Inorganic carbon source for photosynthesis in the aquatic macrophytes Potamogeton natans and Ranunculus fluitans

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Aquatic Botany48 (1994) 109-120

Inorganic carbon source for photosynthesis in the aquatic macrophytes Potamogeton natans and Ranunculus fluitans Maria Bodner lnstitut J~r Botanik, Sternwartestr. 15, A.6020 lnnsbruck, Austria

(Accepted2 March 1994)

Abstract Photosynthetic carbon uptakes of floating and submersed leaves of the pondweed Potamogeton natans L. and submersed leaves of Ranunculusfluitans Lain. were studied in a number of KHCO3 media of varying pH ( 7-9.5 ) and concentration ( 1-12 mM ). Photosynthetic rates of the floating and submersed leaves ofP. natans correlated with free-CO2 concentrations and were independent of HCOF concentrations in the media. The CO2 affinity of submersed leaves was greater than that of floating leaves, but floating leaves showed marked increases in CO2 affinity after submergence at low CO2. R.fluitans clearly has the ability to use HCOF for photosynthesis. The mechanism of HCO~- utilization in relation to leaf forms within the genus Potamogeton is discussed.

1. Introduction In water, inorganic carbon is present in three forms (CO2, HCOF and CO 2- ); the proportions of the carbon species are highly pH dependent apart from dependence on temperature and ionic strength. In freshwaters, dissolved inorganic carbon (DIC) typically varies from close to zerc~in oligotrophic, acid waters to more than 10 mM in waters with catchment areas rich in carbonates. Along with the fact that the diffusion rate of CO2 is 104 times lower in water than in air, CO2 may be a limiting factor for photosynthesis under certain circumstances (Maberly and Spence, 1983; Madsen and Maberly, 1991 ). Therefore, different strategies, which could be seen as adaptations to amelio0304-3770/94/$07.00 © 1994ElsevierScienceB.V. All fights reserved SSD10304-3770 (94)00394-2

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M. Bodner/3quaticBotany 48 (1994) 109-120

rate the carbon constraints, have evolved in submersed macrophytes. These can be separated into physiological and exploitation strategies (Madsen and SandJensen, 1991 ). Among the former, the most widespread is the use of HCOF in photosynthesis (Pfins and Elzenga, 1989), which has been found in approximately 50% of the freshwater macrophytes tested so far. The most widespread exploitation strategy is the development of floating or aerial leaves, which allow access to atmospheric CO2, where CO2 is more readily available owing to the higher diffusion rate and current velocities in air relative to those in water (Boston et al., 1989; Madsen and Sand-Jensen, 1991 ). These strategies may also occur combined, as for example with Ranunculus peltatus Schrank: the submersed leaves are able to use HCOF and the plant can also form emergent leaves, which, however, do not assimilate HCO~- (Sand-Jensen et al., 1992 ). HCOF affinity is highly variable in response to growth conditions and may be induced mainly by low CO2 concentrations (Sand-Jensen and Gordon, 1986). Arens (1937) reported for a tropical Potamogeton species that in leaves intermediate between floating and submersed, leaf polarity could be induced by submergence. However, this special mechanism for using HCOF (e.g. Prins et al., 1982b) could not be induced in the floating leaves. The aims of the present study were: ( 1 ) to compare the photosynthetic mechanism of the floating leaves and submersed plant parts ofPotamogeton na~ans L.; (2) to test plants subjee',.ed to low CO2 conditions by keeping a floating leaf submerged before the experiments; (3) to use Ranunculus fluitans Lam., with its ability to use HCOA-~ to test the competence of the method to distinguish between COs and HCO~- usages.

2. Materials and methods

2.1. Plant material Potamogeton natans L. and Ranunculus fluitans Lam. were planted in small ~onds of the Botanical Garden of Innsbruck in summer 1991. Both species thrived, and flowered in 1992, when the experiments were carried out. The alkalinity of the pond water (tapwater and rain) was about 2 meq 1- ~, whereas the pH varied from 8.1 to above pH 10 near algal mats. Temperatures were between 18°C and 24°C in summer. Young but fully developed plant parts which were minimally colonized by epiphytes or encrusted with carbonates were chosen. Nevertheless, they were gently cleaned by hand and rinsed in tapwater. Samples were always harvested at about 09:00 h some 20 min before the start of an experiment, and were cut into 1.2 cm pieces to fit the sample chamber. The same plant material was transferred succesively into a series of about 18 experimental solutions with decreasing CO2 concentrations. For the last measurement the first solution was used again to check that the plant sample had not suffered damage or undergone alterations owing to

M. Bodner/ Aquatic Botany 48 (1994) 109-120

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the repeated measurements over about 8 h. Between the single measurements, the probe was kept in tapwater in darkness for about 9 min. 2.2. Cultivation at low C02

A small young plant ofP. natans with two linear submersed leaves and the first floating leaf developing from the tip of the rhizome was planted in coarse sand in a glass tank ( V=2.21) with tapwater ( 1.7-1.9 meq l - t ). After about I week the unfolding third floating leaf was kept submersed by a small weight on the petiole. After a measurement with a small piece (0.7 cm× 1.2 cm) of that leaf, the rest was allowed to float on the surface and further measurements with the same leaf were conducted 1 week later. The medium was bubbled with CO2-free air for 1.5 h three times a day, thereby removing CO2 to a final concentration of about 0.26/zM and gradually raising the pH from 8.5 to 9.93; after 1 week the medium was changed and algae were removed from the leaves. Illumination was provided by white light (Powerstar HQIL W400, Germany) at about 200/tmol m -2 s-~ photosynthetically active radiation (PAR) in a 14 h light-10 h dark cycle at 22-26°C. 2.3. Measurement o f photosynthesis

To establish a photosynthetic use of HCO~-, the method outlined by Abel (1984) and Maberly (1992), for media not differing greatly in pH, was used (comparable with the method of Madsen and Maberly, 1991 ). Combinations of pH and total carbon were chosen such that similar free-CO2 concentrations were obtained in media of widely differing HCOF concentrations within small pH steps of 0.5 units. CO2 uptake is indicated where the rates of photosynthesis fall on a single curve when plotted against free CO2 and on pH-dependent curves when plotted against HCOF concentration. If HCOF were the only substrate, a single curve of photosynthetic rate vs. HCOF concentration would be expected, and pH-dependent curves vs. CO2, which would not pass through the origin. If both CO2 and HCOF were utilized to a significant degree, then pH-depcndent curves would be expected for both plots (Abel, 1984). The extent of HCOj- use alone was further estimated by plotting photosynthetic rates against variable CO2 concentrations and calculating the intercept with the ordinate at zero CO2 (PHco3) by linear regression according to Sand-Jensen and Gordon (1986). Photosynthetic rates were determined in 2 ml of solution for at least four samples with a temperature-controlled Clark-type oxygen electrode (DW2/2 Hansatech, Kings Lynn, UK) attached to a chart recorder (Servogor 130, Goerz, Austria). Rates were calculated as/~mol O2 (mg chl) - l h - I from the maximum slope of the curve during the first 25 min. Temperature was maintained at 20_+ 0.15 °C with a circulating water bath. Irradiances of about 1000/lmol m -2 s-~ (PAR) were provided by a Schott KL 1500 cold light source (Schott, Wiesbaden, Germany) fitted with a 150 W halogen bulb through a fibre optic.

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M. Bodner/Aquatic Botany 48 (1994) 109-120

2.4. Experimental solutions KHCO3 was dissolved in distilled water at concentrations of 1, 2, 4, 8 and 12 raM. pH values of 7.5, 8, 8.5, 9 and 9.45 were achieved shortly before each measurement by bubbling the solutions with breath (pH 7.5) or with air made CO2free by pumping it through sodalime (i.e. without any addition of acid or base). No buffer was used, as they have been shown to interfere with the mechanism of HCO;" utilization (Price and Badger, 1985; Prins and Elzenga, 1989 ). However, HCOF itself has some buffering capacity. The solutions were equilibrated at the 20°C temperature used in the experiments. The pH ofthe solutions was determined with an accuracy of 0.01 using a combination pH-electrode for media of low ionic strength media (lngold, Urdorf, Switzerland). Alkalinity was determined by titration with 0.01 N HCI. The relative proportions of CO,, HCO~" and CO~- were calculated from pH, alkalinity, temperature and ionic strength according to Helder (1988) and the tables of Rebsdorf (1972). 2.5. Chlorophyll determination Immediately after the last measurement, plant samples were immersed in 3 ml ofN, N-dimethylformamide for chlorophyll extraction (Moran and Porath, 1980). Chlorophyll concentrations were calculated using the extinction coefficients proposed by Inskeep and B|oora (1985) after measuring absorption with a spectrophotometer (U-3210 Hitachi, Tokyo, Japan ). 3. Results

3.1. Photosynthesis at various pH and total carbon concentrations The effect of pH on the photosynthetic rate of submersed leaves of P. natans and R.fluitans at 2 mM KHCO3, which is close to the concentration at the growing site, is shown in Fig. 1. Whereas the photosynthetic rate ofP. natans decreases to zero at pH 9.48, R. fluitans maintains a higher rate even at the highest pH, which indicates the use of HCO£. As pH also drastically changes the concentrations of free CO2 and HCO£, it is not possible to decide whether pH has any effect other than on the concentrations of carbon species. Fig. 2 shows the response of the photosynthetic rates to HCOA- concentrations (Fig. 2A) and to free-CO2 concentrations depicted on two scales (1.7 and 0.1 mM CO2; Fig. 2B). For floating leaves ofP. natans, photosynthetic rates are pH dependent when plotted against HCO~" concentration, but not when plotted against free-COe concentration, thus indicating that free CO2 was the photosyn/hetic substrate. The results for the submersed leaves are very similar. The submersed leaves of R.fluitans clearly show pH-dependent curves of photosynthesis vs. CO2, and the rates of photosynthesis at CO2 concentrations close to zero are

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M. Bodner I Aquatic Botany 48 (1994) 109-120

substantially higher than with P. natans ( Fig. 2 (B), lowest part, inset ). The curves of photosynthesis plotted against HCOF are pH dependent only at low pH, but fall on one line above pH 9, thus indicating the predominant use of CO2 as long as it is available at lower pH values, but also the ability of the species to assimilate

HCOF. A decrease of photosynthesis at the highest C O 2 concentration (Fig. 2 (B) ) was also found for Elodea densa (Planchon) Caspary and was explained by an inhibitory effect of high CO2 concentrations greatez than 1 mM (Weber et al., 1979; Pokorn~' et al., 1985). 3.2. Photosynthesis o f leaves cultivated at low COe

The floating leaf ofP. natans, which was kept submersed at low C O 2 conditions while unfolding, gave the same result with respect to the pH dependences of the photosynthetic rates (Fig. 3 ) as a 'normal' floating leaf from the pond (Fig. 2) or a floating leaf from the plant cultivated at low CO2 (data not shown). When it was allowed to float and was measured again after 1 week, results were again the same (data not shown). With the conditioned submersed leaf, the flight pH dependence of the photosynthetic rate vs. CO2 is not statistically supported and the photosynthetic rate at the highest pH of 9.46 was still zero, as with the uncondiT

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M. Bodner/AquaticBotany48 (1994) 109-120

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tioned leaf. Madsen ( 1991 ) found a similar small stimulation of photosynthesis at increasing HCOF for Callitriche cophocarpa Sendt. 3.3. Kinetics of inorganic carbon use CO2 concentration at half the maximum photosynthetic rate (Ko.5(CO2)) was much lower for the submersed leaf than for the floating leaf of P. natans;, both leaf types were assayed underwater. Ko.5(CO2) of the floating leaf, which had been cultivated submersed at low CO2, was comparable to that of submersed leaves, i.e. it had markedly increased its CO2 affinity. The submersed leaves from the pond and after conditioning at low CO2 had nearly the same value (Table 1 ), probably because CO2 concentrations in the pond were also very law owing to photosynthesis of algal mats. The Ko.5(CO2) value for Ranunculus, which uses HCO~-, is not a true value when HCOF is present in significant proportions in the medium. Photosynthesis at zero CO2 as an estimate of HCOF use alone (PHCO3) amounts to 25% of maximum photosynthesis at the highest inorganic carbon concentration used (Table 2). Both regression analysis and direct measurement showed that the CO2 compensation points for Potamogeton were between about 1 and 2/zM CO2. Maberly and Spence (1989) found comparable values of 2.1-2.9/zM CO2 for the different leaf forms ofP. natans using the pH-dfift technique. 3.4. Time course of photosynthetic O, production In solutions with high pH the photosynthetic rate of Ranunculus leaves was two-phasic, with an increase after about 13 min after switching on the light (4 mM KHCO3 at pH 9.5; Fig. 4). Prins et al. (1980), who found similar pH and Table 1 CO2concentrationsat half the maximumphotosyntheticrate (Ko.5(COz)) estimatedby fittingdata by Michaelis-Mentenfunction;for Ranunculusfluitans,whichusesHCO£, this is not a true valuein the presenceof HCO~', and is thereforegivenin parentheses Species P. natans Floatingleaf (pond) Floatingleafsubmersedat low CO2 Sameleaf,submersedthen floating Submersedleaf (pond) Submersedleafat lowCO2 R.fluitans Submersedleaf

Ko.s(CO2) (mM CO2)+SD 0.630+ 0.060 0. ! 22+ 0.026 0.170+ 0.029 0.185+ 0.029 0.175+ 0.025 ( O.1O0+ 0.0 i 4)

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M. Bodner / Aquatic Botany 48 (1994) 109-120

Table 2 The photosynthesis of submersed leaves of Ranunculusfluitans in pure HCO£ (Paco3) calculatedby

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Fig. 4. Time course of photosynthetic oxygen release of submersed leaves of Ranunculusfluitans at pH 9.5 and 4 mM KHCOsafter switchingon light, showinga biphasic pattern (increaseafter about 10-13 min). A typicalexampleout of five experimentsis presented.

02 concentration changes, have shown that initially there is a period in the light during which only CO2 assimilation occurs, before HCO~- use starts in the second phase. At low pH values (i.e. high CO2 concentrations) the rate sometimes slowly decreased with time, probably owing to increasing photorespiration when solu.. tions become oversaturated with 02, which results from much higher pho',osynthetic rates than at low COs concentrations (data not shown). Accordingly. ander conditions of high carbon availability and low light no biphasic pattern was found by Madsen and Sand-Jensen ( 1991 ). 4. D i s c u s s i o n

4.1. Occurrence o f r i C O 7 utilization within the genus Potamogeton

The results indicate that photosynthesis of both leaf types in P. natans is dependent on free-CO2 concentration. Madsen ( 1991 ), who found only very restricted stimulating effects of HCOF on net photosynthesis at rate-limiting CO2

M. Bodner / Aquatic Botany 48 (1994) 109-120

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concentrations for Callitriche cophocarpa, concluded that it was not due to direct uptake of HCO~-, but caused by uptake of CO2 supplied by uncatalysed conversion of HCO~- to CO2 within the boundary layer. Pondweeds, which include about 100 species world-wide (Hegi, 1981 ), can be divided into homophyllous species, with entirely submersed foliage, and heterophyllous species, with both floating and submersed foliage. The use of HCO~- for photosynthesis has been shown for all of the homophyllous species tested. The submersed leaves of five heterophyllous species could also use HCO~', whereas those of three further species did not (see Table 3). Among the latter, P. fryeri Benn. is almost exclusively restricted to soft and acid waters with low alkalinity (Kadono, 1984), P. oblongus Viv. grows also in oligotrophic, not calcareous waters (Hess et al., 1976), whereas P. natans has a broader ecological amplitude (Wiegleb et al., 1991 ). Species of the genus Myriophyllum differ also in their ability to assimilate HCO£ (Prins et al., 1982a). Interestingly, Sand-Jensen et al. (1992) found none of 12 amphibious species in streams using HCO~-, but there is a general oversaturation with CO2 in Danish streams (6-28 times). Table 3

Occunence of HCO£ use within the genus Potamogeton in relation to leaf forms Species

Reference

Fully submersed species without floating leaves Arens (1933), Kadono (1980), Mabedy and Spence (1983) P. crispus L. Arens (1933) P. densus L. Maberly and Spence (1983) P. filiformis Pets. Sand-Jensen et al. (1992) P. friesii Rupr. Arens ( 1933); e.g. Prins et al. (1982b) P. lucens L. Kadono (1980) P. maackianus Benn. Kadono (1980) P. malaianus Miq. Kadono (1980) P. oxyphyllus Miq. Sand-Jensen (1983), Spence and Maberly (1985) P. pectinatus L. Kadono (1980), Blacket al. ( 1981 ) P. perfoliatus L. Blacket ai. ( 1981 ), Sand-Jensenet al. (1992) P. praelongus Wulfen Arens (1933), Spenceand Mabedy (1985) P. pusiilus L. (panormitanus ) Biv.Ber. Adamec et al. (1993) Denny and Weeks (1970) P. schweinfurthii Benn. Species with submersed leaves and floating leaves possible (a) Submersed leaves using HCO£ Arens (1933) P. alpinus Balbis P. distinctus Benn. Kadono (1980) Arens (1937) P. sp. (tropical) P. gramineus L. Smits et al. (1988) Arens (1933), Maberlyand Spence (1983) P. × zizii Koch in Roth ( = gramineus 3
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M. Bodner / Aquatic Botany 48 (1994) 109-120

P. lucens L. (along with Elodea) has mainly been used for establishing the mechanism for HCOF assimilation by the so-called polar leaves, which exhibit a remarkable pH polarity in the light, i.e. a H + effiux from the cells of the morphologically lower epidermis, and a H ~ influx (or OH- effiux) by the cells ofthe upper epidermis (Prins et al., 1982; Miedema and Prins, 1992 ). Although these polar leaves are normally only two to three cell layers thick with nearly no intercellular spaces (except around the midveins), the thick linear leaves possess more or less extensive aerenchyma. The mechanism of these thick leaves is not yet as well established; Arens (1933) did not find a polar leaf reaction for P. pectinatus L., i.e. the pH increase occurred mainly on the illuminated side and was not strictly restricted to the upper leaf side independent of the light direction as with a polar leaf. The anatomical structures of the submersed leafofP, pectinatus and that of P. natans are nearly identical (Les and Sheridan, 1990). Therefore, it is not the anatomy of the submersed leafofP, natans which excludes HCO~- use, but rather that the biochemical mechanism is lacking. In a pH-drift experiment, a cr/Alk ratio of 0.97 was found for linear leaves of P. natans, indicating no HCOF use (Maberly and Spence, 1983). 4.2. Photosynthetic behaviour offloating leaves With the normal floating leaves of a tropical Potamogeton species, Arens (1937) did not find polarity. Interestingly, the species forms thinner floating leaves as intermediates to the truly submersed leaves which have only three cell layers; in these intermediate leaves polarity could be induced by submergence. Submergence is also the main factor for H C O ; utilization in Stratiotes aloides L.: leaf tips which emerge in summer lose this ability within a few days after emergence (Prins et al., 1982a). The floating leaves ofP. natans did not show any sign of HCO£ use, even after submersed cultivation at low CO2. A cross-section showed three palisade layers and extensive aerenchyma on the lower leaf side as with a 'normal' floating leaf. Floating leaves of P. distinctus Benn. also do not use HCOF for photosynthesis (Kadono, 1980). The floating and submersed laminae of three nymphaeid species showed no HCOF use, and a very limited HCOF uptake by the seedlings was found (Smits et al., 1988). CO2 affinity of submersed leaves of P. natans was much higher than that of the floating leaves. The same result was obtained by Sand-Jensen et al. (1992) with submersed and emergent leaves of several amphibious and mainly terrestrial species, which occasionally grow submersed. Submersed cultivation at low CO2 increased CO2 affinity of the floating leaves. Similarly, submergence besides photoperiod and temperature (30 ° C- 14 h light) induced the low compensation point state in aerial leaves of Proserpinaca palustris L. (Salvucci and Bowes, 1981 ). The change seems dependent upon physiological factors, as no obvious morphological or anatomical changes have been observed. Madsen ( 1991 ) found that in summer the lower CO2 compensation

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p o i n t o f the C3 plant Callitriche cophocarpa was d u e to a low rate o f d a r k respiration a n d a high initial slope o f t h e CO2 response curve.

Acknowledgements I wish to t h a n k Drs. J a n Pokorn~' a n d L u b o m i r A d a m e c ( D e p a r t m e n t o f H y d r o b o t a n y , T~eboh, Czech R e p u b l i c ) for critical r e a d i n g o f the m a n u s c r i p t a n d valuable suggestions.

References Abel, K.M., 1984. Inorganic carbon source for photosynthesis in the seagrass Thalassia hemprichii (Ehrenb.) Aschers. Plant Physiol., 76:776-781. Adamec, L., Husdk, S., Janauer, G.A. and Otahelov~, H., 1993. Phytosociolo?ical and ecophysiolo?icai study of macrophytes in backwaters in the Danube river inundation area near Palkovicovo (Slovakia). Ekol6gia (Bratislava), 12: 69-79. Arens, K., 1933. Physiolo?isch polaris~erter Massenaustausch und Photosyntbese bei submersen Wasserpflanzen. I. Planta, 20:62 !-658. Arens, K., 1937. Ober eine Funktion des Kaliums bei der Photosyntbese yon Wasser- und Luftbl~ittern. Boi. Fac. Philos. Sci. Lett. Univ. Sao Paulo, 1: 23-38. Black, M.A., Maberly, S.C. and Spence, D.H.N., 1981. Resistance to carbon dioxide fixation in four submerged freshwater maerophytes. New Phytol., 89: 557-568. Boston, H.L., Adams, M.S. and Madsen, J.D., 1989. Photosynthetic strategies and productivity in aquatic systems. Aquat. Bot., 34: 25-57. Denny, P. and Weeks, D.C., 1970. Effects of light vnd bicarbonate on membrane potential in Potamogeton schweinfurthii (Benn). Ann. Bot., 34: 483-496. Helder, R.J., 1988. A quantitative approach to the inorganic carbon system in aqueous media used in biological research: dilute solutions isolated from the atmosphere. Plant Cell Environ., 11:211230. He?i, O., 1981. Illustrierte Flora von Mitteleuropa. Vol. I, Part 2. Parey, Berlin, pp. 1-269. Hess, H.E., Landolt, E. and Hirzel, R., 1976. Flora der Schweiz und angrenzender Gebiete. Birkh~user, Basel. Inskeep, W.P. and Bloom, P.R., 1985. Extinction coefficients of chlorophyll a and b in N,N-dime~hylformamide and 80% acetone. Plant Physiol., 77: 483-485. Kadono, Y., 1980. Photosynthetic carbon sources in some Potamogeton species. Bot. Mag., 93: 185194. Kadono, Y., I q84. Comparative ecology of Japanese Potamogeton: an extensive survey with special reference to growth form and life cycle. Jpn. J. Ecol., 34:161-172. Les, D.H. and Sheridan, D.J., 1990. Biochemical heterophylly and flavonoid evolution in North American Potamogeton (Potamogetonaceae). Am. J. Bot., 77: 453-465. Maberly, S.C., 1992. Carbonate ions appear to neither inhibit nor stimulate use of bicarbonate ions in photosynthesis by Ulra lactuca. Plant Cell Environ., 15: 255-260. Maberly, S.C. and Spence, D.H.N., 1983. Photosynthetic inorganic carbon use by freshwater plants. J. Ecol., 71: 705-724. Maberly, S.C. and Spence, D.H.N., 1989. Photosynthesis and photorespiration in freshwater organisms: amphibious plants. Aquat. Bot., 34: 267-286. Madsen. T.V., 1991. Inorganic carbon uptake kinetics of the stream macrophyte Callitriche cophocarpa Sendt. Aquat. Bet., 40: 321-332. Madsen, T.V. and Maberly, S.C., 1991. Diurnal variation in light and carbon limitation of photosyn-

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