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Thalli discs of the marine macroalga Ulva lactuca were given inorganic carbon in the form of HC03-, and the progression of photosynthetic 02 evolution was followed and compared with predicted 02 evolution as based on calculated extemal formation of CO2 (extracellular carbonic anhydrase was not present in this species) and its carboxylation (according to the Km(CO2) of ribulose-1,5-bisphosphate carboxylase/oxygenase), at two different pHs, assuming a photosynthetic quotient of 1. The Km(inorganic carbon) was some 2.5 times lower at pH 5.6 than at the natural seawater pH of 8.2, whereas V,,, was similar under the two conditions, indicating that the unnaturally low pH per se had no adverse effect on U. lactuca's photosynthetic performance. These results, therefore, could be evaluated with regard to differential CO2 and HC03-utilization. The photosynthetic performance observed at the lower pH largely followed that predicted, with a slight discrepancy probably reflecting a minor diffusion barrier to CO2 uptake. At for a few species (3,5), whereas CO2 uptake under natural conditions has been shown for one species (24).The ubiquitous genus Ulva has been included in relatively numerous physiological marine macroalgal studies. Regarding photosynthetic traits, species ofthis genus have been described as being able to suppress photorespiration. This is indicated by their O2-insensitive photosynthetic rates (2) and low CO2 compensation points (6,15). Still, at least Ulva fasciata features C3 photosynthesis (2), and it has been suggested that (3) is the basis for a system that concentrates Ci internally (4) and, presumably, CO2 to the surrounding of Rubisco, thereby causing the C4-like gas exchange responses. In this work, we have investigated whether extracellular HC03-dehydration could supply CO2 fast enough to explain measured photosynthetic rates of Ulva lactuca as based on CO2 diffusion. This was done given the notion that it, unlike U. fasciata (3), apparently lacks an external/surface-bound CA (9), and assuming a Km(CO2) for Rubisco as measured for the closely related latter Ulva species (4). MATERIALS AND METHODSUlva lactuca was collected from the intertidal just north of Tel Aviv, and maintained in aerated seawater on the laboratory windowsill until used for the following experiments (within 5 d). Circular discs of approximately 5 mg fresh weight were punched with a stopper borer and incubated at 25°C and 600 umol photons m-2 s-' (400-700 nm) in air-sparged synthetic seawater (450 mm NaCl, 10 mm KCI, 10 mm CaCl2) and 30 mm MgSO4 to which NaHCO3 had been added to a final concentration of 1 mm. In some cases, when the Ci storage capacity of the Ulva discs was large, NaHCO3 was omitted in order to reduce the subsequent depletion time of internal Ci. 02 evolution was measured using an 02 electrode system as described earlier (4). Two Ulva discs were taken from the incubation medium, rinsed in synthetic seawater, and transferred into the illuminated (1000 gmol photons m-2 s-')

Evidence of an inorganic carbon concentrating system in a marine macroalga is provided here. Based on an 02 technique, supported by determinations of inorganic carbon concentrations, of experimental media (as well as compensation points) using infrared gas analysis, it was found that Ulva fasciata maintained intracellular inorganic carbon levels of 2.3 to 6.0 millimolar at bulk medium concentrations ranging from 0.02 to 1.5 millimolar. Bicarbonate seemed to be the preferred carbon form taken up at all inorganic carbon levels. Seawater provides an environment of 2.7 mm Ci', only some 12 uM of which is in the form of C02 (at 20°C). Since the Km (C02) of Rubisco from marine macroalgae is in the 30 to 60 Mm range (6,8), it seems likely that these plants have developed a system not only to assure high resupply rates of CO2 (12), but also to actually concentrate this carbon form to the site of fixation via Rubisco. Gas exchange features such as 02 insensitive photosynthetic rates and low C02 compensation points have also indicated the operation of such a system, and it has been suggested that Circular discs of 8 mm diameter, punched with a stopper borer, were used throughout. These discs weighed 5.5 ± 0.6 mg each (n = 17); their volume was further determined by [3H]H20 incubations (0.5-30 min yielding the same results) to 5.3 ± 1.3 ,uL (n = 7).[14C]Sorbitol incubations (90 s followed by a 1 s rinse) yielded a periplasmic volume of 0.83 ± 0.12 1L (n = 5) which is 16% of the total disc volume. Thalli were, on the average, 76 Mim thick, including two cell layers of 30 Mm each. The 79% crosssection area thus occupied by cells was close to the 84% sorbitol impermeable space.Ulva discs were stored in the light for at least 1 h in a simple synthetic seawater medium (500 mm NaCl, 10 mm KCI, 10 mM Ca(Cl)2, and 30 mM MgSO4) lacking HC03-. Thereafter, they were transferred to equal media to which NaHC03 had been added to various final concentrations. All solutions were presparged overnight with outside air whereby stable final pHs (6.6-8.1) were reached; for the lowest Ci level, 5 mM Mes-buffered seawater (without HCOO) was used. Exact Ci levels of these solutions were determined by IRGA as described below. Inorganic carbon was allowed to assimilate in the light (PPFD = 300 uE m-2 s-') for 0.5 to 1 h.

Abstract. The marine macroalga Ulva sp. can take up HCO3 via a process which chemically resembles that of anion exchange in red blood cells (Drechsler et al. 1993, Planta 191, 34~40). In this work we explore the possibility that high-pK amino-acid residues could be functionally involved in the binding/transport of HCO3. It was found that the specific arginyl-reacting agents phenylglyoxal and 2,3-butanedione inhibited photosynthesis of Ulva competitively with inorganic carbon at pH 8.2-8.4 (which is close to the pH of normal seawater), where HCO 3 was the predominant inorganic carbon form taken up. The inhibition by phenylglyoxal was irreversible at 32~ and high pH values, while that of butanedione became irreversible in the presence of borate. These interactions, as well as the protection of the irreversible phenylglyoxal-inhibition by inorganic carbon and by the membrane-impermeant agents 4,4'-diisothiocyanostilbene-2,2'-disulfonate and 4,4'-dinitrostilbene-2,2'-disulfonate indicate that arginine (and possibly also lysine) are involved in the HCO 3 uptake process, probably at the plasmalemma level. The photosynthetic affinity of Ulva to external inorganic carbon gradually decreased with increasing pH from 8.2 to 10.5, and this decrease parallels the decline in protonation of amino acids with a pK of around 10. Based on this information, as well as the inhibition studies, it is suggested that arginine and lysine residues are essential proteinaceous constituents involved in anionic inorganic carbon (HCO3 and possibly also CO32 ) uptake into the Ulva cells.

It was found that DCMU had a differential effect at two concentration ranges on variable fluorescence kinetics in isolated chloroplasts. The increase in fluorescence rate at low concentrations of DCMU was abolished by preincubation of chloroplasts with ferricyanide or formate, treatments which were shown to convert Fe in the PS II reaction center (i.e., the FeQA complex) into a non-oxidizable form, but it was not affected by Tris treatment. Increase in fluorescence kinetics (at the initial linear rate) at high concentrations of DCMU was found to be abolished by Tris treatment but it was only marginally affected by ferricyanide or formate treatments. The effect of Tris could be abolished by addition of hydroquinone-ascorbate, which restored electron flow to the pool of secondary acceptors.Contrary to the effect of DCMU, no such differential concentration dependence of the variable fluorescence kinetics was found for atrazine.The increase in fluorescence kinetics (at the initial linear rate) at a low concentration rate of DCMU is presumably restricted to units which contain an oxidizable Fe in the FeQA complex. Increase in fluorescence kinetics (at the initial linear rate) at high DCMU concentration is probably related to the effect of DCMU at the QB site.

Brewer and Jagendorf (3) observed that dark preincubation of chloroplasts with 0.5 mm FeCN' caused an inhibition of electron transport which they attributed to a step related to 02 evolution.Ikegami and Katoh (10) have found that preincubation of chloroplasts with FeCN slowed down the rise kinetics of fluorescence in the presence of DCMU. This was interpreted as due to oxidation of an internal acceptor during preincubation. Contrary to the interpretation of Brewer and Jagendorf (3), they suggested that the site inhibited is located between the reaction center of PSII and the DCMU-sensitive site.In the present work, the effects of preincubation with FeCN on electron transport and fluorescence were reinvestigated and compared. We conclude that the interpretation of Brewer and Jagendorf (3) is valid and that the inhibition in the rate of the fluorescence rise is also caused by inhibiting electron flow between H20 and PSII.MATERIALS AND METHODS Isolation of Chloroplasts. Chloroplasts were prepared from 2-week-old pea seedlings (Pisum sativum L. cv. Kelvedon) by a modified method of Nakatani and Barber (11). Ten g of leaves were homogenized for 5 s in a homogenizer with 75 ml of medium containing 0.33 M sorbitol, 0.2 mm MgCl2, and 20 mm Mes, pH 6.5. The homogenate was filtrated through 8 layers of gauze and centrifuged at 2500g for 40 s. The pellet was resuspended in the same medium.Tris Treatment. The chloroplast pellet was resuspended in 16 ml of0.8 M Tris, pH 8, and incubated on ice for 25 min. Thereafter, the suspension was centrifuged at 10,000g for 7 min and the pellet was resuspended in the sorbitol-MgCl2-Mes medium.

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