulder on the pH profile should shift to the right, but this does not occur, as the shoulder is at the same pH at 6.5 mM total inorganic carbon (Fig. 1 inset). The data on the apparent K m HCO; are also at variance with this hypothesis, as it appears to be around 20 mM, ten times the estimated satura-

207 tion level of the transporter. The negative potential difference across the cells would also act against such a system. These results indicate that HCO~ uptake cannot be estimated by altering the pH and assuming that all photosynthesis is due to HCO~ use (as assumed by Beer et al., 1977) as the results from Fig. 2 indicate that HCOs uptake is not independent o f pH. Abel (1984) noted problems in assigning HCO; use, based on such data. More detailed investigations on inorganic carbon sources in other seagrasses are indicated. By making a number of assumptions, the results from Fig. 2a are consistent with a pH~lependent HCO~ transporting system. If it is assumed that firstly, the K m CO2 is about 0.20 mM, as reported for both marine and freshwater angiosperms (Abel, 1984; Sand,Jensen and Gordon, 1984), compared with an apparent K m CO2 of 0.128 mM at pH 7.9; and secondly, that the maximum rate of photosynthesis with CO2 is equal to that with HCO;, as there is no evidence for Vrnax being higher at low pH where t h e r e is a greater proportion of CO2-dependent photosynthesis, then the CO 2 concentration curve in Fig. 2a is also an approximation of the proportion of the photosynthetic rate due to CO2. By subtracting this rate f r o m the observed rate, photosynthesis due to HCO~ can be calculated. This is actually a profile of the pH dependence of the transporting system, as the concentration of HCO; is constant. Such a profile is shown in Fig. 2a. The maximum at pH 8.4 also corresponds to, and could account for, the position of the shoulder in Fig. 1. In plants which are capable of using HCO; as a photosynthetic substrate, the affinity for HCO; appears to be substantially higher in marine t h a n in freshwater plants. The Km HCO; is approximately twice that of CO2 in marine plants, compared to 30--120 times that of CO 2 for freshwater macrophytes (Sand~lensen and Gordon, 1984), however the affinity o f b o t h fresh water and marine plants for CO2 is fairly stable, with K m CO2 ranging from 0.07--0.30 mM CO2. The affinity found for Z. mueUeri for HCO; is at variance with t h a t of Z. marina (Sand~lensen and Gordon, 1984), which has a K m HCO; at pH 8.2--8.4 of 0.60 mM compared with 13.4--15.8 mM for Z. muelleri at the same pH. Such high K m values for HCO; resemble those obtained for freshwater vascular macrophytes such as Potamogeton crispus L. and Elodea canadensis (Allen and Spence, 1981) and for Hydrilla verticillata (L.f.) Royle, Ceratophyllum demersum L. and Myriophyllum spicaturn L. {Van et al., 1976). The apparent K m HCO; for Z. muelleri is at variance with the t w o hypotheses proposed by Sand~ensen and Gordon (1984), which suggest that not only should marine plants be able to use HCO; efficiently, but that they should be more efficient than freshwater plants. In this respect, Z. muelleri is more like freshwater angiosperms than other marine plants. The pH profile of photosynthetic rate at constant inorganic carbon for Z. muelleri is similar to those obtained by Beer et al. (1977) and Beer

208

and Waisel (1979) for a number of seagrasses, in that it shows a shoulder in the pH
The authors would like to thank Dr. F.A. Smith for helpful discussion and for reading the manuscript, and Graham Possingham for assistance in calculating calcium carbonate solubilities. J. Millhouse was supported by a Commonwealth Postgraduate Research Scholarship.

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