LIBRARY Michigan State University This is to certify that the thesis entitled I Oxidative Photosynthetic Carbon Cycle In ' Green Algae presented by Sttphen Dietrich has been accepted towards fulfillment of the requirements for Master Of Science degreein Biochemistry new Major professor Date 2-26-87 0-7639 MS U is an Affirmative Action/Equal Opportunity Inuitution )V1531_J RETURNING MATERIALS: Place in book drop to LIBRARIES remove this checkout from .-_—. your record. FINES will be charged if book is returned after the date stamped below. OXIDATIVE PHUTOSYNTHETIC CARBON CYCLE IN GREEN ALGAE BY Stephen Dietrich A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Biochemistry 1987 .47.-.. ABSTRACT OXIDATIVE PHOTOSYNTHETIC CARBON CYCLE IN GREEN ALGAE BY STEPHEN DIETRICH A.survey of some green algae tor the type of glycoiate oxidizing enzyme, the presence of peroxisomes, and the presence of a 602 concentrating mechanism is presented. One group of algae has glycolate dehydrogenase and one group has glycolate oxidase. Catalase is present in all of the algae studied, but in much greater amounts in the algae with glycolate oxidase. The presence of a CO2 concentrating mechanism was also determined in these algae by measuring the £0.5(c02) for photosynthesis and the level of external carbonic anhydrase. Some of the algae have characteristics of a 002 concentrating mechanism and some do not. Theda2 concentrating mechanism of Chlamydomonas moewusii was studied in more detail. The species of inorganic carbon taken into the cell is 002. and this alga lacks an external carbonic anhydrase. Photosynthetic characteristics of Chlamygomonas moewusii were compared to Chlamydomonas reinhardtii, which also takes up co2 but has an external carbonic anhydrase. It was found that these two algae have the same low KO 5(602) and accumulate Hcoa’ internally to the same high level. TABLE OF CONTENTS INTRODUCTION .......................... . ......... . ...... . .......... LITERATURE REVIEW ...... ................ .................... .... The C2 Cycle ..................... ...... ... .......... .......... Algal Glycolate Oxidizing Enzymes ............................. Algal Peroxisomes ................................. ...... ...... Evolution ............................. ....... . ..... ..... ...... C02 Concentrating Mechanisms .................................. PART 1 A Survey of Glycolate Metabolism and C02 Concentration in Green Algae ....... 0.0.000....00.........OOOOOOOIOO... MATERIALS AND METHODS ................. ...... . ................. .... Growth of Algae ................... ........... . ....... ......... Preparation of Cell Free Extracts ..... ..... ..... . ..... ........ Enzyme Assays ........................... ...... . ..... .......... Glycolate Excretion ........................... ..... . ..... ..... Photosynthetic Measurements ......... ..... ........... .......... RESULTS ............................... ................... ......... Enzyme Survey ......................... ........... ............. C02 Concentrating Mechanisms ......... ...... .............. ..... Glycolate Excretion .............. ......... . ....... ....... ..... DISCUSSION ............ . ....... .... ................................ PART 2 The C02 Concentrating Mechanism of Chlamydomonas moewusri .... ....... .................... ...... .. .......... Introduction . ....... ..................... .................... Methods ............... ......... . ...... . ...................... Results .......... ...... ... ................................ ... Discussion ...................... ........ ..................... BIBLIOGRAPHY ... ........ . ...... . ........... . ........ . ......... ii 18 18 18 18 18 19 20 LIST OF TABLES Table No. Page 1 Algae, Source, And Growth Media .............................. 21 2 Glycolate And Lactate Oxidation By Algal Extracts ... ..... L..23-2A 3 The Presence Of A C02 Concentration Mechanism In Algae ....... 27 A Effect Of Acetazolamide On K0.5(C02) At pH 8.3 For Air-Grown Algae 000......I....O....0......00............OOOOOOOOOOOO...O 28 5 Glycolate Excretion By Selected Algae ...... ........ .. ........ 3O 6 Type of Glycolate Oxidation In Algae Orders .............. .... 32 7 Inorganic Carbon Requirement For Photosynthetic 02 Evolution By Air-Grown E. moewusii And The Absence Of An Effect From AcetaZOla-mide (AZ) 00...... ..... O0.00.......OOOOOOOOOOOOIOOOO. “3 8 Internal Carbonic Anhydrase Activity And The Effect Of Ethoxzolamide On The K0.5(C02) Of Air-Grown C. moewusii And 9. reimardtii At pH 7.3. 0............OOOOOOOOOOOOOOOOO ...... “5 9 The Effect Of Proteinase Treatment On The K0.5(C02) Of 2. reinhardtii At pH 8.3. ........ ...... . ..................... M6 1O Inorganic Carbon Requirement For 5% COZ-Grown E. moewusii .... 51 iii LIST OF FIGURES Figure No. Page 1 The Oxidative Photosynthetic Carbon Cycle In Higher Plants ... 5 2 Model Of The 002 Concentrating Mechanism In Chlamydomonas reinhardtii ......OOOOOO ....... 00...... 0000000000 I 000000000000 1S 3 The Rate Of Photosynthetic 02 Evolution At Increasing External C02 Concentration By Air-Grown g. moewusii And 9. reinhardtii At pH 8.3. ...0000............IOOOOOOOOOIOIOOO. '41 A 11‘COZ Fixation And Ci Accumulation By Air-Crown C. moewusii And C. reinhardtii At pH 8.3 And The Effect Of Acetazolamide.. H8 5 MC02 Fixation And Inorganic Carbon Accumulation By High COZ-Grown Q. moewusii And C. reinhardtii At pH 7.3. ..... ..... 52 6 1”002 Fixation And Ci Accumulation By High COZ-Grown C. moewusii At pH 8.3. ....... . ........................... .... 5A iv AOA CA Ci DAB DCPIP FMN PVP RUBISCO LIST OF ABBREVIATIONS Aminooxyacetate Carbonic anhydrase Inorganic carbon 3,3'-diaminobenzidine Dichloropnenoiindophenol Flavin mononucleotide Polyvinylpyrrolidine Ribulose bisphosphate carboxylase/oxygenase .INTRODUCTION The C2 oxidative photosynthetic carbon cycle has been studied extensively in green algae and higher plants. Most aspects of the C2 cycle are the same in algae and plants, however, major differences exist in the nature and location of the glycolate oxidizing enzymes. Plants and some higher algae have a glycolate oxidase located in the peroxi- somes. The electron acceptor is oxygen, and its peroxisomal location with catalase is linked with detoxification of the H202 generated by this oxidase. Many algae have a mitochondrial glycolate dehydrogenase whose primary electron acceptor is unknown, although it is not oxygen. Glycolate oxidase is not inhibited by cyanide and also it oxidizes L-lactate but not D-lactate, while glycolate dehydrogenase is cyanide sensitive and oxidizes D but not L-lactate. The location and type of the glycolate oxidizing enzyme represents a difference between eukaryotic single cell algae and higher plants and algae. Glycolate dehydrogenase has very low activity, especially when grown on high levels of Goa, and because of this, many unicellular algae excrete much of the glycolate formed. Plants and algae with glycolate oxidase, which is much more active, do not excrete glycolate. Thus, carbon is lost by algae that excrete glycolate. Plants and algae with glycolate oxidase also lose carbon through photorespiration (theoreti- cally 1 out of A carbons) but are able to recycle most of the carbon when two glycolates are metabolised to one glycerate. Since glycolate dehydrogenase is located in the mitochondria, glycolate oxidation could be linked to energy production via the electron transport chain (10,17, 55). Glycolate oxidation in the peroxisomes is not linked to electron transport and therefore is an energy wasting process. Many algae apparently contain a few small unspecialized peroxisomes, or micro- bodies, which contain catalase but not glycolate oxidase. The formation and metabolism of glycolate is dependent on the levels of 02 and C02. Since glycolate is formed by the oxygenase reac- tion of ribulose bisphosphate carboxylase/oxygenase, the lower the C02/ 02 ratio the more glycolate is made. The amount of glycolate dehydro- genase may be increased by growth on low levels of C02 because of substrate activation. Some plants and algae have deveIOped ways to reduce glycolate forma- tion. C2 plants reduce the competition between C02 and 02 by fixing 002 as HCO§ into malate and aspartate, and then decarboxylating these acids at the site of ribulose bisphosphate carboxylase, thus increasing the C02 concentration above levels obtained by gas diffusion into the chlorOplasts. C3 plants have no such means to reduce glycolate forma- tion, which is apparently dependent only on atmospheric levels of C02 and 02. Some unicellular algae have also developed a C02 concentrating mechanism to increase 002 fixation and reduce photorespiration. The details for this mechanism are unknown but is thought to involve carbonic anhydrase and a HCO§ transporter on either the plasma membrane or the chloroplast envelope (32). Since it thought that plants may have evolved from the green algae, changes in glycolate metabolism and the development of leaf-type peroxi- somes also may have occurred during evolution. Many questions can be asked about these changes namely, why did plants and higher algae develop a wasteful glycolate oxidase instead of keeping the energy 2 conserving glycolate dehydrogenase; are there advantages to having a particular form of the enzyme; what environmental factors caused the change; and are all algae able to reduce photorespiration with a C02 concentrating mechanism? A survey of some green algae for the type of glycolate oxidizing enzyme and the presence of a C02 concentrating mechanism is presented in Part 1 of this thesis. During the course of this research it was discovered that Chlamydomonas moewusii has a modified C02 concentrating mechanism relative to Chlamydomonas reinhardtii. A more detailed investigation of Chlamydomonas moewusii is presented in Part 2. LITERATURE REVIEW The C2 Cycle: The light-dependent uptake of 02 and release of C02 during photosynthesis is called photorespiration. The metabolic pathway for photorespiration is known as the oxidative photosynthetic carbon cycle or 02 cycle. The reductive photosynthetic carbon cycle, or C3 cycle, is initiated by ribulose bisphosphate carboxylase/oxygenase (RUBISCO), and C02 is reduced to sugar phosphates in the chloroplast. The sugar phosphates are converted to starch for storage or transport out of the chloroplast for energy and sucrose synthesis. The C2 and C2 cycles are linked due to the competition between 002 and 02 for RUBISCO; RUBISCO either converts C02 and ribulose bisphosphate to two molecules of POA or reacts with 02 to form one molecule of PGA and one molecule of phosphoglycolate for the C2 cycle. A diagram of the C2 cycle in higher plants is shown in Figure 1 (18). The first step is the formation of P-glycolate from ribulose bis- phosphate and 02 by the oxygenase reaction of RUBISCO in the chloro- plasts. P-glycolate phosphatase hydrolyzes the P-glycolate to glycolate which is transported from the chloroplasts to the peroxisomes. glyco- late is oxidized in the peroxisomes to glyoxylate and H202 by glycolate oxidase. The H202 is degraded by catalase and the glyoxylate is trans- aminated to glycine, which is transported to the mitochondria. In the mitochondria, glycine is oxidized by glycine oxidase to C02, NH3, and a C1 unit bound to tetrahydrofolate. The C1 unit is added to a second glycine molecule to form serine by serine-hydroxymethyl transferase. Serine is converted to hydroxypyruvate by an aminotransferase in the peroxisome, and hydroxypyruvate is reduced to glycerate by Figure 1. The Oxidative Photosynthetic Carbon Cycle In Higher Plants. From reference 18. .54] CN20® Hommnnic cso ovum “a“ bow a cum ' I/‘u' cuzo® """" “00. / .02 m / ' “‘KETO sauna-Os P (”‘0‘ a 0 . NATIHL capo m7 CNzOCD uutf'wc \ GLUTAMWE (.11)! p. m IMAM! CHLOROPLASTS "- 90w coon coon 2 GLUTAIMTE I 01,0" cute" cwo murmur Sucrose --- -—-Yco|e-P “my, 9-“ ——-—c-~m-oea—e I??? CYTOPLASM Glycolate Mus, am! PM PEROXISOMES coon CO; I MALATE - _____ __.. .. HALATE 9°“ 900:" I / ’ucoow : *0; C"! I, o. ' l t' «no «no J . o. /H2 :02 “- Mow MD" "2° ""'"z°z ’0? "A coon manna mm 60 do 6003 . O :I/ stunner: .-_..GumMATE -----u‘f >d autumn -::/ . \ g a- K510- ___\__. a-KETO- - _ ___ l-KETO- __--_._. . 3‘ stamens GLUTARATE 90°“ ' coon G-UWATE N\ ' 9N1. - \ WTATE ------ -\ - - -. MATE cup; 9‘2” ¥ 1 I _J l 1 _ Q ~ ’ ~~~—-"‘ 9 O l I mrocwouonm goon 900,, 31:39 ,\ cam, “12““: :20: é"20" '< «no )5,- GLUTAMATE "an. \ f“ a-KETO- CO: . cpnra . NH, . _ GLUTARME l \ METHYL accrpiows’ "“0" ‘ncoou hydroxypyruvate reductase. The glycerate can return to the chlorOplast where it is phosphorylated and can reenter the C3 cycle and regenerate ribulose bisphosphate. 02 uptake occurs during the formation of P-glycolate by the oxygenase reaction of RUBISCO, during glycolate oxidation in the peroxi- somes, and during glycine oxidation in the mitochondria. C02 is released during glycine oxidation. The C2 cycle in green algae is very similar to the cycle in plants, but has some differences. Many unicellular and multicellular algae have a mitochondrial glycolate dehydrogenase instead of a peroxisomal glyco- late oxidase. The activities of glycolate dehydrogenase and glycine decarboxylase appear to be too low to account for the measured flow of carbon through the C2 cycle (18). Cells grown on high 002, or treated with aminooxyacetate (AOA) which blocks conversion of glyoxylate to gly- cine, accumulate and excrete glycolate (50). Photorespiration in cyanobacteria is not well understood. Many of the C2 cycle enzymes are absent or detected only in trace levels. RUBISCO from cyanobacteria exhibits oxygenase activity, and much P- glycolate phosphatase activity is present. In fact, P-glycolate phospha- tase is up to 6 times more active in cyanobacteria than green algae (19). Thus, glycolate is probably made but its metabolism is unknown. No glycolate excretion is seen even in the presence of ADA, and little if any glycolate dehydrogenase activity is detectable. The function of the C2 cycle is a subject of continuing investiga- tion. It is considered a wasteful energy burning process that is unavoidable due to the oxygenase reaction of RUBISCO. The C2 cycle can serve as a carbon scavenging system since some of the carbon atoms 7 flowing through the system reenter the C3 cycle, and some of the released C02 can be refixed by the cell. Because the C2 cycle can dispose of excess photosynthetic assimilatory capacity, it may prevent photooxidative damage, especially during periods of 002 shortage. Another function is to provide glycine and serine and C1 units for cell growth. Algal Glycolate Oxidizing Enzymes: The distribution of glycolate oxidizing enzymes was studied by Frederick et al (1“). They assayed several green algae, bryophytes, ferns, and fern allies for the presence of glycolate oxidase or dehydrogenase, the specificity for D or L- lactate, and the peroxisomal marker, catalase. All of the plants and a few of the algae had glycolate oxidase, but most of the algae had glyco- late dehydrogenase. Glycolate oxidase also oxidized L-lactate and occasionally low amounts of D-lactate, while glycolate dehydrogenase oxidized D-lactate and low levels, if any, of L-lactate. Catalase was present in most organisms, but was usually present in much higher levels when glycolate oxidase was present. Tolbert (51) surveyed several marine green algae and land plants for the type of glycolate oxidizing enzyme. All of the algae had glyco- late dehydrogenase. The land plants had glycolate oxidase except for Cymodocea rotundata and Thallassia hemprichii, which had glycolate dehydrogenase. These are the only land plants known to have glycolate dehydrogenase. In a survey of 27 species of green algae representing 16 genera in the Chlorococcolales and Chlorosarcinales, Bullock et al (7) found all but two of the species to contain glycolate dehydrogenase. Some of the species with glycolate dehydrogenase had unusually high rates of 8 L-lactate oxidation, 50-138% of the glycolate activity. However, activity with D-lactate was always higher than with L-lactate in these algae. One of the algae with glycolate oxidase, Planophila terrestris, also had high rates of D-lactate oxidation, 108% of glycolate oxidation, although L-lactate oxidation was much higher. Growth conditions of some of these algae were varied to see if a change in the form of enzyme occured. The type of glycolate oxidizing enzyme did not change when grown on solid or liquid media, increased nitrogen, or when grown heterotrophically or autotrophically. Algal Peroxisomes: Organelles similar to animal and plant peroxisomes have been detected in many green algae (38). These structures are usually termed microbodies because the enzyme content is not known for most of the algae. Almost all algae studied by electron microsCOpy have revealed the presence of microbodies. Silverberg (38) studied 29 species of algae by electron microscopy and found microbodies in most of them. A cytochemical stain, DAB, for catalase was performed on selected algae and was positive for the majority. Several other species have been stained with DAB by other researchers (39,”8) with both positive and negative results. Silverburg states that negative findings in a cytochemical preparation do not necessarily mean the absence of enzyme activity, but may be explained by inactivation by the fixative, solubilization of the enzyme during pre- paration, or inadequacy of the incubation. Many algae, and other tissue, once reacting negatively with DAB, later gave a positive reaction. Thus, it is likely that all green algae have catalase- containing unspecialized microbodies regardless of which type of glycolate oxidizing enzyme is present. 9 Glycolate dehydrogenase has been localized in the mitochondria of a few green algae and diatoms. Using a cytochemical assay for glycolate dehydrogenase, Beezley et al (5) found deposition of a electron opaque material in the outer compartment of the mitochondria of Chlamydomonas reinhardtii. D-lactate gave similar results as glycolate, while L- lactate gave only a weak reaction. Oxamate, which inhibits glycolate dehydrogenase in cell free extracts, inhibited the cytochemical reaction. Other cellular components did not accumulate stain. Intact organelles of a few green algae have been isolated and their enzymology characterized. Organelles from only three algae, Eremosphaera, Chlorogonium, and Euglena, with glycolate dehydrogenase have been characterized. Intact microbodies and mitochondria of Eremosphaera were isolated by Stabenau (A1). The microbodies contained catalase and uricase, while the mitochondria contained the glycolate pathway enzymes, including glycolate dehydrogenase, glutamate-glyoxylate aminotransferase, serine-hydroxymethyl transferase, serine-glyoxylate amino transferase, and hydroxypyruvate reductase. The microbodies of Chlorogonium were also isolated (A6) and only catalase and uricase were present. This alga has glycolate dehydro- genase which is probably in the mitochondria, although the mitochondrial enzymes were not assayed in this study. Euglena represents a more complex distribution of enzymes. Catalase has not been found in the microbodies, but a glycolate dehydro- genase, hydroxypyruvate reductase. and serine-glyoxylate amino- transferase are in the microbodies. Glycolate dehydrogenase was also found in an equal amount in the mitochondria along with glutamate- glyoxylate aminotransferase, glycine oxidase, and serine-hydroxymethyl 10 aminotransferase (11,26). The two glycolate dehydrogenases are under different physiological control, both are present when the algae are grown on air levels of C02, but the microbody enzyme disappears when grown on 5% 002 (55). The peroxisomal glycolate oxidizing system did not take up oxygen and was cyanide sensitive, typical of glycolate dehydrogenase. Glycolate dehydrogenase was found in isolated mitochondria of two marine diatoms (36). The enzyme was shown to link indirectly to oxygen via the electron transport system. Glycolate oxidase has been found in the peroxisomes of all plants studied, and in a few green algae. A cytochemical assay showed glyco- late and L-lactate dependent reaction product in the microbodies of Klebsormidium (15). These microbodies also stained positive for catalase (A8). Intact peroxisomes and mitochondria were isolated from Spirogyra (A5). Glycolate oxidase, catalase, and hydroxypyruvate reductase were found in the microbodies, other enzymes were not looked for. Micro- bodies of Mougeotia were also isolated (A3), and found to contain catalase, glycolate oxidase, alanine-glyoxylate aminotransferase, glutamate-glyoxylate aminotransferase, and hydroxypyruvate reductase. Serine-hydroxymethyl transferase was in the mitochondria. These algae therefore have peroxisomes as in the leaves of higher plants. Other enzyme systems present in algal microbodies as well as plant peroxisomes, are the glyoxylate cycle and fatty acid u-oxidation. These enzymes have been established in only a few algae (A2). Given the limited data, it appears that there are two groups of algal microbodies, unspecialized microbodies containing catalase and uricase, and micro- bodies similar to leaf peroxisomes of higher plants. 11 Evolution: Ideas on the evolution of green algae have been developed from cytological and biochemical evidence. Pickett-Heaps (37) and Stewart and Mattox (A7) divide the green algae into two basic evolu‘ tionary lines. The first line, called the chlamydomonad line, never evolved past the algal stage, and the organisms have the following characteristics: (a) symmetric motile cells; (D) anteriorly attached flagella associated with four crucially arranged, narrow microtubular roots; (0) cell division in which the mitotic spindle disperses after nuclear division with the two daughter nuclei coming close together, another set of microtubules arising perpendicular to the former position of the microtubules of the mitotic spindle, and the new cell wall forming along these microtubules; (d) the presence of glycolate dehydrogenase. The second evolutionary line eventually led to the formation of the higher plants, and it is characterized by the following: (a) asymmetric motile cells with the flagella attached in a lateral position; (b) flagella roots that consist of a single broad band of microtubules; (c) cell division in which the spindle does not disintegrate but holds the two daughter nuclei apart while the new cell wall is formed by a phragmo- plast or by a phragmoplast associated with an infurrowing of the parent plasmalemma; (d) the presence of glycolate oxidase. The origin of the two lines of evolution is unknown, their ancestor is referred to as an "archetypal unicellular flagellate". C02 Concentration: Some unicellular algae are very efficient in their utilization 0f C02. These algae are Gog-saturated for photosynthesis at C02 concentrations less than atmospheric levels, have a very low C02 compensation point, and photosynthesis is not inhibited by 02 (3,6). 12 These effects are seen when the algae are grown on low levels of C02 (air levels). When grown on high (1-55) C02, the algae have high C02 requirements and high C02 compensation points. The ribulose bisphosphate carboxylase-oxygenase of these algae has a high Km(C02) and oxygenase activity as in C3 plants, which do not change when grown on high or low C02. Air grown cells can accumulate inorganic carbon (Ci) internally to levels higher than can be accounted for by passive C02 diffusion. COZ-grown cells do not accumulate Ci: Therefore, it has been proposed that air grown cells develop a C02 concentrating mechanism that increases C02 at the site of RUBISCO. The species of C02 that enters the cell varies. All the blue-green algae tested (A,20,29) and the green algae Scenedesmus (13) take up HCO§ from the environment. On the other hand, Chlamydomonas reinhardtii, Chlorella vulgaris, Dunaliella, and other green algae take up 002 (33.52,1). Two different mechanisms are proposed depending on whether HCO§ or C02 enters the cell. The first mechanism is a carbonic anhydrase facilitated diffusion of C02 across the plasma membrane. Some algae with this mechanism have large amounts of carbonic anhydrase (CA) located in their periplasmic space (1,9). 002 is apparently the only species of Ci that enters the cell, as determined by short term fixation experiments using either C02 or H00; (53), by the dependence of C02 fixation with external pH (33), and by the use of membrane-impermeant inhibitors of CA (32). The membrane-impermeant CA inhibitor, acetazolamide (AZ), severely inhibits €02 fixation and Cl accumulation at high external pH, but has little or no effect at low external pH (32). The mechanism may also involve internal carbonic anhydrases. Figure 2 shows a model pr0posed by 13 Moroney and Tolbert (32). The external CA replenishes the cell with C02 that crosses the plasmalemma, the C02 in the cytoplasm then is converted to HCO§ by a cytoplasmic CA. The HCO§ is then trans- ported into the chloroplast by a HCO§ transporter on the chlorOplast envelope. A chloroplast CA then replenishes the C02 derived from HCO§ in the basic chloroplast stroma for RUBISCO. If the internal CA is inhibited by the membrane-permeant inhibitor, ethoxzolamide, photo- synthesis is reduced even when the external pH is acidic (32). At the same time, though, internal HCO§ builds up. This suggests that although inorganic carbon is still accumulating, it is trapped in the chloroplast in a form not available to RUBISCO. This effect is also seen in a mutant that lacks an internal CA, probably because the mutant lacks the chloroplast CA and not the other CA forms (A0). These results support a location for the accumulation step at the chloroplast enveIOpe, as C02 crosses the plasmalemma yet HCO§ is accumulating within the cell. The second mechanism of C02 concentration involves direct uptake of HCO§ from the environment. No periplasmic CA is present in these algae and they photosynthesize efficiently even at an external pH of 10 where the concentration of C02 in the media is very small. Active trans- port of HCO§ is presumed to be at the plasmalemma and is possibly driven by cyclic electron transport around photosystem I in cyano- bacteria (35). Having a C02 concentrating mechanism is an obvious advantage for an algal cell. The algae could grow at C02 concentrations much below the C02 compensation point of C3 plants, such as is found in alkaline waters. Concentration of C1 would also reduce photorespiration by increasing the 14 Figure 2. A Model Of Tne C02 Concentrating Mechanism In Chlamydomonas reinhardtii. From reference 33. 15 CHLOROPLAST 1 CYTOPLASM Hco;+—+ Hco; ICA la. (:0: (——__') C02 6"— RUBP Corboxylose PGA ENVELOPE l PE RIPLASMIC SPACE HCO;<——r—) HCC ”—9 C02 {—L C02 l. -l MEDCUM PLASMALEMMA CELL WALL 16 carboxylase reaction and reducing the oxygenase reaction of RUBISCO. The carboxylation rate/oxygenation rate was almost 10 times higher for air grown than COZ-grown C. reinhardtii (31). l7 18 PART 1 A Survey of Glycolate Metabolism and C02 Concentrating Mechanisms in Green Algae MATERIALS AND METHODS Growth Of Algae: The algae used in the study are listed in Table 1 with their growth media. One liter of culture was grown in 3 liter Fernbach flasks under illumination of 150 uE m"2 s'1 and constant shaking. The cultures were aerated with either air (0.0A% C02) or air supplemented with 2 to 5% C02. Cells from the log phase of growth were harvested by centrifugation at 1000 g for 5 minutes, washed once in distilled water, and repelleted at 10,000 g for 5 minutes. The cells then were resuspended in either 25 mM HEPES at pH 7.3 or the homogenization buffer. Preparation 0f Cell Free Extracts: The algal cells were broken by passing through a Yeda press at 1500 psi N2. The homogenization buffer for most algae was 50 mM K phosphate at ph 7.0 with 0.01% Triton X-100. Netrium and Mesotaenium were broken in 50 mM tricine at pH 7.5 with 0.011 triton X-100 and 1A PVP. The cell debris was then removed by centrifuging at 500 g for 10 minutes, and the supernatants were used for the assays. Enzyme Assays: Glycolate dehydrogenase and glycolate oxidase were assayed in Thumberg tubes by following the anaerobic reduction of DCPIP at 600 nm with a Gilford recording spectrophotometer (3A). The reaction was done in a 2.5 ml volume containing 0.08 mM Na pyrophosphate at pH 8.5, 0.12 mM DCPIP, 0.1 mM FMN, and either 8 mM glycolate, 20 mM D-lactate, or 20 mM L-lactate. The sensitivity to cyanide was tested by assaying in the presence of 2 mM KCN. Reactions were initiated by addition of substrate after reading the endogenous rates for 5 minutes. Glycolate dependent 02 uptake was also measured in an 02 electrode. The reaction contained 200 pmoles Na perphosphate, 20 pmoles glycolate, and 8 pmoles FMN. Catalase was measured spectrophotometrically by the disappearance of H202 at 2A0 nm (28). Protein was measured by the method of Lowry (27). Glycolate Excretion: Glycolate excretion was measured with harvested cells in buffer at either pH 7.5 (Netrium and Euglena) or pH 6.3 and 8.3 (9. mouewusii). The cells were incubated in 800-1000 pH [11'2 3'1 of light and at a limiting amount of NaHC03. One ml samples were removed at 0, 15, 30, and A5 minutes, the cells spun out in the microfuge, and the supernatants assayed for glycolate. AOA was added at a concen- tration of 2 mM. Glycolate was measured by the Calkins method (8). 19 Photosynthetic Measurements: Photosynthetic 02 evolution was measured in a Rank Brothers 02 electrode. The cells were used at a final concen- tration of 25 pg Chi/ml upon dilution in either 25 mM MES at pH 6.3, 25 mM HEPES at pH 7.3, or 25 mM EPPS at pH 8.3. The buffers were prepared daily and were bubbled with N3 to lower the dissolved C02 and 02. Three ml of the cell suspension in the 02 electrode chamber at 25°C were illuminated with 800 pE m'2 s" of light filtered through CuSOu to remove heat. Two methods were used to measure the KO.5(C1) for photosynthesis. The first method was to follow a single progress curve (56) for 02 evolu- tion after addition of about three times the amount of NaHC03 needed for half-maximal rates of photosynthesis. The Ci concentration when the 02 evolution rate was 50; of maximum was then calculated, assuming that the 02/C02 net exchange ratio was one. The second method was to add known amounts of NaHCO3 and measure the initial rate of 02 evolution for each concentration. This method was used at high pH and for 002 grown cells. Before addition of NaHC03 in both assays, the endogenous 002 was depleted by illuminating the algae until 02 evolution ceased. The presence of periplasmic CA was determined by the addition of the CA inhibitor acetazolamide (AZ) to photosynthesizing cells. AZ was used at a concentration of 50 mM. CA activity was determined by measuring the time required for the pH to drop from 8.0 to 7.0 in a reac- tion mixture containing 1.5 ml of 22 mM sodium barbital at pH 8.3, 1 ml of 002 saturated water, and 200 ul of cell suspension. All reagents were at A°C. To measure endogenous rates, AZ was added to inhibit any CA activity. 20 TABLE 1 Algae, Source, And Growth Media Algae Netrium digitus var.digitus UTEX 1257 Netrium digitus var.lamellosum UTEX 1256 Mesotaenium caldorium UTEX 283 Gonium pectorale Muller UTEX L3826 Chlorococcum minutum Starr UTEX 1259 Ulothrix fimbriata Bold UTEX LB638 Chlamydomonas moewusii Duke CC55 Euglena gracilis Klebs UTEX LB753 Spirogyra varians UTEX LBA79 Chlorella vulgaris 11h Mougeotia s3 UTEX LB758 Chlamydomonas reinhardtii 9O UTEX 21 datt-Fogg + 5} soil extract (22) watt-Fogg + 5% soil extract watt-Fogg + 5% soil extract Minimal (“9) Minimal Watt-Fogg + 5% soil extract Minimal Minimal + vitamins 81 and B12 DyIII (2A) Minimal DyIII Minimal 22 RESULTS Enzyme Survey: The presence of either glycolate dehydrogenase or glyco- late oxidase was determined by the differences in sensitivity to cyanide and D or L-lactate substrate specificity. Glycolate dehydrogenase is inhibited by 2 mM cyanide and oxidizes D-lactate but not L-lactate. Glycolate oxidase is not inhibited by cyanide and oxidizes L-lactate but not D-lactate. DCPIP was used as the electron acceptor in the reaction since the natural acceptor for glycolate dehydrogenase is unknown. Cells grown on air were compared to cells grown on high 002 to see whether any cnange occurred in the type of enzyme. The results of the enzyme survey are shown in Table 2. One group of the algae studied had glycolate dehydrogenase. Besides glycolate they oxidized D-lactate with activities ranging from 27-68 1 that of glycolate. Chlorococcum, however, oxidized D-lactate with a rate 16Ai higher than glycolate. Specific activities in the cell extracts with glycolate ranged from 2.2-167 nmoles/hr/mg protein. No activity with L-lactate was found for any of these algae. Cyanide inhibited the dehydrogenase activities in these extracts. Mesotaenium and two species of Netrium contained glycolate oxidase, since 100% of the activity was retained in the presence of 2 mM cyanide and L-lactate was also oxidized. L-lactate rates were 22-83% that of glycolate as previously observed for peroxisomal glycolate oxidase. However, D-lactate was also oxidized with rates 31-58% that of glycolate, which is not observed with enzymes from C3 plants. Specific activities with glycolate were 1.2-58.0 nmoles/hr/mg protein. 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