EFFECTS OF CARBON DIOXIDE ON THE PHYSIOLOGY AND BIOCHEMISTRY OF PHOTOSYNTHESIS IN SOYBEAN BY WILLIAM J. CAMPBELL, JR. A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1986 It is not too much to say that a comparatively sudden increase of carbon dioxide in the air to an extent of but two or three times the present amount, would result in the speedy destruction of nearly all our flowering plants. H. T. Brown and F. Escombe (1902) ACKNOWLEDGMENTS I wish to express my appreciation to Dr. L. H. Allen, Jr., for serving as chairman of my supervisory committee and for his generous support during my graduate study. I sincerely thank Dr. George Bowes for allowing me to spend two and one-half rewarding years in his laboratory as well as for serving on my supervisory committee. The time spent in his laboratory was most beneficial to my education. I would also like to thank Drs. K.J. Boote, J. W. Jones, and T. R. Sinclair, for their time and efforts as supervisory committee members. I wish to thank Dr. Pierce Jones for assistance during several of the experiments and for years of interesting conversations. The assistance of Dr. Klaus Heimburg in deciphering the leaf gas exchange system was indispensable and is gladly acknowledged. In addition, I would like to thank Drs. Gabriel Holbrook and J. C. V. Vu , for their instruction and discussion concerning laboratory techniques. The assistance of Mr. Paul Lane in calibrating the IR gas analyzers and the helpful suggestions of Dr. Julia Reiskind and Mr. William Spencer are gratefully acknowledged. This research was supported in part by USDOE-USDA Interagency Agreement No. DE-AI01-81ER60001 , and funding for the graduate assistantship was provided in part by USDA-ARS and in part by the USDOE-USDA Interagency Agreement. Finally, I would like to thank Susie for her constant encouragement and patience. TABLE OF CONTENTS PAGE ACKNOWLEDGMENTS LIST OF TABLES LIST OF FIGURES KEY TO ABBREVIATIONS ABSTRACT CHAPTER I INTRODUCTION: A REVIEW OF PHOTOSYNTHETIC CARBON ASSIMILATION IN C3 PLANTS Photosynthetic Carbon Reduction Cycle Photorespiratory Carbon Oxidation Cycle RuBP Carboxylase/Oxygenase Experimental Approach , CHAPTER II THE EFFECTS OF SHORT-TERM EXPOSURES TO C0„ ON LEAF PHOTOSYNTHETIC RATE, RuBP CARBOXYLASE ACTIVITY AND RuBP LEVEL Introduction Materials and Methods Results Discussion CHAPTER III RESPONSE OF PHOTOSYNTHETIC BIOCHEMISTRY AND PHYSIOLOGY TO LONG-TERM EXPOSURE TO SUBATMOSPHERIC AND SUPERATMOSPHERIC C00 CONCENTRATIONS t Introduction Materials and Methods Results Discussion CHAPTER IV EFFECTS OF TEMPERATURE ON PHOTOSYNTHESIS AND RuBP CARBOXYLASE AT TOO GROWTH CO CONCENTRATIONS f Introduction Materials and Methods Results Discussion . 2 10 13 18 22 22 28 37 57 66 71 77 106 116 116 119 123 133 PAGE CHAPTER V GENERAL SUMMARY AND CONCLUSIONS 139 APPENDIX A LEAF AND CANOPY PHOTOSYNTHETIC RATE RESPONSES TO LIGHT AT TOO C02 CONCENTRATIONS 143 APPENDIX B EFFECT OF LEAF SAMPLE SIZE ON IN VITRO RuBP CARBOXYLASE ACTIVITY 150 APPENDIX C LINEAR REGRESSION PARAMETERS 1 58 LITERATURE CITED 161 BIOGRAPHICAL SKETCH 181 LIST OF TABLES TABLE 2.1 Effects of growth CCL concentration on leaf characteristics PAGE 38 2.2 Effects of growth C02 concentration on pod weight and total green leaf area per plant 40 2.3 Effects of growth C02 concentration on RuBPCase activity in leaves collected following 1-hour exposures to six different C02 concentrations 52 2.4 Effects of growth CO concentration on RuBP levels in leaves collected following 1-hour exposures to six different C02 concentrations 58 3.1 Effect of growth (XL concentration on SLW, LAI, chlorophyll, and total leaf soluble protein....' 78 3.2 Effect of growth CO concentration on chlorophyll and total leaf soluble protein expressed on a dry weight basis ^ _ _ o0 3.3 Effect of growth C02 concentration on apparent V00?)' Vmax and dlssolved free C00 at the mesophyll cell wall f _ 105 4.1 Effect of growth air temperature on maximum canopy net photosynthetic rates 125 CI Linear regression parameters (for short-term C02 concentrations) for data in Chapter II C2 Linear regression parameters (for growth CO concentration) for data in Chapter III C3 Linear regression parameters (for growth air temperature) for data in Chapter IV 158 159 160 LIST OF FIGURES FIGURE 3.1. PAGE 12 42 1.1. A non-stoichiometric diagram of the PCR cycle in C chloroplasts (after Bassham, 1979) ?., 1.2. A non-stoichiometric diagram of the integration of the PCR and PCO cycles in C chloroplasts (after Lorimer, 1981) f 2.1. Intercellular CC>2 concentration versus ambient C09 concentration for leaves grown at two C09 concentrations . 2.2. Leaf photosynthetic rate versus intercellular CO concentration for leaves grown at 330 \i\ C0o 1 (A) and 660 yl C02 l~l (B) f 44 2.3. Mean leaf photosynthetic rate versus mean intercellular CO concentration for Reaves grown at 330 ul C02 1 and 660 ul C02 1_1 kl 2.4. Leaf RuBPCase activity versus CO concentration for samples collected following 1 hoOr exposures to six different C02 concentrations 50 2.5. Activation status of RuBPCase versus CO concentration for leaves grown at 330 ul CO 1_1 or 660 Ml C02 1 l 2. ...... 54 2.6. Leaf RuBP levels versus C02 concentration in samples collected following 1-hour exposures to six different C02 concentrations # 56 The soluble protein/chlorophyll ratio versus growth C02 concentration ' fi9 3.2. Canopy net photosynthesis (on a land area basis) versus solar irradiance for canopies grown at 6 different C02 concentrations # 84 3.3. Maximum canopy net photosynthetic rate versus growth C09 concentration _ _ 87 3'A' t' IU!I?Se activlty versus growth CO concentration, b. KuBFLase activation versus growth CO concentration 2 FIGURE PAGE 3.5. Levels of RuBP versus growth C02 concentration 92 3.6. RuBPCase activity versus HCO ~ concentration in leaf tissue grown at 160 /il C0„ 1 95 3.7. RuBPCase activity versus HCO ~ concentration in RUDruase activity versus huj„ conct leaf tissue grown at 280 /jl CCL 1 , 3.8. RuBPCase activity versus HCO ~ concentration in leaf tissue grown at 330 yl C0„ 1 3.9. RuBPCase activity versus HCO..- concentration in leaf tissue grown at 660 yl C0„ 1 3.10. RuBPCase activity versus HCO ~ concentration in 4.1. 97 99 101 103 iMiDr^ase acLivity versus nuj„ conct leaf tissue grown at 990 /jl C09 1 , Initial RuBPCase activity versus growth air temperature for 330 and 660 ul CO.-, 1 grown plants 128 4.2. Total RuBPCase activity versus growth air temperature for 330 and 660 ul C0? 1 grown plants 130 4.3. RuBPCase activation (%) versus growth air temperature for plants grown at 330 or 660 Ml C02 rl 132 4.4. Levels of RuBP versus growth air temperature for plants grown at 330 or 660 pi C02 1 135 A.l. Leaf net photosynthesis versus quantum flux density for plants grown and measured at 330 and 660 Ml C02 1 i46 A. 2. Canopy net photosynthesis versus quantum flux density for canopies grown and measured at 330 and 660 Hi C02 1 148 B.l. Initial and total RuBPCase activity versus leaf sample size used in assay 153 B.2. Percent activation of RuBPCase versus leaf sample size used in assay 155 KEY TO ABBREVIATIONS C Stromal concentration of C0„ CA Carbonic anhydrase Ca CO concentration ambient to leaf Ci CO2 concentration in air in leaf intercellular spaces (p.1 1~ ) Ci' Percent of (X^ in air in leaf intercellular spaces (v/v) ^in ^2 concentrati°n °f air entering leaf chamber CQut CO2 concentration of air leaving leaf chamber CO2 Activator C0„ in Rubisco activation DAP' Days after planting DHAP Dihydroxyacetone Phosphate diPGA 1 ,3-diphosphoglycerate DTT Dithiothreitol E Enzyme E4P Erythrose 4-phosphate EDTA Ethylenediaminetetraacetic acid FBP Fructose 1 ,6-bisphosphate F6P Fructose 6-phosphate GAP Glyceraldehyde 3-phosphate Kc Michaelis constant for C0„ ^c . Enzyme turnover number (s ) K Michaelis constant Ko Michaelis constant for 0„ LAI Leaf area index M Metal cation for enzyme activation 0 Stromal concentration of 0„ P Atmospheric pressure PCO Photorespiratory carbon oxidation PCR Photosynthetic carbon reduction PGA 3-phosphoglycerate P-GLY 2-phosphoglycolate Pi Inorganic phosphate pK' First ionization constant Pn Net photosynthetic rate PVP-40 Polyvinylpyrrolidone R5P Ribose 5-phosphate Rleaf Total leaf resistance to water vapor diffusion RuBP Ribulose 1 ,5-bisphosphate Ru5P Ribulose 5-phosphate S7P Sedoheptulose 7-phosphate SBP Sedoheptulose 1 , 7-bisphosphate SLW Specific leaf weight Tris Tris (hydroxymethyl) aminomethane Tris-HCl Hydrochloride of Tris V Standard molar gas volume Vc vmax of carboxylation reaction "° ^max °f oxygenation reaction vc Velocity of carboxylation reaction max Theoretical maximum velocity of enzyme catalyzed reaction v0 Velocity of oxygenation reaction X5P Xylulose 5-phosphate a Solubility coefficient in water Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECTS OF CARBON DIOXIDE ON THE PHYSIOLOGY AND BIOCHEMISTRY OF PHOTOSYNTHESIS IN SOYBEAN By WILLIAM J. CAMPBELL, JR. December 1986 Chairman: L. H. Allen, Jr. Major Department: Agronomy In three consecutive years (1983, 1984, and 1985) soybeans (Glycine max L. Merr. cv Bragg) were grown from seed to maturity in six outdoor environmentally controlled plant growth chambers under natural solar irradiance. The C02 concentrations inside the chambers were controlled to various levels during these studies. Both field and laboratory measurements were made to investigate the effects of CO2 concentration on photosynthesis. Emphasis was placed on the response to C02 of ribulose 1 , 5-bisphosphate (RuBP) and RuBP carboxylase (RuBPCase), the substrate and enzyme of the carbon fixation reaction in soybean. Following growth at 330 (atmospheric concentration) or 660 ul CO2 1 , leaflet photosynthetic rates were always greater for the elevated Od^ grown plants when measured over a wide range of COn concentrations. This enhanced capacity for photosynthesis was possibly a result of changes in internal leaf anatomy, or to greater assimilate demand, or both, in the high CO- grown plants. The RuBP concentration decreased with increasing CCL, but still appeared to be greater than the active site concentration of RuBPCase. The RuBPCase activity, expressed on an area basis, was not affected by growth CCL concentration. It appears that RuBPCase and RuBP are thus not involved significantly in the enhanced photosynthetic capacity. Evaporative cooling kept leaf temperatures from reaching the higher air temperatures during studies on temperature effects on soybean grown at atmospheric and twice atmospheric concentrations of C0„. Although air temperatures were increased by approximately 5 and 10°C, leaf temperatures were usually not increased more than approximately 2.5 and 4.5°C, respectively. These leaf temperature increases were not great enough to affect canopy photosynthesis or RuPBCase activity (on a chlorophyll basis) in either CCL treatment. Canopy photosynthesis was, however, greater at the higher CCL concentration. The concentration of RuBP was reduced at higher temperatures. Increasing growth CCL concentrations (from 160 to 990 jul CCL 1~ ) resulted in decreasing RuBPCase activities and RuBP levels, when both were expressed on a chlorophyll basis. At the higher C0„ concentrations, the concentration of RuBP appeared to approach the concentration of RuBPCase active sites. Both the apparent K (C0?) and V of RuBPCase showed small, but statistically significant, decreases with increasing C0„ . CHAPTER I INTRODUCTION: A REVIEW OF PHOTOSYNTHETIC CARBON ASSIMILATION IN C3 PLANTS Photosynthesis is the process in which green plants and certain bacteria assimilate inorganic carbon into organic compounds. Light is the source of energy for this process and is absorbed in the plant by various pigments. The photochemical reactions involved in absorbing and transferring light energy are referred to as the "light reactions" while reactions responsible for the fixation of inorganic carbon and its subsequent metabolism are often referred to as the "dark reactions." Since several of the enzymes of photosynthetic carbon assimilation are light-activated, the "dark reactions" are not completely independent of light. Under conditions of high quantum flux density, several processes can be identified as being potentially involved in regulation of photosynthetic carbon assimilation. One of the more marked of these processes is the C0„ fixation reaction. Characteristics of this reaction have been used to assign plants to various photosynthetic categories. Terrestrial plants have been divided into four photosynthetic categories based on the path of carbon during photosynthesis, physiological characteristics, and leaf anatomy. In C3 plants the initial product of the carbon fixation reaction is a three-carbon phosphorylated compound, whereas in C, plants it is a four-carbon organic acid. Crassulacean acid metabolism (CAM) is a photosynthetic pathway in which the initial carbon fixation product is a four-carbon compound, however, most of the carbon fixation occurs at night. Characteristics of these three pathways are reviewed by Black (1973). The fourth category, CyC^ intermediates, exhibit physiological and anatomical characteristics intermediate between C and C4 species. Holaday and Chollet (1984) have recently reviewed the photosynthetic characteristics of plants in this category. One of the main objectives of the research described in the following chapters was to investigate the C0o fixation reaction in soybean, a C3 type plant, by examining the enzyme and substrates involved. Prior to discussing specific objectives and the general experimental approach, C02 fixation in C3 type plants is reviewed. This review covers C02 fixation and the subsequent regeneration of the CC^ acceptor, the competitive photorespiratory cycle, and the enzyme responsible for catalyzing the initial reactions in both pathways. Proposed sites of regulation other than the carboxylation reaction are also discussed. Photosynthetic Carbon Reduction Cycle Description of the Cycle The photosynthetic carbon reduction (PCR) cycle (also known as the reductive pentose phosphate or Calvin cycle) is the biochemical pathway in which C02 ±s converted to a number of sugar phosphates including the regeneration of the C02 acceptor ribulose 1,5- bisphosphate (RuBP) (Bassham et al., 1954). This biochemical pathway is apparently present in all photosynthetic green plants (Bassham, 1979). The 13 enzyme-catalyzed reactions of this cycle occur in the chloroplast. These reactions are catalyzed by 11 different enzymes, as it is currently believed that the two aldolase reactions are catalyzed by the same enzyme as are the two transketolase reactions (Robinson and Walker, 1981; Latzko and Kelly, 1979). A non- stoichiometric schematic diagram of the PCR cycle is presented in Figure 1.1. Carbon enters the cycle when C02 is combined with RuBP to produce two three-carbon compounds. This carboxylation reaction is catalyzed by the enzyme RuBP carboxylase (RuBPCase). Carbon passes through the cycle to regenerate the C02 acceptor. At two key points in the cycle carbon compounds may be removed and either utilized in starch synthesis or exported from the chloroplast to be metabolized in the cytosol. Both of these pathways represent net carbon gain for the photosynthetic cell. The ATP and NADPH consumed in the PCR cycle are generated during photosynthetic electron transport, and production of both requires light energy (Arnon et al., 1954). In addition to combining with C02, RuBP can combine with 0 in an oxygenation reaction catalyzed by RuBP oxygenase (Bowes et al., 1971). The carboxylation and oxygenation reactions are catalyzed by the same enzyme RuBP carboxylase/oxygenase (Rubisco), which functions both as a carboxylase and an oxygenase. Further discussion of this enzyme and its regulation is presented later. Fig. 1.1. A non-stoichiometric diagram of the PCR cycle in Co chloroplasts (after Bassham, 1979). Abbreviations: RuBP, ribulose 1 ,5-bisphosphate; PGA, 3-phosphoglycerate; diPGA, 1,3- diphosphoglycerate; GAP, glyceraldehyde 3- phosphate; DHAP, dihydroxyacetone phosphate; FBP, fructose 1,6-bisphosphate; F6P, fructose 6- phosphate; E4P, erythrose 4-phosphate; SBP, sedoheptulose 1 ,7-bisphosphate; S7P, sedoheptulose 7-phosphate; X5P, xylulose 5- phosphate; R5P, ribose 5-phosphate; Ru5P, ribulose 5-phosphate. Sites of potential metabolic regulation are: (1) RuBP carboxylase; (2) GAP dehydrogenase; (3) fructose 1,6- bisphosphatase; (4) sedoheptulose 1,7- bisphosphatase; (5) phosphoribulokinase; (6) pathway for starch synthesis in the chloroplast; (7) phosphate translocator facilitating exchange of certain metabolites between chloroplast and° cytosol. ADP RuBP R5P VATP diPGA Y NADPH (2)V->NADP + GAP * EXPORT TO (6) CYTOSOL VIA PHOSPHATE - — '^ TRANSLOCATOR STARCH SYNTHESIS Regulation of the PCR Cycle Five of the PCR cycle enzymes have been identified as being light-activated. These are RuBPCase, glyceraldehyde 3-phosphate (GAP) dehydrogenase, fructose 1 ,6-bisphosphatase (FBPase), sedoheptulose 1,7-bisphosphatase (SBPase), and phosphoribulokinase (Buchanan, 1980). These enzymes are located at positions 1 through 5, respectively, in Figure 1.1. A number of roles for light in enzyme activation have been proposed for PCR cycle enzymes. The chloroplast stroma becomes more alkaline in the light, as compared to the dark, as a result of proton transport across the thylakoid membranes (Heldt et al., 1973). The change in pH favors carbon assimilation and is sufficient to increase C02 fixation from zero to" high rates (Werden et al., 1975). In exchange for protons moving out of the stroma, Mg + ions act as counter-ions and enter the stroma thus raising the total Mg concentration (Portis and Heldt, 1976). Portis et al. (1977) have shown that the light-dependent changes in stromal Mg concentration can control FBPase and SBPase activity. The activation of RuBPCase in vitro has also been shown to require Mg + (Laing and Christeller, 1976; Lorimer et al., 1976). Other mechanisms of light-activation of PCR cycle enzymes include the f erredoxin/thioredoxin system (Buchanan, 1980) and the light effect mediator (LEM) system (Anderson, 1979a). These two mechanisms are similar in that both use light energy to reduce disulfide (oxidized) containing compounds to the sulfhydryl (reduced) state. In the reduced state they are able to activate certain enzymes. One difference between the two mechanisms is that the ferredoxin/thioredoxin system requires a soluble protein factor whereas the LEM system does not. Very recent evidence from Salvucci et al. (1985) has shown an apparently different chloroplast protein to be involved in the activation of RuBPCase. Activation, while suggested to be catalyzed by the protein, is regulated by the energization status of the thylakoids (Salvucci et al., 1986b) and is thus light-dependent. Light effects on some PCR cycle enzymes can also be mediated by effectors such as ATP and NADPH, both of which are generated in the light. The relative saturation of the adenylate pool with phosphate (i.e., ATP levels relative to ADP and AMP levels) regulates the activity of phosphoribulokinase and 3-phosphoglycerate (PGA) kinase (Pradet and Raymond, 1983). Both of these enzymes catalyze reactions requiring ATP (Figure 1.1). Also related to light are electron transport rates. Dietz et al. (1984) report, however, that even at high light intensity and saturating CCL, electron transport rates do not play a direct role in limiting photosynthetic rates. Five potential control points associated with the PCR cycle have been identified by Anderson (1979b) to be possible regulatory sites. Two of these points are the export of the triose phosphates GAP and dihydroxyacetone phosphate (DHAP) by the phosphate translocator and the pathway from fructose 6-phosphate (F6P) to starch. These points are discussed later. The remaining three points are the enzymes RuBPCase, FBPase, and SBPase. Dietz and Heber (1984) found even at high light and C02, FBPase did not limit photosynthesis. Likewise, Latzko and Kelly (1979) report all PCR cycle enzymes have been found to possess activity sufficient to support observed rates of C0o fixation with the exception of SBPase. Knowles (1985) has suggested that transketolase may regulate carbon flow through the PCR cycle by restricting regeneration of RuBP. Evidence from Dietz and Heber (1984) also indicates that at some point during the regeneration of RuBP from F6P and triose phosphate, CC>2 fixation appears to be limited under conditions of high CC"2 and high light intensity. The activity of RuBPCase has been suggested to be a limiting factor in photosynthesis even at high C02 concentrations (Dietz and Heber, 1984). Individual reaction rates may be influenced by the accumulation or depletion of reaction products and substrates. Some enzymes are also affected by other chloroplast metabolites. These may be modulated in a positive or negative manner by the binding of a positive or negative allosteric effector at a site on the enzyme distinct from the catalytically active site (Robinson and Walker, 1981). Triose Phosphate Export and Starch Synthesis Export of triose phosphate from the chloroplast via the phosphate translocator and the synthesis of starch are processes which utilize fixed carbon from the PCR cycle (Figure 1.1). The phosphate translocator is the most powerful of several transport systems facilitating exchange between the chloroplast and the cytosol (Heber and Heldt, 1981). It is located at the inner chloroplast envelope membrane and is capable of transporting triose phosphates (DHAP and GAP), PGA, and inorganic phosphate (Pi) (Flugge and Heldt, 1984). The stoichiometry of the transporter is such that export of one molecule of triose phosphate or import of one molecule of PGA is accompanied by the counter transport of one Pi (Heber and Heldt, 1981). Thus, the total amount of phosphate in the stroma is kept constant. The export of triose phosphate is the mechanism whereby carbon fixed in the chloroplast can be transported to the cytosol, where it is metabolized and subsequently translocated to other locations within the plant. The availability of cytosolic Pi to be transported into the chloroplast can affect photosynthesis and starch metabolism (Walker and Sivak, 1986). Triose phosphate can be metabolized to sucrose and Pi in the cytosol, with Pi becoming available for transport back into the chloroplast in exchange for additional triose phosphate. Low rates of sucrose synthesis during photosynthesis may result in decreased cytosolic Pi for transport and therefore a build-up of some PCR intermediates in the chloroplast (Huber and Israel, 1982). Inorganic phosphate is ultimately required for formation of chloroplastic sugar phosphates. When the rate of C02 fixation is greater than the availability of cytosolic Pi for chloroplast import, triose phosphates will not be formed and PGA may accumulate (Heber and Heldt, 1981). The subsequent high PGA/Pi ratio in the chloroplast has been shown to result in starch synthesis (Preiss, 1982). Starch synthesized in the chloroplast is usually degraded during the following night period (Heber and Heldt, 1981). The possibility that photosynthesis may be limited by (1) the inability of the plant to use sucrose at a rate similar to the rate at which it is produced, or 10 (2) accumulation of starch in the chloroplast, is discussed in Chapter II. Photorespiratory Carbon Oxidation Cycle Photorespiration may be defined as the oxygen and light-dependent release of C02 from certain plants (Somerville and Ogren, 1982). The rate of photorespiration is often greater than the rate of dark respiration (Zelitch, 1971). Summarized briefly, RuBP combines with 02 to produce 2-phosphoglycolate (P-GLY) and PGA in a reaction catalyzed by Rubisco, the same enzyme responsible for catalyzing C0„ fixation in the PCR cycle (Bowes et al., 1971; Ogren and Bowes, 1971). The P-GLY produced in the photorespiratory carbon oxidation (PCO) cycle undergoes a series of reactions in the chloroplast, peroxisome and the mitochondrion where photorespiratory C0„ is released (Ogren, 1984; Chollet and Ogren, 1975). The ratio of oxygenase to carboxylase activity is dependent on the relative concentrations of 0„ and C09, Rubisco kinetics (Laing et al., 1974) and temperature. Temperature affects both the kinetics of Rubisco (Jordan and Ogren, 1984) and the relative solubilities of 02 and C02 "(Jordan and Ogren, 1984; Ku and Edwards, 1977). Figure 1.2 shows a non-stoichiometric schematic diagram demonstrating the integration of the PCR and PCO cycles by the common enzyme Rubisco and the common substrate RuBP. Besides CO- NHo is also released in the PCO cycle. Keys et al. (1978) have shown that NH3, like C02, is released in the mitochondrion during the conversion of glycine to serine and NH3 ±s then reassimilated into glutamine in the cytosol. Fig. 1.2. A non-stoichiometric diagram of the integration of the PCR and PCO cycles in C chloroplasts (after Lorimer, 1981). The initial reaction in both cycles is catalyzed by Rubisco and utilizes RuBP. Triose phosphate represents GAP and DHAP. P-GLY is 2-phosphoglycolate. Other abbreviations are as in Fig. 1.1. 12 Ru5P 13 Because the PCO cycle results in a loss of CCL and energy it is often regarded as a wasteful process. Much research has been aimed at understanding photorespiration. Although various roles have been proposed for the PCO cycle, it appears that other than the subsequent metabolism of any P-GLY produced during RuBP oxygenase activity, there is no known requirement for photorespiration (Ogren, 1984). It has been suggested that photorespiration is an unavoidable result of both the nature of the Rubisco active site chemistry and the concentrations of C02 and 02 at the active site (Andrews and Lorimer, 1978). Mutants of Arabidopsis lacking activity of different PCO cycle enzymes have been found to have inhibited photosynthesis in air and are not viable (Somerville, 1986). However, under conditions of high C0? or low 0„ normal photosynthesis was observed. This led Somerville and Ogren (1982) to the conclusion that once carbon enters the PCO cycle it must continue to be metabolized to prevent photosynthetic inhibition. Thus, apparently the only way photorespiration can successfully be reduced is by reducing the oxygenase/carboxylase activity ratio. RuBP Carboxylase/Oxygenase Introduction Under saturating light conditions, the amount and degree of activation of RuBPCase regulates C02 assimilation (Jensen and Bahr, 1977). This emphasizes the importance of Rubisco (used here Rubisco refers to the enzyme RuBP carboxylase/oxygenase while RuBPCase and RuBP oxygenase refer to the carboxylation and oxygenation activities, respectively). This enzyme represents up to 65% of the total soluble 14 leaf protein (Ellis, 1979). It is located in the chloroplast stroma in concentrations of approximately 0.4 to 0.5 mM (Jensen and Bahr, 1977). In higher plants the enzyme is composed of eight large subunits (containing one active binding site per large subunit) and eight small subunits whose function is not yet known (Miziorko and Lorimer, 1983). Thus, the binding site concentration in the chloroplast is approximately 3 to 4 mM. The prodigious amount of this enzyme is countered by its slow rate of catalysis. The turnover number of fumarase (a tricarboxylic acid cycle enzyme) is 50 times greater than spinach RuBPCase (Seemann and Berry, 1982). Compared to spinach carbonic anhydrase (Pocker and Miksch, 1978) spinach RuBPCase is four orders of magnitude slower. Because of its central role in CO2 assimilation and agricultural productivity, Rubisco has been previously and is currently the object of intense investigation. Reactions of Rubisco The two competitive reactions catalyzed by Rubisco are the carboxylation and the oxygenation of RuBP (Bowes et al., 1971; Ogren and Bowes, 1971). As previously described, oxygenation of RuBP is the initial step in photorespiration while carboxylation of RuBP initiates photosynthesis. The ratio of photosynthesis to photorespiration can be described in terms of enzyme kinetics by the equation of Laing et al. (1974), Vc/vo = VcKoC/VoKcO, [1.1] where v^ and vq are the rates of carboxylation and oxygenation, and Vc, Vo, Kc and Ko are the V (theoretical maximum rate of reaction) 15 anC* ^m (Wichaelis constant) values for car boxy lation and oxygenation, respectively. The concentration of CO- and 02 at the reaction site are represented by C and 0. At atmospheric conditions of CCL and 09 and 25°C, the ratio of carboxylation/oxygenation is approximately 4/1 (Ogren, 1984). In spite of much research to identify factors which can alter the vc/vq ratio, only the substitution of Mn2+ for Mg2+ during the enzyme reaction and temperature have proven effective (Ellis, 1979). The K (C^) is decreased when activation and catalysis involves Mn + rather than Mg + (Lorimer, 1981). Temperature has been found to differentially affect Rubisco kinetics. This was shown using the substrate specificity factor defined by Jordan and Ogren (1984), VcKo/VoKc, [1.2] where the variables are defined as in equation [1.1]. At given concentrations of C02 and CL the specificity factor determines the relative rates of carboxylation and oxygenation. A high value indicates a high carboxylase to oxygenase ratio. As temperature increases Vc, Vo, and Kc increase, however, Ko is not temperature dependent. The overall effect of the temperature increase is a decrease in the specificity factor (Jordan and Ogren, 1984). Jordan and Ogren (1984) found the specificity factor of purified enzyme to drop to less than one-third of its value as the temperature increased from 5 to 40°C. A similar response was observed by Brooks and Farquhar (1985) using gas exchange techniques on intact leaves. 16 Activation of RuBP Carboxylase Prior to becoming catalytically competent, RuBPCase undergoes an activation process. The proposed model for activation involves CCL and Mg + in the following manner (Lorimer et al., 1976; Laing and Christeller, 1976), E + AC02 ^± E - AC02 + M ^± E - AC02 - M, [1.3] (inactive) (inactive) (active) where E is enzyme, AC02 is activator C02 (distinct from substrate C02), and M is a divalent metal cation, usually Mg . The formation of the E - C form (E - AC02) is slow while formation of the E - C - M form (E - C02 - M) is rapid. In intact chloroplasts activation has been shown to depend on light and C02 (Bahr and Jensen, 1978). Activation and catalysis are separate phases in the RuBPCase reaction. Lorimer et al. (1977) have described methods for the activation of the enzyme in vitro. Activation of Rubisco is necessary for both carboxylase and oxygenase activities (Lorimer, 1981). Inhibition of RuBPCase by substrate RuBP (Jordan and Chollet, 1983; Laing and Christeller, 1976) and by HC03~ (Machler and Nosberger, 1980) have been reported in in vitro studies. In the light and in air-CCL levels, RuBPCase (in vivo) is typically activated to a substantial degree (Perchorowicz et al . , 1981). Herein lies an enigma in that conditions believed to exist in the stroma in the light (5 to 10 yM C02, 5 to 10 mM Mg + and pH 8.0) are not sufficient to activate RuBPCase in vitro (Miziorko and Lorimer, 1983). A number of metabolites have been shown to affect RuBPCase activation and/or activity. This group of metabolites has been reported to include 17 NADPH, 6-phosphogluconate, ribose 5-phosphate, 3-phosphoglycerate, fructose 1 ,6-bisphosphate and several other compounds (Jordan et al., 1983; Badger and Lorimer, 1981; Hatch and Jensen, 1980; Lorimer et al., 1978; Chollet and Anderson, 1976; Chu and Bassham, 1975). These effectors were suggested to act at allosteric regulatory sites (Chu and Bassham, 1975) but more recent evidence indicates that the effectors bind competitively at the same active site as does RuBP (Jordan et al., 1983; Badger and Lorimer, 1981; McCurry et al., 1981). It has been suggested that the concentration of these effectors in the stroma (Lorimer et al., 1978) and the magnitude of their induced responses (Akazawa, 1979) are inadequate to be physiologically important in vivo. Somerville et al. (1982) have identified a mutant of Arabidopsis thaliana in which RuBPCase is present in a nonactivatable form in vivo. This implies that a factor necessary for activation is absent in the mutant. Recently, Salvucci et al . (1985) have discovered two polypeptides missing from the same Arabidopsis mutant and have linked these polypeptides to a soluble chloroplast enzyme designated Rubisco activase. These data suggest activase may be involved in light- activation of RuBPCase in vivo and that activation is a catalyzed and not a spontaneous process (Salvucci et al., 1986a). An additional regulatory aspect of light on RuBPCase was discovered independently by Vu et al. (1983), McDermitt et al. (1983), and Ku et al. (1982). They found crude extracts of RuBPCase from leaves collected in the dark to be less catalytically active than from leaves collected in the light. This light/dark modulation has been found in a number of different species from different photosynthetic 18 categories (Vu et al., 1984a). Restoration of catalytic ability by ammonium sulfate fractionation of the crude extract of dark collected leaves indicated the potential involvement of an inhibitory compound (Vu et al., 1984b). Subsequent work by Seemann et al. (1985) and Servaites (1985) have shown the inhibitor to be a phosphorylated compound which binds to the active site of RuBPCase. Berry et al. (1986) have identified the inhibitor as carboxyarabinitol 1-phosphate. Non-Catalytic Roles of Rubisco Due to its high concentration in the chloroplast, Rubisco has been suggested to function as a storage protein (Huffaker and Miller, 1978; Huf faker and Peterson, 1974). It also has been suggested to be a major source of protein for animals for the same reason (Huffaker and Peterson, 1974). Another function, that of a metabolite buffer, has been proposed by Ashton (1982). The ability of compounds such as fructose 1 ,6-bisphosphate (FBP) to bind to Rubisco and the relative concentrations of FBP and Rubisco binding sites imply that greater than 98% of the FBP could theoretically be bound to Rubisco in illuminated chloroplasts (Ashton, 1982). The physiological significance of this effect is apparently speculative. Experimental Approach The CC^ in the atmosphere surrounding a leaf , or other photosynthetic organ, is the source of carbon for terrestrial photosynthesis. Manipulation of the C02 concentration and observation of the resulting photosynthetic responses provide insight into the 19 control and mechanism of C02 fixation. This approach has been carried out by a number of investigators (see reviews by Kimball, 1983; Lemon, 1983; Strain and Cure, 1985), not only to learn more about photosynthesis but to study the effects of CC>2 supply on yield and what effect rising atmospheric levels of C02 might have on vegetation. In the experiments reported in the following chapters, soybeans were grown in outdoor, naturally sunlit, controlled environment chambers, in which CC>2 concentration and dry bulb and dew point temperatures were controlled to pre-selected values. Gas exchange techniques were used to measure leaf and canopy photosynthetic rate response to different C02 concentrations. Leaf tissue samples were collected for analysis of RuBP and RuBPCase, the substrate and enzyme involved in C0„ fixation. The purpose of the experiments described in the following chapters was to examine the effects of C02, both in the short-term and the long-term, on the physiology and biochemistry of photosynthesis in soybean. It was hypothesized that long-term exposure (exposure during growth) to different C02 concentrations could result in a change in the capacity for photosynthesis in soybean. To examine this hypothesis, specific objectives were: (1) to determine the leaflet photosynthetic rate response to CO for soybeans grown at atmospheric and twice-atmospheric CO concentrations , (2) to examine the effects of C02 concentration (during short- and long-term exposures) on RuBP levels, 20 (3) to examine the effects of C02 concentration (during short- and long-term exposures) on RuBPCase activity, (4) to determine the effects of growth in subatmospheric and superatmospheric concentrations of CO on kinetics of RuBPCase, (5) to examine the effects of growth air temperature on RuBP levels and RuBPCase activity, and (6) to determine if either the RuBP level or RuBPCase activity may be limiting to photosynthesis under high quantum flux density and various C02 concentrations. In Chapter II, experiments are described in which soybeans were grown at atmospheric and twice-atmospheric concentrations of C0„ . Short-term exposures (1 hr) to various CCL concentrations allowed leaflet photosynthetic rate response to C02 to be measured as well as RuBP levels and RuBPCase activities. In Chapter III, the effects of growth in various subatmospheric and superatmospheric concentrations of C02 on canopy photosynthetic rates are described. The effects of C02 concentration on levels of RuBP and the activity and kinetics of RuBPCase were also determined. The effects of three different day/night air temperature regimes on canopy photosynthesis, RuBP levels, and RuBPCase activity of soybean grown at atmospheric and twice-atmospheric C02 concentrations were investigated and are discussed in Chapter IV. In Appendix A, the photosynthetic rate response to light for leaflets and canopies is discussed. The effect of leaf tissue sample size on the in_ vitro assay of RuBPCase activity is discussed in Appendix B. Parameters from linear regression analyses are tabulated in Appendix C. 21 The long-range goal of research such as described herein is to reach a greater understanding of the fundamental process of photosynthesis. This knowledge may hopefully contribute to improvements in agricultural productivity. CHAPTER II THE EFFECTS OF SHORT-TERM EXPOSURES TO CO ON LEAF PHOTOSYNTHETIC RATE, RuBP CARBOXYLASE ACTIVITY AND RuBP LEVEL Introduction That present day concentrations of atmospheric CO„ are limiting to photosynthesis in C3 plants is widely recognized (Pearcy and Bjorkman, 1983). It is well documented that photosynthetic rates increase when C^ plants are exposed to higher than normal CO- concentrations (Tolbert and Zelitch, 1983; Osmond et al., 1980; Allen, 1979). The increase in CO^ not only provides more substrate for carbon assimilation, but also alters the photosynthetic/ photorespiration ratio by reducing photorespiration (Ogren, 1984). Investigations into the effects of C0„ on photosynthesis have proceeded in several directions including long-term and short-term exposures of plants to various C02 concentrations. Often times long- term exposure involves growing plants from seed to maturity at elevated C02 concentrations. Experiments of this type, in which plants were grown at both atmospheric and elevated C0„ concentrations, have yielded mixed results when leaf photosynthetic rates were measured at the respective growth C02 concentrations. In some experiments, plants grown at atmospheric C0„ had greater photosynthetic rates than high C02 grown plants (Peet et al., 1986; von Caemmerer and Farquhar, 1984; Hofstra and Hesketh , 1975). In other experiments the reverse was found, leaf photosynthetic rates 22 23 were greater in high C02 grown plants when both were measured at their growth C02 concentration (Peet et al., 1986; Havelka et al . , 1984; Huber et al., 1984; Downton et al . , 1980; Wong, 1979; Ho, 1977). In other experiments in which various CL species were grown either from seed or for long periods of time at different CO concentrations, photosynthesis was measured over a range of (XL concentrations. Results from these experiments suggest leaf photosynthetic rate responses appear to fit into one of three categories. These categories may in fact represent a continuum of possible responses that depend on species, growth conditions, stage of growth, and other factors, including experimental conditions. These categories may be described as follows: (1) leaf photosynthetic rates are greater in plants grown at higher rather than lower C0„ when measured at all C02 concentrations, (2) leaf photosynthetic rates are greater in plants grown at lower rather than higher (XL when measured at all C02 concentrations, and (3) leaf photosynthetic rates are greater in plants grown at lower C02 when measured at low C0? but higher in plants grown in high C02 when measured at high C09. Hicklenton and Jolliffe (1980a), working with young tomato plants, found leaf photosynthetic rates, on a fresh weight basis, to be greater in plants grown at 1000 u\ (XL 1_1 than those grown at 300 ul C0o l-1 * 2 when measured over a range of C02 concentrations. With older plants the difference in photosynthetic rate response of the leaves was less. Plants grown at 5000 yl C02 T1, however, were always found to have leaf photosynthetic rates lower than 300 yl (XL 1_1 grown tomato plants. Mauney et al. (1979) grew soybeans at 330 and 630 yl (XL l_1 24 and found that when leaf photosynthetic rates were measured at the lower C02 concentration the rates were the same but at high C02 concentration the rates were greater in the 630 ul (XL l-1 grown plants. The majority of the data in the literature shows leaf photosynthetic rates, when expressed on an area basis, to be greater in plants grown at lower rather than higher C02 when measured over a range of 2 concentrations. This type of relationship has been reported for experiments run under a variety of environmental conditions with various species such as cotton (Delucia et al., 1985; Mauney et al., 1979), Phaseolus vulgaris (Ehret and Jolliffe, 1985; von Caemmerer and Farquhar, 1984), sunflower (Mauney et al., 1979), tomato (Ho, 1977), Nerium oleander and Larrea divaricata (Downton et al., 1980), and waterhyacinth (Spencer and Bowes, 1986). Plants in which the relative rates of leaf photosynthesis shift between low and high C02 grown plants, depending on the (XL concentration during measurement, make up the third response category. Examples of this type of response have been reported with cotton (Wong, 1979), grape (Kriedemann et al., 1976), and Amorphophallus konjac (Imai and Coleman, 1983). The different responses to C02 may be explained in part by the species chosen. However, the species alone cannot account for all of the variation in photosynthetic rates since some species demonstrated more than one type of response. For example, Ho (1977) and Hicklenton and Jolliffe (1980a) both worked with tomato but observed different types of photosynthetic behavior. Vhile their experiments were similar in regard to C02 concentrations, differences existed in plant 25 age, growth photoperiod, growth temperature, and whether the plants were grown from seed or were transferred to a particular CCL concentration at an early age. In addition, Mauney et al. (1979) obtained different results working with the same species under apparently similar experimental conditions in two consecutive years. Measurement protocol as well as growth and measurement conditions, and possibly other factors, apparently influence leaf photosynthetic rate response to CCL (Woo and Wong, 1983). There are numerous reports where long-term growth in high CCL resulted in declining leaf photosynthetic rates, which ultimately became lower than rates of plants maintained at atmospheric CCL concentrations (Kramer, 1981). This reduction in photosynthetic rate has sometimes been shown to be reversible when plants are switched from high to low C02 conditions (Sasek et al., 1985; Kriedemann and Wong, 1984). Sasek et al. (1985) suggest that feedback inhibition of photosynthesis by starch accumulation is responsible for these types of observations, but according to Raven (1981), there is little evidence for feedback inhibition of photosynthetic rates by photosynthetic product accumulation. Growing plants in air enriched with CC^ has often been shown to increase the amount of starch present in the leaf (Cave et al . , 1981; Mauney et al., 1979; Hofstra and Hesketh, 1975; Madsen, 1968). These high starch levels have sometimes been linked to chloroplast disruption (Cave et al . , 1981; Carmi and Shomer, 1979). Neales and Incoll (1968) have reviewed reports that suggest chloroplast disruption may include reduction of light incident to the grana and interference with CCL. diffusion inside the leaf. The 26 relationship between high starch levels and changes in leaf photosynthetic rates is equivocal. There are a number of examples where high levels of starch have been correlated to reduced photosynthetic rates (Delucia et al., 1985; Sasek et al., 1985; Azcon- Bieto, 1983; Mauney et al., 1979; Nafziger and Koller, 1976; Hofstra and Hesketh, 1975), and a number of examples where starch was not observed to affect photosynthetic rates (Potter and Breen, 1980; Carmi and Shomer, 1979; Mauney et al., 1979; Little and Loach, 1973). In fact, Mauney et al. (1979) and Little and Loach (1973) showed positive correlations between starch levels and leaf photosynthetic rates. It has been suggested (Milford and Pearman, 1975) that starch may not inhibit photosynthesis until a threshold level, which is not normally attained under field conditions, is reached. Accumulation of starch in the leaf may be related to, among other things, the assimilate demand of the plant. The role of assimilate demand in leaf photosynthesis has been reviewed by Neales and Incoll (1968) and Geiger (1976). The possible mechanisms involved have been discussed by Herold (1980). Most of the data in the literature suggest high assimilate demand results in high photosynthetic rates (Geiger, 1976). King et al . (1967), however, have reviewed several reports showing both positive and negative influences on photosynthetic rates. Positive correlations between leaf photosynthetic rates and increased assimilate demand have been demonstrated in a variety of depodding and leaf shading experiments (Wittenbach , 1983; Clough et al., 1981; Mondal et al., 1978; Thorne and Koller, 1974; King et al., 1967). 27 In addition to the above mentioned effects on photosynthesis, age or the developmental stage of a plant may influence C09 assimilation rates. The podfilling stage in soybeans can be a period of high photosynthetic activity (Enos et al. , 1982; Hesketh et al., 1981; Woodward and Rawson, 1978; Dornhoff and Shibles, 1970), however, Sinclair (1980) has pointed out that there are substantial differences, among cultivars, in the ability to maintain high photosynthetic rates late in the season. Differences in RuBPCase activity in soybean have been noted between expanding and mature leaves (Vu et al., 1983). Changes in the relative photosynthetic rate responses to COj in atmospheric concentrations and high C0? grown leaves have been shown to occur as plants become older (Peet et al., 1986; Ehret and Jolliffe, 1985; Hicklenton and Jolliffe, 1980a). Baysdorfer and Bassham (1985) have found that as alfalfa progressed from seedling to mature crop, photosynthesis shifted from being source-limited to sink-limited. Different leaf photosynthetic rate responses to C0„ have been obtained with a variety of species and under wide ranging environmental and experimental conditions, which may account for much of the variation in results. Additionally, the diversity of interpretation of the results implies that regulation of photosynthesis is not, as yet, well understood. A confounding possibility is the suggestion (Maggs, 1964) that leaves usually operate below their maximum level. The objectives of this study were to measure leaflet photosynthetic rate response to C02 for soybean grown at atmospheric 28 and twice atmospheric concentrations of C0„ . In addition, the effects of the two C02 growth treatments and short-term response to a range of C02 concentrations on the activity of RuBPCase and the level of RuBP were investigated. The photosynthetic rate response to CO and the response of RuBPCase and RuBP to C02 were examined to determine what role the biochemical parameters may have in regulating leaflet photosynthesis under conditions of high quantum flux density and various concentrations of C0? . Materials and Methods Plant Material and Growth Conditions Soybeans (Glycine max L. Merr. cv Bragg) were planted in outdoor, computer-managed, environmentally controlled plant growth chambers located at the University of Florida's Irrigation Research and Education Park, in Gainesville, on 30 Aug. 1983. The upper part of each growth chamber was constructed of clear acrylic and polyester film, allowing the plants to receive 88% of the natural solar irradiation. The chamber tops measured 2 m by 1 m in cross section by 1.5 m in height. The lower steel part of the chamber was of the same cross section and 1 m in depth. It was filled with a reconstructed Arredondo fine sand profile, which was sealed from the upper aerial part following seedling emergence to prevent the mixing of the soil and aerial atmospheres. The dry bulb temperature of the chamber atmosphere was controlled to 31°C during the day and to 23°C at night. The dewpoint temperature was controlled to 16°C. The CO concentration of the chamber atmosphere was controlled, from the date 29 of planting until final harvest, to either 330 yl C0„ l-1 or 660 Ul C02 * • A general description of growth chamber operation may be found in Jones et al. (1985b), while Jones et al. (1984b) provide a detailed description of the growth chamber design and the computer control system. For the experiments described here, two of six plant growth chambers were used. Within each of these plant growth chambers were placed two leaf chambers, each capable of accommodating one fully expanded soybean leaflet. The leaf chambers were constructed of an acrylic frame covered with a clear polyester film which transmitted 88% of the incident solar radiation. The internal volume of each leaf chamber was 0.375 liters. Chilled water, circulating through the chamber frame, maintained the temperature of the air in the leaf chamber close to the air temperature in the plant chamber. The leaf chambers were controlled by a computer system similar to but separate from the system controlling the plant chambers. The origin of the air circulating through the leaf chamber system was the respective plant chamber. Air was circulated, by diaphragm pumps, from the plant chamber through homogenizing containers and then through the leaf chamber system. The leaf chamber system consisted of two IR gas analyzers (Beckman, model 865), two dewpoint hygrometers (General Eastern, model 1100 DP), one thermocouple (0.25 mm diameter) placed in each leaf chamber to monitor air temperature and three thermocouples (0.076 mm diameter) wired in parallel and placed in contact with the abaxial leaflet surface to monitor leaflet temperature. Each IR gas analyzer and hygrometer was dedicated to two 30 two leaf chambers. Air lines were heated and insulated to help prevent condensation. Air flow rates through the leaf chambers were between 0.318 and 0.468 m per hour (5.3 and 7.8 liters per minute). The dry bulb and dewpoint temperatures and the C0„ concentration in the leaf chambers were similar to conditions in the respective plant chambers. The plants completed germination approximately 4 days after planting (4 DAP). On October 18, 49 DAP, the plants were thinned to a density of 30 plants per m . Throughout the experiments, shadecloth (approximately 50% shading) was attached to the outside of the plant chamber at a height equal to the top of the canopy to approximate a closed canopy and reduce the solar irradiance on the sides of the canopy . C0_2 Concentration Experiments A series of short-term experiments were performed from October 25 to October 30 (56 to 61 DAP), during which time all plants were at the beginning seed or R5 stage of development (Fehr and Caviness, 1977) . During this period the C02 concentrations in the plant chamber, and thus also in the leaf chamber, were controlled to various levels different than the normal C02 growth concentrations. These additional C02 concentrations (110, 220, 330, 550, 660, and 880 yl C02 l-1) were imposed at midday and were maintained for approximately 1 hour. During these exposure times photosynthetic rate data were collected, and immediately following these measurements leaf tissue samples were rapidly collected for subsequent laboratory analysis. Supplementary 31 C02 concentrations (160, 440, and 990 yl C02 1" ) were imposed in the plant and leaf chambers after plant tissue sampling to expand the CO range over which photosynthetic rate measurements were collected. In all cases, when the C02 was changed from one concentration to another, steady state conditions were allowed to return inside the plant and leaf chamber prior to collecting data for analysis. This always represented a period of not less than 10 minutes. All data collected during these C02 experiments were obtained between 1100 and 1430 Eastern Standard Time (EST). During each day this was a cloud-free high irradiance period when the quantum flux density (400 to 700 nm) was measured to be at least 1000 umol quanta m s at the leaf level, which in these experiments was saturating for leaflet photosynthesis. Quantum flux density was measured with a quantum sensor (Li-Cor, model LI-190S) and corrections were made for the transmittance through the plant and leaf chambers. Leaf Photosynthesis Measurements The leaf chamber system was used to collect leaf gas exchange data, at 5-minute intervals, continuously during the photosynthesis experiments. Measurements of C02 concentration and dewpoint temperatures were made on air entering and leaving the leaf chambers. In addition, measurements were made of the dry bulb temperature of the air inside the leaf chambers, the leaflet temperature, and the air flow rate. The net photosynthetic rate (Pn) of the leaflet was calculated using the following equation from Gaastra (1959), 32 C. - C Pn = .J£ 2HI * flow rate, [2.1] where C. and C . are the CCL concentrations of the air stream in out I entering and leaving the leaf chamber, respectively, A is the area of the leaflet, and flow rate is the rate of the air-stream flowing through the leaf chamber system. The concentration of CCL in the air in the leaf intercellular space (Ci), was calculated based on the method of Farquhar and Sharkey (1982), Ci = Ca - (Pn * Rleaf * 1.6), [2.2] where Ca is the CCL concentration of the air ambient to the leaflet, R-, £ is the total leaf resistance to diffusion of water vapor and 1.6 is the ratio of the binary diffusivities of water vapor/air and C02/air (Farquhar and Sharkey, 1982). The product of ^ f * 1.6 is the leaf resistance to diffusion of CCL. This method of estimating Ci was found by Sharkey et al. (1982) to be in close agreement with measured values of the intercellular concentration of CCL. The calculation of R, , was based on the equations of Neumann and Thurtell (1972), using measured values of dewpoint and dry bulb air temperatures, air flow rates, and leaflet area. Photosynthetic rates for leaflets grown at each CCL concentration are the pooled values from two leaflets. Plant Sampling Procedure Leaf tissue samples were collected via access doors located on the rear (north side) of the plant chambers. Inside each door was 33 positioned a curtain of polyester film that reduced disturbance of the atmosphere within the plant chamber during plant tissue sampling. This procedure was found to result in small and only brief disturbances of the atmosphere during sampling events. The plant tissue collected was from the upper canopy and consisted of 20 to 25 fully expanded, non-shaded, and visibly healthy leaflets. These leaflets were selected in part based on visual similarity to the leaflets used in the leaf chambers for photosynthetic rate measurements. Leaflet lamina were removed at the petiolule and immediately plunged into liquid N2. This process was completed in approximately 1 second. The leaf tissue was then ground in a liquid ^2 chilled mortar and the resulting leaf powder was stored in a container in liquid N2. The leaf tissue was kept at liquid N2 temperature from the time of harvesting until laboratory analysis which occurred at a later date. Vu et al. (1984a) have shown this method to preserve enzymatic activity for prolonged periods of time. RuBP Carboxylase Assay A quantity of frozen leaf powder (100 to 170 mg dry weight) was removed from liquid N2 storage and placed in a pre-chilled Ten Broeck tissue homogenizer. Added to the leaf powder was 10 ml of extraction buffer consisting of 50 raM Tris (pH 8.5), 5mM DTT, 0.1 mM EDTA, and 1.5% (w/v) PVP-40. The leaf tissue was homogenized for approximately 60 seconds at 0°C, at which point an aliquot of the homogenate was reserved for chlorophyll determination, and the remainder was centrifuged at 12,000 g for 3 minutes. The supernatant of the crude 34 extract was either assayed immediately or else following a 5-minute activation period at 30°C in 10 mM NaHCCX, and 10 mM MgCU. Assays were carried out in triplicate at 30°C, with continuous shaking (125 strokes min ), in 5-ml glass vials with screw-on septum caps. The assay buffer consisted of 50 mM Tris (pH 8.5), 5 mM DTT, 0.1 mM EDTA, 10 mM MgCl2, 0.5 mM RuBP, and 20 mM NaH14C02 (7.54 GBq mol-1). The sealed vials were purged with N2 for 10 minutes prior to the addition of the Tris buffer and the NaH C02< The total assay volume was 1 ml. Assays of enzyme activity were initiated with the injection, through the septum cap, of 0.1 ml of either nonactivated or HC0 ~/Mg2+ activated crude extract to determine initial or total activity, respectively (Perchorowicz et al., 1981). Assays were terminated after 45 seconds with the injection of 0.1 ml of 6N HC1. A 0.9 ml aliquot of the assay mixture was transferred to a 20-ml glass scintillation vial which was placed on a warm heating plate under an air-stream, and remained there until the contents were dried. When dry, 2.5 ml of water and 5 ml of scintillation cocktail were added to the vials and acid-stable C products were determined by liquid scintillation spectrometry. RuBP Determination The determination of RuBP was based on the method of Latzko and Gibbs (1974) with modifications by Vu et al. (1983). A quantity of frozen leaf powder (85 to 150 mg dry weight) was removed from liquid N2 storage and placed in a pre-chilled Ten Broeck tissue homogenizer. Added to the leaf powder was 10 ml of 0.5N HC1 at 0°C. The leaf 35 tissue was homogenized for approximately 60 seconds at 0°C, an aliquot was reserved for pheophytin determination and the remainder was then centrifuged at 12,000 g for 5 minutes. To 5 ml of the supernatant was added 0.75 ml 2M Tris base and 0.44 ml 4N K0H. The neutralized supernatant (pH 8.3) was then stored on ice. Assays were carried out in triplicate in 5-ml glass vials with screw-on septum caps at 30°C with continuous shaking (125 strokes min ) . The assay buffer consisted of 50 mM Tris (pH 8.5), 5 mM DTT, 10 mM MgCl 20 mM NaH C0>2 (7.54 GBq mol ), and 0.5 ml of the neutralized leaf extract supernatant. The total assay volume was 1 ml. The RuBP determination was initiated with the injection of 0.1 ml of activated crystallized RuBPCase from tobacco (equivalent to approximately 55 jjg protein). The tobacco enzyme had been prepared previously according to the method of Kung et al. (1980), and was reactivated by dissolving the enzyme in 50 mM Tris (pH 8.5), 10 mM MgCl2, 10 mM NaHC0„ and 100 mM NaCl and incubating for 25 minutes at 50°C (Kung et al., 1980). After 60 minutes the assay was terminated with the injection of 0.1 ml 6N HC1. An aliquot (0.9 ml) of the assay mixture was transferred to a 20-ml glass scintillation vial which was dried on a warm heating plate under an air-stream. When dried, 2.5 ml water and 5 ml scintillation cocktail were added to each vial and acid-stable C products were determined by liquid scintillation spectrometry. Chlorophyll, Protein, and Specific Leaf Weight Determinations Chlorophyll determinations were performed on sample aliquots reserved during the RuBPCase assays. Chlorophyll was extracted in 80% 36 acetone and calculations were by the method of Arnon (1949). The chlorophyll in sample aliquots reserved during RuEP determinations was converted to pheophytin during extraction with acid, therefore the original chlorophyll concentration was determined using the method of Vernon (1960). In addition, chlorophyll was determined in leaf disks of known surface area, collected and assayed at the same time that leaf tissue was collected for RuBPCase and RuBP assays. Soluble protein determinations were performed on aliquots of the same supernatant from the crude extracts used to initiate the RuBPCase assays. The dye binding spectrophotometric method of Bradford (1976) was used. Protein standards were prepared from crystallized and lyophilized BSA (bovine serum albumin) dissolved in the same buffer used in extraction of RuBPCase from leaf tissue. Specific leaf weight (SLW) was determined by drying freshly harvested leaves of known surface area, collected 49 DAP from the unshaded upper canopy, to constant weight in a 70°C oven. Pod Load and Leaf Area Measurements On October 18, 1983 (49 DAP), 12 plants were removed from each chamber for determination of pod weight (grams dry weight) and leaf area. To measure pod weight, all viable pods were removed from the plants and dried to constant weight in a 70°C oven. To determine leaf area, all green leaves were removed from the plants and the surface area (one side of each leaf) was measured with an area meter (Lambda, model LI 3000). 37 Analysis of Statistical Significance To determine the statistical significance of experimental results, simple linear regressions were performed using the short-term CO2 concentrations to which plants were exposed as regressor. Comparisons of slopes and intercepts between CCL growth treatments, and comparison of slopes to zero, were used as tests to determine if there were significant differences between treatments and also if there were significant responses to the various short-term CCL concentrations. In addition to simple linear regression, a quadratic regression was also performed on the RuBP data. Both types of regressions gave very similar results regarding the significance of RuBP response to C02. In cases where data were collected following growth at the two CCL treatments (but prior to exposure to the various short-term CC"2 concentrations) t-tests were used to determine the significance of the growth CCL treatments on certain plant characteristics. In all cases, all tests of significance were made at the 5% level unless otherwise noted. Regression parameters are tabulated in Appendix C. Results Response of Leaf Characteristics to C0? Soybean plants were grown from seed at atmospheric and twice atmospheric C02 concentrations. As shown in Table 2.1, specific leaf weight increased significantly at elevated C02. Chlorophyll and total soluble protein (expressed on a leaf area basis) were not significantly different in the two CC>2 treatments. Pod weight, leaf 38 Table 2.1. Effects of growth C02 concentration on leaf characteristics. Specific leaf weight determined on samples collected 49 DAP. All other samples collected 56 to 60 DAP. Mean values ± SD are presented. Growth C0„ concentration Ml C02 l"1 330 Specific leaf weight _2 g dry wt. m 2 Chlorophyll -2 17.00 ± 0.10 0.475 ± 0.005 660 23.70 ± 0.04 0.520 ± 0.017 Total soluble protein" -2 4.03 ± 0.04 4.26 ± 0.14 Protein/Chlorophyll ratio 8.5 8.2 2t = 67.03, df = 2 Ho :y 660-^330 = 0 rejected at 5% level. ;t = 3.39, df = 2 2.06, df = 2 Ho:/j660-jj330 = 0 not rejected at 5% level. Ho:/j660-/j330 = 0 not rejected at 5% level. 39 area per plant, and the ratio of pods to leaf area all increased with C02 (Table 2.2), however, the differences in pod weight and leaf area were not significant. These morphological and biochemical differences reflect the effects of increased C02 concentration which also affects leaf photosynthetic rate response. Leaf Photosynthetic Rate The effects of C02 concentration on leaf photosynthesis were examined following long-term and short-term exposures to different CCL concentrations. Intercellular C02 concentrations (Ci) were calculated as the C02 concentration ambient to the leaf (Ca) was varied from 80 to 1000 ul 2 1" . In Figure 2.1, Ci is plotted against Ca for leaves grown at both C02 concentrations. Linear regression analysis of the data yields slopes, and hence Ci/Ca ratios, of 0.72 (r=0.985) and 0.55 (r=0.965), respectively, for the 330 and 660 yl C02 1_1 grown leaves. The difference in the Ci/Ca ratio was found to be significant. Because the Ci/Ca ratio was lower in high C0„ grown leaves, the Ci calculated at any ambient C02 concentration was greater in leaves grown at 330 n\ C0„ 1 . Leaf photosynthetic rates were greater in high C0„ grown plants at all C02 concentrations in which they were measured (Figure 2.2). When plotted against Ci, plants grown at high C02 had greater maximum leaf photosynthetic rates. Plotting leaf photosynthetic rate against Ci allows evaluation of the C02 assimilation rate response to C02 concentration independent of stomatal influences. Each point in Figure 2.2 represents one photosynthetic rate measurement made at a 40 Table 2.2. Effects of growth C02 concentration on pod weight and total green leaf area per plant on samples collected 49 DAP. Mean value ± SD are presented for leaf area. Pod weight represents total dry weight of pods divided by the number of plants. Pod weight g dry wt. plant Leaf area m plant -1 Growth CCL concentration 330 ui co2 1 -1 660 0.084 ± 0.023 0.125 ± 0.052 660/330 1.48 0.1475 ± 0.0546 0.1855 ± 0.0432 1.25 Pod/Leaf area -2 0.569 0.674 1.18 1.23, df = 4 1.89, df = 32 Ho:/i660-jj330 = 0 Ho:/i660->J330 = 0 not rejected at 5% level, not rejected at 5% level, Fig. 2.1. Intercellular CO,, concentration versus ambient C02 concentration for leaves grown at two C02 concentrations. In leaves grown at 330 ul C02 1 (+), Ci/Ca = 0 72 (r = 0.985). In leaves grown at 660 ul (XL 1 L W\ ca /r„ - n ^ f grown at 660 jul C02 1 1 (a), Ci/Ca = 6.55 (r = 42 KHOK) GROWTH C02 CONCENTRATION + 330 a! co2 r1 800- A 660 Ml co2 r1 **; 600 — - 1 * s*\ ++ 400 — & £ 200- p A # \ l\ C02 I'' ) !888 Fig. 2.2. Leaf photosynthetic rate versus intercellular CCL concentration for leaves grown at 330 Ml C0„ 1 (A) and 660 yl C02 1"J (B). Each data point represents one measurement made at 5 minute intervals. The solid curves were generated by non-linear regression analysis of the data. The regression model was P=Pmax-;;-Ci/(KCi+Ci)+R; where P is leaf net photosynthetic rate, Pmax is the maximum value of P-R, Ci is intercellular C0~ , KCi is the Michaelis constant for Ci and R is the estimated respiration rate at Ci=0. For 330 yl C02 1 grown leaves: Pmax=55.5, KCi=206 and R= -II. 0. For 660 ul CO 1 grown leaves: Pmax=96.1, KCi*223, and R=-13.8. Pmax and R are in jumol C02 m s and KCi is in ul C0? 1 . Photosynthetic measurements were made 56 to 60 DAP. 44 100- 80- 60- O 40H o E 4 20- 55 0- UJ X 100- en o 5 80- X a. GROWTH C02 CONCENTRATION 330 >il C02 I"1 GROWTH C02 CONCENTRATION 660 p.\ co2 r1 4 » 0 200 400 600 800 INTERCELLULAR C02 CONCENTRATION ( p.1 C0„ I"1) 45 5-minute interval, and are the pooled values from two leaflets at each C02 treatment. The highest rate measured for a leaf grown at 330 ul C02 1 was 41 ymol C02 m~ s~ , and for a 660 ^l C02 l-1 grown leaf 69 umol C02 „T2 s_1 (Figure 2.2, A and B). At low Ci, high C02 grown leaves showed greater rate response to increases in CO The solid curves in Figure 2.2 (A and B) were generated by non-linear regression analysis of the data points. The data in Figure 2.2 were divided into 10 discrete groups based on C02 concentration, and the mean Ci and mean leaf photosynthetic rate were calculated. The Ci values in each group varied less than 5% from the mean. These means are plotted in Figure 2.3 (A). Comparison of Figure 2.2 (A and B) with Figure 2.3 (A), indicates that plotting the means of the data did not affect the relationship between photosynthetic rates nor the relationship between photosynthetic rate and Ci. Since there was a difference in SLW between leaves grown at the two C02 concentrations, mean leaf photosynthetic rates were also calculated based on dried leaf weight and are plotted against Ci in Figure 2.3 (B). The difference in photosynthetic rates between high C02 and atmospheric C02 concentration grown leaves was less when expressed on a dry weight basis, particularly at lower C0? concentrations. However, leaf photosynthetic rates were still greater in the high C02 grown leaves at all C02 concentrations, suggesting that the increase in SLW in the high C02 grown leaves did not account for all of the increase in leaf photosynthetic rate. Arrows in Figure 2.3 (A and B) indicate the mean photosynthetic rates obtained when measured at the respective ambient growth C0„ concentrations. The Fig. 2.3. Mean leaf photosynthetic rate versus mean intercellular CO concentration for leaves grown at 330 yl C02 1 (•) and 660 ul C0? l"1 (a). Photosynthetic rates are expressed on a leaf area basis (A) and a leaf dry weight basis (B). Data are from Figure 2.2. Arrows indicate mean photosynthetic rates measured at the respective ambient C02 growth concentrations. Vertical lines represent ± SD. 47 co CO UJ X f- -z. CO o \- o X Q. L±J Li_ < UJ _l 60- fc 40 H CVJ o CJ "5 | 20 CD E CVJ o CJ "o E 0 GROWTH C02 CONCENTRATION ® 330 julI co2 r1 660 Ml C02 I"! B GROWTH C02 CONCENTRATION 9 330 ju.1 co2 I-1 O 660 ul co2 r1 0 200 400 600 800 INTERCELLULAR C02 CONCENTRATION (jjj CO, I'1) 48 photosynthetic rates of leaves grown and measured at 660 jjl CCL 1_1 were greater than in leaves grown and measured at 330 ul (XL 1 . RuBP Carboxylase Activity Assays of RuBPCase activity were performed on leaf tissue sampled from plants at their growth C02 concentration and also following short-term exposure to a range of C02 concentrations. Both initial (nonactivated) and total (HC03~/Mg2+ activated) activities were assayed in samples (collected under high light conditions) that were extracted without added Mg +. The results of these assays are shown in Figure 2.4 (A and B) . Each data point is the mean of triplicate assays. Enzyme activity in Figure 2.4 is expressed on a leaf area basis so a more meaningful comparison can be made with leaf photosynthetic rates. Figure 2.4 (A) shows that initial activity of RuBPCase did not significantly respond to short-term exposure to different CO,, concentrations. There was no significant difference between the two growth C02 concentrations. Total activity was also independent of short-term C02 concentrations [Figure 2.4 (B)]. It also did not significantly respond to increases in C0„. The catalytic rates were quite similar (not significantly different) between the two growth C02 concentrations whether measured as initial or total enzyme activity at all C02 concentrations. On a leaf area basis there was less than a 5% difference between the activities (both initial and total) of RuBPCase when sampled at the respective growth C0„ concentrations. Initial and total enzyme activities were also calculated on a chlorophyll basis and these data are presented in Fig. 2.4. Leaf RuBPCase activity versus CO concentration for samples collected following I hour exposures to six different CO concentrations. Plants, were grown at 330 ul CO^ 1 (•) or 660 ul C0„ 1_1 (a). Both initial activity (A) and total activity (B) were assayed. Mean values of triplicate assays are presented. 50 120- 80- o o _ 401 o E t 0 > o I60- < UJ en < _l x |20- CD < O a. CO cc INITIAL ACTIVITY GROWTH C02 CONCENTRATION 9 330 mI C02 I"' A 660 Ml CO, l~' 80- 40- TOTAL ACTIVITY GROWTH C02 CONCENTRATION • 330 jul co2 r1 A 660 Ml C02 I"' 200 400 600 800 COg CONCENTRATION (jxl C02 I"1) 1000 51 Table 2.3. Due to the difference in the amount of chlorophyll per unit leaf area, the relative enzyme activities shift somewhat when expressed on a different basis. When expressed on a chlorophyll basis, leaves grown and sampled at 330 ul C02 l_1 had initial and total activities 10 and 13% greater than leaves grown and sampled at 660 ul C02 1 . However, the response to C02 of both initial and total RuBPCase activities was not significantly different between the two growth C02 treatments . The activation state of RuBPCase in vivo may be estimated by initial activity/ total activity * 100%. As would be expected based on the independence of initial and total enzyme activities from (X>2 concentration (Figure 2.4), the activation was also independent of C02 (Figure 2.5). The response of activation to C02 concentration was insignificant (at the 1% level) for both C0„ treatments. There was no significant effect of exposure to different short-term C02 concentrations or to long-term growth C02 concentration on activation. RuBP Levels Steady state RuBP levels were measured in the same tissue samples collected for RuBP carboxylase assays. Samples were collected at growth C02 concentrations and also following the short-term exposures to the various C02 concentrations. RuBP data are reported on a leaf area basis in Figure 2.6. Each data point represents the mean of triplicate assays. There was a significant response of RuBP levels to C02 concentration. In both growth C02 treatments, below a (X>2 concentration of 330 ul C02 l"1, RuBP levels increased as C02 52 Table 2.3. Effects of two growth C02 concentrations on RuBPCase activity in leaves collected following 1-hour exposures to six different CCL concentrations. Both initial and total enzyme activity were assayed. Mean values of triplicate assays ± SD are presented. RuBP carbos ylase activity Ambient co2 ition 330 nl co2 i-1 560 ul co2 I"1 Concentre (iraol C0„ mg Chi hr -1 n co2 ] -1 Initial Total Initial Total 110 700 ± 13 731 ± 53 651 ± 26 726 ± 10 220 803 ± 20 880 ± 6 627 ± 18 793 ± 5 330 803 ± 28 890 ± 24 847 ± 9 903 ± 14 550 665 ± 5 771 ± 29 614 ± 18 735 ± 32 660 706 ± 6 800 ± 16 720 ± 19 771 ± 8 880 715 ± 7 809 ± 17 624 ± 18 757 ± 6 Fig. 2.5. Activation status of RuBPCase versus CO concentration for leaves grown at 330 yl CO, (•) or 660 ul C02 1 (a). Mean values of 2 triplicate assays are presented. Percent activation calculated from data in Figure 2.4. 54 100- 80- 2 . i= 60H o < en < > 40 x o CD cc < (J gj 20- 3 CC GROWTH C02 CONCENTRATION • 330 Ml C02 I"1 A 660 Ml C02 lH 200 400 600 COg CONCENTRATION (jllI C02 I"1) 800 1,000 Fig. 2.6. Leaf RuBP levels versus CO concentration in samples collected following 1-hour exposures to six different C0„ concentrations. Leaves were grown at 330 jul CO 1 1 (©) or 660 ul C0? I-1 (A) . Mean values of triplicate assays are presented. 56 100 80 o 60 £ 4 £ 40 cr 20- 0 GROWTH C02 CONCENTRATION 9 330 Al C02 f ' A 660 M-l C02 f ' 200 400 600 800 C02 CONCENTRATION (jil COg I'1) 1000 57 decreased. Above this concentration RuBP was rather insensitive to CC^. The levels of RuBP were higher in leaves grown at high COo regardless of the different short-term C02 concentrations. The RuBP levels showed significant responses to both the short-term CO concentrations and to growth C02 treatment. Due to the difference in chlorophyll content, RuBP levels were also calculated on a chlorophyll basis. The concentration of RuBP in the chloroplast stroma was calculated assuming RuBP is present only in the chloroplast (Heber, 1974) and that the stromal volume is equivalent to 25 ul mg chlorophyll- (Sicher and Jensen, 1979). These data are shown in Table 2.4. As was the case on an area basis, the RuBP levels on a chlorophyll basis were significantly higher in the high C0„ grown leaves. The RuBP level decreased significantly with increasing C0„ concentration when expressed on either a chloropyll basis or as the stromal concentration of RuBP. Discussion Soybean leaflet photosynthetic rates increased with increasing C02 concentration in plants grown at both atmospheric and twice atmospheric C02 concentrations. There are relatively few examples of high C02 grown plants having greater leaf photosynthetic rates than atmospheric C02 grown plants, when both are measured over the same range of C02 concentration. However, at all C02 concentrations in which photosynthesis was measured, rates were greater in leaflets grown at the higher C02 concentration (Figures 2.2 and 2.3). Thus, these results agree with those of Hicklenton and Jolliffe (1980a) and 58 Table 2.4. Effects of growth CO- concentration on RuBP levels in leaves collected following 1-hour exposures to six different C02 concentrations. Levels of RuPB are expressed both on a chlorophyll basis and a chloroplast concentration basis. Mean values of triplicate assays ± SD are presented. R jBP Ambient CO ConcenEration ni co2 i~r 330 nmol mgChl Growth C0? Concentration Ul C09 l"1 _x 660 330 mM 660 110 141 ± 2 158 ± 1 5.6 ± 0.08 6.3 ± 0.04 220 128 ± 1 186 ± 1 5.1 ± 0.04 7.4 ± 0.04 330 108 ± 5 106 ± 2 4.3 ± 0.20 4.2 ± 0.08 550 115 ± 1 103 ± 1 4.6 ± 0.04 4.1 ± 0.04 660 109 ± 1 135 ± 5 4.3 ± 0.04 5.4 ± 0.20 880 113 ± 1 123 ± 1 4.5 ± 0.04 4.9 ± 0.04 59 are similar to the results of Mauney et al. (1979) with soybean. The implication of this type of relationship between leaf photosynthesis and C02 with regard to control of leaf photosynthetic rate is discussed below. Intercellular C02 concentrations were calculated and leaflet photosynthetic rates were then plotted against Ci . Figure 2.1 shows the relationship between Ci and Ca to be linear and therefore the ratio of Ci/Ca was found to be constant across a range of C0? concentrations from 80 to 1000 Hl C02 l"1. Whereas Goudriaan and van Laar (1978) found Ci/Ca to be constant in Phaseolus vulgaris only when Ca was below and not above 300 ul C02 l-1, the results reported here are consistent with those of most other researchers (Spencer and Bowes, 1986; Sharkey et al . , 1982; Wong et al., 1979). While the Ci/Ca ratios were constant at all C02 concentrations, the ratio was significantly lower (by 23%) in plants grown at higher C0? . This could be due to the higher photosynthetic rates or differential stomatal response in the high C02 grown leaves. Either factor might lower the Ci. However, another factor may be responsible for the Ci/Ca ratio difference. Growth of soybean at elevated C0„ concentrations can result in thicker leaves with more palisade cells per unit leaf area (J.C.V. Vu , personal communication; Thomas and Harvey, 1983), and therefore an increased mesophyll cell surface area/external leaf surface area ratio. An increased internal surface area would permit greater uptake of C02 from the leaf intercellular air spaces and result in a lower Ci value. Nobel et al. (1975) and Nobel (1980) have discussed the influence of several environmental 60 variables, other than C02 , on the internal to external surface area ratio. The effects of increased mesophyll cell surface area on leaf photosynthesis are discussed below. Wong et al. (1985) and Spencer and Bowes (1986) did not find a difference in Ci/Ca ratios with different growth C02 concentrations. In plants grown at both atmospheric and elevated C0„ , RuBPCase activity (on a chlorophyll basis) was not significantly greater in the leaves grown and sampled at the lower rather than the higher CO concentration (Table 2.3). An apparently significant effect of CO has been reported in the literature for a variety of C„ plants including cotton (Wong, 1979), Nerium oleander, and Atriplex triangularis (Downton et al., 1980), Phaseolus (von Caemmerer and Farquhar, 1984; Porter and Grodzinski, 1984), soybean (Vu et al., 1983), tomato (Hicklenton and Jolliffe, 1980a), and waterhyacinth (Spencer and Bowes, 1986). When RuPBCase activity is expressed on a leaf area basis (Figure 2.4) there is also no significant difference between C02 treatments in the enzyme response to C02 concentration. In plants that were grown at a particular C02 concentration and then exposed for short periods of time to concentrations of C0„ ranging from 110 to 880 yl C02 l"1, prior to sampling leaves, there was no significant effect of the short-term exposures on initial or total enzyme activity (Figure 2.4). The independence of initial activity from short-term exposure to C02 in the light has also been reported in Arabidopsis (Salvucci et al . , 1986a) and white clover (Schnyder et al., 1984). When the C02 concentration was raised to 5000 fxl C0? 1_1 , Schnyder et al. (1984), however, found a 50% decrease in activity 61 compared to the activity at the C02 compensation point. The percent activation of RuBPCase, an estimation of the in vivo enzyme activation status, like the initial and total activities was essentially not affected by CC>2 (Figure 2.5). Perchorowicz and Jensen (1983) and Schnyder et al . (1984) reported similar results with wheat and white clover, respectively. Although C02 is necessary in the activation of RuBPCase (Bahr and Jensen, 1978; Lorimer et al., 1976), there was no indication that even at CC>2 concentrations as low as 110 yl C02 1_1 (and corresponding Ci value of 60 to 75 yl C0„ l"1) the enzyme suffered a significant decrease in activation. This indicates that a high C02 concentration inside the leaf is not required for a high level of RuBPCase activation at high light intensity. Unlike the apparent lack of effect of C02 concentration on RuBPCase activity in vitro, steady state RuBP levels were found to respond to C02 . Plants grown at both C02 concentrations had the highest levels of RuBP following exposure to low C02 concentrations. The RuBP levels declined as C02 increased (Figure 2.6). Work by other researchers has yielded similar results (Badger et al., 1984; Dietz and Heber, 1984; Mott et al., 1984; Collatz, 1978). The results of Dietz and Heber (1984) indicated approximately two times the concentration of C02 was required with spinach, as compared with the soybean data in Figure 2.6, prior to the onset of the decline in RuBP. Hitz and Stewart (1980) did not find changes in RuBP levels in soybean during steady state photosynthesis in 21% 02 and C02 concentrations ranging from 50 to 500 yl C02 1_1. Levels of RuBP decreased as leaf photosynthetic rate increased with increasing C02 regardless of growth 62 at 330 or 660 jliI C02 1 (Figure 2.6). The lower levels of RuBP (as CKX, concentration was increased) are presumably a result of greater consumption due to higher photosynthetic rates associated with the increased 2 concentration. Although both photosynthesis and RuBP levels were greater in leaves grown at high C02, the turnover time for the pool of RuBP was about the same for leaves grown at either C0„ concentration when calculated at both low and high ambient (XL (110 and 880 yl C02 1 ) . This suggests coordination between leaf photosynthetic rate and RuBP levels. Turnover times were calculated based on the rate of photorespiration being 15% of the rate of photosynthesis (Canvin, 1979), one mole RuBP consumed per mole C0„ assimilated (Bassham, 1979), and two moles RuBP consumed per mole C0„ released during photorespiration (Ogren, 1984). This stoichometry, the leaf photosynthetic rates, and the measured steady state levels of RuBP yielded turnover times of 11.5 and 10.8 seconds for leaves grown at 330 and 660 yl C02 l" , respectively, when measured at 110 yl C02 1 , and 1.1 and 0.8 seconds when measured at 880 yl C0„ 1 . The RuBP concentrations (Table 2.4) were always greater than the estimated RuBPCase binding site concentration for RuBP of 3 to 4 mM (Jensen and Bahr, 1977), indicating that RuBP was probably at saturating concentrations. The similarity of turnover times and the concentration of RuBP greater than the estimated binding site concentration, suggest that RuBP was probably not limiting leaf photosynthetic rates in these experiments. Initial RuBPCase activity [Figure 2.4 (A)] was greater at all C0„ concentrations than the leaf photosynthetic rate [Figure 2.3 (A)] when 63 both were expressed on a leaf area basis. Results of this nature have previously been reported (Bjorkman, 1981; Singh et al., 1974). There are a number of reasons why leaf photosynthetic rate measured in situ would be less than RuBPCase activity measured in vitro. The enzyme assays are performed under saturating inorganic carbon concentrations which not only provides more C02 than is normally available within the leaf chloroplast in the field, but also essentially eliminates the competitive oxygenase reaction. The effects of dark respiration are not measured in the enzyme assay. Also, extraction of the enzyme from its intrachloroplastic location prior to assay will presumably remove metabolic regulation that may normally function in the intact photosynthetic cell. Furthermore, the assay procedure used to determine RuBPCase activity measures both the E-C and E-C-M forms of the enzyme while in the intact leaf only the E-C-M form will be active (Seftor et al., 1986). If the E-C form is present in significant quantities the in vitro enzyme assay will tend to overestimate the active species of RuBPCase in vivo. Farquhar et al. (1980) have proposed a model suggesting leaf photosynthetic rate is limited by RuBPCase at low Ci and by RuBP regeneration at high Ci. Results supporting this model have been reported by von Caemmerer and Farquhar (1981), while Makino et al. (1985) have indicated their results suggest RuBPCase was always limiting to leaf photosynthesis. Results reported here show no significant effect of Ci on RuBPCase activity and suggest that RuBP levels were probably saturating for RuBPCase binding sites at all Ci 64 values. These data, therefore, do not appear to support the model of Farquhar et al . (1980). Since the leaflet photosynthetic rates were greater in leaves grown at twice the atmospheric concentration of CO yet the difference in RuBPCase activity between the two CO growth treatments were not significant, and RuBP appeared to be at saturating levels, three possibilities are suggested which may account for the greater leaflet photosynthetic rates of the high C0„ grown plants. First, as already described, growth at elevated C02 concentration can result in an increase in the mesophyll cell surface area/leaf surface area ratio. Nobel et al. (1975) have shown an increase in this ratio to result in higher photosynthetic rates. This may have occurred in the high C02 treatment. Second, leaflet photosynthetic rates were measured during the pod filling stage, and plants grown at high C0„ had a greater pod weight per plant and per unit leaf area. Long-term growth in high C02 has been shown to increase the number of fruit per plant in several cases (Havelka et al., 1984; Baker and Enoch, 1983; Cooper and Brun, 1967), and these increases represent an increase in assimilate demand. An increase in assimilate demand has often been associated with increased photosynthesis (Gifford and Evans, 1981; Geiger, 1976; King et al., 1967). Plants grown at high C0„ had greater leaf photosynthetic rates as well as greater pod weights per plant. Enos et al . (1982) have also reported higher photosynthetic rates in soybean plants with heavier pods. Third, the C0?-saturated RuBPCase activity in vitro may not be an accurate representation of activity in vivo. There may be differential regulation of RuBPCase in 65 vivo in soybean grown at different CC^ concentrations, however, no evidence of this was observed. An additional factor needs to be addressed with regard to photosynthetic rates; the effects of leaf starch. Although starch was not measured quantitatively in these experiments, visual estimations of relative starch levels performed prior to enzyme assays indicated that leaves grown at high CCL contained more starch. In previous studies, where starch was measured quantitatively, it was found to be higher in soybean leaves grown at elevated CCL concentrations (Allen et al., 1983). In the experiments reported here the results are in agreement with those in the literature that indicate no evidence of photosynthetic rate inhibition by starch accumulation at high CCL. Based on the results presented here from soybean, it is shown that growth at twice the atmospheric concentration of CCL can result in an enhanced capacity for leaflet photosynthesis. Since the response of RuBPCase activities was not significantly different with growth CCL treatment and the levels of RuBP appeared to be saturating with regard to RuBPCase binding sites, the role of either in the enhanced photosynthetic capacity remains unsupported. The increased photosynthetic capacity following growth in elevated CCL may be due to either an increase in the internal/external leaf area ratio or greater assimilate demand or a combination of both. CHAPTER III RESPONSE OF PHOTOSYNTHETIC BIOCHEMISTRY AND PHYSIOLOGY TO LONG-TERM EXPOSURE TO SUBATMOSPHERIC AND SUPERATMOSPHERIC C09 CONCENTRATIONS J2 Introduction Much of the interest in the effects of C02 on vegetation is based on the fact that the atmospheric concentration of C0„ has been increasing for the last century (Baes et al., 1977). Research has focused on predicting how this continuing trend will affect future crop yields and water use. In addition to learning the answers to these questions, experiments with C02 concentrations can enhance our comprehension of plant processes such as photosynthesis. Since the response of plants to C02 is largely mediated by the photosynthetic process, understanding the effects of C02 on photosynthesis is paramount to understanding the effects on whole crop responses. Almost all of the research on the long-term effects of C0„ on plants has involved exposing plants to elevated concentrations of C09 (Lemon, 1983; Kramer, 1981). It appears that long-term research on plants grown at reduced rather than elevated C02 concentrations has previously just involved plants native to high altitudes where they normally grow at C02 partial pressures below those at or near sea- level (Mooney et al., 1966; Billings et al., 1961). Long-term exposure to elevated C02 results in a number of changes in plant characteristics. Leaf area on a whole plant basis has been shown to increase with C02 (Jones et al., 1984a; O'Leary and Knecht, 1981; 66 67 Cooper and Brun, 1967). Stomatal density (stomata mm-^) increased, although not significantly, in soybean grown at high CO- (Thomas and Harvey, 1983). In Phaseolus fewer stomates were found on the abaxial surface of leaves grown at high C02, but the leaves were larger and thus the overall result was more stomates per leaf (O'Leary and Knecht, 1981). Increases in specific leaf weight (SLW) following growth at elevated CC>2 have been reported in tomato (Madsen, 1968), Nerium oleander (Downton et al., 1980), Phaseolus (Jolliffe and Ehret, 1985), and soybean (Havelka et al., 1984; Jones et al., 1984a; Thomas and Harvey, 1983; Hofstra and Hesketh, 1975). Chlorophyll content of leaves has been shown to either increase (Downton et al., 1980), decrease (von Caemmerer and Farquhar, 1984), or stay the same (Havelka et al., 1984) in plants grown at elevated C02> Other cytological responses to long-term high C02 exposure include increased cell water content (Madsen, 1968), and changes in cell volume (Gates et al., 1983; Madsen, 1968). In soybean, the presence of a third layer of palisade cells not found in plants grown at atmospheric C0„ concentration was observed in high C02 grown plants (Thomas and Harvey, 1983). Carbon dioxide concentration has been shown to affect the concentration of proteins as well as enzyme activities. In soybean grown at elevated C02 seed protein was found to decrease as C02 increased (Rogers et al., 1984), but in another study there was no effect of C02 on pod nitrogen levels (Hardy and Havelka, 1976). The response of total soluble leaf protein to C0„ varies. It has been shown to increase (Downton et al., 1980), decrease (Wong, 1979), and not change (Havelka et al., 1984; Porter and Grodzinski, 1984) with 68 long-term exposure to elevated C02. Most reports indicate that growth at high CC>2 results in reduced activity of RuBPCase when compared to plants grown at atmospheric C02 concentrations, when activity is expressed on either a chlorophyll basis (Spencer and Bowes, 1986; Vu et al., 1983; Downton et al., 1980) or a leaf area basis (von Caemmerer and Farquhar, 1984; Wong, 1979). However, Fair et al . (1973) have reported higher activity, when expressed on a fresh weight basis, in young barley plants grown at 10,000 to 50,000 ul CO- l-1. The difference in activity became less as the plants aged. The proportion of leaf soluble protein composed of RuBPCase (mg RuBPCase/g soluble protein) decreased 22% in Nerium oleander when the growth C0? concentration was increased from 330 to 660 ul C0„ 1 (Downton et al., 1980). The effects of C02 on a variety of other enzymes have also been reported. Carbonic anhydrase activity increased in oat when grown at 80 ul C02 1 and decreased when grown at 600 ul C0„ 1 (Cervigni et al., 1971). In Phaseolus, carbonic anhydrase activity decreased following growth at 1200 ul C02 1~ (Porter and Grodzinski, 1984). Phosphoenolpyruvate carboxylase activity decreased when waterhyacinth was grown at 600 ul C02 1~ (Spencer and Bowes, 1986), as did nitrate reductase in barley grown at 10,000 to 50,000 ul C0„ 1 (Fair et al., 1973). There was no difference in fructose 1, 6-biphosphatase activity in Nerium oleander grown at atmospheric and twice atmospheric C02 concentrations (Downton et al., 1980). Glycolate oxidase activity decreased when grown at high C0„ in both Phaseolus (Porter and Grodzinski, 1984) and barley (Fair et al., 1973), but in tomato no well-defined response to CO.-, was apparent 69 (Hicklenton and Jolliffe, 1980a). Catalase activity was lower in barley grown at high C02 (Fair et al., 1973). There were no significant differences in sucrose phosphate synthase activity in soybeans grown at atmospheric or elevated CCL (Huber et al . , 1984) or in soybean proteolytic enzyme activity (Havelka et al., 1984). Whether the differences in the activities of these enzymes from plants exposed to various C02 treatments are always significant is not clear. The physiological significance of the responses to C0? of all of these enzymes is not always evident. There are reports of plant damage, sometimes extreme, as a result in of growth at high concentrations of C02. Accumulation of starch plants grown at 1000 /jl C02 1_1 was found to cause chloroplast disruption (Cave et al., 1981). Chlorosis occurred in Phaseolus grown at 1400 yl C02 1_1 (Ehret and Jolliffe, 1985) and in tomato (Thomas and Hill, 1949). Thomas and Hill (1949) also reported the appearance of necrotic areas on tomato leaves at high C0„. Brown and Escomb )e (1902) reported a variety of disorders in plants grown at 1100 ul C02 1 . These included loss of leaves, reduced number of flowers and lack of fruit formation. According to Ehret and Jolliffe (1985), it has been suggested that the injuries reported by Brown and Escombe (1902) may have been due to the impurities in the air in the enclosed greenhouse. While ethylene contamination of compressed CO^ cylinders was demonstrated by Morrison and Gifford (1984), presumably most reports of plant injury are not the result of tainted air. There are numerous examples of plants exposed to high C0„ with no injurious effects, including exposure for 14 days to C0„ as high as 50,000 ul 70 C02 1 (Hicklenton and Jolliffe, 1980b), suggesting that exposure to high C02 per se is not damaging to all plants. Whole canopy photosynthetic rate responses to C0„ of canopies grown at atmospheric and elevated OX, have been reported for soybeans by Acock et al. (1985) and Jones et al. (1984a). In both cases soybeans were grown in outdoor sunlit chambers for an entire season. Both Acock et al. (1985) and Jones et al. (1984a) showed greater photosynthetic rates, at all levels of solar irradiance, in canopies grown at elevated (X>2 when compared to canopies grown at 330 yl C02 1 . Jones et al. (1984a) reported maximum canopy photosynthetic rates, measured at the respective growth C02 concentration and approximately 1900 /jmol quanta m~2 s"1, were 50% greater in the canopy grown at 800 ul C02 l"1 compared to the 330 yl C02 1_1 grown canopy. In Chapter II a study was described in which soybeans were grown continuously from seed at atmospheric and twice atmospheric concentrations of (X>2 to investigate the effects on photosynthesis. In the study presented here, the range of growth (XL concentrations was expanded. Soybeans were grown at three subatmospheric , atmospheric, and two superatmospheric concentrations of CO . The objective of this study was to investigate the effects of long-term growth in various concentrations of C02, ranging from subatmospheric to superatmospheric levels, on soybean. Specific objectives were to determine the effects on the activity and kinetics of RuBPCase and on the levels of RuBP. In addition, the effects of OL growth concentration on several plant characteristics and on canopy photosynthesis were investigated. 71 Materials and Methods Plant Material and Growth Conditions Soybeans (Glycine max L. Merr. cv Bragg) were planted in six outdoor environmentally controlled plant growth chambers (described in Chapter II) on 14 Sept. 1984. The C02 concentration was controlled to 160, 220, 280, 330, 660, or 990 Ml C02 l"1 in each chamber from the date of planting until harvest. The chamber dry bulb and dewpoint temperatures were controlled to 31 and 16°C, respectively. The chambers received natural solar irradiation. The quantum flux density (400-700 nm) values reported here are measurements made at the upper canopy level (the chambers transmit 88% of the incoming solar radiation) . These values were integrated over 5 min intervals from data collected every 20 s. Photosynthetic rate measurements and collection of all plant material for analysis were made on 18 October (34 DAP). At this time the plants had not yet started reproductive development and had been thinned to a density of 30 plants per m . The canopies were at the V7 to V8 stage of development (Fehr and Caviness, 1977). Leaf tissue for biochemical analysis was collected and stored in liquid N„ as previously described. For each canopy, leaf area index (LAI) was estimated from the measured leaf area of four plants harvested from each chamber on 18 October. 72 Canopy Photosynthesis Measurements Measurements of net photosynthetic rate of whole canopies were made based on a whole chamber carbon mass balance which was corrected for leakage of C02 from the system (Jones et al., 1985b). The desired C02 concentration in a chamber was maintained by injecting pure C09 into the chamber to replace the C02 assimilated by the canopy. The C02 injections were based on light response algorithms determined for each canopy. The algorithms were updated as the canopies developed. Corrections for drift in this procedure were made every 5 min by making chamber C02 concentration measurements with an IR gas analyzer (Jones et al., 1984a). Canopy photosynthetic rate response to light was measured as the solar irradiation varied throughout the day. Measurements were made every 5 min over a 10.5 h period (0750 to 1800 EST) which was cloud free. During this time period, irradiance at the upper canopy level —2 —1 varied from 145 pimol quanta m s in the morning, to a midday —2 —1 — ? —1 maximum of 1370 ymol quanta m s , to 15 umol quanta m s in the evening . RuBP Carboxylase Assay and RuBP Determination The methods for sampling leaf tissue and for the assay of RuBPCase activity and the determination of RuBP levels were the same as those described in Chapter II. For the determination of the Michaelis constant, K (C09), and Vmov. of RuBPCase, the assay ill ^ IilaX J procedures were modified and are described in the following section. 73 Determination of Apparent K (C0o)and V ' nn — M max Assays of RuBPCase activity were performed to determine the Michaelis constant, Km(C02), and V^. The Km(C02) reported here is actually the apparent Kra(C02) as assays were performed on a crude extract from the leaf rather than the purified enzyme. The assay procedures were similar to those described in Chapter II with some modification and are described as follows. A quantity of frozen leaf powder (70 to 150 mg dry weight) was removed from liquid N storage and placed in a pre-chilled Ten Broeck tissue homogenizer. Added to the leaf powder was 5 ml of extraction buffer consisting of 100 mM Tris-HCl (pH 8.0), 5 mM DTT, 10 mM isoascorbate , 5 mM MgCl and 1.5% (w/v) PVP-40. The leaf tissue was homogenized for approximately 60 s at 0°C. An aliquot of the homogenate was reserved for chlorophyll determination, and the remainder was centrifuged at 12,000 g for 3 min. Following centrifugation the supernatant was activated and used to initiate the assays (described later) and the pellet was discarded. The buffer media used in the assay consisted of 50 mM Tris-HCl, 5 mM DTT, 5 mM MgCl2, and 10 mM isoascorbate. The media was prepared C0~- free by purging at pH 3.1 for 15 min with N2 then raising the pH to 8.0 with C02-free NaOH solution. To a 5-ml assay vial was added the C02_free buffer media, 0.5 mM RuBP, and 54 Wilbur-Anderson units of carbonic anhydrase (CA) (from bovine erythrocytes). The carbonic anhydrase was added to prevent depletion of C02 during the assay, particularly at the lower concentrations of HC0~ (Bird et al. , 1980). The vials were capped and then purged with N2 for 10 min. Through the cap septum NaH14C02 (7.54 GBq/mol) was added in eight different final 74 concentrations ranging from 0.25 to 10 mM. The consumption of substrate H C03~ was always less than 20% and usually less than 10% during each of the assays. The assays were initiated by the injection of activated supernatant from the homogenized crude extract. The supernatant was activated at 0°C for 45 min in 50 mM Tris (pH 8.0), 5 mM DTT, 10 mM isoascorbate, 5 mM MgCl2, and 10 mM NaH1AC03. Following activation, the supernatant was kept at 0°C while the assays were being performed. The injection of 25 yl of activated supernatant carried over 0.25 mM H14C03" into the assay vials and this quantity was taken into consideration when the final H14C0 ~ concentration calculations were made. Assays were performed in triplicate, at 30°C, with continuous shaking (125 strokes min-1), in a total volume of 1 ml. The assays were terminated after 45 s with 6N formic acid in methanol. An aliquot (0.4 ml) of the assay mixture was then transferred to a 5-ml plastic scintillation vial which was placed under an air-stream until all remaining 14C not fixed into acid-stable products was driven off. This required leaving the vials in the air- stream overnight. To the approximately 0.4 ml remaining in each vial was added 4 ml of scintillation cocktail. Acid-stable 14C products were determined by liquid scintillation spectrometry. Since C02 is the form of inorganic carbon used as a substrate by RuBPCase (Cooper et al., 1969), it was necessary to calculate the concentration of dissolved C02 in the assay mixture based on the added quantities of H C03~. At the assay temperature of 30°C the solubility coefficient of C02 (a) in water is 0.665 ml ml-1 (Umbreit et al., 1972) and the pK' of carbonic acid is 6.327 (Harned and 75 Bonner, 1945). Using these values, the gas space volume above the liquid in the assay vial, and the Henderson-Hasselbach equation, the partitioning of inorganic carbon between dissolved C02, C02 in the gas space and bicarbonate was calculated (Ogren and Hunt, 1978). No corrections were made for the effect of ionic strength on a or pK' . While the effect of salts on a appears to be minor in the concentration range encountered in these assays (Umbreit et al., 1972) the effect on pK' is more substantial (Harned and Bonner, 1945). However, since all assays had essentially the same salt concentration the relative effects on the kinetic values are insignificant. The 14 concentration of H CCL in each vial was corrected for the 14 consumption of H CO., during the assay. This required the assumption that the velocity of the reaction catalyzed by RuBPCase was constant during the 45 s assay. The corrected substrate concentrations and the reaction velocities were used to calculate K (CCL) and V values m z max using Lineweaver-Burke plots and the least squares method (Cleland, 1979). These kinetic values were also calculated using Eadie-Kof stee plots (data not shown) and were found to be very similar to the values presented here. Estimation of Dissolved Free C0? at the Cell Wall The dissolved free C02 at the cell wall of the mesophyll tissue was assumed to be in equilibrium with the C0„ in the air in the leaf intercellular spaces. Data from Figure 2.1 (Chapter II) yields a value of 0.72 for the ratio of the concentrations of intercellular to ambient C02, Ci/Ca, for plants grown at 330 yl C02 1 . The Ci/Ca 76 ratio for plants grown at 660 Ml C02 i"1 differed from 330 nl C09 1 grown plants by 23% (Figure 2.1). Since Ci/Ca ratios were not determined for all the growth C02 concentrations used in this study, and the exact nature of the relationship between the Ci/Ca ratio and growth C02 concentration is not known, the value for 330 ul CO- l"1 grown leaves, Ci/Ca = 0.72, was used for all calculations. Other assumptions included an atmospheric pressure of 760 mm Hg and a solubility coefficient, a, for C02 in water of 0.665 ml ml-1. All calculations were based on a temperature of 30°C. The calculation of free C02 dissolved in the cell wall was by the method of Umbreit et al., 1972), C02 . """" '°0°, [3.!] 760 * V * 100 where C02 is in units of moles liter-1 (M, molar concentration), the term P/760 converts atmospheric pressure to standard conditions, Ci? is the intercellular C02 concentration in percent (v/v), the term 1000/V converts a from ml ml-1 to moles liter-1, and 100 converts percent C02 to pC02 (partial pressure of C02 in mm Hg) . Chlorophyll, Protein, and Specific Leaf Weight Determinations The measurements of chlorophyll, total soluble leaf protein, and specific leaf weight (SLW) were made using the same methods described in Chapter II, with the exception that leaves for the SLW determination were collected from either nodes 5 and 6 or 6 and 7. In each canopy leaves from these nodes represented two of the most recently fully-expanded leaves in the upper canopy. All plant 77 material used for these measurements was collected on 18 Oct. 1984 (34 DAP). Analysis of Statistical Significance Simple linear and quadratic regression analyses were performed to determine the statistical significance (at the 5% level) of experimental results. In this chapter, the C02 concentration during growth was used as regressor. The methods used are described further in Chapter II. Regression parameters are tabulated in Appendix C. Results Response of Plant Characteristics to CO Continuous exposure during growth of soybeans to a range of CO from 160 to 990 yl C02 1_1 resulted in changes in leaf and canopy characteristics. There was a significant, almost linear increase in SLW with increasing C02 (Table 3.1). The plants grown at the highest C02 concentration had leaves with SLW 50% greater than those grown at the lowest concentration. Below atmospheric concentration of C09 (330 Ml C02 1 ) there was a minor response of SLW to C09. The greatest response occurred as C02 was increased from 330 to 990 Ml C0„ l"1. The LAI increased two-fold as C02 increased from 160 to 990 yl C02 I-1 (Table 3.1). The LAI generally increased with increasing CO showing a significant response to C02 concentration. The LAI values were similar for canopies grown at 160 and 220 yl C02 l_1, and although higher, similar for canopies grown at 280, 330, and 660 Ml CO l-1. 78 Table 3.1. Effect of growth CCL concentration on SLW, LAI, chlorophyll, and total leaf soluble protein. The SLW was calculated based on fully-expanded leaves collected from the upper canopy level. Canopy LAI was estimated from the total leaf area of four representative plants. All measurements were made on leaf samples collected 18 October (34 DAP) when plants were in the V7 to V8 vegetative stage. Growth C02 Specific Leaf Leaf Area Total Soluble Concentration Weight Index Chlorophyll Protein ,-1 °2 X g dry wt. m 160 20.3 ± 2.1a 220 20.9 ± 1.7 280 21.4 ± 2.4 330 21.4 ± 2.4 660 26.6 ± 5.2 990 30.5 ± 5.2 -2 2 -2 m ra -2 g m -2 1.63 ± 0.11 0.204 ± 0.001 2.53 ± 0.01 1.61 ± 0.03 0.261 ± 0.002 3.23 ± 0.02 2.40 ± 0.42 0.248 ± 0.002 2.58 ± 0.02 2.54 ± 0.20 0.214 ± 0.001 2.31 ± 0.01 2.40 ± 0.42 0.205 ± 0.004 2.28 ± 0.04 3.25 ± 0.33 0.234 ± 0.001 2.29 ± 0.01 Mean values ± SD. 79 The canopy grown at the highest CC>2 concentration had an LAI at least 28% greater than each of the other canopies. On a leaf area basis, the chlorophyll and leaf soluble protein levels showed similar responses to Q,Q>2 (Table 3.1). The general trend was a decrease in value with increasing C02, but the response to C02 of both chlorophyll and soluble protein was not significant. Because of the variation in SLW, chlorophyll and soluble protein are also expressed on a dry weight basis in Table 3.2. When expressed on this basis, the response of chlorophyll and soluble protein to CCL is significant. On a dry weight basis the levels of both chlorophyll and soluble protein in the 330 jliI C02 1~ grown leaves were approximately midway between the highest and lowest values, found in the 220 and 990 yl C02 1 grown canopies, respectively. Soluble protein on a dry weight basis decreased 50% as (XL was increased from 220 to 990 ul C02 1 . While the direction of responses to C0„ was similar for both chlorophyll and soluble protein, the magnitude of these responses varied. This is shown in Figure 3.1 where the protein/chlorophyll ratio is plotted against (X>2 concentration. The ratio is highest at low C02# The response to C02 of the protein/chlorophyll ratio was found to be significant. Canopy Photosynthetic Rate Canopy photosynthetic rate responses to sunlight for plants grown at each of the six C02 concentrations are shown in Figure 3.2. Data points represent measurements made at 5 min intervals as solar irradiance varied throughout the day. When canopy photosynthetic 80 Table 3.2. Effect of growth C02 concentration on chlorophyll and total leaf soluble protein expressed on a dry weight basis. Values are calculated from data in Table 3.1. Growth C02 Total Soluble Concentration Chlorophyll Protein ul C02 X m8 (g dry wt.) 1 mg (g dry wt.) X 160 10.05 ± 0.05a 124.7 ± 0.6 220 12.49 ± 0.09 154.5 ± 1.1 280 11.59 ± 0.09 120.5 ± 0.9 330 10.00 ± 0.05 107.9 ± 0.5 660 7.71 ± 0.15 85.7 ± 1.7 990 7.67 ± 0.03 75.1 ± 0.3 "Idean values ± SD. Fig. 3.1. The soluble protein/chlorophyll ratio versus growth C02 concentration. Data were calculated from the mean values in Table 3.1. Vertical lines through data points represent ± SD. 82 1000 Fig. 3.2. A-F. Canopy net photosynthesis (on a land area basis) versus solar ir radiance for canopies grown at 6 different CCL concentrations. A) 160 ul C0„ 1 , B) 220 Ul COj I"1, C) 280 ul C0„ 1"\ D) 33§ /il CO l \ E) 666 ^1 CO I"1, F) 990 yl C0„ l"1. Each data point represents a measurement made at a 5 min interval. Data were collected over a 10.5 h period (0750-1800 EST) on October 18 (34 DAP). Maximum solar irradiance occurred at midday when quantum flux density was approximately 1370 «nol quanta m s~ . Light levels are values for the upper canopy surface. Growth chambers transmit 88% of incoming solar irradiance. The canopy LAI's varied two-fold across the CC^ concentration range. 84 60 40 160 ill C02 I"1 D 330 mi co, r1 E 20 O o 1 60 en co 40 UJ X h- « 20 o h- o X Q. H 60 UJ Q. 40 O < o .>■**» .?}"*&'' 1^,+f.l ■+****+.* I ' ' ' ' I i I I I I I i i i I i rrr 220 p.\ co2 r1 * _ ...I i -' i> Hi ■ 8»^* » rTTT,:.,J_L,_!_L'_L'.LlJ_l_L-,T 280 Ml co, r1 20- ...-*•*#- .#•• i i i i i i i i i i i 1 1 1 1 i 660 Ml CO, I ..jV^ I ' ' ' ■ I 990 Ml co2 r1 •|.*"i<** 1 ' i ' ' ' ' i ' ' i ' i > i > i i 500 1000 1 ' i ' ' ' i i i i i ' i i ' '• 500 1000 1500 QUANTUM FLUX DENSITY ( u.mol quanta m"2 s"1 ) 85 rates were measured (at 34 DAP) canopies grown at 160 and 220 jul C02 1 were light saturated at light levels lower than 1000 /imol -2 -1 quanta m s (Figure 3.2 A and B). The canopy grown at 280 «1 C02 1 (Figure 3.2 C) did not appear to light saturate at midday light levels of 1370 ymol quanta m~2 s_1, but did not respond with increasing photosynthetic rates as high as the 330 ul CO l_1 grown plants (Figure 3.2 D). The photosynthetic rate response increased continuously with increasing irradiance in plants grown at 330, 660, and 990 ul C02 T1 (Figure 3.2 D, E, and F) . At two and three times atmospheric C02 concentration the photosynthetic rate response to light was clearly still increasing, even at maximum midday irradiance, showing no indication of light saturation. Based on the visually estimated intercept of response curves in Figure 3.2, the canopy light compensation points did not appear to be strongly C0„ dependent. Compensation points for each canopy were in the range of 50 to 150 -2 -1 ymol quanta m s The maximum photosynthetic rates of the canopies are plotted against growth C02 concentration in Figure 3.3. Each data point is the mean of between 7 to 10 measurements made at the growth CO concentration at midday when irradiance inside the chambers was at its peak of 1250-1370 jumol quanta m s . The maximum rates were greater as the C02 concentration during growth increased. The slope of the response is steeper at the lower C02 concentrations. Because the total leaf area of a canopy varied by two-fold over the range of C0„ concentrations, the canopy photosynthetic rates in Figures 3.2 and 3.3 are a reflection, in part, of the differences in LAI. Fig. 3.3. Maximum canopy net photos ynthetic rate versus growth C02 concentration. Photosynthesis is on a land area basis. Each data point is the mean of 7-10 measurements made at midday when the.quantum flux density was 1250-1370 jumol quanta m~2 S~T Data are from Fig. 3.2. Vertical lines through data points represent ± SD. 87 0 200 400 600 800 1000 C02 CONCENTRATION ( jllI C02 I'1 ) 88 RuBP Carboxylase Activity and RuEP Levels The RuBPCase activity was assayed from fully-expanded leaves collected from the upper part of each canopy. The means of triplicate assays are plotted in Figure 3.4 (A). Both initial and total activities decreased significantly as the C02 concentration increased, with the highest activities occurring at the lowest CO The initial activity decreased by 28% as C02 increased from 160 to 990 yl C02 1_1, while the total activity decreased by 23% over the same CO range. The activation of RuBPCase was calculated from data in Figure 3.4 (A) and was found to be quite high, particularly at low C02 [Figure 3.4 (B)]. Activation did show a significant but not a great response to C02, however, the highest activation (greater than 95%) occurred at the lower C02 concentrations. Above atmospheric concentrations of C0? there was not much activation response to C02 . The initial and total RuBPCase activities tended to parallel each other regardless of CO concentration. The RuBP levels were determined in a subset of the same leaf samples used for RuBPCase assays. The means of triplicate assays are shown in Figure 3.5. The level of RuBP decreased significantly as CO increased, however, at C02 concentrations greater than 660 ul CO I-1 the measured levels of RuBP did not appear to respond strongly to CO The RuBP at 660 Ul C02 i"1 was oniy 30% Qf the level at 160 »l C02 1_1. Assuming that RuBP is present only in the chloroplast (Heber, 1974), and that the stromal volume of the chloroplast is 25 ul mg Chi-1 (Sicher and Jensen, 1979), chloroplast concentrations of RuBP can be Fig. 3.4. A. RuBPCase activity versus growth CO concentration. Both initial (•) and total (o) activities are shown. Assays were performed at 30 C at pH 8.0 for 45 s. Data points are the means of triplicate assays. Plant samples were collected October 18 (34 DAP). B. RuBPCase activation versus growth C0? concentration. Percent activation calculated from data in A. 90 1000 > \- > _ o -C 750 < 1 III -C en o < _i E 500 X o CO m O cr U ~o 250 Q_ 00 3 V CT 0 -2. O 1- £ 75 \- ( ) < U! (.0 0^ fSO - X O CD cc 25 < o Q_ co =3 0 cr o— o TOTAL ACTIVITY •--• INITIAL ACTIVITY B i i -i 1 0 200 400 600 800 1000 C02 CONCENTRATION (jllI C0o I"1 ) Fig. 3.5. Levels of RuBP versus growth CO concentration. Chloroplast concentration of RuBP (mM) assumes 25 ^1 stroma volume mg chlorophyll- . Data points are means of triplicate assays. Leaf samples were subsamples of tissue used for RuBPCase assays in Fig. 3.4. 92 300 200 400 600 800 C02 CONCENTRATION (jx\ C02 l_l) 1000 93 estimated. These values were determined for each CO concentration and are also presented in Figure 3.5. The RuBP concentration ranged from 2.9 to 8.3 mM as the CO,, concentration decreased from 990 to 160 ui co2 r1. Effects of C0„ on K,(C0Q and V 1 n^ &* — — max Assays of total RuBPCase activity were performed at various HCO ~ concentrations to determine the Michaelis constant, K (COO and V m 2 y ' max " Total activity (Mg +/HC03" activated) was assayed for determination of Km(C02) and V^ to separate activation from catalysis kinetics. The mean enzyme activity of triplicate assays are plotted against HCO _ concentration, for each growth C02 concentration, in Figures 3.6 to 3.10. The HC03~ concentrations have been corrected for consumption of the substrate during the assays. The assumption was made that the reaction rate was constant during the 45 s assay. The solid curve in each of the Figures 3.6 to 3.10 represents the predicted response of total RuBPCase activity to HCO^ based on the Michaelis-Menten equation, V * [HCO " max L 3 [3.2] Km(HC03 ) + [HCO3-] where v£ is the velocity of the carboxylation reaction (total RuBPCase activity), V^^ is the theoretical maximum rate of reaction, [HCO.-] is the concentration of the substrate, and K^HCOg-) is the Michaelis constant for HCO3-. The kinetic parameters of equation [3.2] [Km(HC03 ) and Vmax] were calculated from the data in Figures 3.6 to 3.10 by the least squares method of Cleland (1979). The inset in each Fig. 3.6. RuBPCase activity versus HCO concentration in leaf tissue grown at 160 yl C0„ 1 i. Assay vials were flushed with N2 prior to assay. All reagents were prepared CO -free. Assays were performed at 30°C and pH 8.0 for 45 s in the presence of CA. The HCOg- concentrations are corrected for substrate consumption during assay. Data points are the means of triplicate assays. The solid curve is the predicted response based on Michaelis-Menten kinetics. The Km and Vmax were calculated from the data. Leaf samples were subsamples of tissue used for RuBPCase assays in Fig. 3.4 Inset. Double reciprocal plot of RuBPCase activity versus HC0~ concentration. 95 1000 V \- BOO > -~s y- o < 1 UJ (.n O 600 < o> _i >- E X (N o O CD rr CJ 400 < o o E Q_ ^ QQ » — 3 q: POO 4 6 HC03" (mM) 8 10 Fig. 3.7. RuBPCase activity versus HCO ~ concentration in leaf tissue grown at 280 yl CCL i_I. Assay vials were flushed with N2 prior to assay. All reagents were prepared CO -free. Assays were performed at 30°C and pH 8.0 for 45 s in the presence of CA. The HC03~ concentrations are corrected for substrate consumption during assay. Data points are the means of triplicate assays. The solid curve is the predicted response based on Michaelis-Menten kinetics. The Km and Vmax were calculated from the data. Leaf samples were subsamples of tissue used for RuBPCase assays in Fig. 3.4 Inset. Double reciprocal plot of RuBPCase activity versus HCO..- concentration. 97 1000 4 6 HC03" (mM) 10 Fig. 3.8. RuBPCase activity versus HCO concentration in leaf tissue grown at 330 jul CCL l"1. Assay vials were flushed with N2 prior to assay. All reagents were prepared CO -free. Assays were performed at 30°C and pH 8.0 for 45 s in the presence of CA. The HC03 concentrations are corrected for substrate consumption during assay. Data points are the means of triplicate assays. The solid curve is the predicted response based on Michaelis-Menten kinetics. The Km and Vmax were calculated from the data. Leaf samples were subsamples of tissue used for RuBPCase assays in Fig. 3.4 Inset. Double reciprocal plot of RuBPCase activity versus HCO ~ concentration. 99 1000 V h- 800 > *-*• \- jC u < i LU in O 600 < cp >- E X OJ o o CD O 400 < o U E CL d. QQ "> — Z3 cr ?()() 4 6 HC03" (mM) 8 10 Fig. 3.9. RuBPCase activity versus HCO ~ concentration in leaf tissue grown at 660 ul CCL 1 . Assay vials were flushed with N2 prior to assay. All reagents were prepared CO -free. Assays were performed at 30°C and pH 8.0 for 45 s in the presence of CA. The HC03" concentrations are corrected for substrate consumption during assay. Data points are the means of triplicate assays. The solid curve is the predicted response based on Michaelis-Menten kinetics. The Km and Vmax were calculated from the data. Leaf samples were subsamples of tissue used for RuBPCase assays in Fig. 3.4 Inset. Double reciprocal plot of RuBPCase activity versus HC03" concentration. 101 1000 4 6 HC03" (mM) 10 Fig. 3.10. RuBPCase activity versus HCO " concentration in leaf tissue grown at 990 ill (XL 1_1. Assay vials were flushed with N2 prior to assay. All reagents were prepared (XL-free. Assays were performed at 30°C and pH 8.0 for 45 s in the presence of CA. The HC03~ concentrations are corrected for substrate consumption during assay. Data points are the means of triplicate assays. The solid curve is the predicted response based on Michaelis-Menten kinetics. The Km and Vmax were calculated from the data. Leaf samples were subsamples of tissue used for RuBPCase assays in Fig. 3.4 Inset. Double reciprocal plot of RuBPCase activity versus HCO,, concentration. 103 H 800- • < L_ • ^^~ 990 jllI C02 •' $ <-> 600- g 2 l" leaf photosynthetic rates still declined in the 25 to 35°C range (Jurik et al., 1984; Sionit et al., 1984; Enoch and Hurd, 1977). However, at a C02 concentration of 1910 to 1960 ul C02 l"1 leaf photosynthetic rates of bigtooth aspen increased as leaf temperature was increased to 35°C but then decreased at higher temperatures (Jurik et al., 1984). This implies that leaf temperatures up to 35 °C were increasing photorespiration relative to photosynthesis but not causing heat damage to those plants. In studies with soybean, Hofstra and Hesketh (1969) reported an increase in leaf photosynthetic rates as leaf temperature increased from 20 to 40°C. Gourdon and Planchon (1982), working with two cultivars, observed either no effect or a decrease in maximum leaf photosynthetic rates as air temperature was increased from 25 to 30°C. In addition to the above mentioned response of enzyme kinetic parameters, temperature may have other effects on RuBPCase. Growth at low temperatures (2 to 4°C during cold hardening of rye plants) for a few weeks resulted in an RuBPCase that had an increased activity and a greater specific activity when compared to plants grown at 25°C (Huner and Macdowall, 1979). These changes were found to be related to a conformational change in the enzyme (Huner and Macdowall, 1978). Growth temperature, unlike the assay temperature, did not affect RuBPCase kinetics in Nerium oleander (Bjorkman et al., 1978). It may, however, be responsible for changes in the quantity of RuBPCase in N. 119 oleander (Osmond et al., 1980). In several experiments where plants were grown at different temperatures ranging from 20 to 45°C (and assayed at a common temperature) the activity of the enzyme was found to be little affected in cotton (Downton and Slayter, 1972) but decreased as the growth temperature of N. oleander and the C, Atriplex lentiformis increased (Bjorkman et al., 1978; Pearcy, 1977). There are little data concerning the effect of growth temperature on levels of RuBP. Berry and Downton (1982) cited work by J. Collatz showing decreased levels of RuBP at temperatures above the photosynthesis temperature optimum. According to Berry and Bjorkman (1980), a substantial decline in the rate of electron transport at higher temperatures could result in a reduction in photophosphorylation or NADPH production leading to the lower levels of RuBP. The objectives of this study were to determine the effects of air temperature during growth on the activity of RuBPCase and the level of RuBP in soybean grown at atmospheric and twice atmospheric concentrations of C02. Air temperature effects on canopy photosynthesis were also investigated. Materials and Methods Plant Material and Growth Conditions Soybeans (Glycine max L. Merr. cv Bragg) were planted in six outdoor environmentally controlled plant growth chambers (described in Chapter II) on 8 Sept. 1985. Three of the chambers had atmospheric CO concentrations controlled (from seed to harvest) to 330 /il CO l_1 120 the remaining three chambers were controlled to 660 jul CO 1_1. Three day/night air temperature regimes were maintained at each CO treatment. The regimes were 26/19°, 31/24°, and 36/29°C and are referred to by the daytime temperatures throughout this chapter. The dew point temperatures maintained in the chambers were 12, 16, and 20°C for the 26, 31, and 36°C air temperature regimes. As in previous experiments (Chapters II and III) the chambers received natural solar irradiation. Leaf tissue was collected at midday, on November 4 (57 DAP), for RuBPCase assays and RuBP determinations. This sampling day was cloud-free. The plant density was 32 plants m~2. Estimates of the canopy LAI's for plants grown at 330 ul CO 1_1 were 4.13, 4.89, and 5.62 and for 660 ul C02 1_1 grown plants 5.52, 7.92, and 7.94 for growth temperatures of 26, 31, and 36°C, respectively. The plant sampling procedures described in Chapter II were followed. Canopy Leaf Temperature and Vapor Pressure Deficit Leaf temperatures within the upper canopies were measured using an IR temperature transducer (Everest Interscience , series 4000) mounted inside the plant growth chambers. Temperature measurements from three days, November 9, 10, and 12 (62, 63, and 65 DAP), were used to calculate leaf temperature response to air temperature and C0? concentration. The vapor pressure deficit (VPD) was calculated using the dry bulb and dew point temperatures of the air inside the growth chambers, according to Murray (1967). The VPD calculations were performed using data collected during the same time periods in which leaf temperature 121 measurements were made. The plants were non-water-stressed during these measurements . Canopy Photosynthesis Measurements Canopy net photosynthesis was measured as described in Chapter III. Measurements were made on November 4 (57 DAP), between the hours of 1100 and 1230 EST when quantum flux density (400 to 700 nm) at the upper canopy level was 1250 to 1350 umol quanta m~2 s_1. The reported photosynthetic rates are the means of six measurements at each treatment. Canopy net photosynthesis is expressed on a land area basis. RuBP Carboxylase Assay The assay procedure for RuBPCase activity was similar to the previously described methods with some modifications. A quantity of frozen leaf powder (70 to 150 mg dry weight) was removed from liquid N2 storage and placed in a pre-chilled Ten Broeck tissue homogenizer. Added to the leaf powder was 5 ml of extraction buffer consisting of 100 mM Tris-HCl (pH 8.0), 5 mM DTT, 10 mM isoascorbate , and 1.5% (w/v) PVP-40. The leaf tissue was homogenized for approximately 60 s at 0°C. An aliquot of the homogenate was reserved for chlorophyll determination and the remainder was centrifuged at 12,000 g for 3 min. The supernatant of the crude extract was either used immediately to initiate the initial RuBPCase assays or was activated (as described later) and then used to initiate the total RuBPCase assays. Assays were performed in triplicate at 26, 31, or 36°C in a waterbath with 122 continuous shaking (125 strokes min-1). The assay buffer consisted of 50 mM Tris-HCl (pH 8.0), 5 mM DTT, 5 mM MgClj, and 10 mM isoascorbate. To the assay buffer was added 0.5 mM RuBP. The 5-ml glass vials were sealed with screw-on septum caps and through the septum was added 20 mM NaH (X>2 (7.54 GBq/mol). Initial RuBPCase assays were initiated with the injection of 50 yl of crude extract. For total RuBPCase activity assays 50 yl of crude extract was injected into the assay vials minus RuBP. The enzyme was allowed to activate for 5 min at the assay temperature. Following activation the assay was initiated with the injection of RuBP. For all assays, the total assay volume was 5 ml. Assays were terminated after 45 s with the injection of 0.1 ml of 6 N formic acid in methanol. Determination of acid-stable C products was as described in Chapter III. RuBP Determination The assay procedure for RuBP was similar to that described in Chapter II with some minor modifications. A quantity of frozen leaf powder (70 to 150 mg dry weight) was removed from liquid N„ storage and placed in a pre- chilled Ten Broeck tissue homogenizer. Added to the leaf powder was 5 ml of 0.5 N HC1 at 0°C. The leaf tissue was homogenized for approximately 60 s at 0°C and an aliquot was reserved for chlorophyll determination. The remaining homogenate was centrifuged at 12,000 g for 5 min. To 2.5 ml of the supernatant was added 0.37 ml 2M Tris base and 0.22 ml 4 N K0H. The neutralized supernatant (pH 8.3) was then stored on ice. The assay buffer consisted of 50 mM Tris-HCl (pH 8.0), 10 mM MgCU, and 5 mM DTT. The 123 assay buffer was added to 5-ml glass assay vials which were capped with screw-on septum caps. Through the septum was injected 20 mM NaH C02 (7.54 GBq/mol) and 0.25 ml neutralized leaf extract supernatant. The assay was initiated by the injection of 50 /jl of activated RuBPCase from tobacco (as described in Chapter II). Assays were performed in triplicate at 26°C in a waterbath with continuous shaking (125 strokes min~ ). Total assay volume was 0.5 ml. After 60 min the assay was terminated with the injection of 0.1 ml of 6 N formic acid in methanol. Determination of acid-stable C products was as described in Chapter III. Chlorophyll Determination Chlorophyll was determined in sample extracts used for RuBPCase activity and RuBP assays by the methods described in Chapter II. Analysis of Statistical Significance Simple linear and quadratic regression analyses were performed to determine the statistical significance (at the 5% level) of experimental results. In this chapter, air temperature during growth was used as regressor. The methods used are described further in Chapter II. Regression parameters are tabulated in Appendix C. Results Canopy Leaf Temperature The temperature of leaves in the upper canopies was always several degrees below the air temperature, regardless of the C0„ or 124 air temperature treatment. Increases in air temperature were accompanied by increases of a lesser magnitude in leaf temperature. The measured difference in dry bulb temperature between chambers at the two lowest control temperatures (26 and 31°C) were actually 4.9 and 4.6°C for the 330 and 660 Ul C02 l"1 treatments, whereas the difference in leaf temperatures were only 2.2 and 1.9°C, respectively. The measured difference in dry bulb temperatures between the lowest and highest control temperatures (26 and 36°C) were actually 9.6 and 9.0°C for the 330 and 660 yl C02 l"1 treatments, whereas the difference in leaf temperatures were only 3.5 and 4.4°C, respectively. The lower temperature of the leaves, with respect to the air, was due to evaporative cooling. The mean leaf to air temperature differential ^TL~Ta) and VPD were calculated and were found to be closely related. A linear regression of TL-TA with VPD as regressor yielded a correlation coefficient of -0.993. At 330 yl CO l-1 T -T and VPD were, for the 26°C treatment, -3.1°C and 1.78 kPa, for the 31°C treatment, -5.8°C and 2.53 kPa, and for the 36°C treatment, -9.2°C and 3.87 kPa. At 660 yl Co2 1_1 TL-TA and VPD were, for the 26°C treatment -3.4°C and 1.96 kPa, for the 31°C treatment, -6.1°C and 2.77 kPa, and for the 36°C treatment, -8.0°C and 3.56 kPa. Canopy Net Photosynthesis^ Within the range of temperatures used in the experiments reported here, air temperature during growth of soybeans had no effect on the maximum midday rates of canopy net photosynthesis (Table 4.1). At each C02 concentration the differences in canopy net photosynthesis at 125 Table 4.1. Effect of air temperature on maximum canopy net photosynthetic rates. Plants were grown at 330 or 660 111 (XL 1 . Measurements were made between 1100 and 1230 EST on November 4 (57 DAP). The quantum flux density was 1250 to 1350 nmol quanta m s at the upper canopy level. The plant density at all treatments was 32 plants m- . Growth temperature Canopy Net Photosynthesis °C 330 ul C02 l-1 _2 60 nl C02 l"1 "jLimol C0„ m s" 26 31 36 29.3 ± 1.8 a 28.1 ± 1.7 30.6 ± 1.7 55.4 ± 1.2 55.0 ± 1.0 54.5 ± 2.3 ^ean ± SD. 126 growth air temperatures of 26, 31, and 36°C were not significant. The canopy net photosynthetic rates averaged 87% greater in the 660 compared to the 330 ul (X>2 l"1 grown canopies. RuBP Carboxylase Activity Initial and total RuBPCase activities were assayed at both 26°C and at the growth temperature for plants grown at 330 and 660 yl C02 1 . Initial RuBPCase activity (Figure 4.1) showed no significant response to growth air temperature or C02 treatment when assayed at 26°C. When assayed at the growth temperature initial activity increased with temperature. Initial activity increased 76% when assay temperature was raised from 26 to 36°C in the 330 ul C0„ l"1 grown plants and by 53% in the 660 ul C02 l"1 grown plants. Total RuBPCase activity (Figure 4.2) also showed no significant response to growth air temperature or C02 treatment when assayed at 26°C. Total activity increased with temperature when assayed at the plant growth temperature. Total activity increased 37% when assay temperature was raised from 26 to 36°C in the 330 ul C02 1_1 grown plants and by 23% in the 660 yl C02 1" grown plants. These increases correspond to Q,„ values (between 26 and 36°C) of 1.7 and 1.4 for 330 ul CO l"1 grown plants and 1.5 and 1.2 for 660 yl C02 l"1 grown plants for initial and total RuBPCase activity, respectively. The percent activation of RuBPCase was calculated from data in Figures 4.1 and 4.2 and is shown in Figure 4.3. When assayed at 26°C (Figure 4.3 A) activation was in the range of 73 to 81% for all growth temperatures in both C0„ treatments. The response of RuBPCase activation to growth air Fig. 4.1. Initial RuBPCase activity versus growth air temperature for 330 (•) and 660 (o) yl C0„ 1_1 grown plants. RuBPCase was assayed at either 26 °C (broken line) or at the growth temperature (solid line). Assays were performed at pH S.O for 45 s. Data points are the means of triplicate assays. Plant samples were collected November 4 (57 DAP). 128 >■ > b < iLl CO < -J >- g 00 CZ < o Q_ GD tr 1000 800 ° 600- E < g 400 E * 200- 0 - = = ^-^ 330 Ml C02 I'1 ► ASSAYED AT GROWTH TEMPERATURE •--•ASSAYED AT 26° C 660 Ml CO |-l °"~ °ASSAYED AT GROWTH TEMPERATURE 0--0 ASSAYED AT 26° C 25 i r 30 "I r 35 GROWTH TEMPERATURE ( °C ) Fig. 4.2. Total RuBPCase activity versus growth air -1 temperature for 330 (•) and 660 (o) yl CO 1 grown plants. RuBPCase was assayed at either 26°C^ (broken line) or at the growth temperature (solid line). Assays were performed at pH 8.0 for 45 s. Data points are the means of triplicate assays. Plant samples were collected November 4 (57 DAP) . 130 > I- O < UJ if) < _J g CD < o Q. CD ■3 QC _J O I- 1000 800 1 o 600 0 £ CM O O 400 "5 £ 200 - ""***"•*#-- **■ 330 Ml co2 r • 9 ASSAYED AT GROWTH TEMPERATURE • --•ASSAYED AT 26° C )ASSAYED AT GROWTH TEMPERATURE 660 Ml CO, f ' 2 O - - OASSAYED" AT 26° C l 1 1 1 1 r 25 30 1 1 1 r 35 GROWTH TEMPERATURE (°C) Fig. 4.3. A, B. RuBPCase activation (%) versus growth air temperature f^r plants grown at 330 (•) or 660 (o) yl CO2 1 . Activation was calculated from data in Figs. 4.1 and 4.2. Assays were performed at A) 26°C, and B) the respective growth temperatures. 100 132 75- 5^ O < UJ CO < 50- i= 25 0 >- x o m 75 < o m 50 cr 25- ASSAYED AT 26° C 330 jllI C02 I* 660 jllI C02 I" ASSAYED AT GROWTH TEMPERATURE • 330 jllI COg I"1 o 660 jiil C02 I"1 0 25 30 35 GROWTH TEMPERATURE (°C) 133 temperature was minimal, however, it was found to be statistically significant in the 330 til C02 i"1, but not the 660 «1 C02 l"1, treatment. When assayed at growth temperatures (Figure 4.3 B) activation was between 74 and 80% for assay temperatures of 26 and 31°C. When assayed at 36°C the activation climbed to greater than 93%. RuBP Levels The levels of RuBP are plotted against growth air temperature for both C02 treatments in Figure 4.4. The highest levels of RuBP were found in plants grown at 26° C. In both C02 treatments the RuBP level decreased significantly as growth air temperature increased above 26°C. There was not a significant difference in the response of RuBP to growth air temperature between the two growth C0„ concentrations. Based on assumptions made in Chapter II (concerning cellular location of RuBP and stromal volume) the chloroplast concentration of RuBP (mM) in leaves grown at air temperatures below 31°C was quite a bit greater than the 3 to 4 mM active site concentration of RuBPCase (Jensen and Bahr, 1977). At 31 and 36°C, RuBP levels were approaching, but still greater than, the RuPBCase active site concentration. Discussion The lack of response of maximum canopy photosynthetic rates, within a C02 treatment, to growth air temperature is due, in part, to the fact that leaf temperatures remained several degrees cooler than air temperatures. There was a smaller differential in leaf Fig. 4.4. Levels of RuBP versus growth air temperature for plants grown at 330 (•) or 660 (o) ul m i-1. Chloroplast concentration of RuBP (mM) assumes 25 ul stroma volume mg chlorophyll . Data points are the means of triplicate assays. Leaf samples were subsamples of tissue used for RuBPCase assays in Figs. 4.1 and 4.2. 135 250 - 200 o en E o E c Q. CD 150 100- 50- 0 25 • 330 jllI C02 I"1 ° 660 jul C02 I"1 "i 1 1 — 30 -i 1 1 r 35 GROWTH TEMPERATURE (°C) 10 -8 "6 Q_ 4 ? 0 136 temperatures, between treatments, than in air temperatures. At 330 yl C02 1 , when air temperature was increased by 4.9 and 9.6°C, leaf temperature only increased by 2.2 and 3.5°C, and at 660 yl CO l_1, when air temperature was increased by A. 6 and 9.0°C, leaf temperature only increased by 1.9 and 4.4°C. The leaf temperature remained below the air temperature due to evaporative cooling of the leaves. As the dry bulb temperatures were increased the magnitude of Ty-TA became greater. This was due to an increasing VPD, as a result of greater increases in dry bulb temperatures relative to dew point temperatures. There was a strong correlation between increasing VPD and the increasing magnitude of TL-TA, due to evaporative cooling. Generally speaking, TL-TA becomes more negative as VPD increases (Ehrler, 1973; Idso, 1982). The value of TL~TA is typically negative when plants are well-watered (Idso, 1982),. as was the case with the plants in this study. Thus, as the dry bulb temperatures were increased (both the absolute temperature and relative to the dew point temperature) VPD increased, as did evaporative cooling, resulting in a greater differential between leaf and air temperature. The effect of this relationship was that leaf temperatures were not as high, nor spanned as wide a range, as did air temperatures. Jones et al. (1985a) also reported a lack of response of canopy photosynthetic rates when soybeans were exposed to different air temperatures. In the experiments of Jones et al. (1985a), plants were grown at 31°C and canopy photosynthesis was measured at air temperatures of 28 and 33°C (and constant dew point temperature) with no apparent difference in rates. 137 Higher photosynthetic rates in the high C02 canopies (Table 4.1) is a widely observed response and is similar to results in Figure 3.3 (Chapter III). Both initial and total RuBPCase activities, when assayed at 26°C, were independent of growth air temperature and C0„ concentration. These results (with regard to temperature) agree with the results expressed on a leaf area basis of Downton and Slayter (1972) working with cotton. However, when Downton and Slayter (1972) expressed their results on a chlorophyll basis (as are the data in Figures 4.1 and 4.2), increases in growth temperature from from 25 to 40°C caused a decrease in RuBPCase activity. This was the result of an increase in chlorophyll per unit leaf area as growth temperature increased. Phillips and McWilliam (1971) found a small decrease in the specific activity of RuBPCase (assayed at 25°C) in wheat as the growth temperature increased over the range of approximately 25 to 35°C. When RuBPCase was assayed at the respective growth temperature (Figures 4.1 and 4.2) the activities were greater at higher temperatures. This is not surprising as higher temperatures typically increase reaction rates. The greater RuBPCase activity at increasing temperatures is not contradictory with observed decreases in leaf photosynthesis as temperature increases (Jurik et al., 1984; Monson et al., 1982; Enoch and Hurd, 1977). As Jordan and Ogren (1984) have shown, photosynthesis decreases relative to photorespiration at higher temperatures, but increasing C02 can overcome this decline (Osmond et al., 1980). When RuBPCase was assayed in the experiments reported here, inorganic carbon was present at saturating levels and thus the 138 oxygenase reaction was essentially eliminated. Hence, the higher temperatures during the enzyme assays increased carboxylase reaction rates but probably did not increase oxygenase reaction rates. While RuBPCase activity assayed at growth temperatures indicates the potential CC^ assimilation rates, they should not be regarded as accurate reflections of in situ leaf photosynthetic rates. The activation of RuBPCase was not greatly affected by CCL or air temperature during growth. The significant decrease in RuBP levels with increasing growth air temperature was observed in both CCL treatments. Berry and Bjorkman (1980) have suggested that reduced rates of photophosphorylation and NADPH production could cause decreased RuBP levels at higher temperatures. The increased rates of RuBP consumption as both the RuBP carboxylase and RuBP oxygenase activities increase in vivo with temperature is probably also a factor. The results presented here indicate that air temperature during growth had no significant effect on RuBPCase activity when assayed at a common temperature. The RuBP levels decreased significantly as temperature increased, possibly as a result of greater consumption due to higher rates of both carboxylation and oxygenation occurring in vivo. At a given CC^ concentration, air temperature had no effect on maximum canopy photosynthetic rates. The lack of effect of air temperature on RuBPCase activity and canopy photosynthesis is due, partially, to the fact that evaporative cooling kept the leaves from reaching temperatures as high as the air. The fact that RuBP levels did respond to the increases in temperature suggests that RuBP may be more sensitive to moderate changes in the environment. CHAPTER V GENERAL SUMMARY AND CONCLUSIONS The studies described in the previous chapters were initiated to examine the effects of C02 concentration on photosynthesis in soybean. The focus of these studies was placed on the enzyme (RuBPCase) and the substrate (RuBP) responsible for the C02 fixation reaction. In addition, leaf characteristics such as chlorophyll and protein content were also examined for response to the CCL treatments. The photosynthetic rate response of soybean leaflets to a wide range of C02 concentrations indicated plants grown at twice atmospheric concentrations of C02 had higher rates than plants grown at atmospheric C02 concentrations. These measurements, made under conditions of high solar irradiance and during the pod filling stage, indicate an enhanced leaf photosynthetic capacity for plants grown at an elevated C02 concentration. This supports the hypothesis stated in Chapter I. There was no significant effect of the high C0„ treatment on chlorophyll or protein content nor on the activity of RuBPCase (all expressed on a leaf area basis). Furthermore, RuBP was present in what appeared to be saturating concentrations for photosynthesis and therefore may not have been limiting photosynthetic rates in either C02 treatment. These results apparently preclude these characteristics from playing a significant role in the enhanced photosynthetic capacity. Previously published and non-published results of other researchers have shown growth at high C0„ 139 140 concentrations to result in changes in the leaf mesophyll of soybean. These changes can lead to increased photosynthetic rates, and may have occurred in the studies reported here. It is concluded that the enhanced photosynthetic capacity of leaflets grown at elevated CCL may be a result of an increase in the mesophyll cell surface area/external leaf surface area ratio, or, as a result of the increased assimilate demand of plants grown at elevated CC>2 with their greater pod weights. Alternatively, a combination of these two factors or possibly an as yet to be described factor may also be involved. Long-term growth of soybeans at C02 concentrations ranging from subatmospheric to superatmospheric provided information on the response to CC>2 of RuBPCase activity and kinetics and RuBP, as well as on canopy photosynthesis. The level of RuBP in leaves (on a chlorophyll basis) decreased at the higher CCL concentrations. It appeared that RuBP was probably at saturating concentrations for was photosynthesis, however, at high C02 the concentration of RuBP approaching the concentration of RuBPCase active sites. The initial and total activities of RuBPCase (on a chlorophyll basis) decreased following growth at increasing concentrations of CCL . The apparent Kn/C02-) and Vmax of RuBPCase a!so demonstrated small decreases with increasing C02 concentrations. Since initial RuBPCase activity is an estimate of in vivo enzyme activity, these data suggest that RuBPCase activity in the leaf may be reduced following exposure to high concentrations of CCL.. Whether this reduction in activity is a result of a lowered specific activity or a decrease in the quantity of RuBPCase protein was not determined. Rates of canopy photosynthesis 141 increased with C02> Since the LAI also increased two-fold over the C02 range, at least part of the canopy photosynthetic response was due to a greater photosynthetic surface area. At high CO light becomes more important as a limiting factor for maximum canopy photosynthesis. The effects of temperature on RuBPCase, RuBP, and canopy photosynthesis were investigated in soybeans grown at atmospheric and twice atmospheric concentrations of CO,,. Canopy photosynthetic rates were independent of air temperature but were greater at the higher CO concentration. The activity of RuBPCase (on a chlorophyll basis) was also independent of air temperature, although the level of RuBP (on a chlorophyll basis) did decrease with increasing temperature. Due to the increased VPD at higher air temperatures, and as a result of evaporative cooling, leaf temperatures were not as high as air temperatures. The actual temperature treatments were apparently not great enough to cause significant effects with respect to RuBPCase activity or canopy photosynthesis. It is concluded that in addition to the previously mentioned enhanced leaflet photosynthetic capacity, growth of soybeans at elevated C02 concentrations may result in reduced RuBPCase activity (expressed on a chlorophyll basis). The physiological significance (if any) of a small decrease in Km(C02) is not known. It is also concluded that the response of RuBP to short-term changes in the CO concentration, increases in temperature, and its rapid turnover rate, suggest that the leaf RuBP concentration may be more sensitive to moderate changes in the environment than RuBPCase activity or photosynthetic rates. 142 Because of the importance of the process of photosynthesis, and to better understand the response of plants to CCL, future work on these problems could proceed into a number of areas. Several questions remain unanswered following the studies presented here. These questions can be used to define areas for future research. Specifically, four such areas are described. 1 . Determine if the decrease in RuBPCase activity with increasing CC^ concentration represents a reduction in the quantity of RuBPCase protein. 2. Once RuBPCase is quantified, examine the relationship between the concentration of RuBPCase active sites and the concentration of RuBP in the leaf at elevated CCL concentrations. 3. Pursue an investigation of the effects of CCL on internal leaf anatomy and how any changes may affect photosynthetic rates. 4. Continue efforts to describe the relationship between carbon fixation rate, translocation, and assimilate demand. This is an area that has received the attention of researchers in many areas of plant physiology. Because of the effects of CC>2 on photosynthesis and yield, this relationship should be examined with respect to the concentration of CCL. These areas of research should yield information helpful in better understanding photosynthesis and potentially useful in increasing agricultural productivity. APPENDIX A LEAF AND CANOPY PHOTOSYNTHETIC RATE RESPONSES TO LIGHT AT TWO C02 CONCENTRATIONS Introduction Increasing irradiation typically results in higher photosynthetic rates. Carbon dioxide interacts with the photosynthetic rate response to light via the quantum yield. Quantum yield is essentially the efficiency of utilization of absorbed light (Radmer and Kok, 1977). At high C02 the quantum yield increases due to the reduction in RuBP oxygenase activity (Ehleringer and Bjorkman, 1977). Increasing the supply of C02, therefore, not only provides more substrate for assimilation, it also improves the efficiency of use of light energy. Materials and Methods Plant Material and Growth Conditions The plants and growth conditions used in this study were the same as described in Chapter II, the only difference being that this study was conducted later in the same season. Estimations of the canopy LAI were made prior to and following the photosynthesis measurements and indicated the LAI was 25 to 30% greater in the 660 compared to the 330 Ul C02 L grown canopy. 143 144 Leaf and Canopy Photosynthesis Measurements Both leaf and canopy photosynthesis measurements were made on 14 Nov. 1983 (76 DAP). This was a cloud-free day with a maximum quantum flux density, as measured outside of the growth chambers, of 1350 jumol -2 -1 quanta m s . The growth and leaf chambers each transmitted 88% of the light incident to their upper surfaces. The appropriate corrections for transmission of solar irradiance were made in Figures A.l and A. 2. Leaf photosynthetic rates were measured as described in Chapter II. At each CO,, concentration they are the combined responses of two leaflets. Canopy photosynthetic rates were measured as described in Chapter III. Both leaf and canopy measurements were made at the respective growth C02 concentrations. The different quantum flux densities represent the natural daily variation in solar irradiance. Leaf photosynthetic rates are expressed on a leaf area basis while canopy rates are on a land area basis. Results and Discussion The leaf photosynthetic rate increased with light in both C0„ treatments (Figure A.l). At all light intensities, the high C0„ grown leaves had greater photosynthetic rates. The absolute difference in rates was greatest at high light intensity. Leaves from both C02 treatments appeared to light saturate at 900 to 1000 umol quanta m~ 2 s . Canopy photosynthetic rates also increased with light intensity at both C02 concentrations (Figure A. 2). As was the case with leaves, the absolute difference in rates was greatest at high light intensity. Fig. A.l. Leaf net photosynthesis versus quantum flux density for plants grown and measured at 330 (•) and 660 (o) ul CO 1 . Measurements were made on November 14 (75 DAP). Data points represent mean values of two leaflets at each C0„ concentration. Vertical lines through points are ± SD for photosynthetic rates and horizontal lines are ± SD for quantum flux density. Photosynthesis is expressed on a leaf area basis. 146 45 30- E 3,5 III 1 lto >- CM (/) o b h- o o o-t GROWTH C02 CONCENTRATION • 330 Ml C02 I"1 O 660 Ml C02 I"1 300 600 900 QUANTUM FLUX DENSITY (Mmol quanta m'2 s"1) 1200 Fig. A. 2. Canopy net photosynthesis versus quantum flux density for canopies grown and measured at 330 (•) and 660 (o) u\ m 1 . Measurements were made on November 14 (76 DAP). Data points represent mean values. Vertical lines through points are ± SD for photosynthetic rates and horizontal lines are ± SD for quantum flux density. Photosynthesis is expressed on a land area basis. 148 60- c/) UJ X K ■z. > 45- u CL o < CVJ O O o E 30- 15- o- GROWTH C02 CONCENTRATION • 330 Ml C02 I"1 660 Ml C02 I 300 600 900 1200 QUANTUM FLUX DENSITY (jumol quanta nf2 s"1 ) 149 Light saturation of canopy photosynthesis was not observed in either canopy . The responses to light of leaf and canopy photosynthesis are similar except for light saturation in the leaves at high light intensity. This is not surprising considering the leaf rates were from leaves in the upper unshaded layer of the canopy. The leaves in the upper layer of a canopy contribute, by far, the majority of the photosynthetic response (Acock et al., 1978; Hatfield and Carlson, 1977). Hatfield and Carlson (1977) reported that 80% of the C0? uptake in a soybean canopy occurred in the upper 20% of the canopy. The higher photosynthetic rates in leaves grown and measured at 660 nl C02 1 are due to the greater supply of substrate for assimilation, increased quantum yield, and the inherent capacity for increased photosynthesis in soybean leaves grown in elevated C09 (discussed in Chapter II). Canopy photosynthetic rates are increased by the same factors described above for leaves. Additionally, increased light intensity and C02 concentration improves the supply (to the lower levels of the canopy) of energy and substrate that normally become attenuated with depth in the canopy. APPENDIX B EFFECT OF LEAF SAMPLE SIZE ON IN VITRO RuBP CARBOXYLASE ACTIVITY Introduction The degree of activation as well as the quantity of RuBPCase are key factors in the regulation of C02 assimilation in vivo (Jensen and Bahr, 1977). It has been shown that activation of RuBPCase is dependent on Mg + and C02 (Laing and Christeller, 1976; Lorimer et al., 1976). Assays of RuBPCase following incubation of the enzyme 2+ with added Mg and HC03 yield "total" activity, while assays without 2+ added Mg and HC0„ yield "initial" activity (Perchorowicz et al., 1981). The initial activity is often used as an estimate of the in vivo RuBPCase activity (Perchorowicz et al., 1982). The percent activation is calculated by the ratio of initial to total activity times 100%. It thus provides an idea of the activation status of RuBPCase in_ vivo. Materials and Methods Plant Material and Growth Conditions Field grown soybeans (Glycine max L. Merr.) were used to supply leaf tissue in two successive years for this study. On 15 Aug. 1984 (75 DAP), leaves of soybean (cv Braxton) were collected and on 15 May 1985 (55 DAP), leaves of soybean (cv Biloxi) were collected. On both 150 151 dates leaves were sampled rapidly and were immediately plunged into liquid N2, ground to a powder, and stored in liquid No as described in Chapter II. RuBP Carboxylase Assay Assays of RuBPCase were performed as described in Chapter II with the exception that 10 mM MgCl2 was added to the extraction buffer where noted. Results and Discussion Initial and total RuBPCase activities are shown in Figure B.l as a function of leaf sample size used in the assay. Total activity was not affected by sample size. Below a leaf sample size of approximately 150 mg (dry weight), initial activity decreased with decreasing sample size. The percent activation (Figure B.2) shows the same dependence on sample size below 150 mg (dry weight). All samples were homogenized in 10 ml of extraction buffer, hence, the smaller sample sizes were effectively diluted to a greater degree. Apparently with small amounts of leaf material the endogenous Mg present in the tissue becomes too dilute and RuBPCase deactivation occurs prior to the assay. Incubation of RuBPCase with Mg2+ during the activation process in the total activity assay prevents this deactivation. To determine if Mg2+ added during RuBPCase extraction would prevent deactivation in the tissue samples, 10 mM MgCl„ was added to the extraction buffer of some assays. The addition of MgCl„ to small tissue samples increased the percent activation over two-fold. With Fig. B.l. Initial ( ) and total ( ) RuBPCase activity versus leaf sample size used in assay. RuBPCase was extracted without added Mg . Assays were performed at 30°C at pH 8.5 for 45 s. Leaves were collected from field grown plants at 75 DAP, 153 1,000- 800- «2 6 600- _| o> >• £ x 1 8 3 | 0-3 m 400- 200- TOTAL ACTIVITY INITIAL ACTIVITY 50 100 150 200 250 LEAF SAMPLE SIZE (mg dry weight) 300 350 Fig. B.2. Percent activation of RuBPCase versus leaf sample size used in assay. The 1984 results (•) were calculated from activities in Fig. B.l where no MgCl2 was added to the extraction buffer. The 1985 results (a) were calculated from activities with and without 10 mM MgCl2 added to the extraction buffer. Except tor added MgCl? all assays followed the same procedure. The 1985 results are from leaves collected from field grown plants at 55 DAP. 100- 155 ^80H o ▲ (+MgCI2) 60- x 40- o Q. cd 20- • 1984 FIELD GROWN A 1985 FIELD GROWN 50 100 150 200 250 LEAF SAMPLE SIZE (mg dry weight) 300 350 156 the added Mg the activation level was similar to that measured in the larger tissue samples without added MgCl2. With added MgCl9 activation also increased in samples weighing more than 250 mg (dry weight) although not as dramatically as with the smaller samples. Servaites (1984) showed a similar dependence of the initial activity on the ratio of leaf tissue weight to extraction buffer volume. While there may be factors other than Mg2+ concentration involved in the loss of initial activity, use of an appropriate leaf sample size is recommended to avoid problems of excess dilution of Mg2+ and possibly other endogenous leaf effectors. APPENDIX C LINEAR REGRESSION PARAMETERS Table C.l. Linear regression parameters (for short-term CO, concentrations) for data in Chapter II. i Figure/Tabl e Dependent variable Regression parameters Model 60 eco2 R2 Fig. 2.1 Ci (330) -17.37**1 0.721** 0.971 Fig. 2.1 Ci (660) - 9.01NS 0.546** 0.932 Fig. 2.4A initial RuBPCase (330) 100.79** -0.010NS 0.143 Fig. 2.4A initial RuBPCase (660) 104.36** -0.010NS 0.056 Fig. 2.4B total RuBPCase (330) 107.90** -0.001NS 0.003 Fig. 2.4B total RuBPCase (660) 113.61** -0.002NS 0.004 Fig. 2.5 RuBPCase activation (330) 93.69** -0.009* 0.315 Fig. 2.5 RuBPCase activation (660) 91.72** -0.008NS 0.142 Fig. 2.6 RuBP (330) 84 . 29** -0.030** 0.267 Fig. 2.6 RuBP (660) 63.07** -0.014** 0.469 Table 2.3 initial RuBPCase (330) 763.67** -0.O77NS 0.142 Table 2.3 initial RuBPCase (660) 722.52** -0.077NS 0.065 Table 2.3 total RuBPCase (330) 817.58** -0.014** 0.003 Table 2.3 total RuBPCase (660) 786 . 49** -0.016NS 0.004 Table 2.4 RuBP (330) 132.66** -0.031** 0.479 Table 2.4 RuBP (660) 161.40** -0.057** 0.262 Regression parameter = 0 rejected at a probability level of 0.05 (**) or 0.01 (*), or not significant (NS). 158 Table C.2. Linear regression parameters (for growth C0o concentration) for data in Chapter III. 2 159 Figure/Table Dependent variable Regression parameters Table 3.1 SLW Table 3.1 Table 3.1 Table 3.1 Table 3.2 Table 3.2 Fig. 3.1 Fig. 3.4A Fig. 3.4A Fig. 3.4B Fig. 3.5 Tabl b 3.3 Tabic 2 3.3 LAI chlorophyll protein chlorophyll protein protein/chlorophyll ratio initial RuBPCase total RuBPCase RuBPCase activation RuBP max So 78": 1 1 . 58** 0.02** 0.28** 0.01** 0.14** 12.18** 789.42** 812.35** 97 . 10** 191.89** 16.51** 1040.44** *1 eco2 0.001** 0.001** -7*10-7NS -6*10-5NS -5*10-6** -7*10-5** -0.002** -0.243** -0.189 -0.009** -0.144** -0.002** -0.181** Model R 0.559 0.565 0.009 0.310 0.674 0.748 0.287 0.692 0.712 0.226 0.584 0.423 0.645 Regression parameter = 0 rejected at a probability level of 0 05 ("'0 or not significant (NS). 160 Table C.3. Linear regression parameters (for growth air temperature) for data in Chapter IV. Figure/Table Dependent variable Regression parameters Model eo Bco2 R2 Table 4.1 canopy photosynthesis (330) 25 . 58-""* 1 0.121NS 0.069 Table 4.1 canopy photosynthesis (660) 57.62** -0.085NS 0.051 Fig. 4.1 initial RuBPCase 26° (330) 399.87** 2.233NS 0.043 Fig. 4.1 initial RuBPCase 26° (660) 503.07** -1.300NS 0.033 Fig. 4.2 total RuBPCase 26° (330) 694.41** -2.966NS 0.041 Fig. 4.2 total RuBPCase 26° (660) 733.12** -3.900NS 0.246 Fig. 4.3A RuBPCase activation 26°(330) 55.96** 0.700** 0.633 Fig. 4.3A RuBPCase activation 26° (660) 68.10** 0.233NS 0.102 Fig. 4.4 RuBP (330) 414.73** -8.066** 0.643 Fig. 4.4 RuBP (660) 267.17** -4.066** 0.980 Regression parameter = 0 rejected at a probability level of 0.05 (**) or not significant (NS). LITERATURE CITED Acock, B., D. A. Charles-Edwards, D. J. Fitter, D. W. Hand, L. J. Ludwig, J. Warren Wilson, and A. C. Withers. 1978. 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BIOGRAPHICAL SKETCH William James Campbell, Jr. was born on March 24, 1951, in Elmira, New York. He attended schools in New York and Florida prior to enrolling at the University of South Florida in 1968. Mr. Campbell received the Bachelor of Arts degree, with a major in zoology, in 1972. Following graduation, he was employed by the State of Florida Division of Health and the Agricultural Engineering Department at the University of Florida. While employed at UF he enrolled in the Graduate School, and in 1979 received the Master of Science degree from the Agricultural Engineering Department. In 1981, Mr. Campbell entered the Agronomy Department and began work toward the Ph.D. degree. 181 I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. L. H. Allen, Jr. , Chairman Associate Professor of Agronomy I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Bqtj ^Professor of Botany I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. ^ K. Professor of Agronomy I certify that I have read this study and that in ray opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. "A. J./W. Jones/ Professor of Agricultural Engineering I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. T. R. Sinclair Professor of Agronomy This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. December 19£ Dean, College of Agriculture Dean Graduate School UNIVERSITY OF FLORIDA 3 1262 08553 3700