. Mr 4v ”“5".35‘52 .," 1.41%":3‘1111 . «3' 5‘ “$131333 "th31, . '. ' wé . "3 r fig“ “2' 1* . 11' ’1‘“: .41" ‘13? 33 3.12.1; .M‘w. '33:!31459. gig.“ ti 1.; 2:4 1;: H, ”('33: 1631‘." r! if; 11"}. W11: .2 11w; 2; yéélflugihj. 1 11:1: :»*<~.a.r* '_ ”LL-.9 S A A "‘1 W 21!“:gfitflt. “"5131: K» . (Iii/1113,! “fly, ... ‘ -,1,1,1Ir1¢‘,!. 2" i :24; if.) '6‘“ '. ' w$§§ LL51 m,’ - _, an: ‘1. 1' 1’ can * n 4!: q q ' \n y. . 1r}: '.. :13“: [:0 ““343“,": ‘- . 1.1%. . '5' J 3“ ‘ " M 7:34, ‘1'“:fiE‘1E! '1‘ :3 ‘11“"“}‘:.. S». ”-15-.” 4'. 15;.) ’ I «i ‘ng u' 3“ ,u 3‘ 1k: ’11", {3'1“ 2.1, . u» '2‘ «'1' mm 1131. “'51st 1 "‘1; par” 3 .1"; . :1‘ ‘11:, . 'wrg 3' “11:1 .5: , ' . 21“.; mfg". ".31, my: 3 ”31:“; "2:33” .~ 3“ 3?? f "5 #13th 31:25: “until J‘.“"*' ‘~ firm» " ”13% m? x: " s 'I\’~ ”“3 1km. 2 :‘E "L‘ flaw-47.6. ‘ .. 3541:3353; ‘5": 'a‘32fiih13§1‘1““"' "V. Vim a,» ,3” awlr we m: :1“. .‘V . ’1‘. It "{5}" 7g *— 33:3? 3 5w 3;, ,, , .;, ‘9‘ ‘.’ afiWW' ‘2'"7113 fig? 1* “st"? ‘ mg." '1. f 93;", my. :3: ‘ 4.... m'x—‘xh . V r&§3‘. "‘- . ’X a ~."1'\ ‘1 .5115"? ~"- ‘ " V ” , , .. Inn; 12 ~45 “4120!“ $1,, .1 w .. 3. .93.: ’ ' ‘2 '3‘? “.3333". ‘x ”3.33.2‘ k 150‘ 2“" «‘99 This is to certify that the dissertation entitled STIMULATION OF GROWTH AND PHOTOSYNTHETIC CARBON METABOLISM IN CHLAMYDOMONAS REINHARDTII WITH TRIACONTANOL presented by Robert L. Houtz has been accepted towards fulfillment of the requirements for Ph.D. Horticulture degree in game Ma; _ 1/ Major professor l 1e . "es Date 2'/'7 8? Stan YK R1 MSU is an Affirmative Anion/Equal Opportunity Institution 0-12771 MSU RETURNING MATERIALS: Place in book drop to LIBRARIES remove this checkout from -3—-_ your record. FINES will be charged if book is returned after the date stamped below. NOV 2 0 2006 11039.6, STIMULATION OF GROWTH AND PHOTOSYNTHETIC CARBON METABOLISM IN CHLAMYDOMONAS REINHARDTII WITH TRIACONTANOL By Robert L. Houtz A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1985 DEDICATION This dissertation is dedicated in memory of Alice w. Price and Harry James Houtz. ii ACKNOWLEDGEMENTS The author gratefully acknowledges the financial support provided by Dr. Stanley Ries, Dr. Ed Tolbert, Dr. Gordon Guyer, and the Department of Horticulture. I would also like to extend my thanks and appreciation to Dr. John Kelly, Dr. David Dilley, Dr. Anton Lang, Dr. Martin Bukovac, Dr. Ed Tolbert, and Dr. Stanley Ries for their patience and guidance during the completion of my graduate program, especially during the last few months. Special thanks goes to professors Ries,.Dilley, Tolbert and Lang. To Dr. Anton Lang for improving my compositional skills; to Dr. Ed Tolbert for teaching me the biochemistry of photosynthesis; to Dr. David Dilley for facilitating my growth as a person as well as a scientist; and to Dr. Stanley Ries for cultivating my creativity. Last, but most, my thanks and dedication go to Pamela K. Houtz, for her love and support which has been instrumental in the completion of this work. ABSTRACT STIMULATION 0F GROWTH AND PHOTOSYNTHETIC CARBON METABOLISM IN CHLAMYDOMONAS REINHARDTII WITH TRIACONTANOL By Robert L. Houtz Treatment of Chlamydomonas reinhardtii Dangeard cells (-, strain N. 90), cultured at 5% C02, with l to 1000 ug/L triacontanol (TRIA) resulted in a 2l% to 35% increase in cell density, 7% to 3l% increase in total chlorophyll, and 20% to 100% increase in photosynthetic C02 asshnilation. The increase'h1photosynthetic.002 assimilation with TRIA treatment occurred before, and was independent of the increase in total chlorophyll or cell number. Chlamydomonas cells responded to a broad range of TRIA concentrations that were at least 10-fold above the optimum concentration for higher plants. The necessity for higher concentrations of TRIA may be due to destabilizing effects of Ca++ and _..._., .__...._‘....... _._., __ ' ”1- v i- “‘ —- H-tu—-—-.‘.—-——-"’ K+ present in the Chlamydomonas growth medium on the TRIA formulation. ~.-.~——,.1+._,_i _. TRIA particles were bound to Chlamydomonas cells treated with colloidally dispersed [l4CJTRIA. Octacosanol inhibited the effect of TRIA on photosynthetic C02 assimilation. TRIA treatment did not alter the distribution of l4C-label among photosynthetic products. The effect of TRIA on photosynthetic C02 assimilation increased with time up to 3 days after treatment. Chlamydomonas cells cultured in low C02 (air) did not respond to TRIA. Transfer of high C02 (5%)tn1ltured cells that had responded to TRIA to a low 002 atmosphere resulted in a loss of the effect of TRIA. The effect of pH on photosynthetic 002 assimilation indicated that the maximum increase by TRIA treated Chlamydomonas cells was between pH 5.0 and 7.0. TRIA did not alter glycolate excretion, the coz compensation point or sensitivity of photosynthetic 002 assimilation to 02 in Chlamydomonas. Kinetic analysis of TRIA-treated cells showed that the increase in photosynthetic €02 assimilation was a result of an increase in the whole-cell apparent Vmax. The activity of RuBP carboxylase/oxygenase was significantly higher in cell lysates from TRIA-treated cells than those from control cells. However, quantification of RuBP carboxylase/oxygenase levels by 14CABP binding did not show increased enzyme levels in TRIA-treated cells. Therefore, there was an increase in the specific activity of RuBP carboxylase/oxygenase extracted from Chlamydomonas cells treated with TRIA. TRIA alone had no effect 13 iiEEQ on the activity of RuBP carboxylase/oxygenase purified from spinach (Spinacia oleracea) leaves or from cell lysates of Chlamydomonas. RuBP levels were significantly higher in TRIA-treated ced'ls at high and low C02. Increased RuBP levels in TRIA-treated Chlamydomonas cells were also observed in the absence of 002 with atmospheres 01‘ N2 and 2l% 02. The increase in photosynthetic 002 assimilation by TRIA-treated Chlamydomonas cells was associated‘with an increase in the specific activity of RuBP carboxylase/oxygenase and RuBP levels. TABLE OF CONTENTS PAGE INTRODUCTION ........................... 1 CHAPTER 1. GROWTH AND PHOTOSYNTHETIC C0 ASSIMILATION IN CHLAMYDOMONAS REINHARDTII A AFFECTED BY TRIACONTONAL ................... 4 ABSTRACT .......................... 4 MATERIALS AND METHODS ................... 7 Algae Culture and Treatment ............... 7 Photosynthetic C02 Assimilation Assays ......... 7 [l4CJTRIA Binding Measurements ............. 8 Flocculation of Colloidally Dispersed TRIA ....... 9 MC-Label Distribution ............... . . 9 Chemicals ............ . ........... 10 Statistical Procedures ................. l0 RESULTS .......................... l0 TRIA Stimulation .................... 10 TRIA Formulation and Dose Response ........... 16 Effect of Culture Age on Photosynthetic C0 Assimilation in Control and TRIA Treated ells ..... 29 Octacosanol Inhibition ................. 29 Effect of TRIA on Photosynthetic 14C02 Fixation Products ........................ 34 Effect of pH and Low C02 on TRIA Stimulated Photosynthetic C02 A551milation ............ 34 DISCUSSION ......................... 41 iv PAGE LITERATURE CITED ...................... 44 CHAPTER 2 CHARACTERISTICS OF PHOTORESPIRATION RuBP LEVELS AND THE SPECIFIC ACTIVITY OF RuBP CARBOXYLASE/OXYGENASE IN CHLAMYDOMONAS REINHARDTII CELLS TREATED WITH TRIACONTANOL .................... 47 ABSTRACT .......................... 48 MATERIALS AND METHODS ................... 49 Algae Culture and Treatment ............... 49 RuBP Carboxylase Extraction and Assay .......... 49 RuBP Carboxylase Specific Activity and Active Site Concentration ..................... 49 RuBP Determinations ................... 50 PEP Carboxylase Activity ................ 5l C02 Compensation Point Determinations .......... 51 Effect of 02 on Photosynthetic C02 Assimilation ..... 52 Chemicals. . .l ..................... 53 Statistical Procedures ................. 53 RESULTS .......................... 54 Characteristics of the Oxidative Photosynthetic Carbon Cycle ......................... 54 Photosynthetic COZ Assimilation Kinetics ........ 55 .lfl.!il£ and IQ 113:9 RuBP Carboxylase Activity ..... 59 RuBP Carboxylase Levels ................. 65 RuBP Levels ...................... 65 Glycolate Levels .................... 68 DISCUSSION ......................... 73 LITERATURE CITED ...................... 79 SUMMARY ............................. 83 BIBLIOGRAPHY ........................... 86 CHAPTER I Table I. II. III. IV. LIST OF TABLES PAGE The Effect of TRIA (lOO ug/L) on Cell Density, Chlorophyll and Photosynthetic C02 Assimilation by High C02 Grown Chlamydomonas Cells ........ 11 Increase in Chlorophyll, Cell Density, and Photosynthetic C02 Assimilation in Chlamydomonas Cells 3 Days After Transfer of an Aliquot of TRIA-treated (100 ug/L) and Control (TAS, l ug/L) Cells to Fresh Growth Medium ..... l2 Dose Response for TRIA and Photosynthetic C02 Assimilation by Chlamydomonas Cells ......... 17 Effect of Flocculated TRIA Dispersions on Photosynthetic C02 Assimilation by Chlamydomonas ................... l8 vi Table VI. VII. CHAPTER 2 Table I. II. III. The Effect of Octacosanol (l00 ug/L) on the Increase in Photosynthetic C02 Assimilation by TRIA-Treated (100 ug/L) Chlamydomonas Cells . . . The Distribution With Time of 14C Incorporated From Photosynthetic 14C02 Assimilation in Control and TRIA-Treated Chlamydomonas Cells ........ The Effect of TRIA on Photosynthetic C02 Assimilation by Chlamydomonas Cells Grown at High or Low C02 ................. C02 Compensation Levels and Photosynthetic ‘COZ Assimilation with 2% and 2l% 02 by Control (TAS, 0.l ug/L) and TRIA-Treated (l0 ug/L) Chlamydomonas Cell Cultured at 5% C02 ........ The Absence of TRIA and TAS Activity on RuBP CarbOxylase from Cell Lysates of Chlamydomonas . . . Activation and Activity of RuBP Carboxylase Purified from Spinach (Spinacia oleracea) Leaves With and Without TAS and TRIA ........ PAGE 33 36 56 6O 6l Table IV. VI. PAGE Photosynthetic C02 Fixation by Intact Cells and the Activity of RuBP Carboxylase and PEP Carboxylase in Lysates from Control (H20) and TRIA-Treated (l mg/L) Chlamydomonas Cells ...... 62 Specific Activity and Level of RuBP Carboxylase in Lysates from Control (TAS, l ug/L) and TRIA- Treated (lOO ug/L) Chlamydomonas Cells ....... 66 Levels of RuBP in Control (TAS, l ug/L) and TRIA- Treated (TOO ug/L) Chlamydomonas Cells . . . . . . . 67 viii CHAPTER I Figure l. LIST OF FIGURES PAGE Effect of TRIA on Cell Density and Photosynthetic 002 Assimilation in Chlamydomonas With Time . . . . . . . . . . . . . . . . . . . . . . . . l4 The Effect of Chlamydomonas Growth Medium on The Particle Size of Colloidally Dispersed [l4ClTRIA ...................... 20 Calcium (El ) and Potassium (I ) Induced Flocculation of Colloidally Dispersed [l4ClTRIA ...................... 22 The Binding of [l4CITRIA to Chlamydomonas Cells ........................ 25 Effect of TRIA Concentration on the Number of TRIA Particles Bound to Chlamydomonas Cells ..... 27 ix Figure CHAPTER 2 Figure 1. PAGE The Effect of TRIA (1 or 100 ug/L) on Photosynthetic C02 Assimilation by Chlamydomonas as Affected by Culture Age ...... 30 Photosynthetic C02 Assimilation in Control (TAS) (.Ol ug/L) and TRIA-Treated (l.0 ug/L) Chlamydomonas Cells Before and After Transfer to Low-C02 (air) . . ................ 37 Photosynthetic C02 Assimilation in Control (TAS, l ug/L) and TRIA-Treated (l00 ug/L) Chlamydomonas Cells ................. 39 Kinetics of Photosynthetic C02 Assimilation by Control (TAS, l ug/L) and TRIA-Trated (l00 ug/L) Chlamydomonas cells ................. 57 RuBP-Carboxylase Activity in Combinations of Extracts From Control (H20) and TRIA-Treated (lOO ug/L)Chlamydomonas Cells ............ 63 Photosynthetic C02 Assimilation and the Levels of RuBP in Control (TAS, l0 ug/L) and TRIA- Treated (l mg/L) Chlamydomonas Cells ........ 69 PAGE Figure 4. RuBP Levels and Glycolate Excretion by Control (TAS, l0 ug/L) and TRIA-Treated (1 mg/L) Chlamydomonas Cells ................. .71 xi LIST OF ABBREVIATIONS Bicine, N,N'-bis(2-hydroxyethyl(glycine) CABP, 2-carboxyarabinitol l,5-bisphosphate CHES, 2-(N-cyclohexylamino)ethanesulfonic acid C1, inorganic carbon (HC03' + C02) CV, coefficient of variation DTT, dithiothreitol EDTA, ethylene diaminetetraacetate HEPES, N-2-hydroxyethylpiperazine—N'-2-ethanesulfonic acid IRGA, infrared gas analyzer . LSD, least significant difference MES, 2-(N-morpholino)ethanesulfonic acid NADH, nicotinamide adenine dinucleotide PEP, phosphoenol pyruvate PGA, 3-phosphoglycerate pi, inorganic orthophosphate POPOP, l,4-bis[2-(5-phenyloxazolyl)Jbenzene PPO, 2,5-diphenyloxazole RuBP, ribulose l,5-bisphosphate RuBP carboxylase, ribulose l,5-bisphosphate carboxylase/oxygenase TAS, sodium tallow alkyl sulfate TRIA, triacontanol [CH3(CH)28CH20HI xii INTRODUCTION During this century there have been considerable increases in crop productivity in the United States. This has not been due to increases in tillable land, but increases in cropping efficiency per hectare (5, ll, l2). The introduction of new cultivars and mechanization have contributed greatly to these increases. However, during the past few decades increases in crop yields per hectare have leveled off (28). This may reflect the limitations of the techniques, and knowledge of plant biology that facilitated those increases. Substantial increases in crop productivity are presently envisioned by many plant scientists because of advances in molecular biology. Although the use of plant growth regulators and hormones for increasing crop yields has not met with much success in the past, there is still interest in discovering and developing yield-enhancing chemicals. Triacontanol (TRIA), a compound with plant growth regulator properties, has potential for increasing the biomass (total dry weight) of plants (25% Although increases in plant biomass do not necessarily translate into increases in harvested yield (l0, ll), there have been significant increases in the yield of several crop species when treated with TRIA (23). However, inconsistencies under field conditions limit the recommendation of TRIA for use in commercial agriculture. 2 Laboratory results with TRIA are more encouraging; corn (Zea mays L.) and rice (Oryza sativa L”) plants respond well to TRIA under controlled environmental conditions, with up to 15 to 20 percent increases in dry weight evident 24 h after application (22, 23). Although rice plants respond to TRIA in the absence of light, the percent increase in dry weight is considerably less than that in the presence of light(l, 24). Therefore, even though TRIA affects some processes in plants that are independent of light, there is also a substantial interaction with light dependent processes. TRIA has been shown to affect the growth oflnanyinlant species from basidiomycetes and blue-green algae to higher plants. A review of the effects of TRIA on plants was recently published (23L TRIA stimulation of dryinatter accumulation in plants has lead several researchers to investigate the effects of TRIA on photosynthesis and photoassimilate partitioning (4, 7, T4). The results suggest that TRIA may affect photoassimilate partioning and the photosynthetic carbon oxidation cycle in plants. These two processes have been thoroughly investigated in plants with the goal of increasing crop productivity. Understanding the potential of TRIA for increasing crop productivity requires a critical evaluation of the effects of TRIA on plant processes fundamental to crop yield. One of these processes is photosynthetic C02 assimilation, initiated by the enzyme RuBP carboxylase/oxygenase. This enzyme is responsible for the annual input of approximately 5&))< l016 9 of atmospheric C02 into the global biomass (29), and is probably the most abundant protein in the world (6). The oxygenase reaction catalyzed by RuBP carboxylase is regarded by many plant scientists as the most important metabolic constraint on 3 plant productivity (TO, l3). The objective of these studies was to investigate the effects of TRIAcniphotosynthetic:C02 assimilation, photorespiration, and factors affecting these processes. Chlamydomonas reinhardtii, a unicellular green algal species, was chosen as the test organism, since it has been shown to respond to TRIA, and possesses a biochemical pathway of photosynthetic C02 assimilation similar to that in higher plants. CHAPTER 1 GROWTH AND PHOTOSYNTHETIC C02 ASSIMILATION IN CHLAMYDOMONAS REINHARDTII AS AFFECTED BY TRIACONTANOL ABSTRACT Treatment of Chlamydomonas reinhardtii cells, cultured at 5% C02, with 1 to 1000 ug/L triacontanol (TRIA) resulted in a 21% to 35% increase in cell density, 7% to 31% increase in total chlorophyll, and 20% to 100% increase in photosynthetic C02 assimilation. The increase in photosynthetic C02 assimilation with TRIA treatment occurred before, and was independent of, increases in total chlorophyll or cell number. Chlamydomonas cells responded to a broad range of TRIA concentrations that were at least lO-fold above the optimum concentration established for higher plants. The necessity for larger concentrations of TRIA may be due to destabilizing effects of Ca++ and K+ present in the Chlamydomonas growth medium on the TRIA formulation. TRIA particles were bound to Chlamydomonas cells treated with colloidally dispersed [14CITRIA. Octacosanol inhibited the effect of TRIA on photosynthetic C02 assimilation. TRIA treatment did not alter the distribution of 14C-label among photosynthetic products. The effect of TRIA on photosynthetic C02 assimilation increased with time up to 3 days after treatment. Chlamydomonas cells cultured in low levels of C02 (air) did not respond to TRIA. Transfer of high C02 (5%) cultured cells that had responded to TRIA to a low C02 atmosphere resulted in a loss of the effect of TRIA. The effect of pH on photosynthetic C02 assimilation indicated that the maximum increase by TRIA treated Chlamydomonas cells occurred between pH 5.0 and 7.0. 6 TRIA is a 30-carbon, straight-chain primary alcohol, possessing plant growth regulator properties discovered in 1977 (19). The effects of TRIA on plant growth, development, and metabolism have been summarized recently (17). Some of the inconsistencies in reproducing the effects of TRIA have been attributed to inadequate formulation and/or inhibition by traces of aliphatic hydrocarbons and phthalate- esters (13, l4, T7, 20). Acceptable procedures for formulation and application of TRIA are now available (14, 20). The most profound effect of TRIA on plants is an increase in dry weight (l0, l3, T4, 18). Therefore, it follows that photosynthetic C02 assimilation may be a factor involved in the response of plants to TRIA. With tomato (Lycopersicon esculentum), and a unicellular green alga (Chlamydomonas reinhardtii), treatment with TRIA resulted in a decrease in the 02 inhibition of photosynthetic C02 assimilation (7, ll). At atmospheric levels'of C02 and 02 the carboxylase'reaction catalyzed by RuBP carboxylase in C-3 plants and Chlamydomonas cells cultured at high-C02, is inhibited by about 15 to 30% by 02 (4, 8, 9), which is similar to the maximum percent increase in dry weight induced by TRIA (17). Therefore, it was postulated that an alleviation of the 02 inhibition of photosynthetic C02 assimilation could be a mechanism of action of TRIA. However, an increase in photosynthetic C02 assimilation in tomato or Chlamydomonas in response to TRIA treatment was not observed (7, ll). The following investigations on the effects of TRIA on Chlamydomonas showed a stimulation of photosynthetic C02 assimilation, and confirmed an increase in cell number, which has been observed with Chlamydomonas, Anacystis nidulans, and Scenedesmus acutus (6, ll) as well as with tissue cultures of several higher plant species (10). MATERIALS AND METHODS Algae Culture and Treatment. Axenic cultures of Chlamydomonas reinhardtii Dangeard, (-) strain (N. 90) from the algal collection at the University of Texas (R.C. Starr), were cultured in 3-L Fernbach flasks containing 1.0 L of growth media (2l) or in 0.25-L Erlenmeyer flasks with 0.1 L of growth medium. The cultures were continuously mixed with a reciprocating shaker and aerated with 50 to 100 ml/min of COZ-enriched air (5% C02) or air alone. PAR from fluorescent lamps .was 100 umol/S'mz, and the temperature was maintained between 21 and 23°C with fans to move room air over the flasks. Cell densities were determined with a hemaCytometer after suspending the cells in 5% glycerol. Starting cell density after inoculation was typically 100 to 500 cel ls/ul. The density. reached approximately 1x104 cells/ul after 3 d growth. Chlorophyll was determined by the method of Arnon (l). TRIA was applied as a sterile aqueous colloidal dispersion (800 to 1000 ug/ml stock concentration) containing sodium tallow alkyl sulfate (TAS) as the dispersive agent, present at 1% of the level of TRIA (l4). TAS alone, or distilled water were used as controls. As reported previously for higher plants (14), TAS lacked biological activity at the concentrations used on Chlamydomonas. Photosynthetic C02 Assimilation Assays. Cells were harvested by centrifugation at 10009, washed once with an equal volume of 50mM Hepes-KOH buffer (pH 7.5), suspended in the same buffer to give a 8 chlorophyll concentration of 10 to 50 ug/ml, and placed on ice. In experiments where the pH was varied the cells were resuspended in 3 mM Hepes-KOH (pH 7i” and later added to a buffer consisting of 20 mM Hepes, 20 mM Ches and 20 mM Mes adjusted to the desired pH with HCl or KOH. Aliquots of resuspended cells were placed in 1.5 cm x.5 cm flat bottom glass vials which were held in a circulating water bath at 25°C. The cell suspension was stirred with a small magnetic stirring bar. Illumination from a light projector was filtered through 6xlcmlof a 0.1% solution of‘ CuSO4 to remove heat and provided 1200 umol/5°m2 of PAR at the top of the cell suspension. After addition of 1 to 10 mM KH14co3 (0.14 to 0.54 uCi/umol) to the cell suspensions, photosynthetic C02 assimilathn1wa5‘hfitiated with light. Aliquots (0.1 to 0.51nl) of the cell suspensions were removed at various intervals and mixed with an equal volume of 2N HCl. These samples were evaporated to dryness at 85°C. ' After cooling, 0J5 ml H20 and 4.5 ml of scintillation cocktail (2 L toluene, l L Triton X-l00, l2 9 PPO, 0.l5 g POPOP) were added for measurement of acid-stable 14C. When photosynthetic C02 assimilation was measured at different pH values, the vials with the cell suspensions were capped with rubber serum stoppers to prevent loss of 14C02. Under these conditions the cell suspensions were illuminated from the side and a water bath was not used. Instead, aliquots of the cell suspensions on ice were removed and acclimated in aiwater bath to room temperature (23°C) for 5 min prior to use. [14C1TRIA Binding Measurements. Centrifugal silicone oil filtration (2) was used to measure binding of [14CJTRIA to Chlamydomonas cells. The incubations were carried out in 400 ul plastic microfuge tubes in 9 the light (100 umol/snnz) at 25°C. The tubes contained from bottom to top 20 ul of 1 M glycine, 65 ul silicone oil (1:1,v/v, Wacker AR 20 and AR 200), and 250 ul cell suspension. Incubations were initiated by the addition of colloidally dispersed [14CJTRIA and terminated by centrifugation for 1 min with an Eppendorf model 5414 centrifuge. After centrifugation the cell pellem was removed by cutting the centrifuge tube with a razor blade. The pellet was placed into a scintillation vial containing 0.51nl of distilled H20, and resuspended with 4c51nl of scintillation cocktail. Flocculation of Colloidally Dispersed TRIA. Flocculation of the TRIA formulation was measured by determining the amount of colloidally' dispersed [14CITRIA that would pass through alliliporelU’ZS filter with an 8 um pore size. Aliquots of CaCl2 or KCl solutions (0.1 to. 1.0 M) were added to colloidally/dispersed [14ClTRIA (450 UT), and after incubation at 25°C for 41nin, the dispersion was filtered and samples (150 ul) were removed from the filtrate for determination of [14CITRIAC'The stability of the TRIA formulation in the presence of the Chlamydomonas growth medium was determined in a similar manner. Colloidally dispersed [14CJTRIA was added to growth media (20 ml) and samples CLZ ml) were removed for filtration and determination of [14CITRIA in the filtrate. 14C--label Distribution. The distribution of 1(J'Calabel incorporated during photosynthetic C02 assimilation was measured by adding aliquots «L5 ml) of cells which were actively assimilating 14C02, to an equal volume of methanol. After centrifugation the supernatant was removed 10° (soluble fraction) and the pellet (insoluble fraction) was resuspended in 200 ul of distilled H20. Both fractions were counted for acid-stable 14C. Measurement of excreted 14C and analysis of 14C-labeled products by 2-dimensional paper chromatography was conducted as previously described (21). Chemicals. Octacosanol, TRIA, TAS, and [15,16-14CITRIA (23.2 uCi/umol) were provided by the Proctor and Gamble Company (Miami Valley Laboratories, Cincinnati, Ohio 45247L The octacosanol and TRIA were provided as aqueous colloidal dispersions (800 to 1000 ug/ml) with approximately 3x1011 particles per ml (05L4L6 um diameter). NaH14C03(40 to 60 uCi/umol) was obtained from New England Nuclear and Wacker AR 20 and AR 200 silicone oils from Wacker Chemie GmBH, Munich, West Germany. Statistical Procedures. All experiments were replicated at least once. Variation among replicates was removed in the analysis of variance as blocks. The null hypothesis, that the treatment variance was equal to the error variance, was tested in each investigation with an F-ratio. When appropriate, an F ratio was also determined for treatment variance with trend analysis or non-orthogonal comparisons. In some tests treatment means were also separated with an LSD value. RESULTS TRIA Stimulation. TRIA treatment resulted in significant increases in cell density, total chlorophyll, and photosynthetic C02 assimilation of Chlamydomonas cultures (Tables I and II). The cell density was not ll Table I. The Effect of TRIA (100 ug/L) on Cell Density, Chlorophyll and Photosynthetic C02 Assimilation by High C02 Grown Chlamydomonas Cells. The total growth medium for control and TRIA-treated cultures was inoculated with Chlamydomonas cells at approximately 575 cells/ul and 0.1 to l.0 L aliquots of inoculated media were added to 3-L Fernbach (”~(L25-L Erlenmeyer flasks respectively. The cells in one flask were treated with TRIA and the cells in the control flask with TAS (1 ug/L) or distilled H20. Cultures were aerated for 3 d with an atmosphere of air supplemented with 5% C02. Photosynthetic C02 assimilation was determined with l0 mM KHl4C03. Each observation is the mean of 7 experiments with duplicate determinations. Photosynthetic Treatment Cell Density Chlorophyll C02 Assimilation cel ls/ul ug/ml pg/cell umol/h'mg chlorophyll x l04 Control 1.13 10.0 0.88 51.5 TRIA 1.37 10.7* 0.78 72.8** *,**F ratio for comparison of treatments was significant at 5% and 1% level respectively; 12 Table 11. Chlorophyl'l, Cel I Density, and Photosynthetic C02 Assimilation in Chlamydomonas Cells 3 Days After Transfer of an Aliquot of TRIA-Treated (100 ug/L) and Control (TAS, 1 ug/L) Cells to Fresh Growth Medium. Aliquots of 3-d-old control (500 ul) and TRIA-treated (400 ul) cultures, that were equivalent in total chlorophyll content, were transferred to fresh growth medium (100 ml) without TRIA and incubated with an atmosphere supplemented with 5% cog. After 3 (1 growth, cell density, chlorophyll and photosynthetic C02 assimilation (l0 mM KHl4C03) were determined as described in Materials and Methods. Each observation is the mean of 3 experiments with duplicate determinations. Photosynthetic Treatment Cel l Density Chlorophyll C02 Assimilation Cells/ul ug/ml pg/cell umol/hing(flilorophyll x 104 Control 1.20 10.5 0.88 40.5 TRIA l.62* 13.8* 0.85 55.8* *F ratio for comparison of treatments was significant at 5% level. 13 higher in the TRIA-treated cultures after growth for 3 d (Table I), but the error variance in these tests was sufficiently high (CV of 37%) to eliminate any statistical significance. The hemacytometric method used to determine cell number probably contributed to this variation. Chlamydomonas cells were present in different states of division (diad, tetrad, octad) and thus the total cell number could not be determined accurately using a hemacytometer. Under these culture conditions Chlamydomonas cells began to enter the stationary phase of growth after about 48 h, when nutrients and other factors may limit the potential for further growth that would be expected with increased photosynthetic CO2 assimilation. When aliquots of 3-d-old TRIA-treated and control cells were transferred to new media, the stimulation of cell growth with TRIA was greater (Table II). The increase in chlorophyll was small (7%) but significant during the first culture (Table I) and, in a manner similar to cell density, became greater after a second culture of the-cells (Table II). The increases in chlorophyll were a result of increased cell number since the amount of chlorophyll per cell did not change (Tables I and II) The largest and earliest effect of TRIA was on photosynthetic 002 assimilation. The increase in photosynthetic C02 assimilation in Chlamydomonas cells treated with TRIA could be measured after 1 h of treatment, before any change in cell density was demonstrable, and was evident on a chlorophyll basis (Fig. 1). After treatment of newly inoculated cultures there was a rapid increase in the rate of photosynthetic C02 assimilation by cells in control and TRIA cultures. However, this increase was larger in TRIA treated cells than in the control. In the majority of the following studies this increase Figure 1. 14 Effect of TRIA on Cell Density and Photosynthetic €02 Assimilation in Chlamydomonas With Time. .After treatment 100 ml samples were removed for determination of cell density (closed symbols) and photosynthetic C02 assimilation with 10 mM KHl4co3 (open symbols) with time in control (TAS, 1 ug/L) (EJ) and TRIA-treated (100 ug/L) (0) cultures. Each observation is the mean of two experiments with duplicate determinations. The F ratio is significant at the 1% level for the interaction of TRIA with linear time. There was no significant difference in cell number between control and TRIA- treated cultures. The LSD shown is for comparison of any two data points for photosynthetic C02 assimilation. 15 omm . oov . 2355?:sz duo ovm 0mm? omw em? owm OWN ow; / \ owm 2.5 OWN 2K 02 (2.: om ifieozo me. {3&1 20:42: .8: T6 in photosynthetic C02 assimilation was used as an indicator of a response to TRIA. TRIA Formulation and Dose Response. Dose response studies showed that the response of Chlamydomonas cells to TRIA leveled off at 10 ug/L without further increases hiphotosynthetic C02 assimilation (Table III). The lowest concentration of TRIA that resulted in an increase in photosynthetic C02 assimilation (10 ug/L), was above the optimum dose of TRIA established for higher plants by two orders of magnitude. The necessity for this relatively large concentration of TRIA led to the investigation of the stability of the TRIA formulation in the presence of the Chlamydomonas growth medium, and the binding of TRIA to Chlamydomonas cells. Laughlin et al. (14) reported that the optimum dose for the TRIA- elicited increase in dry weight in corn (Lea gays) plants, was two orders of magnitude lower when the particle size of the TRIA formulation was decreased. The colloidal dispersion of TRIA used in these experiments was not stable in the presence of the Chlamydomonas growth medium. When TRIA at a concentration of 1~0 mg/L or higher was added to the culture medium, the small particles (mean diameter 0.1 to 0131m0 originally present in the colloidal dispersion aggregated to form larger particles which were visible to theluunded eye. Chlamydomonas cells did not show increased photosynthetic C02 assimilation when treated with flocculated TRIA (Table IV). 'Therefore, it was postulated that the necessity for high concentrations of TRIA, and the ineffectiveness of the concentrations of TRIA greater l7 TabhaIII. Dose Response for TRIA and Photosynthetic C02 Assimilation by Chlamydomonas Cells. Cultures were inoculated, then treated and after 3 d growth, l00 ml samples were removed for determination of photosynthetic C02 assimilation (l0 mM KH14C03). Each observation is the mean of five experiments with duplicate determinations. Photosynthetic Treatment C02 Assimilation Chemical (ug/L) ‘ p umol/h’mg Chlorophyll TAS 10 31.91 TRIA l . 46.0 TRIA TO 48.8 TRIA lOO 48.5 TRIA lOOO 52.4 5% LSD l4.8 1 The F ratio was significant at the 5% level for the linear trend of increasing photosynthetic C02 assimilation with increasing concentration of TRIA. 18 Table IV. Absence of an effect of Flocculated TRIA Dispersions on Photosynthetic C02 Assimilation by Chlamydomonas. TRIA dispersions were added to 11K)nfl of growth media in 0.1 L Erlenmeyer flasks, and after incubation for 6 h at 25°C inoculated with Chlamydomonas. After 2 d growth samples were removed for determination of photosynthetic C02 assimilation (10 mM KHl4C03hr Each observation is the mean of 2 experiments with duplicate determinations. There were no significant differences in photosynthetic C02 assimilation as a result of treatments. Photosynthetic Treatment C02 Assimilation Chemical (ug/L) umol/h°mg Chlorophyll TAS 10 61.6 TRIA- 1 59.8 TRIA 10 61.6 TRIA lOO . 64.3 TRIA 1000 53.0 19 than 10 ug/L at eliciting further significant increases in photosynthetic C02 assimilation, may be a result of an increase in the particle size of the TRIA dispersion upon addition to the Chlamydomonas growth medium. Experiments designed to test this hypothesis showed that the concentration of colloidally'dispersed [14CITRIA capable of passing through an 8 um filter, decreased with time in a logarithmic manner after addition to the Chlamydomonas growth medium (Fig. 2). This increase in particle size was dependent on the starting concentration of TRIA. Half of the colloidally dispersed [14CITRIA in the Chlamydomonas growth medium was present as particles larger than 8 um in diameter after 165 min and after 50 min with 100 ug/L and 1000 ug/L TRIA respectively; With the lower concentration of TRIA (10 ug/L) the percentage of TRIA present as particles larger than 8 um in diameter did not fall below 50% even after 256 min of exposure to the algae growth medium. Although the TRIA dispersions were not completely flocculated by the Chlamydomonas growth media (Fig. 2) apparently the increase in particle size was sufficient to decrease the effectiveness of the formulation (Table IV). Since particle size was not quantitated in these experiments, it is possible that the TRIA remaining as particles less than 8um in diameter (Fig.2n are still considerably larger than the initial particle size “Ll-4L6 um diameter) and are inactive. K+ and Ca++ in the growth medium could be responsible for the instability of the colloidally dispersed TRIA. When colloidally dispersed [14CITRIA was exposed to K+ or Ca++ there was a rapid increase in the particle size (Fig. 3). The slopes of the two regression lines showed that calcium ions were approximately 12 times Figure 2. 20 The Effect of Chlamydomonas Growth Medium on the Particle Size of Colloidally Dispersed [14CJTRIA. [14CITRIA was added to 201nl of Chlamydomonas growth medium at time zero, and samples (l.2 ml) removed after different times for determination of the amount of TRIA capable of passing through a filter with an 8 um pore size. Each observation is the mean :SE of dUplicate determinations. 80 100 60 TRIA(% of zero time) 40 20 l I 1 _ 2T IlO 155 TlME(min) j. 220 L1 275 Figure 3. 22 Calcium ([3 )and Potassium (ll) Induced Floccu lation of Colloidally Dispersed [14CITRIA. Aliquots (0.5ml) of the TRIA dispersion (210 ug/ml for Ca++ and 180 ug/ml for K+) were incubated with CaCl2 or KCl at 25°C for 4 min, filtered, and the [14CJTRIA remaining in the filtrate determined as described in Materials and Methods. Each observation is the mean of three determinations. The range in the SE measurments was 0.001 to 0.140. The r values from linear regression analyses for the loss of TRIA in the filtrate with increasing ionic strength are significant at the 1% level. The slopes (m, ug TRIA/I) of the two lines are shown for comparison of the effectiveness of the two cations. 16 J""'II 23 e—CoCIZ r=~-.998" m=-2275 "‘ <-KC| r=-.996" m=-194 C0.00 . T 0101 0:02 0:03 0.04 ‘ MOLAR IONIC STRENGTH (I) O T .05 24 more effective at initiating this flocculation than potassium ions. The level of these two cations.in the growth medium UL2 mM Ca++ and 29.41nM KT) are within the range of Ca++ and K+ levels.used in these studies to initiate flocculation. This increase in particle size is probably the result of an interaction of cations with the negatively charged surface of the TRIA particles. Since the Chlamydomonas growth medium can initiate flocculation of the colloidally'dispersed TRIA, and Flocculated TRIA fails to elicit an. increase in photosynthetic C02 assimilation, it might seem that Chlamydomonas cells should not respond at all to TRIA. However, when Chlamydomonas cells are treated with [14ClTRIA under conditions favorable for 'flocculation (TJL cells suspended in growth medium) TRIA particles became bound to the cells (Fig. 4). The binding of TRIA particles to Chlamydomonas cells reached saturation in 20 min (Fig. 4) with approximately 62% bound in the first 10 min of incubation. Chlamydomonas cells cultured at low C02 (air) did not bind as much [l4CITRIA as cel Ls cultured at high 002 (5%) (Fig. 4). This difference in ability to bind [l4CITRIA may be related to the absence of an effect of TRIA1000 ug/L TRIA resulted in significant increases in cell density, total chlorophyll, and photosynthetic C02 assimilation. The increase in chlorophyll was dependent upon increases in cel l density since there was no change in the amount of chlorophyll per cell. During the first 3-d culture period cefl I density was not significantly increased, but upon transfer of an aliquot of TRIA-treated cells to fresh medium, a significant increase hi cell density occurred. The earliest and largest response of . Chlamydomonas cells to TRIA was an increase in photosynthetic C02 assimilation which was independent of increases in chlorophyll or cell density. TRIA treatment reduced the decrease in photosynthetic C02 assimilation in Chlamydomonas cells entering stationary phase growth. The increase in photosynthetic C02 assimilation was specific for TRIA and was inhibited by octacosanol. TRIA treatment did not cause a change in the distribution of fixed l4C label between soluble, insoluble, and excreted 14C-products. Chlamydomonas cells cultured at low C02 levels did not respond to TRIA, possibly due to changes in the binding affinity for TRIA and/or decreased adsorption. Transfer of cells grown at high C02 that had responded to TRIA treatment, to a low C02 atmosphere, resulted in a loss of the effect of TRIA after 6 h. The effect ofinion photosynthetic C02 assimilation by control and TRIA-treated Chlamydomonas cells suggests that C02 is the species of inorganic carbon utilized, and that this species is also utilized tn! 42 TRIA treated cells. The col loidal ly dispersed formulations of TRIA, which are probably the best formulations of TRIA available for experimental purposes (l4, 17), were not stable when added to the Chlamydomonas culture medium. The instability is probably the result of an increase in particle size due to an interaction of cations present in the growth medium with the negatively charged surface of the TRIA particles. Calcium ions were more effective than potassium ions at initiating flocculation of TRIA dispersions. Since this increase in particle size and/or flocculation is detrimental to the effects of TRIA on higher plants and Chlamydomonas cells, the presence of contaminating cations in the water used for preparing and diluting colloidally'dispersed TRIA may be a crucial factor in achieving consistent and reproducible results. Although the TRIA dispersions were not stable in Chlamydomonas growth medium Chlamydomonas cells.apparently'bound sufficient TRIA during treatment to elicit a lasting response. The binding is fairly rapid with the majority of [14CJTRIA becoming bound within the first 10 min of treatment. Since the specific activity of the [14CITRIA was low, it was not feasible to conduct the binding experiments within the range of TRIA concentrations and Chlamydomonas cell densities used in the dose- response and other studies. Therefore, it is not possible based on these data to accurately describe the relationship between bound TRIA and the response of Chlamydomonas cells to TRIA. However, the evidence presented suggest that the response of Chlamydomonas cells to TRIA is influenced by the instability of the TRIA formulation in the presence of Chlamydomonas growth medium and the binding of TRIA to the cells. Since these two processes occur simultaneously during treatment of 43 Chlamydomonas cultures with col loidal ly dispersed TRIA, there is in all likelihood competition between the binding of TRIA particles to Chlamydomonas cells and self-aggregation of the particles to form inactive TRIA floccules. The partitioning of TRIA particles between that bound to Chlamydomonas cells and that aggregating to form floccules may determine the magnitude of the response of Chlamydomonas cells to TRIA. 'These physical factors could also influence the binding of TRIA to the roots or shoots of higher plants, and therefore, play an important role in determining the response of higher plants to TRIA. TRIA treatment may affect some process associated with the C02- concentrating mechanisms in Chlamydomonas cells, but the evidence presented is not conclusive. There may be other biological processes involved in the development of a COZ-concentrating mechanism and TRIA may have affected one of these and not the Goa-concentrating mechanism per se. The effect of TRIA on photosynthetic 002 assimilation was greatest in Chlamydomonas cells entering stationary phase growth. Under conditions of high C02, RuBP levels would be expected to play an (important role in determining the maximum rate of photosynthetic C02 assimilation in algae and higher plants. RuBP levels are increased in Chlamydomonas cells by treatment with TRIA, which may explain the increased photosynthetic C02 assimilation observed'hiTRIA- treated cells (12). LITERATURE CITED Arnon DI 1949 Copper enzymes in isolated chloroplast. Poly- phenoloxidase in Beta vulgaris. .Plant Physiol 24:1-15 Badger MR, A Kaplan, JA Berry 1980 Internal inorganic carbon pool of Chlamydomonas reinhardtii. .Plant Physiol 66:407—413 Berry'J, J Boynton, A Kaplan, M Badger 1976 Growth and photo- I synthesis of Chlamydomonas reinhardtii as a function of C02 concentration. Carnegie Inst wash Yearbook 75:423-432 Bowes G, JA Berry 1972 The effect of oxygen on photosynthesis and glycolate excretion in Chlamydomonas reinhardtii. Carnegie Inst wash Yearbook 71:148-158 Coleman JR, JA Berry, RK Togasaki, AR Grossman 1983 Location and identification of carbonic anhydrase in<flnlamydomonas reinhardtii. Carnegie Inst wash Yearbook 82:99—105 Erhard w 1981 Inventor. Process for removing substances from solutions. Fed Rep Germany Offenleg ungsschrift 29 620 AL May 27, 20 pp Int. C12 CIAN 1/1oo Appl. 1979, December 19, 1979 Ericksen AB, G Sellden, D Skogen, S Nilsen 1981 Comparative analysis of the effect of triacontanol on photosynthesis, photorespiration and growth of tomato (C3-plant), and maize (C4-plant). Planta 152:44—49 44 lo. 11. 12. l3. 14. 15. 16. 45 Farquhar GD, S von Caemmerer, JA Berry 1980 A biochemical model of photosynthetic C02 assimilation in leaves of C3 species. Planta 149:78-90 , Gifford RM, JH Thorne, WD Hitz, RT Giaquinta 1984 Crop productivity and photoassimilate partitioning. Science 225:801-808 Hangarter R, SK Ries, P Carlson 1978 Effect of triacontanol. on plant cell cultures _i_rl vita. Plant Physiol 61:855—857 Haugstad M, LK Ulsaker, A Ruppel, S Nilsen 1983 The effect of triacontanol on growth, photosynthesis and photorespiration in Chlamydomonas reinhardtii and Anacystis nidulans. Physiol Plant 58:451-456. Houtz RL, SK Ries, NE Tolbert 1984 The effect of triacontanol on photorespiraton, RuBP levels and the specific activity of RuBP carboxylase/oxygenase in Chlamydomonas reinhardtii. (accompanying manuscript) Jones.J, VF Wert, SK Ries 1979 Specificity of l-triacontanol as a plant growth stimulator and inhibition of its effect by other long-chain compounds. Planta 144:277-292 Laughlin RG, RL Munyon, SK Ries, VF Wert 1983 Growth enhance- ment of plants by femtomole doses of colloidally dispersed triacontanol. Science 219:1219-1221 Lesniak AP, SK Ries 1984 An increase in plasma membrane associated ATPase activity in barley after triacontanol treatment. J Amer Soc Hort Sci 19:580 Moroney'hh, NE Tolbert 1984 Inorganic carbon uptake by Chlamydomonas reinhardtii Plant Physiol (in press) 17. 18. 19. 20. 21. 46 Ries S, R Houtz 1983 Triacontanol as a plant growth regulator. HortScience 18:654—662 Ries SK, VF Wert 1982 Rapid effects of triacontanol _i_n vivo and _i_n vitro. Plant Growth Regulation 1:117-127 Ries SK, VF Wert, CC Sweeley, RA Leavitt 1977 Triacontanol: a new naturally occuring plant growth regulator. Science . 195:1339-1341 Ries SK, VF Wert, JA Biernbaum 1984 Interference of triacon— tanol activity by chemical constituents present in experi- mental and field sprayers. J Amer Soc Hort Sci 109:145-150 Tolbert NE, M Harrison, N Selph 1983 Aminooxyacetate stimulation of glycolate formation and excretion by Chlamydomonas. Plant Physiol 72:1075-1083 CHAPTER 2 THE SPECIFIC ACTIVITY OF RuBP CARBOXYLASE/OXYGENASE, RuBP LEVELS, AND CHARACTERISTICS OF PHOTORESPIRATION IN CHLAMYDOMONAS REINHARDTII CELLS TREATED WITH TRIACONTANOL ABSTRACT Increased photosynthetic C02 assimilation by Chlamydomonas reinhardtii cells treated with TRIA was not due to changes in glycolate excretion, C02 compensation point, or the sensitivity of photosynthetic C02 assimilation to 02. Kinetic analysis of TRIA-treated cells showed that the increase in photosynthetic C02 assimilation was a result of an increase in the whole-cell apparent Vmax. The activity of RuBP carboxylase/oxygenase was higher in cell lysates-from‘TRIA-treated cells than control cells. Quantification of RuBP carboxylase/oxygenase levels by 14CABP binding did not show increased enzyme levels in TRIA- treated cells. Therefore, there was an increase in the specific activity of RuBP carboxylase/oxygenase extracted from Chlamydomonas cells treated with TRIA. TRIA alone had no effect in vitro on the activity of RuBP carboxylase/oxygenase purified from spinach (Spinacia oleracea LJ leaves or in cell lysates from Chlamydomonas. RuBP levels were 50% to 60% higher in cells treated with TRIA at high, and low C02.TRIA.also increased RuBP levels hithe absence of C02 with atmospheres of N2 or N2 with 21% 02. 47 48 Previously it was demonstrated that TRIA stimulated photosynthetic C02 fixation by Chlamydomonas cells cultured at 5% C02 by 20 to 100% (l4). The distribution of 11%: label among the products of photosynthetic 002 fixation and glycolate excretion were not affected by TRIA. Increased photosynthetic C02 fixation was measured 1 h after treatment with TRIA and persisted for 3 d. Transfer of cells cultured at 5% C02, that had responded to TRIA, to a low C02 environment (air), resulted in a loss of the stimulation from TRIA. Control and TRIA-treated cells responded similarly to changes in external pH, but the largest effect of TRIA on photosynthetic C02 assimilation occurred at pH 5 to 7. Several species of algae, including Chlamydomonas, possess a mechanism for. concentrating Ci within the cells when they are cultured at ambient levels of C02 (2,1h.9, ll, 22)- In the same species of -cells this mechanism does not exist when cultured at high C02 (5%). Chlamydomonas cells that possess this C1 pump exhibit three changes in photosynthetic C02 assimilation with respect to cells that do not have this mechanism: (a) photosynthetic C02 assimilation is insensitive to 02; (b) the apparent Km(C02) of the algae cells is substantially decreased, and (c) the apparent Vmax for photosynthetic C02 assimilation occurs at low levels of external C1 (Zs‘4fi Together, these changes contribute to increased photosynthetic C02 assimilation and efficiency at low levels of external C1 by algae cells cultured at low C02 compared to cells cultured at high C02. TRIA-treated Chlamydomonas cells exhibit some properties suggesting that TRIA may effect the Ci accumulation mechanism (l4). Other researchers reported that TRIA treatment reduced the 02 inhibition of photosynthetic C02 49 assimilation in Chlamydomonas cells (10). The object of this research was to investigate the possibility that the Ci accumulation system in Chlamydomonas cells is affected by TRIA treatment. MATERIALS AND METHODS Algae Culture and Treatment. Chlamydomonas reinhardtii Dangeard (-) strain (N.90) were cultured, treated and photosynthetic C02 assimilation assayed as described earlier (14). Chlorophyll was determined by the method of Arnon (l) and glycolate excretion by the Calkins assay with the procedure described previously (27L RuBP Carboxylase Extraction and Assay. Chlamydomonascel ls were harvested by centrifugation at l000g. The cell pellet was washed twice with and resuspended in cold (4°C) 50 mM Bicine-KOH (pH 8.2) buffer, to a final chlorophyll concentration of 50 to 100 ug chlorophyll/ml. The cell suspension was passed twice through a Yeda press (Linca Science Instruments, Tel-Aviv, Israel) with compressed N2 (l0,400 kPaL The crude cell extract or the supernatant solution obtained after centrifugation for 2 min at approximately 20009 with an Eppendorf model 5414 centrifuge, was used as the enzyme source for RuBP carboxylase. The activation and assay of RuBP carboxylase were as described previously'(21). Activation time at 30°C was 30 min and assay incubation time was 15 s. RuBP Carboxylase Specific Activity and Active Site Concentration. The level of RuBP carboxylase in lysates of Chlamydomonas cells was quantified by measuring the binding of [14CJCABP (2l). Aliquots (l to 50 3 ml) of the extract from Chlamydomonas cells were incubated with 5 uM [14CJCABP. After incubation at 30°C for 45 min nonradioactive CABP was added and any exchange with [14CICABP allowed to proceed for l h. The [14CICABP-RUBP carboxylase-complex was precipitated with 20% PEG 4000 containing 20inM MgClZ and the precipitate-collected by centrifugation at 30,0009 for 20 min. The pellet was washed twice with 20% PEG 4000 containing 20 mM MgC12 and resuspended in 0.51nl H20. The 0.51nl sample was mixed with 4.5inl of scintillation cocktail and 14C was determined by liquid scintillation counting. The specific activity of RuBP carboxylase was calculated assuming the enzyme had a molecular weight of 550,000 daltons with 8 catalytic sites per mol, each of which bound one molecule of CABP. RuBP Determinations. RuBP was determined by'a modification of the procedure described previously (l5, l6). Aliquots (430 ul) of Chlamydomonas cells suspended in 50 mM HEPES-KOH (pH 7i” buffer were added to 70 ul of ice cold 70% HCl04 in l.5 ml plastic microfuge tubes on ice. After 20 min the samples were centrifuged for l min with an Eppendorf centrifuge and most of the supernatant (450 ul) was removed. The supernatant samples were placed in l.5 ml microfuge tubes on ice and 50 ul of l M Bicine-KOH (pH 8.2) and 85 ul of IO N KOH were added to each tube. Following incubation fur 5 min with occasional mixing, the tubes were centrifuged to remove the insoluble KClO4 and SANPIGS 0f the supernatants (250 ul, pH 8.0 to 8.2) were removed for determination of RuBP. RuBP was determined by incubating samples with activated RuBP carboxylase purified from spinach (Spinacia oleracea LJ leaves by measuring the incorporation of H14C03‘ into acid-stable 14C. The RuBP 5l assay media contained l00 mM Bicine-KOH (pH 8.0 to 8.2) buffer, 0.2 mM Nag EDTA, 0.5 mM on, 20 mM MgClz, 10 mM KH‘4003 (1.0 uCi/umol) and 50 ug of activated RuBP carboxylase in a final volume of 0.5 ml in serum- stoppered 8 ml glass scintillation vials. After incubation at 30°C for l h the assay was terminated with 200 ul of 2 N HCl and the samples were dried at 80°C. After cooling 0.5 ml of H20 and 4.5 ml of scintillation cocktailwere added and radioactivity determined by liquid scintillation counting. This method of RuBP determination gave reliable results and good recoveries (95%) of RuBP added to Chlamydomonas cell extracts. PEP Carboxylase Activity. The Chlamydomonas cell extracts used for RuBP carboxylase determinations were also used to assay PEP carboxylase activity. Aliquots of the cell extract (20 to 50 ul) were added to assay media in serum stoppered 8 ml' glass scintillation vials held in a water bath at 30°C. The assay medium contained 5 mM PEP, 10 mM KH‘4co3 (1.0 uCi/umol), 1 mM NADH, 2000 units of malic dehydrogenase, l00 mM Bicine-KOH (pH 8.0) buffer, and enzyme in a final volume of 0.5 ml. The reaction was initiated with enzyme and terminated after 30 s with 200 ul of 2 N HCl. Blanks consisted of identical samples minus PEP. The samples were allowed to stand at 25°C for l2 h to facilitate the exchange of 14C02 from the samples with atmospheric C02. After adjusting the volume to 0.5 ml with H20, 4.5 ml of scintillation cocktail were added and 1“a determined. C02 Compensation Point Determinations. Chlamydomonas cells suspended in 50 mM HEPES-KDH (pH 7.5) buffer (50 ml, 20 to 40 ug chlorophyll/ml), were placed in a glass chamber (150 ml) fitted with 52 inlet and outlet ports. The inlet port extended to the bottom of the chamber so that gas entering the chamber bubbled through the cell suspension. The glass chamber was held in a water bath at 21°C and. PAR (600 umol/s’mz) was provided by a metal halide lamp. Air containing C02 (5l ul/L) was circulated with a piston pump (200 ml/min) through the chamber until the level of C02 in the gas exiting the chamber was stable at 5l ul/L (approximately l0 min). The system was closed and the internal atmosphere was circulated (200 ml/min) through a Beckman Model 865 IRGA. The IRGA was calibrated with air containing known levels of C02 such that the output voltage was linear 1" response to 002 levels from 0 to l00 ul/L. After closing the system the C02 compensation point of'the cell suspension was reached in the next 20 to 30 min. Effect of-Oz on Photosynthetic C02 Assimilation. Photosynthetic C02 assimilation by Chlamydomonas cells was measured in an atmosphere of air (2l% 02) and an atmosphere of air diluted 1:9 v/v with N2 (approximately 2% 02). The C02 in the air was removed with a column (2 cm x l8 cm) of Ascarite (Arthur H. Thomas Co., Philadelphia, PA). Chlamydomonas cells suspended in 20 ml of 50 mM HEPES-KOH (pH 7.5) buffer were placed in a water-jacketted (25°C) lollipop tube (75 ml) and the tube was sealed with a rubber serum stopper fitted with two teflon tubes. The inlet tube extended to the bottom of the lollipop tube so that inlet gas bubbled through the Chlamydomonas cell suspension. After sealing the lollipop tube, the cell suspension was flushed with the appropriate atmosphere (l00 ml/min) for 10 min in the light (l000 umol/smz) prior to the initiation of photosynthetic 53 C02 assimilation with 1 mM KH14C03 (l.0 uCi/umol). .After addition of KH14C03 the inlet and outlet tubes were pinched closed and samples were removed (l00 ul) by opening the inlet tube and withdrawing a sample with a 250 ul Hamilton syringe. Acid stable 14C was determined as described previously. Prior to sampling, 100 ul of the same gas that was used to flush the cell suspension was injected into the lollipop tube to maintain constant pressure. Chemicals. RuBP, [2'-]4C]CABP (l.0 uCi/umol) and RuBP carboxylase were prepared by Dr. M. Mulligan as described ‘by (12, 20, l8), respectively, and were available from the laboratory of Professor N. E. Tolbert (Department of Biochemistry, Michigan State University). NaH14CO3 (40 to 60 uCi/umol) was obtained from New England Nuclear. TRIA and TAS were obtained from the Proctor and Gamble Company. Phosphoenol pyruvate (tri-monocyclohexyl—ammonium salt), NADH (disodium salt, grade III),Inalic dehydrogenase (porcine, mitochondrial), and polyethylene glycol (PEG 4000) were obtained from Sigma Chemical Co. (St.Louis, M0 63l78). Statistical Procedures All experiments were replicated. Variation between replicates was removed in the analysis of variance as blocks. The null hypothesis, that the treatment variance was equal to the error variance, was tested in each investigation with an F ratio. When appropriate, an F ratio was also determined for treatment variance with trend analysis or non-orthogonal comparisons. Treatment means were also compared with an LSD value in some tests. RESULTS Characteristics of the Oxidative Photosynthetic Carbon Cycle. Glycolate excretion, C02 compensation point, and the inhibition of photosynthetic C02 assimilation by 02 are all reduced in Chlamydomonas cells cultured under low C02(air) as compared to cells grown at high C02(5%). These observations are consistent with reports showing that air-grown cells concentrate Ci relative to the external medium. Consequently, measurement of the aforementioned parameters, as well as other parameters, can be indicative of the presence or absence of the Ci accumulation system. In these considerations it is necessary to designate the C02 level during growth. Cells grown on air(low C02) d0 not respond to TRIA but have the Ci pump. Cells grown at high C02 levels do not have a C1 pump but show increased photosynthetic C02 assimilation when treated with TRIA (14). The rate of photosynthetic C02 assimilation by control and TRIA- treated high-COZ- grown (Hilamydomonas cells under atmospheres containing 2% and 2l% 02 was measured (Table 1). One milimolar KHC03 was used so that the C02 concentration would be limiting. When the partial pressure of(hgwas reduced, photosynthetic C02 assimilation increased approximately 20% hiboth control and TRIA-treated cells. Regardless of the 02 concentration, TRIA treated cells had higher rates of photosynthetic C02 assimilation. 54 55 The C02 compensation point represents the balance between the reductive photosynthetic carbon cycle and the oxidative photosynthetic carbon cyCIEL Changes in the internal steady-state level of C02 in Chlamydomonas cells will result in changes in the C02_'compensation point. In Chlamydomonas cells grown with air, the presence of the C]- accumulation system results in low-C02 compensation points (5 5 ul/L), since for any given external level of C02 the internal level is higher and the inhibition of RuBP carboxylase by 02 is reduced (2). There was no effect of TRIA on the compensation point of Chlamydomonas cells grown on high C02 (Table I). From these results it appears that TRIA does not affect the sensitivity of photosynthetic C02 assimilation to inhibition by 02 in Chlamydomonas cells cultured at high C02. Thus the TRIA stimulation of photosynthetic C02 assimilation does not appear to be associated with stimulation of the Ci pump. Photosynthetic C02 Assimilation Kinetics. Chlamydomonas cells adapted to low C02 have a whole-cell apparent Km (C02) for photosynthetic C02 assimilation of 3uM, which is below the Km (C02) of 46uM for the isolated RuBP carboxylase enzyme. Cells cultured with high C02 have an apparent photosynthetic Km (C02) approximating that for isolated RuBP carboxylase (4L. Kinetic analysis of control and TRIA-treated high- Cog-grown Chlamydomonas cells showed that the increase in photosynthetic C02 assimilation by'HRIA-treated cells was due to an increase in the whole cell apparent Vmax (Fig. lA, B). There was no change in the apparent Km(002) with TRIA treatment. Therefore, it does not appear that TRIA treatment affects the internal level of Ci in Chlamydomonas, i.e. the C1 pump. 56 Table 1- C02 Compensation Levels and Photosynthetic C02 Assimilation with 2% and 21% 02 by Control (TAS, 0.l ug/L) and TRIA- Treated (lO ug/L) Chlamydomonas cells cultured at 5% C02. Photosynthetic C02 assimilation was determined with 1 mM KH14C03 at pH 7.5. C02'compensation levels were determined with an IRGA as described in Materials and Methods. Each observation is the mean of 2 experiments with duplicate determinations. Photosynthetic C02 Assimilation Treatment 2% 02 2l% 02 % Increase C02 Compensation Level umol/h°mg (2% 02/21% 02)-l ul C02/L chlorophyll Control 54.5 45.5 20 5l.3 TRIA 76.6** 65.4** l7 54.8 ** F ratio for difference between treatments was significant at the l%level. Figure l. 57 Kinetics of Photosynthetic C02 Assimilation by Control (TAS, l ug/L) and TRIA-Treated (l00 ug/L) Chlamydomonas Cells” (A), Photosynthetic C02 asshnilation by control and TRIA-treated Chlamydomonas cells cultured at high-C02 with increasing concentrations of KHC03. (B),Ljneweaver-Burk plot of data in (A). Two-day-old Chlamydomonas cells were assayed for photosynthetic C02 assimilation as described in Materials and Methods. The endogenous level of HC03" in equilibrium with atmospheric C02 in the buffer (50 mM HEPES-KOH pH 7.5) was included in the total HC03‘ concentration. A pKa value of 6.3‘was used for calculating the whole cell Km(C02). The Vmax for control and TRIA- treated cells was 48.3 and lll.4 umol/h‘mg chlorophyll, respectively. The F ratio for the effect of TRIA on Photosynthetic C02 assimilation was significant at the 1% level. Each observation is the mean of 2 experiments with duplicate determinations. 58 2: 8 48 me mm mm. m. mm are. 9 ifieozo 9:. .325 2933 .8: mM HCO __Eaeozo 9:. {.25 20:52... .00..) a... u x t m. 6 7. 4 ‘ = : in \u .. w m. 3 ( ( n n K K 12 B coho . mono - 5.... 85 8.30 59 I Vivo and In Vitro RuBP Carboxylase Activity. ‘TRIA at several concentrations did not affect the carboxylase activity of RuBP carboxylase activity in extracts from Chlamydomonas cells (Table II). When RuBP carboxylase is activated at less than saturating levels of C02, the total activity measured is sensitive to compounds that influence the activation state of the enzyme (l7). TRIA did not influence the activity of RuBP carboxylase hi extracts from Chlamydomonas cells that were activated at 1 mM KH14C03. ”Therefore, it does not appear that TRIA affects the activation reaction of RuBP carboxylase (Table II). A similar experiment with purified RuBP carboxylase from spinach leaves was also negative for TRIA stimulation of activation or activity of the enzyme (Table III). When 3-d-old control and TRIA-treated Chlamydomonas cells were ruptured and RuBP CarboxylaSe assayed before and after activation, TRIA treated cells showed significantly higher rates of RUBP carboxylase activity (Table IV). There were no differences in PEP carboxylase activities in the lysates from control and TRIA-treated Chlamydomonas cells. P The possibility that the extract from TRIA-treated Chlamydomonas cells contained a promotor of RuBP carboxylase activity, possibly synthesized in response to TRIA treatment, was tested by assaying combinations of the extracts from control and TRIA-treated cells (Fig. 2). Total RuBP carboxylase activity increased linearly with increasing levels of the extract from TRIA-treated cells. This indicates that a negative or positive effector'of RuBP carboxylase activity'does not exist in the cell extracts from control or TRIA-treated Chlamydomonas cells, respectively. 60 Table II. The Absence of TRIA and TAS Activity on RuBP Carboxylase from Cell Lysates of Chlamydomonas. The lysate from 3-d-old Chlamydomonas cells were treated with TRIA or TAS. The enzyme was activated with l or 10 mM KH14C03 at 30°C for 30 min, and the activity of RuBP carboxylase was measured with 10 mM KH14C03. Each observation is the mean of 3 experiments with triplicate determinations. There were no significant differences in 14C02 fixation rates as a result of treatments. Activation Level of H14C03' 1 mM 10 mM Treatment l4coz Fixation Rate Chemical ug/L umol/h‘mg chlorophyll None 100.8 195.6 TAS 10 98.7 201.3 TRIA 1 102.5 199.6 TRIA 10 100.7 189.2 TRIA 100 99.8 199.0 TRIA 1000 103.6 201.8 61 Table III. Activation and Activity of RuBP Carboxylase Purified from Spinach Leaves With and Without TAS and TRIA. RuBP carboxylase (50 ug)1~as activated with 1 or 10 mM KH‘4CO3 at 30°C for 30 min and then assayed with 10 mM KH14C03. TRIA or TAS were present during activation. Each observation 'h; the mean of 2 experiments with triplicate determinations. There were no significant differences in 14(:02 fixation rates as a result of treatments. Activation Level of H14C03' 1 mM 10 mM Treatment- 14(:02 Fixation Rate Chemical ug/L umol/min'mg protein None 0.82 1.66 TAS 10 0.82 1.65 TRIA 1 0.83 _ 1.67 TRIA 10 0.82 1.58 TRIA 100 - 0.81 1.59 TRIA 1000 .O.82 1.61 62 Table IV. Photosynthetic C02 Fixation by Intact Cells and the Activity of RuBP Carboxylase and PEP Carboxylase in Lysates from Control (H20) and TRIA-Treated (1 mg/L) Chlamydomonas Cells. High—COZ-grown Chlamydomonas cells (3-d-old)‘were lysed and the lysate was assayed for RuBP carboxylase and PEP carboxylase activity, with 10 mM KH14C03. RuBP carboxylase activity was determined before and after activation. Each observation is the mean of 3 experiments with triplicate determinations. RuBP Carboxylase Treatment Whole Cells Not Activated Activated PEP-Carboxylase 14C02 Fixation Rate (umol/h°mg Chlorophyll) Control 32.3 19.5 277.2 6.9 TRIA 62.3** 29.6** 301.4* 7.5 *,** F ratio for difference between treatments was significant at the 5% and 1% level respectively. Figure 2. 63 RuBP Carboxylase Activity in Combinations of Extracts From Control (H20) and TRIA-Treated (100 ug/L) Chlamydomonas Cells. The lysate from 3d-old control and TRIA—treated cells cultured at high C02 was mixed at varying proportions and RuBP carboxylase activity assayed without prior activation. The r value from linear regression analysis was significant at the 1% level. The rate of photosynthetic C02 assimilation by intact TRIA-treated cells was significantly higher (1% level) than that for control cells. Each observation is the mean of 3 experiments with triplicate determinations. 1‘CO, FIXATION pmol/h omg chlorOphyll 20 [—J l 64 32 l 30 l T 28 1% LSD 26 l 24 l 22 \ V l j I 7 j 100:0 75:25 50:50 25:75 0:100 EXTRACT RATIO (CONTROLzTRlA) _65 RuBP Carboxylase Levels. Increased RuBP carboxylase activity in extracts from TRIA-treated Chlamydomonas cells suggests that either the amount of RuBP carboxylase or its activation potential could be higher in TRIA-treated cells. Binding of [14CICABP to activated RuBP carboxylase showed 1“) significant changes 'hi the active site concentration of RuBP carboxylase in TRIA-treated Chlamydomonas cells (Table V). However, there was an increase in the specific activity of RuBP carboxylase from TRIA-treated cells. This increase in specific activity was substantial (40%), but could not entirely account for the 100% percent increase in photosynthetic C02 assimilation observed with TRIA-treated intact Chlamydomonas cells (Fig. 1). This led to the investigation of other changes in Chlamydomonas cells treated with TRIA that were stimulating photosynthetic C02 assimilation. RuBP Levels. Chlamydomonas cells cultured at 5% C02 should be RuBP- limited with respect to photosynthetic C02 assimilation (7,5D.‘The levels of RuBP in TRIA-treated Chlamydomonas cells at saturating levels (10 mM KHC03, pH 7i” of C02 were 46% to 55% higher than control cells (Tables V, VI). The levels of RuBP in both treatments were below or similar to the active site concentration of RuBP carboxylase. At less than saturating levels of C02 and high light 10t9051t195 RuBP levels would not be expected to influence the rate of photosynthetic C02 assimilation since RuBP carboxylase should be saturated with RuBP. The increase in photosynthetic C02 assimilation by TRIA treated Chlamydomonas cells at less than saturating levels of C02 (Fig. 1) could be a result of increased specific activity of RuBP carboxylase (Table V). However, RuBP levels in TRIA-treated 66 Table V. Specific Activity and Level of RuBP Carboxylase in Lysates from Control (TAS, ‘1 ug/L) and TRIA-Treated (100 ug/L) Chlamydomonas Cells. Lysates from 24Lold control and TRIA-treated Chlamydomonas cells cultured at high C02 were assayed for RuBP carboxylase activity, and enzyme levels determined by [MC] CABP binding. The specific activity was calculated assuming a‘molecular weight for RuBP carboxylase of 550,000 daltons and 8 catalytic sites per mol of enzyme. Each observation is the mean of 3 experiments with triplicate determinations. RuBP Carboxylase Active Site Activity Specific Activity Concentration umol COz/h' umol COZ/mln° Treatment nmol/mg chlorophyll mg chlorophyll mg protein Control 13.4 261.3 1.43 TRIA 12.9 351.8** 2.00** ** F ratio for difference between treatments was significant at the 1% level. 67 Table VI. Levels of RuBP in Control (TAS, 1 ug/L) and TRIA-Treated (100 ug/L) Chlamydomonas Cells. Two or 3-d-old Chlamydomonas cells grown with high-C02 that were actively assimilating C02 (10 mM KH14CO3, pH 7&0 were assayed for RuBP levels as described in Materials and Methods. In each experiment the effect of TRIA on photosynthetic C02 assimilation was significant. Each mean is the average of 2 experiments with duplicate determinations. Treatment Test 1 Test 2 Test 3 RuBP nmol/mg Chlorophyll Control 11.7 2.4 10.3 TRIA 18.2** 3.5** 15.8** ** F ratio' for difference between treatments is significant at the 1% level. 68 Chlamydomonas cells.were 50% higher than control cells.with and without 1 mM KHC03 (Fig. 3). Since the levels of RuBP that were determined before addition of KHC03 were from cells under a N2 atmosphere and high light intensity, these levels may reflect the pool of phosphorylated metabolites in the chloroplast available for RuBP synthesis. Since the increase in RuBP levels with TRIA treatment was maintained under steady state carboxylation conditions, after the addition of KHCO3. the RuBP regeneration rate may also be affected. In a similar experiment, without the addition of KHC03, RuBP levels were again higher in TRIA-treated Chlamydomonas cells (Fig. 4). Overall, the levels of RuBP were lower in Chlamydomonas cells withzuiatmosphere of C02 free air than with 1 mM KHCO3. This was expected since the oxygenase activity of RuBP carboxylase results in a loss of chloroplast metabolites available for RuBP synthesis. The levels of RuBP increased with time in both control and TRIA-treated cells and may have eventually reached higher levels. The increase in the levels of RuBP in TRIA-treated cells after addition of 02 was larger than the increase in control cells. Since these conditions (air minus C02) could facilitate the loss of chloroplast metabolites in Chlamydomonas cells, _the question arises as to the origin of the precursors for the increased RuBP levels in TRIA-treated cells. Glycolate Levels. Chlamydomonas cells excrete glycolate, a product of the oxygenase activity of RuBP carboxylase (26, 27). This probably reflects an inability to metabolize glycolate, because of inadequate glycolate dehydrogenase activity (25). Glycolate formation and excretion may reflect a loss of photosynthetic carbon metabolites from Figure 3. 69 Photosynthetic C02 Assimilation and the Levels of RuBP in Control (TAS, 10 ug/L) and TRIA-Treated (1 mg/L) Chlamydomonas Cells. Chlamydomonas cells cultured at high C02 for 3 d were suspended in 50 mM HEPES—KOH (pH 7.5) buffer and gassed for 15 min with N2 (50 ml/min) in the light (1000 umol/5°m2) prior to the initiation of the experiment. At zero time and at l or 2 min intervals samples were removed for determination of RuBP (aK-) levels. Three min after zero time 1 mM KHC03 was added and the N2 gassing stopped. Photosynthetic C02 assimilation ([3) by another portion was determined with 1 mM KH14CO3 under identical conditions begining with the addition of KH14c03. The F-ratio for the effect of TRIA on RuBP levels is significant at the 1% level. The F ratio for the interaction of TRIA with linear time for 14C02 fixation is significant at the 1% level. Each observation is the mean of 2 experiments with duplicate determinations. 7O Sm TN SN m; o; me .o F b p p _ p 0 A m _l. 8 0.. C 5 H M m m .. 1 w---.h....\\q 4 a . D I L S O i L R 2 T. % N Ill C 8m 8m SN 08 of OS 8 mo __.£ao..o._._o 933:3 20:33 .8: 350.326 mE\_oEfi mmzm TlME(min) Figure 4. 71 RuBP Levels and Glycolate Excretion by Control (TAS, 10 ug/L) and TRIA-Treated (1 mg/L) Chlamydomonas Cells. Initial conditions were the same as described for Figure 3. After gassing with N2 for 15 min, control (<>) and TRIA (ED treated cells were gassed with COz-free air. RuBP (open symbols) and glycolate (closed symbols) determin- ations were as described in Materials and Methods. Photo- synthetic C02 assimilation was measured under identical conditions, except 1 mM KH14CO3 was added instead of C02- free air after termination of_N2 aeration. The F ratio for the interaction of TRIA with cubic time for RuBP levels is significant at the 1% level. There were no significant differences, in glycolate levels. Each observation is the mean of 2 experiments with duplicate determinations. 72 =28.ch SEE: E5850 ONH oofi om ow 0v ON 0 F p p p _ 0 6 D M B 10 / m u. 7. o S . , Tllll 10 4 10 .v 3 2 T m m all... 1111‘)... ._ N 0 ul- R 10 T. 1 N O C A d A . q d d . )xii ONH o: OOH om om or om om DO zfaocofo mE\_oE: mmzm TlME(min) 73 Chlamydomonas chloroplasts. Absolute levels of excreted glycolate were not altered in Chlamydomonas cells treated with TRIA (Fig. 4). However, the levels of glycolate should be interp-eted with caution because they were low, and the initial determinations (10,15, 17 min) were at or near the detection limit of the assay. These low levels of glycolate are not unusual considering the initial absence of 02. The levels of glycolate determined 12, 22, and 32 min after addition of 02 are more reliable and may reflect increased glycolate synthesis by TRIA-treated cells (2.6 vs 1.5 ug glycolate/mimmg chlorophyll). This would be expected since the rate of photosynthetic C02 assimilation 1'11 these same cells was increased by TRIA (96.1 vs 60.5 umol COZ/h°mg chlorophyll, at 10 mM KHCO3) and TRIA does not appear to affect the photorespiratory metabolism of glycolate (Table I) (14). DISCUSSION Treatment of Chlamydomonas cells with TRIA resulted in significant increases in photosynthetic C02 assimilation (14). Previously it was reported that photosynthetic C02 assimilation by TRIA-treated Chlamydomonas cells exhibited reduced sensitivity to inhibition by 02 (10). This could explain the increased photosynthetic C02 assimilation by cells treated with TRIA. However, in this study reduced sensitivity of photosynthetic C02 assimilation to 02, or a lower C02 compensation point by TRIA-treated Chlamydomonas cells was not detected. Kinetic analysis of photosynthetic C02 assimilation by TRIA- treated Chlamydomonas cells supports these observations since the whole cell apparent Km(C02) was not affected. A low apparent Km(C02) for 74 Chlamydomonas cells is associated with the ability to actively accumulate internal inorganic carbon(HC03' + C02) (2). Since TRIA- treated Chlamydomonas cells do not show significant decreases in the apparent Km(C02) over control cells, the inorganic carbon accumulation process is probably not involved in the response of Chlamydomonas cells to TRIA. When TRIA-treated Chlamydomonas cells are placed in an atmosphere low in C02 (air) the increase in photosynthetic C02 assimilation over control cells is lost within 6 h, a period of time similar to that for induction of the inorganic carbon accumulation mechanism (14). This suggests that there may be other changes in Chlamydomonas cells during the development of the inorganic carbon accumulation mechanism, 'hi addition to their aquiring the ability to accumulate inorganic carbon. TRIA may have affected one of these processes. The presence of the C1 accumulation mechanism alone does not necessarily correlate with increased photosynthetic C02 assimilation (2). The activity of RuBP carboxylase assayed with activiting and non activating conditions was higher in extracts from TRIA-treated Chlamydomonas cells. This increase was not a result of increased enzyme levels, but was due to an increase in the specific activity of the enzyme. Variations in the specific activity of RuBP carboxylase in plant species has been postulated to be associated with the presence or absence of a C02 concentrating mechanism (5, 24). The value reported for Chlamydomonas cells (24) cultured at low C02 (air) “17 :_0.18 umol Cozflnhrmg enzyme) is substantially higher than the values determined here for Chlamydomonas cells cultured at 5% C02 (Table V). These results suggest that during the induction of the inorganic carbon 75 accumulation mechanism in Chlamydomonas cells there may be changes in the specific activity of RuBP carboxylase. The loss in the TRIA stimulation of photosynthetic C02 stimulation with high C02 cultured Chlamydomonas cells.upon transfer to low C02 (14) could be due to an increase in the specific activity of RuBP carboxylase in control cells. The increased specific activity of RuBP carboxylase in TRIA-treated Chlamydomonas cells may be due to differences in inactivation of the enzyme during extraction and assay. Although proteolytic digestion of RuBP carboxylase decreases catalytic activity, it does not necessarily decrease the binding of CABP and hence determinations of the level of active RuBP carboxylase (22). Substantial variations in the specific activity of RuBP carboxylase could be a result of differing proteolytic activity in crude extracts of plants. If TRIA treatment reduces proteolytic activity in Chlamydomonas cells this could account for the increased specific activity of RuBP carboxylase observed in these cells. However, TRIA has not been reported to affect the activity'of proteolytic enzymes. Changes in the levels of chloroplast metabolites, many of which can affect RuBP carboxylase activity (17), may also influence the specific activity of the enzyme from Chlamydomonas by maintaining the enzyme in the active ternary complex during extraction.‘ The extract from TRIA-treated Chlamydomonas cells did not influence the RuBP carboxylase activity in extracts from control cells in an anomalous manner. Thus, there does not appear to be a positive effector of RuBP carboxylase activity in the extract from TRIA-treated Chlamydomonas cells. RuBP will also stabilize the RuBP carboxylase enzyme in an active state under conditions of low C02. Since the level of RuBP in Chlamydomonas cells was increased with TRIA, regardless of the C02 76 concentration, perhaps these increased levelssof RuBP resulted in a higher specific activity of extractable RuBP carboxylase. Based on a current model of photosynthesis (7,£D the limiting steps in photosynthesis may vary between either RuBP carboxylase activity or electron-transport capacity (hence RuBP levels), and the level of RuBP should reflect the rate-limiting step. For Chlamydomonas cells cultured at 5% C02 the i situ rate of photosynthetic C02 assimilation should In; limited by the RuBP regeneration rate. TRIA- treated Chlamydomonas cells assayed for photosynthetic C02 assimilation at saturating levels of C02 show 40% to 60% higher levels of RuBP. Therefore, the increased growth of Chlamydomonas cells cultured at 5% C02 (14) may be due to increased levels of RuBP. At less than saturating levels of 002, photosynthetic C02 assimilation is limited by the activity of RuBP carboxylase and not RuBP. Under these conditions, TRIA-treated Chlamydomonas cel'hs would stil I show increased photosynthetic C02 assimilation, since the specific activity of RuBP carboxylase is higher than that in control cellsn However, RuBP levels were also significantly increased in TRIA-treated Chlamydomonas cells at less than saturating levels of C02 where the level of RuBP was in considerable excess of the active site concentration of RuBP- carboxylase. This percent increase in RuBP levels was equal or similar to the percent increase in photosynthetic C02 assimilation with TRIA- treatment. Rather than discount this observation as coincidence, a search for similar observations by other researchers showed that RuBP levels may influence the rate of photosynthetic C02 assimilation even when in excess of the RuBP carboxylase active site concentration (3% Observations on the levels of RuBP and photosynthetic C02 assimilation 77 in bean (Phaseolus vulgaris L.) leaves, suggested that RuBP limitation of photosynthetic C02 assimilation can occur at RuBP levels that are in considerable excess of RuBP carboxylase active site concentrations. The increase in the specific activity of RuBP carboxylase and level of RuBP in TRIA-treated Chlamydomonas cells can explain the increase in photosynthetic C02 assimilation of intact cells over a large range of C02 levels. TRIA treatment may affect photosynthetic electron transport in Chlamydomonas cells, since the rate of RuBP synthesis is dependent on this process. However, the increase in RuBP levels in TRIA-treated Chlamydomonas cells in the absence of C02 also suggests that the level of chloroplast metabolites available for RuBP synthesis may also be increased in these cells. TRIA-treated Chlamydomonas cells may have an increased ability to mobilize carbon reserves within the chloroplast for synthesis of RuBP. In rice (ngza sativa L.) and soybean (Glycine max L.) leaves TRIA increased the activity of starch phosphorylase and decreased levels of starch (13% If TRIA causes changes in higher plants. similar to those described here for Chlamydomonas, these observations may explain the increase in dry weight associated with TRIA treatment. However, photosynthetic C02 assimilation by higher plants is controlled by some mechanisms not present in algae, such as stomatal conductance. Therefore, changes in RuBP levels and specific activity of RuBP carboxylase similar to those observed in Chlamydomonas cells, may not lead to increased photosynthetic C02 assimilation in higher plants treated with TRIA. Furthermore, the increase in dry weight by higher plants treated with TRIA isrunzentirely light dependent (231. Our results with Chlamydomonas cells treated with TRIA may not be the same as the effects of TRIA on higher plants, but Chlamydomonas cells proved 78 to be a valuable tool for studying the biological effects of TRIA on plants. LITERATURE CITED Arnon 01 1949 Copper enzymes in isolated chloroplast. Poly- phenoloxidase in Beta vulgaris. Plant Physiol 24:1-15 Badger, MR, A Kaplan, JA Berry 1980 Internal inorganic carbon pool of Chlamydomonas 'reinhardtii. Plant Physiol 66:407-413 Badger, MR, TD Sharkey, S von Caemmerer 1984 The relationship between steady-state gas exchange of bean leaves and the levels of carbon-reduction-cycle intermediates. Planta 160:305-313 Berry, J, J Boynton, A Kaplan, M Badger 1976 Growth and photosynthesis of Chlamydomonas reinhardtii as a function of C02 concentration. Carnegie Inst Wash Year Book 75:423-432 Berry JA, M Nobs, B Osorio, JD Plamer, J Tepperman, WF Thompson 1983 Genetic control (Hi the kinetic parameters of RuPZ carboxylase: Studies of a C3 and a C4 species and their F1 hybrid. Carnegie Inst Wash Year Book 82:96-99 Evans JR, JR Seemann 1984 Differences between wheat genotypes in specific actixrity of ribulose - l,5-bisphosphate carboxylase and the relationship to photosynthesis. Plant Physiol 74:759- 765 Farquhar GD 1979 Models describing the kinetics of ribulose bisphosphate carboxylase/oxygenase. Arch Biochem Biophy 193:456-468 Farquhar GD, S von Caemmerer, JA Berry 1980 A biochemical model of photosynthetic C02 assimilation in leaves of C3 species. Planta 149:78-90 79 10. ll. 12. 13. 14. 15. l6. 17. 80 Findenegg GR. 1976. Correlations between accessibility of carbonic anhydrase for external substrate and regulation of photosynthetic use of C02 and HCO3" by Scenedesmus obliquas Z Pflanzen physiol 79:428-437 Haugstad M, LK Ulsaker, A Ruppel, S Nilsen 1983 The effect of triacontanol on growth, photosynthesis and photorespiration in Chlamydomonas reinhardtii and Anacystis nidulans. Physiol Plant 58:451-456 Hogetsu D, S Miyachi 1977 Effects of C02 concentration during growth