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Photosynthesis, photorespiration and related aspects of CO2 exchange by wheat, corn and Amaranthus edulis Jolliffe, Peter Alfred 1970

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PHOTOSYNTHESIS, PHOTORESPIRATION AND RELATED ASPECTS OF C0 2 EXCHANGE BY WHEAT, CORN AND AMARANTHUS EPULIS by PETER ALFRED JOLLIFFE B.Sc. , Queen's University, Kingston, Ontario, 1965 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Botany accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1970 In present ing th is thesis in p a r t i a l f u l f i lmen t of the requirements for an advanced degree at the Un ive rs i t y of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f r ee l y ava i l ab le for reference and Study. I fur ther agree that permission for extensive copying of th is thes is for s cho la r l y purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l i c a t i on of th is thes.is for f i nanc i a l gain sha l l not be allowed without my wr i t ten permiss ion. Department The Un ivers i t y of B r i t i s h Columbia Vancouver 8, Canada i i ABSTRACT Certain aspects of CO2 exchange by wheat (Triticum aestivum L.), corn (Zea mays L.) and the grain Amaranth (Amaranthus edulis Speg.) were investigated. The effects of 0 2 concentration on apparent photosynthesis of juvenile wheat and corn shoots were measured at different temperatures, CO2 concentrations and l i g h t i n t e n s i t i e s using infra-red CO2 analysis and both open and closed gas-flow systems. The only condition i n which apparent photosynthesis of wheat was not inhibited by 0 2 was in 20.8% 0 2 when the C0 2 concentration was saturating and the temperature was 30° C or le s s . The degree of i n h i b i t i o n increased with increasing 0 2 concentration, increasing temperature, and decrea-sing C0 2 concentration and was independent of the l i g h t i n t e n s i t y . During some of the growing season i n temperate regions, the effect of atmospheric 0 2 on the photosynthetic productivity of wheat may be negli g i b l e . The effect of 0 2 on wheat was ascribed to both a stimulation of photorespiration and an i n h i b i t i o n of photosynthesis by 0 2. In corn, which apparently lacks photorespiration, photosyn-thesis was also i n h i b i t e d by 99% 02- No i n h i b i t i o n occurred at 20.8% 0 2, however, and the s t a b i l i t y and r e v e r s i b i l i t y of the i n h i b i -t i o n observed at 99% 0 2 d i f f e r r e d from that found with wheat. These differences between wheat and corn are correlated with differences i n leaf anatomy and photosynthetic carbon metabolism and with differences i n the response of apparent photosynthesis and the C0 2 compensation point to environmental conditions. Many of the gas i i i exchange characteristics of corn and s i m i l a r plants seem to be advantageous for the warm dry areas they often inhabit. I n i t i a l l y high rates of CO2 production are exhibited by wheat immediately following i l l u m i n a t i o n , and i t has been suggested that this post-illumination CC>2 burst i s an extension of photo-respiration into the dark period. In accord with t h i s , CO2 concentration was found to influence both the post-illumination CO2 burst and the i n h i b i t i o n of apparent photosynthesis by O2 i n a si m i l a r way. Except for Amaranthus e d u l i s , plants which lack photorespiration also lack s i g n i f i c a n t post-illumination CO2 bursts. On the basis of i t s response to O2 concentration, however, the burst of Amaranthus edulis i s concluded to be unrelated to photorespiration. Measurements of "^C02 and ^C02 exchange were used to estimate the quantity of carbon i n wheat and corn shoots which was i n free exchange with atmospheric CO^ . The free exchange pool s i z e was found to be very small and i t cannot be an important factor i n the CO2 concentration response of photosynthesis or i n the post-illumination COo burst. TABLE OF CONTENTS PREFACE . . . LITERATURE CITED CHAPTER I. The Effects of Temperature, CO2 Concentration and Light Intensity on the Oxygen I n h i b i t i o n of Apparent Photosynthesis i n Wheat INTRODUCTION 1 MATERIALS AND METHODS 2 RESULTS 1. CO2 Exchange by Intact and Excised Wheat Shoots. . 11 2. Effects of Low O2 Concentration on CO2 Exchange by Excised Wheat Shoots (Experiment I) (a) Effects of Low O2 Concentration and Temperature on Apparent Photosynthesis 13 (b) Effects of Low O2 Concentration and Temperature on the CO2 Compensation Point . . 15 (c) Effects of Low O2 Concentration, Temperature and CO2 Concentration on Apparent Photosynthesis 15 3. Further Studies on the Effects of O2 Concentration on CO2 Exchange by Excised Wheat Shoots (Experiment II) (a) Effects of O2 Concentration and Temperature on Apparent Photosynthesis and the CO2 Compensation Point . . . . . . . „ . . . v . . . 21 (b) Effects of O2 Concentration, Temperature, CO2 Concentration and Light Intensity on Apparent Photosynthesis 23 4. Effect of O2 on Photosynthetic Productivity . . . 34 DISCUSSION . 38 LITERATURE CITED 46 CHAPTER I I . Some Comparative Aspects of the Physiology of CO2 Exchange by Wheat and Corn Shoots INTRODUCTION 50 i v Page x i i i • xv V Page MATERIALS AND METHODS 51 RESULTS AND DISCUSSION 1. The C0 2 Compensation Point 55 2. Effect of O2 Concentration on the Apparent Rate of Photosynthesis 57 3. Effect of Temperature on Apparent Photosynthesis . 62 4. Effect of CO2 Concentration on Apparent Photosynthesis 65 5. Effect of Light Intensity on Apparent Photosynthesis 67 6. General Discussion 71 LITERATURE CITED . . . 77 CHAPTER I I I . Photorespiration and the Post-illumination C0 2 Burst i n Wheat and Amaranth us edulis INTRODUCTION 84 MATERIALS AND METHODS . . 85 RESULTS AND DISCUSSION 1. Kinetics of the Post-illumination CO2 Burst . . . 86 2. Effect of 0 2 and CO2 Concentrations on the Post-illumination CO2 Burst 91 3. Effect of 0 2 Concentration on the Post-illumination CO2 Burst of Amaranthus edulis 101 LITERATURE CITED .' . '. 104 CHAPTER IV Estimation of the C0 2 Free exchange Pool Size i n Wheat and Corn Leaves INTRODUCTION 106 MATERIALS AND METHODS 107 RESULTS AND DISCUSSION 114 LITERATURE CITED 118 v l Page CONCLUSIONS 120 APPENDIX I ^Calculation of the C0 2 Concentration i n the A i r Entering the IRGA during the Post-illumination C0 2 Burst 122 APPENDIX I I Calculation of the Absorption of C0 2 by 0.2 M Phosphate Buffers 124 v i i LIST OF TABLES Table Page I I - l . Comparison of the Apparent K i n e t i c Constants of Photosynthetic CO2 Assimilation of Excised Wheat and Corn Shoots 65 IV-1 Magnitude of CO2 Free-Exchange Pools i n 0.2 M Phosphate Buffer 115 IV-2 Magnitude of CO2 Free-Exchange Pools i n Wheat and Corn Shoots . . . 116 v i i i LIST OF FIGURES Figure Page 1-1. Plant Chamber Used for Excised Shoots i n Experiment I 3 1-2. Plant Chamber Used for Excised Shoots i n Experiment I I 4 1-3. Lamp Tower and Freezer Assembly Used for Control of Light Intensity and Temperature 6 1-4. Spectral Energy Di s t r i b u t i o n of Light from a General E l e c t r i c "Cool Beam" Lamp as Measured by an ISCO spectroradiometer 7 1-5. Plant Chamber Used for Intact Shoots 10 1-6. Effects of C0£ Concentration and Temperature on the Apparent Rates of Photosynthesis of Intact and Excised Wheat Shoots 12 1-7. Effects of Temperature on the Apparent Rates of Photosynthesis of Excised Wheat Shoots i n Atmospheres Containing 300 u l . / l . CO2 and 20.8% 0 2 or 3± 1% 0 2 14 1-8. Effects of Temperature on the CO2 Compensation Points of Excised Wheat Shoots i n Atmospheres Containing 20.8% O2 or 3± 1% 0 2 16 1-9. Effects of CO2 Concentration and Temperature on the Apparent Rates of Photosynthesis of Excised Wheat Shoots i n Atmospheres Containing 20.8% 0 2 or 3± 1% 0 2 18 I-10. Effects of Temperature and C0 2 Concentration on the Per Cent I n h i b i t i o n of Apparent Photosynthesis by 20.8% O2 i n Excised Wheat Shoots 20 1-11. Effects of O2 Concentration and Temperature on the Apparent Rate of Photosynthesis of Excised Wheat Shoots i n 300 - u l . / l . C0 2 . . . . 22 1-12. Effects of O2 Concentration and Temperature on the CO2 Compensation Point of Excised Wheat Shoots 24 i x Figure Page 1-13. Effects of 0 2 Concentration _= and CO2 Concentration on the Apparent Rate of Photosynthesis of Excised Wheat Shoots at 13° C 25 1-14. Effects of O2 Concentration and CO2 Concentration on the Apparent Rate of Photosynthesis at 20° C 26 1-15. Effects of 0 2 Concentration and C0 2 Concentration on the Apparent Rate of Photosynthesis at 25° C 27 1-16. Effects of O2 Concentration and CO2 Concentration on the Apparent Rate of Photosynthesis at 30° C 28 1-17. Effects of O2 Concentration and CO2 Concentration on the Apparent Rate of Photosynthesis at 35° C 29 1-18. Effects of O2 Concentration and CO2 Concentration on the Apparent Rate of Photosynthesis at 40° C 30 1-19. Effects of 02 Concentration and CO2 Concentration on the Per Cent I n h i b i t i o n of Apparent Photosynthesis i n Excised Wheat Shoots at 25° C 32 1-20. Effects of CO2 Concentration and Light Intensity on the Per Cent I n h i b i t i o n of Apparent Photosynthesis i n Excised Wheat Shoots at 25° C and 20.8% 0 2 or 60.9% 0 2 33 1-21. The Carbon D e f i c i t i n Excised Wheat Shoots Caused by 20.8% 0 2 at Different Temperatures and CO2 Concentrations (Experiment L) . 35 1-22. The Carbon D e f i c i t i n Excised Wheat Shoots Caused by 20.8% 0 2 at Different Temperatures and CO2 Concentrations (Experiment II) 36 I I - l . Open System Used to Generate Constant CO2 Concentrations i n an A i r Stream Containing Different O2 concentrations . 54 X Figure Page II-2. Time Course of CO2 Exchange by Excised Wheat Shoots i n Atmospheres Containing 300 u l . / l . C0 2 and Different 0 2 Concentrations 58 II-3 . Time Course of C0 2 Exchange by Excised Corn': Shoots i n Atmospheres Containing 300 u l . / l . C0 2 and Different 0 2 Concentrations 59 II-4. Effects of 0 2 Concentration, Temperature, C0 2 Concentration and Light Intensity on the Apparent Rate of Photosynthesis of Excised Corn Shoots 61 II-5. Effects of Temperature on the Apparent Rates of Photosynthesis of Excised Wheat and Corn Shoots i n A i r Containing 20.8% O2 and 335 u l . / l . C0 2 63 II-6. Effects of Light Intensity on the Apparent Rates of Photosynthesis of Excised Wheat and Corn Shoots i n A i r Containing 20.8% 0 2 and 335 u l . / l . C0 2 68 II-7. Effects of Exposure to High Light Intensity on the Apparent Rate of Photosynthesis of Excised Corn Shoots 69 I I I - l . Effects of Darkening on C0 2 Exchange of Excised Wheat Shoots i n .20.8% 0 2 87 II I - 2 . Relative Response of the IRGA to Pulse Injections of CO2 into the A i r Stream i n the Plant Chamber and at the Entrance to the IRGA Sample Cylinder 90 I I I - 3 . Post-illumination CO2 Exchange'by Excised Wheat Shoots i n 1.8% O2 and 20.8% 0 2. The C0 2 Concentration of the A i r Entering the Plant Chamber was 100 u l . / l . C0 2 92 I I I - 4 . P o s t - i l l u m i n a t i o n CO2 Exchange by Excised Wheat Shoots i n 1.8% 0 2 and 20.8% 0 2. The CO2 Concentration of the A i r Entering the Plant Chamber was 200 y l . / l . C0 2 93 III - 5 . Post-illumination CO2 Exchange by Excised Wheat Shoots i n 1.8% 0 2 and 20.8% O2. The CO2 Concentration of the A i r Entering the Plant Chamber was 300 y l . / l . C0 2 94 x i Figure Page III-6. Post-illumination CO2 Exchange by Excised Wheat Shoots i n 1.8%-0, and 20.8% 0 2. The C0 2 Concentration of the A i r -Entering the Plant Chamber was 400 u l . / l . C0 2 95 III-7. Post-illumination C0 2 Exchange by Excised Wheat Shoots i n 1.8% 0 2 and 20.8% 0 2. The C0 2 Concentration of the A i r Entering the Plant Chamber was 500 u l . / l . C0 2 96 III- 8 . Effects of C0 2 Concentration on the Size of the Post-illumination C0 2 Burst and the Magnitude of the Depression of Apparent Photosynthesis by 20.8% 0 2 97 III-9. Effects of C0 2 Concentration on the Per Cent Inh i b i t i o n of Apparent Photosynthesis by 20.8% 0 2 and on the Analogous Burst Function % I b 100 111-10. The Post-illumination C0.2 Burst of Amaranth us edulis i n 1.8% 0 2 and 20.8% 0 2 102 IV-1. Plant Chamber Used for Measurements of the C0 2 Free-exchange Pool Size with Excised Wheat and Corn Shoots 108 IV-2. Gas Flow Systems Used for 1 2 C 0 2 Exchange and -^C02 Exchange Measurements with Excised Wheat and Corn Shoots 110 IV-3. Gas Flow Systems Used for 1 2 C 0 2 Exchange and 1^C02 Exchange Measurements with 0.2 M Phosphate Buffers 113 x i i ACKNOWLEDGEMENTS The author wishes to express his sincere appreciation for the counsel and support given by Dr. E. B. Tregunna throughout these studies. Thanks are also extended to his former graduate students, Dr. W. J . S. Downton and Dr. J . A. Berry for many he l p f u l discussions during the development of this research. The assistance and advice given by Dr. T. Bisalputra, Dr. B. A. Bohm,.::D£. W. B. Schofield, Dr. T. A. Black and Dr. N. R. Bulley i s gratefully acknowledged. Dr. G. R. L i s t e r of Simon Fraser University kindly supplied the Gallenkamp gas mixing pumps and ISCO spectroradiometer used i n these investigations. The use of the airflow planimeter of Dr. D. P. Ormrod, formerly of the Department of Plant Science, i s acknowledged. The author was a recipient of a University of B r i t i s h Columbia Scholarship i n 1966-67, and of National Research Council of Canada Scholarships during 1967-68 and 1968-69. F i n a l l y , this thesis i s dedicated to my wife Ann, who assisted i n drafting many of the diagrams, and whose encouragement made this thesis possible. x i i i PREFACE Two r e l a t i v e l y recent developments have focussed new attention on the CO2 exchange processes of plants. Starting about 10 years ago, Krotkov, Egle,and others carried out a number of studies on the effects of atmospheric 0 2 on plant C0 2 exchange (2, 3, 4, 10, 11). These investigations culminated i n the suggestion that many plants release C0 2 during photosynthesis by a process called photorespiration. The d i s t i n c t i v e feature of photorespiration i s that i t i s enhanced by 0 2 concentrations greater than 2% 0 2 while respiration by leaves i n the dark is not. Meanwhile, i n 1965 i t was reported that malate and aspartate, instead of the phosphorylated sugars t y p i c a l of the Calvin cycle, were the i n i t i a l products of photosynthetic C0 2 f i x a t i o n i n sugar cane (9). Hatch, Slack and t h e i r collaborators extended these results and developed the C4-dicarboxylic acid pathway to account for these observations (5, 6, 8). In addition, they discovered numerous other " t r o p i c a l " species which possessed the same type of photosynthetic carbon metabolism (6, 7). These two trends i n plant research provide the basis for the present studies. They are linked by the observation that plants which exhibit the C^-dicarboxylic acid pathway apparently lack photores-p i r a t i o n , while plants which possess only the Calvin cycle can rapidly photorespire (1). This thesis includes experiments designed to indicate the a c t i v i t y of photorespiration i n different environmental conditions as w e l l as to indicate the differences i n C0 2 exchange characteristics x i v of plants which possess and lack photorespiration. In addition, the re l a t i o n s h i p between photorespiration and p o s t - i l l u m i n a t i o n CO2 exchange transients was studied, and an attempt was made to estimate the quantity of carbon within plants which can exchange with atmospheric CO2• LITERATURE CITED Downton, W. J . S. and E. B. Tregunna. 1968. Carbon dioxide compensation - i t s r e l a t i o n to photosynthetic carboxylation reactions, systematics of the Gramineae, and leaf anatomy. Can. J . Botany 46: 207-215. Egle, K. and H. Fock. 1966. Light respiration - correlations between C0 2 f i x a t i o n , 0 2 pressure and glycollate concentration. In: T. W. Goodwin (editor). Biochemistry of Chloroplasts. Academic Press, New York. Vol. 2, pp. 79-87. Fock, H. and K. Egle. 1966. Uber die "Lichtatmung" bei grunen Pflanzen. I. Die Wirkung von Sauerstoff und Kohlendioxid auf den C0 2 - Gaswechsel wahrend die Licht-und Dunkelphase -B e i t r . B i o l . Pflanzen 42: 213-239. Forrester, M. L., G. Krotkov and C. D. Nelson. 1966. Effect of oxygen on photosynthesis, photorespiration and respiration i n detached leaves. I. Soybean. Plant Physiol. 41: 422-427. Hatch, M. D. and C. R. Slack. 1966. Photosynthesis by sugar-cane leaves. A new carboxylation reaction and the pathway of sugar formation. Biochem. J. 101: 103-111. Hatch, M. D., C. R. Slack and H. S. Johnson. 1967. Further studies on a new pathway of photosynthetic carbon dioxide f i x a t i o n i n sugar-cane and i t s occurrence i n other plant species. Biochem. J. 102: 417-422. Johnson, H. S. and M. D. Hatch. 1968. D i s t r i b u t i o n of the C4-dicarboxylic acid pathway of photosynthesis and i t s occurrence i n dicotyledonous plants. Phytochem.7: 375-380. Johnson, H. S. and M. D. Hatch. 1969. The C^-dicarboxylic acid pathway of photosynthesis. I d e n t i f i c a t i o n of intermediates and products and quantitative evidence for the route of carbon flow. Biochem. J. 114: 127-134. Kortschack, H. P., C. E. Hartt and G. 0. Burr. 1965. Carbon dioxide f i x a t i o n i n sugarcane leaves. Plant Physiol. 40: 209-213. Krotkov, G. 1963. Effect of l i g h t on respiration. In: Photosynthetic Mechanisms of Green Plants, Publication 1145, National Academy of Science, National Research Council, Washington, D.C, pp. 452-454. Tregunna, E. B., G. Krotkov and C. D. Nelson. 1966. Effect of oxygen on the rate of photorespiration i n detached tobacco leaves. Physiol. Plantarum 19: 723-733. 1 Chapter I THE EFFECTS OF TEMPERATURE, C0 2 CONCENTRATION AND LIGHT INTENSITY ON THE OXYGEN INHIBITION OF APPARENT PHOTOSYNTHESIS IN WHEAT INTRODUCTION The apparent rate of photosynthesis, whether i t i s measured i n terms of C0 2 assimilation or 0 2 evolution, i s often found to be inhibi t e d i n the presence of molecular oxygen. This i n h i b i t i o n was f i r s t demonstrated by Warburg (46) and has come to be known as the "Warburg effect". The effect has been observed i n many photosynthetic organisms, including algae, mosses, liverworts, ferns, gymnosperms and angiosperms (31, 37, 43,'.44, 46). It i s known that the inhibitory effect of 0 2 i s influenced by the environmental conditions, but information on the environmental relations of the 0 2 effect i s limited and often inconsistent. The effect of 0 2 i s reduced and may be absent at high C0 2 concentrations (7, 40, 44), and i s increased at C0 2 concentrations below atmospheric levels (40) . In Chlorella at low C0 2 concentration, temperature did not affect the degree of i n h i b i t i o n of photosynthesis over the range 4° to 25° C (40) . Similar results were obtained with the moss Funaria between 20° and 30° C (43). In wheat, cotton and tobacco, however, recent results indicate that i n h i b i t i o n increases between 30° and 40° C (23). Most studies have shown that the degree of i n h i b i t i o n i s not affected by moderate to saturating l i g h t i n t e n s i t i e s (3, 31, 43, 47). In several other cases, the degree of i n h i b i t i o n increased with 2 increasing l i g h t intensity (43, 47). Numerous suggestions have been advanced concerning the mechanism(s) by which 0 2 i n h i b i t s apparent photosynthesis (12, 43). Both the l i g h t (3, 32, 34, 36) and dark (10, 40, 45) processes of photosynthesis, as w e l l as photorespiration (14, 17, 18, 41) have been implicated as possible s i t e s for the 0 2 e f f e c t . In the present investigation, the effect of 0 2 concentration on the apparent rate of photosynthesis of wheat shoots i n different conditions of temperature, C0 2 concentration and l i g h t intensity was examined. The results permit us to assess the significance of the 0 2 effect on photosynthetic productivity, and add perspective to current proposals on the mechanism of the 0 2 e f f e c t . MATERIALS AND METHODS Seeds of wheat (Triticum aestivum L. var. Spring Thatcher), obtained from Buckerfield's Seed Co., Vancouver, were planted i n f l a t s of vermiculite, watered d a i l y , and grown i n a ventilated room, at 21,600 lux of f lubres^eentrf and... incandescent! .light and 16 hr. photoperiod. At the st a r t of experiments with excised shoots, 1.5 g. fresh weight of 8 to 14 day-old shoots were detached by severing the bases of the shoots with a razor blade. The cut ends of the shoots were immediately immersed i n water and recut beneath the water surface about 5 mm. below the lowest node of the sh'oo;t.. The excised shoots were then weighed and transferred to one of the plexiglass chambers shown i n Figures I - l and 1-2. Within these chambers, the cut ends of the shoots were submerged i n approximately 5 mm. of water. After the shoots were enclosed i n a chamber, they were allowed to adjust to the Figure I - l ant Chamber Used for Excised Shoots i n Experiment inlet FRONT VIEW shoot chamber-water jacket plant shoot water trough wing nuts and bolts air outlet / r 2 4 cm water inlet and outlet SIDE VIEW Figure 1-2 Plant Chamber Used for Excised Shoots i n Experiment FRONT VIEW SIDE VIEW 5 experimental conditions for 30 to 45 minutes before CO2 exchange measurements were started. The enclosed shoots were illuminated by 1 to 3 General E l e c t r i c "Cool Beam" 750 watt incandescent lamps. The lamp tower, shown i n Figure 1-3 was used i n experiments with excised shoots. The chamber was placed on the freezer immediately below the lamp tower or, when more e f f i c i e n t cooling was required, within the freezer supporting the lamp tower. Before the l i g h t was incident on the shoots, i t was passed through 14 cm. of water to remove much of the infra-red radiation. Figure 1-4 shows the spectral energy d i s t r i b u t i o n of the un-f i l t e r e d radiation as measured by an Instrumentation Specialties Co. (ISCO) spectroradiometer. Light intensity was measured by a Gossen "Tri-Lux" footcandle meter and converted to lux by multiplying by 10.764. An i l l u m i n a t i o n of 32,300 lux (3000 f t - c ) was equivalent to a radiant intensity of 1.6 x 10^ erg./cm./sec. between 400 and 700 nm. when no water f i l t e r was used. The l i g h t intensity was varied by changing the number of lamps used, by varying the distance of the lamps from the shoots, and by interposing layers of cheesecloth between the lamps and the chamber. The temperature within the chambers was detected by a ther-mistor placed i n contact with some of the shoots. A Yellow Springs Instruments Co. Telethermometer was connected to the thermistor and indicated the temperature. The chamber temperatures were controlled by c i r c u l a t i n g water from a thermoregulator through jackets surrounding the chambers. A simple closed system was used for the CO2 exchange studies reported here. This system contained the plant chamber, a CO2 release Figure 1-3 Lamp Tower and Freezer Assembly Used for Control Light Intensity and Temperature 6 Figure 1 - 4 e c t r a l Energy Di s t r i b u t i o n of Light from a General E l e c t r i c Cool Beam" Lamp as Measured by a ISCO Spectroradiometer 8 flask, a Beckman IR 215 infra-red gas analyzer (IRGA), a Fisher "Dynapump" a i r pump, and a Matheson R-2-15-B flowmeter, which were linked i n series by Tygon tubing. A Bausch and Lomb VOM-5 s t r i p chart recorder was connected to the IRGA and recorded the CC^ concentration changes i n the system. Two studies were made of the effect of 0^ concentration on CO^  exchange by excised wheat shoots . Experiment I was carried out i n September 1967 to determine the effects of 20.8% (v./v.) and 3+ 1% 0 2 on the apparent rate of photosynthesis and on the CO^  compensation point of wheat. For this experiment, the plants were grown at 22-26/18-22 °C day/night temperatures. The chamber shown i n Figure 1-1 was used, and the volume of the closed system was 860 ml. The a i r flow rate i n the closed system was maintained at 2 l./min., and the l i g h t i n tensity at the chamber surface was 10,760 lux. A Clark oxygen electrode, connected to a Beckman Oxygen Adapter and a millivoltmeter, was included i n the system to measure the 0^ concentration i n the a i r stream. Laboratory a i r or ^  from a compressed gas tank was flushed through the system to adjust the 0^ concentration i n the a i r stream. Experiment I I was carried out from February to May 1969 to i n -vestigate the effects of 0^ concentrations higher than atmospheric on CO^  exchange by excised wheat shoots. In this case, the plants were grown at day/night temperatures of 26-31/22-26 °C, and the chamber shown i n Figure i-2 was used. The volume of the closed system was 230 ml., and the a i r flow rate was 3 l./min. Except for the l i g h t i ntensity study reported i n Figure 1-15, the l i g h t i ntensity was 32,300 lux. The 0 2 concentrations were established by flushing the closed system with laboratory a i r or with gas from compressed gas tanks. The 0 9 concentrations i n the a i r stream were not measured during this experiment, but separate measurements with a Picker MS-10 mass spec-trometer indicated that the 0 2 concentrations used were 1.8%, 20.8%, 60.9%, 78.6% and >99% 0 2. Between 11 and 24 measurements of the apparent rate of photosynthesis were made at any one combination of 0 2 concentration, temperature, C0 2 concentration and l i g h t intensity used i n Experiments I and I I . When intact plants were required, the wheat seeds were planted i n a row so that a number of shoots could be sealed into the 135 ml. plexiglass chamber shown i n Figure 1-5 by a rubber gasket coated with s i l i c o n e grease. The environmental conditions during the growth of these plants, and during the measurement of th e i r C0 2 exchange were comparable to those described for Experiment I. When intact plants were used, the illumination system was si m i l a r to that described above except that the l i g h t was directed horizontally at the upright chamber. The following sequence of operations was carried out during the C0 2 exchange studies reported i n this Chapter. After the system was flushed to establish:.:the 0 2 concentration, i t was closed. A hypodermic syringe was then used to i n j e c t less than 0.5 ml. of a i r containing a high C0 2 concentration into the release flask to elevate the C0 2 concentration i n the system above 600 u l . / l . The C0 2 concen-tr a t i o n i n the system then decreased because of net C0 2 assimilation by the enclosed shoots u n t i l the C0 2 compensation point was reached. Thereafter, this sequence of operations was repeated u n t i l measurements with one sample of shoots was terminated. No sample of shoots was used for more than 3.5 hours following the i r enclosure i n a chamber. Figure 1-5 Plant Chamber Used for Intact Shoots-F R O N T V I E W w a t e r o u t l e t s h o o t c h a m b e r w a t e r j a c k e t p l a n t s h o o t " w i n g n u t s a n d b o l t s v e r m i c u l i t e 4 c m O S I D E V I E W 11 Apparent rates of photosynthesis were calculated from the time required for the shoots to reduce the CO2 concentration i n the system by 50 y l . / l . or by 25 y l . / l . and from the system volume. The rates were expressed on a per fresh weight basis. Separate measurements of shoot area, using an airflow planimeter s i m i l a r to that of Jenkins (27), established that the area of 1 g. fresh weight of 10 day-old wheat shoots was 0.40 dm2. Thus, an approximate i n d i c a t i o n of the apparent rates of photosynthesis on a leaf area basis (mg. C02/hr/dm^) can be obtained by multiplying the reported rates by 2.50. RESULTS 1. CO^  Exchange by Intact and Excised Wheat Shoots To establish whether excised shoots were suitable experimental material for the current studies, preliminary tests were done to compare the CO2 exchange characteristics of i n t a c t and excised shoots. Figure 1-6 i l l u s t r a t e s the s i m i l a r i t y of the CO2 concentration res-ponse of apparent photosynthesis by intact and excised shoots at high and low temperatures. The v e r t i c a l bars which extend above and below the averages at each CO2 concentration indicate the 95 per cent confidence l i m i t s about the sample means. Where si m i l a r v e r t i c a l bars occur on other figures i n this thesis, they also show the 95 per cent confidence l i m i t s about the sample means. I f the excised shoots were well-supplied with water, t h e i r apparent rates of photosynthesis remianed constant between 0.5 and 3.5 hours after excision, even when the temperature was as high as 40° C. Several other investigations have indicated that gas exchange by leaves i s l i t t l e affected for several hours after excision (25, 35, 42), except for transient Figure 1-6 Effect of CO2 Concentration and Temperature on the Apparent Rates of Photosynthesis of Intact and Excised Wheat Shoots. The Light Intensity Was 10,760 Lux 13 changes i n CC>2 exchange (8, 28) or transpiration (33) when petioles or leaves are severed. Because t h e i r CO2 exchange behavior resembled that of intact shoots, and because of t h e i r convenience, excised shoots were used for the rest of the research reported i n this thesis. 2. Effects of Low O2 Concentration!on CO2 Exchange by Excised Wheat Shoots (Experiment I) (a) Effects of Low O2 Concentration and Temperature on Apparent Photosynthesis. An i n i t i a l series of measurements was carried out to compare the effects of 3± 1% and 20.8% O2 on the apparent rate of photosynthesis of wheat shoots exposed to 300 y l . / l . CO2 and temperatures ranging from 2° to 43° C. Figure 1-7 shows that there was an optimum tempera-ture for apparent photosynthesis, below and above which the rate of net C0 2 assimilation declined. I t appears from these results that O2 concentration exerted an influence on the response of apparent photosyn-thesis to temperature. In 20.8% the apparent rate of photosynthe-s i s was greatest between 20° and 26° C, but i n 3± 1% O2 the optimum temperature was between 26° and 34° C. In 20.8% O2, the apparent rate of photosynthesis at 34° C was s i g n i f i c a n t l y less than at 20° C, while i n 3± 1% O2, the rate was s i g n i f i c a n t l y greater at 34° C than at 20° C. Therefore, a decrease i n O2 concentration from 20.8% O2 to 3± 1% O2 appeared to cause an increase i n the temperature optimum for apparent photosynthesis. In addition, i t isnofceworthy that at 13° C or lower temperatures, there was no s i g n i f i c a n t difference between the apparent rates of photosynthesis i n 20.8% O2 and 3± 1% O2. At higher temperatures, however, apparent photosynthesis i n 20.8% 0 2 was greatly inhibited compared to the rates of CO2 assimilation observed i n 3± 1% O2. Thus, these results show that 0 2 concentration can a l t e r the temperature respone,of apparent photosynthesis, and that the , Figure 1-7 Effects of Temperature on the Apparent Rates of Photosynthesis of Excised Wheat Shoots i n Atmospheres Containing 300 y l . / l . C0 2 and 20.8% 0 2 or;3± 1% 0 2 15 inh i b i t o r y effect of 0 2 on apparent photosynthesis i s modified by the temperature. (b) Effects of Low 0 2 Concentration and Temperature on the C0 2 Compensation Point. When plants are placed i n a sealed chamber and illuminated, C0 2 i s assimilated u n t i l a C0 2 concentration i s reached at which the rate of C0 2 uptake i s equal to the rate of C0 2 production. The C0 2 concentration at which this equilibrium occurs i s known as the C0 2 compensation point (15). Figure 1-8 shows the effect of temperature on the C0 2 compensation point of wheat shoots i n 3± 1% and 20.8% 0 2 • The C0 2 compensation point was always much lower i n 3± 1% 0 2 than i n 20.8% 0 2, but at both r 0 2 concentrations the C0 2 compensation point increased with increasing temperature. At 32° C or l e s s , the rat i o of the C0 2 compensation point i n 3± 1% 0 2 to the C0 2 compensation point i n 20.8% 0 2 was constant. Also, this r a t i o was equivalent to the r a t i o of the 0 2 concentrations used. Above 32°, however, the ra t i o increased sharply. (c) Effects of Low 0 2 Concentration, Temperature, and C0 2 Concentration on Apparent Photosynthesis. It was clear from Figure 1-7 that at 13° C or lower tempe-ratures the apparent rate of photosynthesis was the same i n 3± 1% O2 and 20.8% 0 2. At the same temperatures, however, Figure 1-8 indicated that the C0 2 compensation point was proportional to the 0 2 concent-ration. Because of this -anomaly, a thorough study was made of the effects of 3± 1% 0 2 and 20.8% 0 2 on apparent photosynthesis at different temperatures and C0 2 concentrations-. Rates of C0 2 assimilation were measured at 4 temperatures from 13° C to 34° C and at C0 2 concentrations between 500 y l . / l . C0 2 and the C0 2 compensation Figure 1-8 Effects of Temperature on the CC>2 Compensation Points of Excised Wheat Shoots i n Atmospheres Containing 20.8% 0 2 or 3± 1% 0 2 point. The results of this study are presented i n Figures 1-9 and 1-10. At CO2 concentrations just above the CO2 compensation point, the apparent rate of photosynthesis was limited by the CO2 since the rate increased rapidly as the CC>2 concentration increased. At more elevated CO2 concentrations, the response of^apparent photosynthesis to a change i n CO2 concentration became less pronounced. Often, i f the CO2 concentration was s u f f i c i e n t l y high, the apparent rate of photosynthesis became insensitive'to changes i n CO2 concentration. When this occurred, apparent photosynthesis was saturated by CO2. I t i s evident from Figure 1-9 that the CO2 concentration required to saturate apparent photosynthesis increased as the O2 concentration and temperature increased. In 20.8% O2 at 25.9° C and 34.3° C, apparent photosynthesis was not saturated by CO2 concentrations below 500 y l . / l . Under a l l other conditions, apparent photosynthesis was saturated by 500 y l . / l . CO2 or l e s s , and the apparent rate of photosynthesis at saturating CO2 concentrations increased with increasing temperature. Except at high CO2 concentrations at 13° C and 19.7° C, the apparent rate of photosynthesis was less i n 20.8% O2 than i n 3± 1% O2• The CO2 concentration required to eliminate the inh i b i t o r y effect of 20.8% 0 2 was higher at 19.7° C than at 13° C and appears to correspond to the minimum CO2 concentration required to saturate apparent photosynthesis i n 20.8% O2 i n these cases. Under no condition of temperature'or CO2 concentration was the apparent rate of photosynthesis s i g n i f i c a n t l y enhanced i n the presence of 20.8% 0 2 compared to the rate i n 1.8% O2. In addition, the slope of the CO2 concentration response curves immediately above the CO2 compensation Figure 1-9 Effects of CO2 Concentration and Temperature on the Apparent Rates of Photosynthesis of Excised Wheat Shoots i n Atmospheres . Containing 20.8% 0 2 or 3± 1% 0 2 1 8 4= 4.0 ^3.0 O CJ £ 2.0 CO UJ CD 1.0 i 1 1 1— o o3±1 % 0 2 13.CTC A * 2 0 . 8 % 0 r O.0.0t> 1 O X QL O 5.0 w 4.0 3.0 £ 2.0 Q_ < 1.0 25.9°C *. — — 0 i / r o i / O.OH-A—J. J I 1 L T r ~ — i r 19.7°C O /A o _/ ft-i I I - J S - L 34.3°C o—* 0 100 200 300 400 500 0 100 200 300 400 500 C 0 2 CONCENTRATION (JJI/I) 19 points appeared to be less i n 2 0 . 8 % than i h 3 ± 1 % O 2 . Inspection of Figure 1-9 reveals that the in h i b i t o r y effects of 2 0 . 8 % 0 2 on apparent photosynthesis were greatest at high tempe-ratures and low C 0 2 concentrations. A useful quantitative index of the effect of O 2 on apparent photosynthesis -is".-that of per cent i n h i b i t i o n (% I p ) , which may be calculated ( 3 , 18, A 3 ) using the expression: * XP = p l " P 2 1 0 0 P l ( 1 ) i n which P-^  and P 2 represent the apparent rates of photosynthesis at a low (e.g. 3 ± 1 % O 2 ) and a high (e.g. 2 0 . 8 % O 2 ) concentration of O 2 respectively. Examination of this formula shows that 1 0 0 % i n h i b i t i o n must occur at the C O 2 compensation point i n the higher concen-t r a t i o n , since at that point P 2 equals zero. Figure 1 - 1 0 shows the per cent i n h i b i t i o n of apparent photosynthesis by 2 0 . 8 % O 2 at different temperatures and C 0 2 concentrations. These data are derived from the results presented i n Figure 1-9. I t i s clear that the per cent i n h i b i t i o n of apparent photosynthesis increased with increasing temperature. Except where the C O 2 concentration approached the C O 2 compensation point, the increase i n degree of i n h i b i t i o n appeared to be linea r with the same slope at a l l C O 2 concentrations. In this l i n e a r portion of the figure, the temperature at which a certain per cent i n h i b i t i o n o occurred appears to be related to the logarithm of the C O 2 concen-t r a t i o n . At any one temperature, the per cent i n h i b i t i o n decreased i n a non-linear fashion with increasing C O 2 concentration. Figure I-10 Effects of Temperature and CO2 Concentration on the Per Cent In h i b i t i o n of Apparent Photosynthesis by 20.8% O2 i n Excised Wheat Shoots PER CENT INHIBITION 21 3. Further Studies on the Effects of 0 2 Concentration on C0 2 Exchange by Excised Wheat Shoots (Experiment I I ) (a) Effects of 0 2 Concentration and Temperature on Apparent Photosynthesis and the C0 2 Compensation Point. A second series of tests was carried out to extend these results to include higher 0 2 concentrations. Figure 1-11 i s comparable to Figure 1-7 and shows the temperature response of apparent photo-synthesis of wheat shoots i n 300 y l . / l . C0 2 and 0 2 concentrations ranging from 1.8% 0 2 to >99% 0 2. Throughout Experiment I I , the apparent rates of photosynthesis were much higher than those obtained i n Experiment I. This difference can be accounted for largely by the higher l i g h t intensity used i n Experiment I I . In accord with the previous r e s u l t s , at low tempera--tures there was no s i g n i f i c a n t difference i n the apparent rates of photosynthesis i n 20.8% 0 2 and 1.8% 0 2. In this case, however, the apparent rates of photosynthesis at these 0 2 concentrations were s i m i l a r at 20° C as w e l l as at 13° C. At high temperatures apparent photosynthesis i n 20.8% 0 2 was again i n h i b i t e d compared with the rates observed at the low 0 2 concentration used. At a l l the temperatures examined, apparent photosynthesis was i n h i b i t e d by the presence of 0 2 concentrations from 60.9% 0 2 to >99% 0 2, and the degree of i n h i b i t i o n was enhanced as the 0 2 concentration and temperature increased. Although this time there was no s i g n i f i c a n t difference i n the optimum temperature for apparent photosynthesis i n the 20.8% 0 2 and 1.8% 0 2, Figure-.IftlL.confirms, the e a r l i e r i n dication that the optimum temperature tends to decrease as the 0 2 concentration increases. In this case, the apparent rate of photosynthesis was greatest at 30° C Figure 1-11 Effects of C>2 Concentration and Temperature on the Apparent Rates of Photosynthesis of Excised Wheat Shoots i n 300 ul. / l . C0 2 22 23 and 35° C when the 0 2 concentration was 1.8% 0 2 or 20.8% 0 2. When the 0 2 concentration was raised to >99% 0 2, however, the optimum occurred between 13° C and 25° C. Figure 1-12 shows the effect of 0 2 concentrations from 1.8% 0 2 to >99% 0 2 on the C0 2 compensation point. Once again, the C0 2 compensation point was affected by the 0 2 concentration and temperature. At any one temperature, the C0 2 compensation point was d i r e c t l y proportional to the 0 2 concentration. The slope of the l i n e a r C0 2 compensation point-0 2 concentration relationship increased as the temperature increased. I f the data are extrapolated to zero 0 2 concentration, the C0 2 compensation points appear to become less than 15 y l . / l . C0 2. (b) Effects of 0 2 Concentration, Temperature, C0 2 Concen-t r a t i o n and Light Intensity on Apparent Photosynthesis. Figures 1-13 to 1-18 show the results of additional measurements which were made to assess the effects of 0 2 concentrations from 1.8% 0 2 to >99% 0 2 on apparent photosynthesis i n different conditions of C0 2 concentration and temperature. The general pattern of the results obtained at 1.8% 0 2 and 20.8% 0 2 was quite s i m i l a r to the e a r l i e r results from Experiment I which were presented i n Figures 1-9 to 1-10. In Figures 1-13 to 1-18 i t can be seen that at higher 0 2 concentrations the trends observed between 1.8% 0 2 and 20.8% 0 2 were continued. At 60.9% 0 2 to >99% 0 2 the apparent rate of photosynthesis was s i g n i f i c a n t l y i n h i b i t e d i n a l l conditions except at high C0 2 concentrations at 13° C and 60.9% 0 2. The degree of i n h i b i t i o n increased as the 0 2 concentration and temperature increased and the C0 2 concentration decreased. The slopes of the C0 2 concen-t r a t i o n response curves immediately above the C02 compensation points Figure 1-12 Effects of C>2 Concentration and Temperature on the CO2 Compensation Point of Excised Wheat Shoots Figure 1-13 Effects of Concentration and CC^ Concentration on the Apparent Rates of Photosynthesis of Excised Wheat Shoots at 13° C 25 C0 2 C O N C E N T R A T I O N C L J I / I ) Figure 1-14 Effects of 0^ Concentration and CC^ Concentration on the Apparent Rates of Photosynthesis of Excised Wheat Shoots at 20° C "T T T Figure 1-15 Effects of Concentration and CO^  Concentration on the Apparent Rates of Photosynthesis of Excised Wheat Shoots at 25° C 27 412.0 CN O ( J £ 1QC CO 00 LU f 8.0 >-oo O § 6.0 ° 4.0 or 2.0 o A-0 -o 25 C - O l . 8 % 0 2 -A20.8°/ oO 2 ~A6Q9° / 0 0 2 -Q78.6° / O 0 2 - • > 9 9 ° / 0 0 2 /T / " 5 a / A _ _ LU m ol 00"c>- A ' < 0 . , S*/ 100 200 a -L 300 400 500 CONCENTRATION (ul/l) 600 Figure 1-16 Effects of Concentration and CO^  Concentration on the Apparent Rates of Photosynthesis of Excised Wheat Shoots at 30° C 28 Figure 1-17 Effects of 0^ Concentration and CO2 Concentration on the Apparent Rates of Photosynthesis of Excised Wheat Shoots at 35° C O 1 . 8 % 0 2 A 2 0 . 8 % 0 2 A 6 0 . 9 % 0 2 E 7 8 . 6 % 0 2 • > 9 9 ° / 0 0 2 A / - f ° / / " -A / / A-— -_ B — .•a A ' S 0 / S 1 3 ^ 2 " 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 C 0 2 CONCENTRATION (ul/l) 6 0 0 Figure 1-18 Effects of O2 Concentration and CC^ Concentration on the Apparent Rates of Photosynthesis of Excised Wheat Shoots at 40° C 30 1 4 . 0 1 2 . 0 1 0 . 0 8 . 0 6 . 0 4 . 0 2 . 0 0 . 0 40°C o — - — O 1 . 8 % 0 2 A — — — A 2 0 . 8 % 0 2 A — A 6 0 . 9 ° / O O 2 n-— — B 7 8 . 6 % 0 2 D — - ^ • > 9 9 % 0 2 -Q" 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 C 0 2 CONCENTRATION ( p l / l ) 31 were s i g n i f i c a n t l y decreased as the 0 2 concentration increased from 1.8% 0 2 to >99% 0 2. At 13° C and 78.6% 0 2, and at 20° C and 60.9% 0 2, apparent photosynthesis was saturated by the highest C0 2 concentrations used but was s t i l l i n h i b i t e d to some extent. Therefore i t appears that J saturating C0 2 concentrations may not be able to reverse the in h i b i t o r y effects of very high 0 2 concentrations on apparent photosynthesis. The data at 25° C were used for Figure 1-19 which shows the per cent i n h i b i t i o n of apparent photosynthesis by O2 concentrations ranging from 1.8% 0 2 to >99% 0 2 at C0 2 concentrations from 100 y l . / l . e©2 to 500 y l . / l . C0 2. At each C0 2 concentration, the degree of i n h i b i t i o n increased with increasing 0 2 concentration. At low CO2 concentrations at l e a s t , this increase appears to be non-linear. When the results are extrapolated to zero i n h i b i t i o n , i t appears that there would be no i n h i b i t i o n of apparent photosynthesis i n 300 u l . / l . C0 2 below about 14% 0 2. S i m i l a r l y , i n 400 y l . / l . C0 2 zero i n h i b i t i o n would occur below about 22% 0 2, and i n 500 y l . / l . C0 2 below about 30% 0 2. This lack of i n h i b i t i o n by some 0 2 concentrations above 1.8% 0 2 i s equivalent to that observed at high C0 2 concentrations i n Figures 1-9 and Figures 1-13 to 1-17. Figure 1-20 summarizes other measurements which were made to test the influence of l i g h t intensity on. the per cent i n h i b i t i o n of apparent photosynthesis at 25° C. Although the data are somewhat scattered, l i g h t intensity had no apparent effect on the degree of i n h i b i t i o n caused by 20.8% 0 2 or 60.9% 0 2. Changes i n l i g h t intensity between 6460 lux and 107,640 lux affected the apparent rate of photosynthesis (see Chapter I I ) , but the per cent i n h i b i t i o n was not altered because the rati o P i / P 2 w a s constant over this range i n l i g h t Figure 1-19 Effects of 0 2 Concentration and CO2 Concentration on the Per Cent Inhibi t i o n of Apparent Photosynthesis i n Excised Wheat Shoots at 25° C 32 Figure 1-20 Effects of CO2 Concentration and Light Intensity on the Per Cent Inhibition of Apparent Photosynthesis i n Excised Wheat Shoots at 25° C and 20.8% 0 9 or 60.9% 0 9 34 int e n s i t y . Figures 1-14 and IT-15 again show the decrease i n per cent i n h i b i t i o n as the C O 2 concentration increased. 4. Effect of C>2 on Photosynthetic Productivity Per cent i n h i b i t i o n i s a r e l a t i v e measure of the O 2 effect and does not d i r e c t l y indicate the influence of O 2 on the quantity of Carbon assimilated i n photosynthesis. The expression: Carbon D e f i c i t = 12 (P x - P 2) (2) 44 can be used to calculate the decrease i n photosynthetic productivity caused by the in h i b i t o r y effect of O 2 . The data from Experiment I were used to calculate the carbon d e f i c i t produced by 20.8% 0 2 i n different conditions of temperature and C O 2 concentration, and these results are presented i n Figure 1-21. I t i s apparent that there was a general increase i n carbon d e f i c i t with each r i s e i n temperature. At 300 y l . / l . C O 2 or higher C O 2 concentrations at 13° C and 450 u l . / l . C O 2 or above at 19.6° C, the carbon d e f i c i t was zero. The absence of a carbon 'deficit under these conditions i s related to the zero per cent i n h i b i t i o n of apparent photosynthesis which was. noted i n Figure 1-9. Also, according to Figure 1-21, the carbom:deficit tended to decrease as the C O 2 concentration approached 50 y l . / l . C O 2 . As we have seen i n the preceding sections, the degree of i n h i b i t i o n of apparent photosynthesis increases as the C O 2 decreases. The carbon d e f i c i t does not p a r a l l e l this increase i n per cent i n h i b i t i o n because the apparent rate of photosynthesis i s reduced by low C O 2 concen-trations . The carbon deficit'values calculated from the results of Experiment I I are presented i n Figure 1-22. Once again, the carbon Figure 1-21 The Carbon D e f i c i t i n Excised Wheat Shoots Caused by 20.8% 02 at Different Temperatures and C O 2 Concentrations (Experiment I) CARBON DEFICIT (mg C/hr/g fr wt) o p o o o Figure 1-22 The Carbon D e f i c i t i n Excised Wheat Shoots Caused by 20.8% 0 2 at Different Temperature and C O 2 Concentrations (Experiment II) d e f i c i t s were greatest at high temperatures and at C O 2 concentrations intermediate between the C O 2 compensation point and saturating C O 2 concentration. At the CC^ concentration to which plants are normally exposed, about 300 y l . / l . C O 2 , the carbon d e f i c i t ranged from zero to 0.67 mg. C/hr./g. f r . wt., and there was no s i g n i f i c a n t carbon d e f i c i t at 20° C or le s s . The greatest carbon d e f i c i t observed i n these studies was 0.76 mg. C/hr./g. f r . wt. and this occurred at 40° Ccand 450 y l . / l . C O 2 i n Experiment I I . Therefore, the presence of 20.8% O 2 i n the atmosphere can greatly reduce the photosynthetic productivity of wheat. I t i s obvious from the results presented i n Figures 1-13 to 1-18 that the presence of higher O 2 concentrations would reduce photosynthetic productivity even more. High C O 2 concentrations and low temperatures, however, tend to reduce the carbon d e f i c i t . 38 DISCUSSION These results provide a comprehensive description of the inhibitory effects of 0 2 on apparent photosynthesis by wheat under different environmental conditions. The degree of i n h i b i t i o n of apparent photosynthesis by 0 2 was enhanced by high 0 2 concentrations, high temperatures and low C0 2 concentrations, and under some conditions the presence of 0 2 can cause s i g n i f i c a n t reductions i n photosynthetic productivity. These results extend and are i n general accord with the findings of most previous studies on the effect of 0 2 on apparent photosynthesis. The relationship between the 0 2 concentration and the per cent i n h i b i t i o n of apparent photosynthesis, shown i n Figure 1-12, i s s i m i l a r to that obtained by other researchers using algae (40) and t e r r e s t r i a l plants (18). For the f i r s t time, except i n algae (40), i t has been demonstrated that high C0 2 concentrations can eliminate the inhib i t o r y effects of 20.8% 0 2 on apparent photosynthesis. Some previous investigations have indicated that the degree of i n h i b i t i o n of apparent photosynthesis by 0 2 i s greatest at low C0 2 concentrations (40, 43), but the details of the response of the 0 2 effect to C0 2 concentration are now clearly evident. The observation that l i g h t intensity does not affect the per cent i n h i b i t i o n of apparent photo-synthesis by 0 2 coincides with most other reports (3, 31, 43, 47). A few experiments with algae and mosses (43, 47) which found the per cent i n h i b i t i o n of apparent photosynthesis to increase with increasing l i g h t i ntensity do not agree with the present r e s u l t s . A c o n f l i c t between the present results and those of others also exists with respect to the influences of temperature on the 02 effect. In this 39 study with wheat, temperature had a marked effect on the per cent i n h i b i t i o n of apparent photosynthesis by 0 2, but early investigations with Chlorella (40) and Funaria (43) revealed no such effect. Some recent results with angiosperms, however, are i n agreement with the present observations (23). Whenever 21% 0 2 was found to i n h i b i t apparent photosynthesis, the degree of i n h i b i t i o n increased between 30° C and 40° C. There i s l i t t l e previous information on the effect of 0 2 concentration on the optimum temperature for apparent photosyn-thesis. Contrary to the results i n Experiment I but i n agreement with Experiment I I , however, BjBrkman found no increase i n the optimum temperature when Marchantia polymorpha was placed i n 2% 0 2 (4). The difference between the present observations and those of others cannot now be resolved, but i t i s possible that some of the discrepancies are due to the different species or different growing conditions u t i l i z e d i n different investigations. The effects of 0 2 on apparent photosynthesis are reflected i n the rates of plant growth at different 0 2 concentrations. The dry matter productivity of Phaseolus vulgaris, Mimulus cardinalis and Marchantia polymorpha was found to be enhanced when the plants were grown i n 2.5 to 5% 0 2 compared with those grown i n 21% 0 2 (5, 6). This enhancement of growth at low 0 2 concentrations near the C0.2:cO;mpen-sation point.was more than when the C0 2 concentration was 320 y l . / l . or 640 y l . / l . C0 2. Therefore, the response of plant growth to 0 2 and C0 2 concentrations corresponds we l l to, andmaybe explained by, the present observations on apparent photosynthesis. I t i s expected that the present results may be useful i n predicting the effect of atmos-pheric 0 2 on the productivity of wheat i n conditions where 0 2 40 concentration, temperature, C O 2 concentration and l i g h t i n t e n s i t y are l i m i t i n g productivity through t h e i r effects on apparent photosynthesis. The CO.2 compensation point results confirm e a r l i e r studies which demonstrated that the C0 2 compensation point i s d i r e c t l y proportional to the O 2 concentration (18, 41). This relationship i s now extended to include a wide range of temperatures, and the increase with increasing temperature i n the slope of the li n e a r response of the C 0 2 compensation point to O 2 concentration i s now apparent. In most previous studies, the C 0 2 compensation point has been found to extra-polate to zero at zero O 2 concentration (18, 41). In the present investigation, this was observed only at 25° C or lower temperatures. Above 30° C, the extrapolated C O 2 compensation point appears to be s i g n i f i c a n t l y greater than zero at zero 0 2 - Heath and Orchard (22) have reported that the extrapolated C O 2 compensation points of Pelargonium and Hydrangea were much higher than zero C O 2 at zero O 2 when the temperature was 27° C. I t would be interesting to learn whether these plants are capable of attaining much lower C O 2 compensation points i n the absence of O 2 at lower temperatures. The l i n e a r relationship between the C O 2 compensation point arid O 2 concentration and the finding that i t extrapolates to zero at zero O 2 has previously been used as evidence that during photosynthesis, dark respiration i s replaced by a different process of C O 2 production called photorespiration (18, 41). In contrast to dark r e s p i r a t i o n , which i s saturated by about 2% 0 2 (2, 18, 48), photorespiration i s distinguished by a lower a f f i n i t y for 0 2 and i t continues to increase i n rate with increasing O 2 concentration up to 1 0 0 % O 2 (18, 41). The observations that the C O 2 compensation point-0 2 concentration relationship sometimes extrapolates to greater than zero at zero 0 2 , however, i s consistent with the p o s s i b i l i t y that some CC^ production by dark respiration may occur during photosynthesis. I t i s possible that during illumination the rate of dark respiration may appear to be small because much of the C O 2 produced i s quickly reassimilated by photosynthesis before i t can escape from the leaf. At 25° C or less the rate of C 0 2 release by dark respiration during photosynthesis may be too low to cause a s i g n i f i c a n t increase i n the C O 2 compensation v point i n wheat. The operation of a c l a s s i c a l t r i c a r b o x y l i c acid cycle has been demonstrated i n the alga Scenedesmus obliquus during photosynthesis, but illumination did reduce the quantity of carbon entering the cycle (29, 30). Therefore, C O 2 production by dark respiration may occur i n illuminated photosynthetic tissue, but probably at a reduced rate. The C O 2 compensation point and the apparent rate of photosyn-thesis are both determined by the opposing processes of C O 2 uptake and C O 2 production. I t follows from this that the observed response of C O 2 exchange to O 2 concentration could be due to an i n h i b i t i o n by O 2 of photosynthetic C O 2 uptake, or to a stimulation by O 2 of C O 2 production, or to a combination of these two effects. The results of recent investigations do not support e a r l i e r suggestions (3, 43) that O 2 i n h i b i t s apparent photosynthesis by acting on photosynthetic electron transport i n the chloroplast. For example, a t y p i c a l O 2 effect has been observed i n C O 2 f i x a t i o n by a chloro-plast-free f r a c t i o n from Euglena g r a c i l i s (16). The a c t i v i t i e s of the isolated Calvin cycle enzymes (NADH and NADPH) glyceraldehyde-3-phosphate dehydrogenase (19, 45) and ribolose-5-phosphate kinase (19) have been shown to be inhi b i t e d by These enzymes could be the primary sit e s for the 0 2 e f f e c t , but the significance of t h e i r i n h i b i t i o n by 0 2 i n terms of the rate of photo-synthesis, and whether the i n h i b i t i o n occurs i n vivo remains to be determined. Direct evidence that photorespiration i s a component of the 0 2 effect has been provided by estimates of the i n d i v i d u a l rates of C0 2 uptake and production obtained from simultaneous measurements of 1U 12 C0 2 and C0 2 exchange by illuminated leaves. Bulley and Tregunna (9) found that, at the C0 2 compensation point, about 80% of the t o t a l effect of 21% 0 2 on apparent photosynthesis i n soybean was due to photorespiratory C0 2 production. The remainder was due to an i n h i b i -t i o n of photosynthetic C0 2 uptake. In addition, D'Aoust and Canvin (11) have reported that a large portion of the 0 2 effect i n sunflower was due to photorespiratory C0 2 production. I t i s i n t e r e s t i n g to note that there was a noticeable i n h i b i t i o n at the saturating C0 2 concentration i n Figure 1-13 at 80% 0 2 at 13° C and at 60% 0 2 at 20° C. This may correspond to the residual non-photorespiratory portion of the 0 2 e f f e c t . Further support for the correlation between photorespiration and the 0 2 effect comes from studies of C0 2 production by illuminated plants into a C0 2-free atmosphere. The effect of 0 2 concentration on the rate of C0 2 production by illuminated barley leaves (48) i s s i m i l a r to i t s effect on the per cent i n h i b i t i o n of apparent photosynthesis shown i n Figure 1-19. Also, the large i n h i b i t o r y effects of O2 on apparent photosynthesis at high temperatures corresponds with the high (about 35° C) optimum temperatures which 43 have been reported for CC^ evolution by illuminated leaves (24, 26). The absence of an 0 2 effect at high CO^ concentrations does not by i t s e l f prove the absence of photorespiration under those conditions. I t i s conceivable that, at C0 2 saturation, photores-p i r a t i o n may not influence the apparent rate of photosynthesis. This si t u a t i o n would occur i f photorespiration involves the oxidation of an intermediate between the C0 2 f i x a t i o n step and the l i m i t i n g step i n photosynthesis. Results which w i l l be presented i n Chapter I I I , however, demonstrate that the size of the post-illumination CC^ burst, and therefore the rate of photorespiration, i s decreased and may be negligible at high C0 2 concentrations. I t i s possible that the portion of the 0 2 effect which i s not due to photorespiratory CC^ production may be associated i n another way with the photorespiratory process. For example, photorespiration could depress the apparent rate of photosynthesis through competition for the photosynthetic C0 2 acceptor or a precursor of i t (19) as w e l l as by the production of C0 2. Of course, that component of the 0 2 effect which cannot be accounted for by photorespiratory C0 2 production could be e n t i r e l y unrelated to photorespiration. It has been suggested that g l y c o l i c acid or a related metabolite i s the substrate for photorespiration (13, 20, 49, 50, 51). The present results are consistent with this hypothesis. The excretion of glycolate by algae i s greatest at low C0 2 concentrations and high 0 2 concentrations (1, 38) and i t i s i n these conditions that the degree of i n h i b i t i o n of apparent photosynthesis by 0 2 i s greatest. Glycolate may be formed from intermediates of the Calvin cycle (1) and i t s formation may i n h i b i t photosynthesis by lowering the levels of photosynthetic intermediates as just described above. An alternative to the g l y c o l i c acid hypothesis has recently been advanced by Samish and Roller (39). They suggest that C>2 acts to increase the leaf mesophyll resistance to C0 2 uptake. As a r e s u l t , increased quantities of the C0 2 produced wit h i n the leaf by dark respiration are released from the leaf at high 0 2 concentrations instead of being reassimilated i n photosynthesis. This i s an intere-sting concept and can account for the present, data equally as w e l l as the g l y c o l i c acid hypothesis. Recent results on the behavior of C0 2 uptake and production rates at the C0 2 compensation point ( 9 ) , however, tend to support the hypothesis that the action of 0 2 i s to stimulate i n t e r n a l photorespiratory C0 2 production rather than to d i r e c t l y i n h i b i t photosynthetic C0 2 f i x a t i o n . At the present time, C0 2 production by photorespiration does not appear to be b e n e f i c i a l to the plant. Growth can be reduced by this a c t i v i t y and no ATP or reduced pyridine nucleotide formation has been associated with the C0 2 production. Goldsworthy (21) has recently speculated that photorespiration has been inherited from primitive photosynthetic and non-photosynthetic microorganisms which underwent symbiotic union early i n the history of l i f e . He has suggested that u n t i l recently photorespiration was not detrimental to plants because the C0 2 concentration of the earth's atmosphere was too high for an 0 2 effect to occur. Now, however, photorespiration i s becoming disadvantageous, especially for plants growing i n dense stands i n the tropics. There i s much to be learned about photorespiration, however, and I hesitate to condemn as e n t i r e l y detrimental a plant a c t i v i t y which can involve a carbon f l u x s i m i l a r to that involved i n photosynthesis. Nevertheless, the function of photorespiration i n the l i f e of a plant i s puzzling and requires further investigation. I t should be re c a l l e d , however, that i n a i r at temperatures of less than 13° C to 20° C, the apparent rate of photosynthesis i n wheat was not inh i b i t e d . Thus, photorespiration and the 0 2 effect may not be di s -advantageous to a plant l i k e x^heat during some of i t s l i f e cycle i n temperate conditions. 46 LITERATURE CITED 1. Bassham, J. A. and M. Kirk. 1962. The effect of oxygen on the reduction of C O 2 to g l y c o l i c acid and other products during photosynthesis by Chlorella. Biochem. Biophys. Res. Comm. 9: 376-380. 2. Beevers, H. 1960. Respiratory metabolism i n plants. Row, Peterson and Co., White P l a i n s , N.Y. 3. Bjorkman, 0. 1966. The effect of oxygen concentration on photo-synthesis i n higher plants. Physiol. Plantarum 19: 618-633. 4. Bjorkman, 0. and E. Gauhl. 1969. Effect of temperature and oxygen concentration on photosynthesis i n Marchantia poly- morph a. Carnegie Inst. Year Book 67: 479-482. Carnegie Inst, of Wash. 5. Bjorkman, 0., E. Gauhl, W. M. Hiesey, F. Nicholson and M. A. Nobs. 1969. Growth of Mimulus, Marchantia and Zea under different oxygen and carbonO. dioxide l e v e l s . Carnegie Inst. Year Book 67: 477-479. Carnegie Inst, of Wash. 6. Bjorkman, 0., W. M. Hiesey, M. Nobs, F. Nicholson and R. W. Hart. 1968. Effect of oxygen concentration on dry matter produc-tion i n higher plants. Carnegie Inst. Year Book 66: 228-232. Carnegie Inst, of Wash. 7. Briggs, G. E. and C. P. Whittingham. 1952. Factors affecting the rate of photosynthesis of Chlorella at low concentrations of carbon dioxide and i n high illumination. New Phytol. 51: 236-249. 8. Brun, W. A. 1961. Photosynthesis and transpiration of banana leaves as affected by severing the vascular system. Plant Physiol. 36: 577-580. 9. Bulley, N. R. and E. B. Tregunna. 1970. Photosynthesis and photorespiration rates at the C O 2 compensation point. Can. J . Botany, i n press. 10. Coombs, J. and C. P. Whittingham. 1966. The mechanism of i n h i -b i t i o n of photosynthesis by high p a r t i a l pressures of oxygen i n Chlorella. Proc. Roy. Soc. B. 164: 511-520. 11. D'Aoust, A. L. and D. T. Canvin. 1969. Effect of oxygen concen-t r a t i o n on the C O 2 exchange of sunflower leaves. XI Internat, Botan. Congr. (Abstract). 12. Detchev, G. , M. Setchenska and N. Tomova. 1969. On the possible enzymatic pathways of the electron to the molecular oxygen i n electron transport reactions of photosynthesis. Progr. i n Photosynthesis Res. 1: 503-507. 47 13. Downton, W. J. S. and E. B. Tregunna. 1968. Photorespiration and glycolate metabolism: A reexamination and correlation of some previous studies. Plant Physiol. 43: 923-929. 14. Egle, K. and H. Fock. 1966. Light respiration - correlations between C O 2 f i x a t i o n , 0 2 pressure and glycollate concentra-t i o n . In: Goodwin, T. W. (editor). Biochemistry of Chloroplasts. Academic Press, New York. Vol. 2.~ pp.79-87. 15. Egle, K. and W. Schenk. 1953. Der Einfluss.der temperature, auf die Lage des COj- K e ^ " e n s a t T c ^ p l " u n k t e s i . P l a n t a 43: 83-97. V " ' " "' 16. E l l y a r d , P. W. and A. San Pietro. 1969. The Warburg effect i n a chloroplast-free preparation from Euglena g r a c i l i s . Plant Physiol. 44: 1679-1683. 17. Fock, H. and K. Egle. 1966. Uber die "Lichtatmung" bei grunen Pflanzen. I. Die Wirkung von Sauerstoff und Kohlendioxyd auf den C02-Gaswechsel wahrend der Licht - und DunkelphasB. B e i t r . B i o l . Pflanzen 42: 213-239. 18. Forrester, M. L., G. Krotkov and C. D. Nelson. 1966. Effect of oxygen on photosynthesis, photorespiration and respiration i n detached leaves. I. Soybean. Plant Physiol. 41: 422-427. 19. Gibbs, M., P. W. E l l y a r d and E. Latzko. 1968. Warburg effect: control of photosynthesis by oxygen. In: Shibata, K., Takamiya, A., Jagendorf, A. T. and F u l l e r , R. C. (editor). Comparative Biochem. and Biophysics of Photosynthesis. U n i -ve r s i t y Park Press, Pennsylvania, pp. 387-399. 20. Goldsworthy, A. 1966. Experiments on the o r i g i n of C0 2 released by tobacco leaf segments i n the l i g h t . Phytochem. 5: 1013-1019. 21. Goldsworthy, A. 1969.' Riddle of photorespiration. Nature 224: 501-502. 22. Heath, 0. V. S. and B. Orchard. 1957. Carbon assimilation at low carbon dioxide l e v e l s . I I . The process of apparent assimilation. J . Exp. Bot. 19: 176-192. 23. Hesketh, J . 1967. Enhancement of photosynthetic C02 assimilation i n the ; absence of oxygen, as dependent upon species and temperature. Planta 76: 371-374. 24. Hew, C-S., G. Krotkov and D. T. Canvin. 1969. Effects of tem-perature on photosynthesis and C O 2 evolution i n l i g h t and darkness by green leaves. Plant Physiol. 44: 671-677. 25. Hoffman, P. and I. Ticha. 1969. Der Gaswechsel von Phaseolus vulgaris - und Pisum sativum - Keimpflanzen nach der Entfer-nung des Wurzelsystmes. Photosynthetica 3: 73-78. 48 26. Hofstra, G. and J . D. Hesketh. 1969. Effects of temperature on the gas exchange of leaves i n l i g h t and dark. Planta. 85: 228-237. 27. Jenkins, H. V. 1959. An airflow planimeter for measuring-the area of detached leaves. Plant Physiol. 34: 532-536. 28. Koch, W. and T. K e l l e r . 1961. Der Einfluss von Alterung und Ab'schheiden auf den CO2 - Gaswechsel von Pappelblattern. Ber. Deutsch bot. Ges. 74: 64-74. 29. Marsh, H. V. J r . , J . M. Galmiche and M. Gibbs. 1965. Respiration during photosynthesis. In: Krogman, D. W. and Powers, W. H. (editors). Biochemical Dimensions of Photosynthesis. Wayne State Univ. Press, Detroit, pp. 95-107. 30. Marsh, H. V., J . M. Galmiche and M. Gibbs. 1965. Effect of l i g h t on the t r i c a r b o x y l i c acid cycle i n Scenedesmus. Plant Physiol. 40: 1013-1022. 31. McAlister, E. D. and J . Myers. 1940. The time course of photo-synthesis and fluorescence observed simultaneously. Smithsonian Inst. Misc. Collections 99: 1-37. 32. Mehler, A. H. 1951. Studies on reactions of illuminated Chloro-plasts. I. Mechanism of the reduction of oxygen and other H i l l reagents. Arch. Biochem. Biophys. 33: 65-77. 33. Meidner, H. 1965. Stomatal control of transpirational water loss. Symp. Soc. Exp. B i o l . 19: 185-204. 34. Miyachi, S., S. Izawa and H. Tamiya. 1955. Effect of oxygen on the capacity of carbon dioxide f i x a t i o n by green algae. J. Biochem. (Tokyo) 42: 221-244. 35. Mortimer, D. C. 1959. Some short-term effects of increased carbon dioxide concentration on photosynthetic assimilation i n leaves. Can. J . Bot. 37: 1191-1201. 36. Oh-Hama, T. and S. Miyachi. 1959. Effects of illumination and oxygen supply upon the levels of pyridine nucleotides i n Chlorella c e l l s . Biochem. Biophys. Acta 34: 202-210.;' 37. Poskuta, J. 1968. Photosynthesis, photorespiration and respira-t i o n of detached spruce twigs as influenced by oxygen concentration and l i g h t i n t e n s i t y . Physiol. Plantarum 21: 1129-1136. 38. Pritchard, G. G., W. J. G r i f f i n and C. P. Whittingham. 1962. The effect of carbon dioxide concentration, l i g h t intensity and i s o n i c o t i n y l hydrazide on the photosynthetic production of g l y c o l l i c acid by Chlorella. J . Exp. Botany 13: 176-184. 49 39. Samish, Y. and D. Ko l l e r . 1968. Estimation of photorespiration of green plants and of t h e i r mesophyll resistance to C O 2 uptake. Ann. Bot. (N.S.) 32: 687-694. 40. Tamiya, H. and H. Huzisige. 1949. Effect of oxygen on the dark reaction of photosynthesis. Acta Phytochemica 15: 83-104. 41. Tregunna, E. B., G. Krotkov and C. D. Nelson. 1966. Effect of oxygen on the rate of photorespiration i n detached tobacco leaves. Physiol. Plantarum 19: 723-733. 42. Turner, W. B. and R. G. S. Bidwell. 1965. Rates of photosyn-thesis i n attached and detached bean leaves and the effect'of spraying with indoleacetic acid. Plant Physiol. 40: 446-451. 43. Turner, J . S. and E. G. B r i t t a i n . 1962. Oxygen as a factor i n photosynthesis. B i o l . Rev. 37: 130-170. 44. Turner, J . S., M. Todd and E. G. B r i t t a i n . 1956. The i n h i b i t i o n of photosynthesis by oxygen. I . Comparative physiology of the effect. Aust. J. B i o l . S c i . 9: 494-510. 45. Turner, J . S., J. F. Turner, K. D. Shortman and J. E. King. 1958. The i n h i b i t i o n of photosynthesis by oxygen. I I . The effect of oxygen on ^gly.ceraldehyde phosphate dehydrogenase from chloroplasts. Aust. J. B i o l . S c i . I I : 336-342. 46. Warburg, 0. 1920. Uber die Geschwindigkeit der photochemischen Kohlensaurezersetzung i n lebenden Zellen. I I . Biochem. Z. 103: 188-217. 47. Wassink, E. C., D. Vermeulen, G. H. Remen and E. Katz. 1938. On the r e l a t i o n between fluorescence and assimilation i n photosynthesizing c e l l s . Enzymologia 5: 100-109. 48. Yemm, E. W. 1969. Photorespiration and photosynthesis i n young leaves of barley. Progr. i n Photosynthesis Res. 1: 474-481. 49. Z e l i t c h , I. 1958. The role of gly c o l i c acid oxidase i i i the respiration of leaves. J. B i o l . Chem. 233: 1299-1303. 50. Z e l i t c h , I. 1959. The relationship of g l y c o l i c acid to respira-tio n and photosynthesis i n tobacco leaves. J . B i o l . Chem. 234: 3077-3081. 51. Z e l i t c h , I. 1966. Increased rate of net photosynthetic carbon dioxide uptake caused by the i n h i b i t i o n of glycolate oxidase. Plant Physiol. 41: 1623-1631. 50 Chapter I I SOME COMPARATIVE ASPECTS OF THE PHYSIOLOGY OF C 0 2 EXCHANGE BY WHEAT AND CORN SHOOTS INTRODUCTION In recent years, certain plants have been distinguished from others on the basis of th e i r characteristics of C 0 2 exchange, photosynthetic carbon metabolism, and leaf anatomy. The chloridoid-eVagrbstoidand some panicoid grasses, such as corn, as w e l l as some members of the Cyperaceae, Amaranthaceae, Chenopodiaceae, Portulacaceae and Euphorbiaceae exhibit C 0 2 compensation points below 5 y l . / l . C 0 2 i n the presence of 2 0 . 8 % 0 2 (17, 66, 79, 80), and are capable of very rapid photosynthesis at warm temperatures and high l i g h t i n t e n s i t i e s (11, 23, 24, 25, 35, 39, 41, 44, 67, 68). These plants possess the C 4 - d i c a r b o x y l i c acid pathway of photosynthesis (17, 36, 38, 51, 52) i n which C 0 2 i s i n i t i a l l y combined with phosphoenolpyruvic acid by the enzyme phosphopyruvate carboxylase. In this thesis, these plants w i l l be called C^  plants since the four-carbon acids oxaloacetate, malate and aspartate are th e i r i n i t i a l products of photosynthetic C 0 2 f i x a t i o n (36, 54). The leaves of C 4 plants contain a well-developed parenchyma bundle sheath surrounded by palisade mesophyll tissue. The s i z e , structure, and a b i l i t y to accumulate starch differsnbetween chloroplasts of the bundle sheath and palisade mesophyll (5, 16, 17, 55, 56, 57) . In contrast, wheat i s representative of other plants which exhibit C 0 2 compensation points much above 5 y l . / l . C 0 2 i n the 51 presence of 2 0 . 8 % 0 2 (17), and have r e l a t i v e l y low apparent rates of photosynthesis at warm temperatures and high l i g h t i n t e n s i t i e s ( 1 2 , 23, 24, 40, 41, 42, 44, 67, 68). For convenience, plants l i k e wheat which carryco.ut photosynthetic C 0 2 f i x a t i o n by the Calvin cycle i n which the three-carbon acid 3-phosphoglycerate i s the i n i t i a l product ( 2 ) w i l l hereafter be called C 3 plants. In leaves of C 3 plants, the parenchyma bundle sheath i s usually poorly defined and the palisade mesophyll can be much more extensive. Chloroplasts i n the palisade mesophyll and the parenchyma bundle sheath are not s t r i k i n g l y different i n appearance (5). The research presented here was carried out to survey the differences i n the C 0 2 exchange characteristics of C 3 and C^ plants, as exemplified by wheat and corn. Forrester e^ t _al. have shown that 0 2 i n h i b i t s apparent photosynthesis i n corn as w e l l as i n several C 3 plants (28, 29). In the present study, further tests were made to determine whether the in h i b i t o r y effects of 0 2 are si m i l a r i n wheat and corn. Measurements were made of C 0 2 exchange by corn i n different 0 2 concentrations, temperatures, C 0 2 concentrations and l i g h t i n t e n s i t i e s . Additional measurements were made with wheat to supp-lement the information already given i n Chapter I. Major differences were observed i n the pattern of C 0 2 exchange by wheat and corn shoots. The relationship of these differences to photorespiration and ecology offCg and C^  plants w i l l be discussed. MATERIALS AND METHODS Wheat (Triticum aestivum L. var. Spring Thatcher) and corn (Zea mays L. var/ Pioneer) plants were grown and excised as described for Experiment I I i n Chapter I. The chamber shown i n Figure 1 - 2 was 52 used for a l l CC>2 exchange measurements reported i n this Chapter. The methods used to control and measure the l i g h t and temperature environment of enclosed shoots were i d e n t i c a l to those used i n Chapter I. Also, the closed system described i n Chapter I was used to obtain the C O 2 compensation point and C O 2 concentration results presented i n Figure;;11-4. A simple open system was used for the temperature and l i g h t intensity studies reported i n Figures II-5, II-6 and II-7. Outside a i r was drawn into this system and passed i n series through the reference c e l l of the IRGA, the plant chamber, the sample c e l l of the IRGA, a Fisher "Dynapump" a i r pump, a Matheson R-2-15-B flowmeter, and then out of the system. The IRGA was calibrated to giveuzero to f u l l - s c a l e deflection between 250 y l . / l . and 350 y l . / l . C O 2 . As i n the closed system, the a i r flow rate through this simple open system was 3 l./min. In this open system, the apparent rates of photosyn-thesis were calculated from the a i r flow rate and the C O 2 concentration differences between the reference and sample c e l l s of the IRGA. When measurements were made of the response of apparent photosynthesis to temperature, the excised shoots were preconditioned for an i n i t i a l 30 minutes at 25° C and 32,300 lux. Then, the temperature i n the chamber was gradually reduced to about 5° C and increased to more than 40° C. The rate of temperature change was less than 2° C/min. The shoots were preconditioned the same way for the l i g h t intensity studies. When the l i g h t intensity was altered, approximately 20 minutes elapsed before apparent photosynthesis became stable, and measurements were made only after this time. Throughout these temperature and l i g h t i ntensity studies, the C O 2 concentration of the a i r entering the system was measured at about 15 minute intervals and was 335± 1 0 - u l . / l . C© 2 « A more complex open system, i l l u s t r a t e d i n Figure I I - l , was used for the time course studies reported i n Figures I I - 2 and I I - 3 . This system was developed to provide constant C O 2 concentrations inya gas stream containing different O 2 concentrations. Pure C O 2 from a compressed gas tank was diluted with gas containing 1 . 8 % , 2 0 . 8 % or 1 0 0 % 0 2 , balance N 2, by mixing these gases together using two Gallenkamp gas mixing pumps and a Matheson gas proportioner i n series An IRGA measured the C O 2 concentration which resulted from these d i l u t i o n s . Once the desired composition of the mixed gas was established, the gas was passed i n series through the reference c e l l of a second IRGA, the plant chamber, the sample c e l l of the second IRGA, a Matheson R-2-15-B flowmeter, and out of the system. The second IRGA was calibrated to give a f u l l scale deflection for a 1 0 0 y l . / l . C O 2 concentration difference between the reference and the sample c e l l s . Once again, the apparent rate of photosynthesis was calculated from the observed C O 2 concentration difference between the reference and sample c e l l s , and the gas flow rate, which was 1 l./min In Figures I I - 2 to II-7, each point i s the result of a single deter-mination of the apparent rate of photosynthesis. As i n Chapter I, th apparent rates of photosynthesis are expressed i n a per unit fresh weight basis. Airflow planimeter measurements (49) determined that 1 g. fresh weight was equivalent to 0.37 dm of corn shoots or 0 0.40 dm of wheat shoots. These values can be used to approximate the apparent rates of photosynthesis on a leaf area basis. Figure I I - l Open System Used to Generate Constant,CO2 Concentrations i n an A i r Stream Containing Different 0 2 Concentrations 54 pressure release flasks ascarite CO2-absorbers plant chamber lowmeter 55 RESULTS AND DISCUSSION 1 . The C O 2 Compensation Point It has been reported that i n a i r containing 2 1 % O 2 , the C 0 £ compensation point of C^  plants i s close to zero y l . / l . C O 2 (17, 29, 60, 64, 66). In Chapter I , i t was observed that the C O 2 compensation point of wheat was always greater than 5 y l . / l . C O 2 except at very low O 2 concentrations. Also, the C O 2 compensation point of wheat was increased by increasing temperatures and O 2 concentrations. Other C 3 plants also exhibit high C O 2 compensation points which respond to temperature and O 2 concentration (17, 2 0 , 28, 81, 89). To determine whether a high C O 2 compensation point could be obtained with corn by modifying the temperature or O 2 concentration, measurements were made with corn shoots at 32,300 lux, at temperatures from 13° to 40° C and at O 2 concentrations from 1.8% to 1 0 0 % 0 2 - In a l l conditions, the C O 2 compensation point of corn was found to be less than 5 y l . / l . C O 2 , which was indistinguishable from zero y l . / l . C 0 2 by techniques used. Therefore, within the l i m i t s tested, the low C O 2 compensation point of corn does not depend on the ambient temperature or O 2 concentration. This difference between the C O 2 compensation points of C 3 and C 4 plants i s important since i t i s indicative of a difference i n the rates of C O 2 production by two types of plants during i l l u m i n a t i o n . The high C O 2 compensation points of C 3 plants are evidence that substantial amounts of C O 2 are released by these plants by photores-p i r a t i o n , and that the r e l a t i v e rates of C O 2 production and release are altered by the temperature and the O 2 concentration. On the other hand, the low C 0 ~ compensation points of corn and other C 4 56 plants indicate a low rate of C O 2 release during photosynthesis by these plants. In fact, i t has proven d i f f i c u l t to demonstrate that C4 plants release any C O 2 at a l l during i l l u m i n a t i o n . When the leaves of C4 plants are exposed to l i g h t and placed i n a stream of CC^-free a i r , no detectable release of C O 2 into the a i r stream i s observed (25). Irvine (47) recently carried out an experiment i n which a ^ C - l a b e l l e d and an unlabelled corn plant were enclosed together i n a sealed chamber and illuminated. After 1 hour, he was able to detect very small but 14 measurable quantities of C accumulating i n the i n i t i a l l y unlabelled plant. He concluded that corn does release small amounts of C O 2 during photosynthesis. The s e n s i t i v i t y of this CC>2 release process to different concentrations of O 2 was not tested, so i t i s not known whether the C O 2 originated from photorespiration or some other source. Meidner (60, 61) was able to obtain an elevated C O 2 compensation point with corn by placing illuminated leaves under water stress. This C O 2 compensation point, however was not responsive to changes i n O 2 concentration above 2% O 2 , and therefore was not due to an increased rate of photorespiration. Instead, i t was attributed to a reduction i n the rate of photosynthetic C O 2 f i x a t i o n by the high water d e f i c i t s . Corn also exhibits a high C O 2 compensation point when the l i g h t i n tensity i s very low (9, 18). Once again, i t does not appear that photorespiration i s implicated i n this r e s u l t , which seems to be due to a simple balance between the opposing processes of C O 2 f i x a t i o n and dark respiration. The C O 2 compensation point studies therefore demonstrate a major difference between C 3 and C 4 plants. During photosynthesis, C 3 plants can release substantial quantities of C O 2 by an 0 2 - s e n s i t i v e 57 photorespiratory process, while plants apparently lack photores-p i r a t i o n . 2 . Effecf-of © 2 Concentration on the Apparent Rate of Photosynthesis Apparent photosynthesis may be inhibited by the presence of O 2 i n both wheat and corn, but the characteristics of the i n h i b i t i o n appear to d i f f e r between the two species. This difference i s i l l u s -trated by Figures I I - 2 and II-3 which show the time course of apparent photosynthesis of wheat and corn shoots which were exposed to atmospheres containing 300 y l . / l . C 0 2 ; 1.8%, 2 0 . 8 % or >"99% 0 2 ; balance N 2 . These measurements were carried out at 25° C and 32,300 lux. Figure I I - 2 shows that, as i n Chapter I , apparent photosynthesis i n wheat was inh i b i t e d by O 2 concentrations above.1.8% O 2 . At any part i c u l a r O 2 concentration, however, the apparent rate of photosyn-thesis of wheat was constant following the i n i t i a l 30 minute period of photosynthetic induction. Also, i f the O 2 concentration was changed from 1.8% or >99% O 2 to 2 0 . 8 % O 2 at any time, the apparent rate of photosynthesis of wheat changed rapidly to become the same as that of shoots which had been exposed to 2 0 . 8 % O 2 a l l the time. Thus, the effect of O 2 on apparent photosynthesis of wheat i s constant i n time and i s reversible. As discussed i n Chapter I , at least part of the effect of O 2 on apparent photosynthesis of wheat appears to be due to the response of photorespiration to O 2 • Figure II-3 shows that the apparent rate of photosynthesis of corn was the same i n 2 0 . 8 % O 2 as i n 1.8% 0 2 ^ When the O 2 concentration was 1 0 0 % , however, C O 2 assimilation by corn was greatly i n h i b i t e d . Also, the degree of i n h i b i t i o n was not constant but increased with the time the corn shoots were exposed to >99% 0 2 . When the corn shoots Figure I I - 2 Time Course of CO2 Exchange by Excised Wheat Shoo i n Atmospheres Containing 300 u l . / l . CO2 and Different O2 Concentrations The Shoots were Excised at Time = 0 TIME (min) Figure II-3 Time Course of CC>2 Exchange by Excised Corn Shoots i n Atmospheres Containing 300 y l . / l . CO2 and Different O2 Concentrations The Shoots were Excised at Time = 0 APPARENT RATE OF PHOTOSYNTHESIS (mg G0 2 . /hr/g fr wt) O —< r o w -N- oi o >j co vo p b b b b b b b b b o b 60 were returned to 20.8% 0 2 after one or two hours i n >99% 0 2, the reversal of the i n h i b i t i o n was much more gradual than with wheat. Forrester et_ al. (29) have previously shown that high 0 2 concentrations i n h i b i t apparent photosynthesis i n corn. The present results reveal that the inhibitory effect of >99% 0 2 i s time-dependent and only slowly reversible. These results confirm the findings of several other studies which have also indicated that photosynthesis of plants i s not enhanced when the 0 2 concentration i s reduced from 20.8% 0 2 to 2% 0 2 or less (15, 29, 40). Additional measurements were made to determine whether 20.8% 0 2 i n h i b i t s apparent photosynthesis of corn under other conditions of temperature, C0 2 concentration or l i g h t i n t e n s i t y . Figure II-4 shows that the apparent rates of photosynthesis i n 1.8% 02 and 20.8% 0 2 were s i m i l a r i n a l l conditions tested. Therefore, 20.8% 0 2 does not appear to be s u f f i c i e n t to affect apparent photosynthesis i n corn. This absence of an i n h i b i t o r y effect with corn does not resemble that found with wheat i n Chapter I when the i n h i b i t i o n was absent only at 1 low temperaturessand high C0 2 concentrations. Since the C0 2 compensation point of corn was always less than 5 y l . / l . C0 2 i n these studies, photorespiration does not appear to be implicated i n the response of apparent photosynthesis of corn to high 0 2 concentrations. I t i s possible that >99% 0 2 i n h i b i t e d photosyn-thesis i n corn by causing stomatal closure. There i s no explanation, however, why stomata i n wheat would not be s i m i l a r l y affected. I t i s perhaps more l i k e l y that the results i n Figure II-3 should be i n t e r -preted i n terms of an i n h i b i t o r y effect of >99% 0 2 on f i x a t i o n i t s e l f . Because photosynthesis involves many steps, there are numerous si t e s Figure II-4 Effects of O2 Concentration, Temperature, CO2 Concentration and Light Intensity on the Apparent Rate of Photosynthesis of Excised Corn Shoots 61 SZ CN O u CO CO LU X >-CO o t-O X D_ 12 11 10 < cn LU cn D_ < O 1 . 8 % 0 2 * 2 0 . 8 % 0 2 ® ^ o o A A ^ O - O 40 C 32,300 lux A A A A o o o o o o~ 25°C 32,300 lux ^ - 9 - 6 - 4 2 5 U C 6,460 lux o o 5 6 ' 9 " •A—-A—/-v— 13 C 32,300 lux 9 6 100 CO. 200 300 CONCENTRATION 400 Cul/I) 500 which could be susceptible to i n h i b i t i o n by 0 2 (13, 83). In plants, i t i s possible that high 0 2 concentrations may i n h i b i t one or several steps i n the C^-dicarboxylic acid pathway, although l i t t l e evidence i s now available to specify the par t i c u l a r s i t e ( s ) of action. One enzyme of the C^-dicarboxylic acid pathway, pyruvate, phosphate dikinase, deserves some investigation, however, since i t i s reversibly inactivated i n the presence of 0 2 i n v i t r o (1). 3. Effect of Temperature on Apparent Photosynthesis There are marked differences i n the responses of C 3 and plants to temperature. In a i r , C 3 grasses generally have optimum temperatures for apparent photosynthesis and for growth which are d i s t i n c t l y lower than those of C 4 grasses (11, 12, 21, 25, 26, 30, 31, 44, 62, 63, 67, 68). For example, Figure II-5 shows that, at 20.8% 0 2 and 335 u l . / l . C O 2 , the optimum temperature for apparent photosyn-thesis i n wheat was about 25° C, while for corn i t was almost 40° C. At the optima, and at a l l temperatures above about 18° C, the apparent rate of photosynthesis of corn was greater than that of wheat, Below 18° C, however, apparent photosynthesis of wheat was more rapid. These results are very s i m i l a r to thos of De Jager (12) who used the C 3 plants Lolium perenne and Lolium multiflorum, and the plant Paspalum dilatatum. The temperature curve for wheat i n Figure II-5 resembles the results of Experiment I i n Chapter I but d i f f e r s from the results of Experiment I I . The data from Experiment I I were collected over a period of 3 months while Experiment I was performed during a 3 week period and the measurements i n Figure II-5 were obtained on one day. Some d r i f t i n the apparent rate of photosynthesis or i n the temperature Figure II-5 Effects of Temperature on the Apparent Rates, of Photosynthesis of Excised Wheat and Corn Shoots i n A i r Containing 20.8% 0 2 and 335 u l . / l . C0 2 response may account for the different results found i n Experiment I I . Since they were obtained over a short period of time, the results i n Figure II-5 probably represent the best estimate of the within plant variation i n apparent photosynthesis i n response to changes i n temperature. Photorespiration may contribute to this difference i n temperature response between C 3 and plants. In Chapter I i t was observed that the optimum temperature for apparent photosynthesis i n wheat decreased as the 0 2 concentration increased. This decrease i n temperature optimum was noticeable i n only one case i n 20.8% 0 2, however, and was not rioted i n recent studies with Marchantia poly- morph a (6). Therefore, conclusive evidence i s not available to indicate whether or not photorespiration i s a major cause of the differences i n temperature response between C 3 and C 4 plants i n 20.8% 0 2. Treharne and Cooper (82) have suggested that these differences may be the consequence of differences i n the temperature responses of the major carboxylating enzymes of C 3 and C^  plants. They observed that the optimum temperature for the ribulose-1, 5-diphosphate carboxylase of the C 3 plants Lolium perenne and Avena sativa was 20° C to 25° C. In contrast, the a c t i v i t y of the phosphoenolpyruvate carboxylase of the C 4 plants Zea mays and Cenchrus c i l i a r u s was greatest between 30° and 35° C. In a different study, the combined a c t i v i t i e s of the phosphoenolpyruvate carboxylase and NAD-malate dehydrogenase enzymes of Bryophyllum tubiflorum were greatest at 35° C (8). Therefore the carboxylating enzymes may be a major source of the d i f f e r e n t i a l effect of temperature on C0 2 exchange by C 3 and C 4 plants. 65 4. Effect of C O 2 Concentration on Apparent Photosynthesis The effect of C O 2 concentration on apparent photosynthesis of wheat was presented i n Chapter I , Figures 1-9 and 1-13 to 1-18. Corresponding results with corn are shown i n Figure 1-4. The effects of CC>2 concentration are generally s i m i l a r except that the C O 2 compensation point of wheat i s higher than that of corn at O 2 concentrations above 1.8% O 2 . As a r e s u l t , corn can carry out net CC>2 assimilation at much lower C O 2 concentrations than can wheat. The CC>2 concentration response of apparent photosynthesis of wheat and corn appears to resemble the results obtained i n studies of the effect of substrate concentration on reaction rate i n isolat e d enzyme systems. This s i m i l a r i t y has led some investigators to apply methods analogous to those used i n the study of enzyme ki n e t i c s to the analysis of the C O 2 concentration response of photosynthesis. In this approach, the rate of CC>2 assimilation at CC>2 saturation i s termed the apparent Vmax of photosynthesis, and the CC>2 concentration at which Table II-1 Comparison of the Apparent K i n e t i c Constants of Photosynthetic C O 2 Assimilation of Excised Wheat and Corn Shoots Plant Temperature (°C) Light Intensity (lux) "02' Cone. (%) (mg. Apparent Vmax C0 2/hr/g f r wt) Apparent Km (y l C02/1) Wheat 25 32,300 1.8 8.50 119 25 32,300 20.8 8.50 177 Corn 25 32,300 1.8 8.72 128 25 32,300 20.8 8.72 121 6 6 the rate of CC^ assimilation i s one-half the apparent Vmax i s called the apparent (32) . Table I I - l and the results of Goldsworthy (32) demonstrate that at low 0 2 concentrations the apparent K m for C 0 2 assimilation by C 3 plants i s simi l a r to that of C 4 plants. In 2 0 . 8 % 0 2 , the apparent of wheat was much higher than i t was at 1 . 8 % 0 2 , while the apparent K m of corn remained low. The s i m i l a r apparent 1^ values for C 3 and plants at low 0 2 concentrations indicates that under these conditions the a f f i n i t y for C 0 2 of the ove r a l l C0 2 assimilation systems of these two plant types i s s i m i l a r . In 2 0 . 8 % 0 2 , the apparent a f f i n i t y of C 3 plants for C O 2 i s much lower than i n 1 . 8 % 0 2 because of photorespiration and other components of the 0 2 effectr:. As before, 2 0 . 8 % 0 2 did not reduce the apparent Vmax of wheat or corn. Goldsworthy (32) has argued that the s i m i l a r K m values at low 0 2 concentrations i s evidence that the internal C 0 2 f i x a t i o n processes i n C 3 and plants are s i m i l a r i n th e i r a f f i n i t y for C 0 2 . This conclusion could be correct i f the resistances to C0 2 d i f f u s i o n into leaves of C 3 and plants were equal. Unfortunately, several other studies have indicated that there i s a greater resistance to C 0 2 d i f f u s i o n into leaves of plants than into leaves of C 3 plants (14, 44, 70). Because of th i s difference i n C 0 2 d i f f u s i o n resistance, the apparent k i n e t i c constants cannot i n themselves be used to describe the characteristics of the in t e r n a l C 0 2 f i x a t i o n systems of C 3 and C 4 plants. Studies with isolated enzymes have indicated that phosphoeno-lpyruvate carboxylase has a higher a f f i n i t y for C 0 2 than ribulose-1, 5-diphosphate carboxylase (50, 8 6 ) . These i n v i t r o results are d i f f i - v . cult to relate to the observations with leaves, however, since the l a t t e r are the consequence of the a c t i v i t i e s of many other enzymes as w e l l as physical processes. 5. Effect of Light Intensity on Apparent Photosynthesis Figure II-6 shows the response of apparent photosynthesis by wheat and corn to different l i g h t i n t e n s i t i e s . These measurements were carried out at 25° C, 335 u l . / l . C0 2 and 20.8% 0 2. In wheat, apparent photosynthesis was close to l i g h t saturation at l i g h t i n t e n s i t i e s above about 32,000 lux. This result i s i n agreement with other studies which have found that l i g h t saturation of apparent photosynthesis of.C^iplants often occurs i n the range from 20,000 to 35,000 lux (10, 12, 39, 41, 67, 84). Although the apparent rates of photosynthesis were higher for corn, the response to l i g h t intensity was s i m i l a r to that of wheat at i n t e n s i t i e s below 36,000 lux. When the l i g h t intensity was increased above that value, however, an unusual response was observed. Figure II-7 shows that after a change i n l i g h t intensity from 32,000 lux to 100,000 lux there was a temporary increase i n the apparent rate of photosynthesis by corn followed by a gradual decline i n the rate. This decline continued after the rate became less than that observed at the preceding lower l i g h t i n t e n s i t y . The temporary increase i n the apparent rate of photosynthesis after the increase i n l i g h t intensity may have been due to the accompanying increase i n tempe-rature from 25° C to 31.5° C. The i n h i b i t o r y effect of high intensity l i g h t i n corn was not reversible since the apparent rate of photosyn-thesis remained below the i n i t i a l rate when the l i g h t intensity was restored to 32,300 lux. The occurrence of this high l i g h t intensity Figure I1-6 Effects of Light Intensity on the Apparent Rates of Photosynthesis of Excised Wheat and Corn Shoots i n A i r Containing 20.8% 02 and 335 u l . / l . C0 2 68 Figure I I - 7 Effects of Exposure to High Light Intensity on the Apparent Rate of Photosynthesis of Excised Corn Shoots APPARENT RATE OF PHOTOSYNTHESIS (mg C 0 2 / h r / g fr wt ) response of corn was v e r i f i e d with f i v e different samples of corn shoots. The behavior seems to be a pe c u l i a r i t y of the plants used, since the author has not encountered other descriptions of high l i g h t intensity i n h i b i t i o n of corn photosynthesis. I t i s possible that the photosynthetic apparatus of these corn shoots was susceptible to damage by high l i g h t intensity because the plants were grown at a r e l a t i v e l y low l i g h t i n t e n s i t y , 21,600 lux. In the plant sugar-cane, however, leaves which develop at low l i g h t i n t e n s i t i e s are s t i l l capable of photosynthesis at f u l l e f f i c i e n c y i n f u l l sunlight (85). Since the optimum temperature for photosynthesis of these corn shoots was about 40° C, i t seems unlikely that the i n h i b i t i o n of photosynthesis was due to a high temperature le s i o n . Therefore, there i s no convenient explanation for this high l i g h t intensity effect on the basis of the available evidence. A l l other experiments with corn shoots were performed at 32,300 lux or lower, where there was no noticeable i n h i b i t i o n of apparent photosynthesis by l i g h t . The high l i g h t intensity results with corn were unexpected since most studies with corn and other plants have shown that the apparent rate of photosynthesis by in d i v i d u a l leaves continues to increase, although not l i n e a r l y , up to l i g h t i n t e n s i t i e s of 60,000 lux or more (10, 12, 21, 22, 35, 39, 41, 42, 67, 74, 84, 85). The dotted l i n e on Figure II-6 i l l u s t r a t e s this t y p i c a l response. Thus, most investigations have indicated that plants are much more e f f i c i e n t than Cg plants i n u t i l i z i n g high intensity l i g h t for photosynthesis. This difference i n effic i e n c y may be related to different ATP requirements for the photosynthesis of C3 and plants. Two moles of ATP are required for the pyruvate, phosphate, dikinase reaction of the C 4 ~ d i c a r b o x y l i e acid pathway (10, 37). I f this pathway operates i n series with the reductive pentose phosphate pathway i n C 4 plants (36, 52) , then these plants w i l l have a higher ATP requirement:-'for photosynthesis than do C 3 plants which possess only the second path-way. Thus, plants may require a high rate of photosynthetic phosphorylation during C O 2 f i x a t i o n . In accord with t h i s , Chen et a l . (10) have recently demonstrated that chloroplasts of the C 4 plant Cynodon dactylon have a higher a f f i n i t y for inorganic phosphate and ADP than chloroplasts of the C 3 plants which have been tested. The leaf anatomy of C 4 plants may also contribute to t h e i r e f f i c i e n c y at u t i l i z i n g high intensity l i g h t . The high chlorophyll content of the bundle sheath of C 4 plants may require higher l i g h t i n t e n s i t i e s for saturation of photosynthesis than are necessary with leaves of C 3 plants i n which the chlorophyll i s more evenly distributed (5). 6. General Discussion I t i s clear from the preceding sections that there are pronounced differences i n the C O 2 exchange characteristics of wheat and corn. The COp compensation point of corn was o r d i n a r i l y i n d i s -tinguishable from zero y l . / l . C O 2 and was not dependent on the O 2 concentration and temperature as was the C O 2 compensation point of wheat. Apparent photosynthesis of corn was not affected by 20.8% O 2 , but > 9 9 % O 2 i n h i b i t e d C O 2 assimilation, and the degree of i n h i b i t i o n increased with time and was only slowly reversible. The i n h i b i t i o n of apparent photosynthesis of wheat by 20.8% O 2 or by > 9 9 % O 2 was constant i n time and was rapidly reversible. In 20.8% O 2 , corn was v 72 more e f f i c i e n t at carrying out C O 2 assimilation at low CC>2 concentra-tions and high temperatures than was wheat, and there i s evidence from other studies that corn i s more e f f i c i e n t at u t i l i z i n g high intensity l i g h t for photosynthesis. On the other hand, apparent photosynthesis was more rapid with wheat than with corn at low temperatures. Photorespiration appears to contribute greatly to these differences between wheat and corn. At 1.8% O 2 , where photorespira-tion i s v i r t u a l l y suppressed, wheat resembled.com i n that i t possessed a low C O 2 compensation point, a high apparent rate of photosynthesis, and possibly an elevated optimum temperature for apparent photo-synthesis (Chapter I ) . I t would be an oversimplification, however, to conclude that differences i n photorespiration are the source of a l l the differences i n C O 2 exchange between wheat and corn. I t i s evident from the preceding sections that many of the differences i n ^ 2 ' exchange can be related to differences i n photosynthetic carbon metabolism or i n leaf anatomy. For example, i n Section I I - 3 , i t was suggested that the d i f f e r e n t i a l effect of temperature on apparent photosynthesis by wheat and corn may arise from differences i n the temperature response of the major enzymes responsible for C 0 2 f i x a t i o n i n the two species. Although corn and other C^  plants do not release appreciable quantities of C O 2 during photosynthesis, they do seem to contain some components which may be involved i n photorespiration.by C 3 plants. I t has been suggested'that photorespiratory C O 2 production by C 3 plants may arise from the oxidation of g l y c o l i c acid (18, 19, 61, 65, 87, 88, 89, 90). Glycolic acid oxidase occurs i n corn (69, 75, 76), sugar cane and Amararithus e d u l i s , although the a c t i v i t y i s low i n these species (78). Glycolic acid has been shown to accumulate when corn i s treated with an i n h i b i t o r of g l y c o l i c acid oxidase (87). In addition, C0 2 release i s stimulated when g l y c o l i c acid i s fed to corn leaves (18). Peroxisomes, which may be associated with photorespiratory metabolism (53, 77, 78), have been detected i n species (78). F i n a l l y , mass spectrometric measurements have indicated that large quantities of 0 2 are assimilated by illuminated corn plants (48). This l a s t r e s u l t , however, may not be due-; to photorespiration, but may have another cause, such as the a c t i v i t y of phenol oxidase which i s abundant i n chloroplasts of the C^  plant sugar cane (33). I t i s possible that C0 2 may be produced i n s i g n i f i c a n t quantities during photosynthesis i n corn, but i t may be prevented from leaving the plant because of an e f f i c i e n t internal C0 2 recycling mechanism (24). Phosphoenolyruvate carboxylase has a high a f f i n i t y f r for C0 2 (86) and i t i s located i n the mesophyll surrounding the paren-chyma bundle sheath (4, 72). I f CC>2 i s produced by the photores-p i r a t i o n of compounds i n the parenchyma bundle sheath of C^  plants, i t may be refixed into C^  dicarboxylic acids i n the mesophyll before i t . can escape from the leaf. Therefore,, although the C0 2 compensation point of C^  plants i s o r d i n a r i l y indistinguishable from zero, i t does not necessarily follow that C^  plants lack the internal capability of photorespiratory C0 2 production during photosynthesis. I f photores-p i r a t i o n does occur i n corn, however, i t does not cause a carbon d e f i c i t . The general geographical d i s t r i b u t i o n of and plants i s correlated with t h e i r C0 2 exchange properties, photosynthetic 74 metabolism and leaf anatomy. C 3 grasses, such as members of the Festuceae, Aveneae and Agrosteae, are abundant i n temperate regions. The C 4 Andropogoneae, Eragrosteae and Paniceae, however, are widespread i n the tropics (34). In the. dicotyledonous C 4 groups, the Amaran-thaceae are thought to be of t r o p i c a l o r i g i n (58). Also, species of At r i p l e x which possess the characteristic C^-type of leaf anatomy are native to the hot and arid regions of Au s t r a l i a and the United States (55). plants, therefore, thrive i n locations which have warm temperatures, high l i g h t i n t e n s i t i e s and often seasonal dry periods (55). The physiological characteristics of plants, p a r t i c u l a r l y t h e i r a b i l i t y to u t i l i z e high intensity l i g h t and high temperatures for rapid photosynthesis, would seem to be w e l l suited for a warm well-illuminated environment. Their a b i l i t y to assimilate C O 2 at low C O 2 concentrations may also be advantageous i n closely packed stands on calm days when the C O 2 concentration within the canopy may decrease appreciably. There i s also evidence that C^ plants are e f f i c i e n t i n their use of water. Results of Schantz and Piemeisel (71) indicate that C 4 plants use about one-half as much water during the production of 1 g. of dry matter as do plants. Since C 4 plants transpire at lower rates than C 3 plants (14, 44), this difference i n water requirement does not simply r e f l e c t differences i n C 0 2 assimilation rates between the two types of plants. In temperate conditions, C 4 characteristics may not be favoured. In Figure II-5 i t was seen that, at temperatures below 18° C, the apparent rate of photosynthesis of wheat was higher than that of corn. Also, chlorophyll accumulation i s retarded i n corn leaves i f 75 the day temperatures are below 15° C (59). No information i s available on whether this effect of low temperature occurs i n other C4 species. These correlations between some physiological characteristics and the geographical d i s t r i b u t i o n of C 3 and plants are not absolute. The Bambuseae, for example, have C 3 characteristics (17), but are abundant and grow rapidly i n the tropics. So do many other plants which are not C^ . Factors other than the apparent rate of photo-synthesis can Control the abundance of a species i n a l o c a l i t y . A plant which exhibits low apparent rates of photosynthesis per unit leaf area may s t i l l dominate i f i t produces a large leaf area. Many plants are common weeds (7). In f a c t , of the ten species l i s t e d by Holm (45) as the most serious weed pests throughout the world, seven belong to genera and the others have not been tested. Certainly, the discovery of a selective herbicide for C 4 species would be of great value for weed control. Black ejt _al. (7) have suggested that the high competitive a b i l i t y of many C^  species may be based on the C O 2 exchange characteristics outlined i n the preceding sections. There appear to be additional differences between C 3 and C 4 plants which may be related to some characteristics already discussed. Compared with C 3 species, C^  plants are composed of carbon which i s enriched i n (3, 73) . This difference could arise from the difference i n the photosynthetic carboxylation pathway or the difference i n apparent photorespiratory a c t i v i t y between the two types of plants. Carbonic anhydrase a c t i v i t y i s low i n C^  plants (27)uarid this may be associated with the C^-dicarboxylic acid pathway. The ac i d i c compounds produced by photosynthetic C O 2 f i x a t i o n i n C^  plants may be responsible for the s l i g h t l y greater ac i d i t y of sap expressed from plants compared with sap obtained from C 3 plants (46). Also, translocation of recent photosynthate may be more rapid i n C 4 plants than i n Cg plants (43). Whether or not these differences as w e l l as the others noted i n this Chapter exist between a l l C 3 and C 4 plants remains to be proven. Future investigations may discover additional differences between these two plant groups and should prove helpful i n r e l a t i n g their physiological properties to t h e i r d i s t r i b u t i o n and behavior i n nature. 77 LITERATURE CITED 1. Andrews, T. J. and M. D. Hatch. 1969. Properties and mechanism of action of pyruvate, phosphate 'dikinase from leaves. Biochem. J. 114: 117-125. 2. Bassham, J . A. and M. Calvin. 1958. The path of carbon i n photosynthesis. Prentice-Hall, Englewood C l i f f s , New Jersey. 3. Bender, M. M. 1968. Mass spectrometric studies of carbon 13 variations i n corn and other grasses. Radiocarbon 10: 468-472. 4. Berry, J. A., W. J. S. Downton and E. B. Tregunna. 1970. 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Irvine, J . E. 1969. Photorespiration i n t r o p i c a l grasses. XI. Internat. Botan. Cong., Seattle, pp. 99 (Abstract). 48. J.acksohqj. W. A. and R. J. Volk. 1969. Oxygen uptake by i l l u m -inated maize leaves. Nature 222: 269-271. 49. Jenkins, H. V. 1 9 5 9 . An airflow planimeter for measuring the area of detached leaves. Plant Physiol. 34: 532-536. 50. Jensen, R. G. and J. A. Bassham. 1966. Photosynthesis by isolated chloroplasts. Proc. Nat. Acad. S c i . U.S. 56: 1095-1101. 51. Johnson, H. S. and M. D. Hatch. 1968. Di s t r i b u t i o n of the C 4 -dicarboxylic acid pathway of photosynthesis and i t s occurrence i n dicotyledonous plants. Phytochem. 7: 375-380. 81 52. Johnson, H. S. and M. D. Hatch. 1969. The C^dxcarboxylic acid pathway of photosynthesis. I d e n t i f i c a t i o n of intermediates and products and quantitative evidence for the route of carbon flow. Biochem. J. 114: 127-134. 53. K i s a k i , T. and N. E. Tolbert. 1969. Glycolate and glyoxylate metabolism by isolat e d peroxisomes or chloroplasts. Plant Physiol. 44: 242-250. 54. Kortschack, H. P., C. E. Hartt and G. 0. Burr. 1965. Carbon dioxide f i x a t i o n i n sugar cane leaves. Plant Physiol. 40: 209-213. 55. Laets:ch, W. M. 1968. Chloroplast s p e c i a l i z a t i o n i n dicotyledons possessing the C4-dicarboxylic acid pathway of photosynthetic C02 f i x a t i o n . Amer. J. Botany 55: 875-883. 56. Laetsch, W. M. 1969. Specialized chloroplast structure of plants exhibiting the dicarboxylic acid pathway of photo-synthetic C02 f i x a t i o n . Progress i n Photosynth. Res. 1: 36-46. 57. Laetsch, W. M. and I . Pr i c e . 1969. Development of the dimorphic chloroplasts of sugar cane. Amer. J. Botany 56: 77-87. 58. Lawrence, G. H. 1951. Taxonomy of vascular plants. Macmillan Co., New York. 59. McWilliam, J. R. and A. W. Naylor. 1967. Temperature and plant adaptation. I. Interaction of temperature and l i g h t i n the synthesis of chlorophyll i n corn. Plant Physiol. 42: 1711-1716. 60. Meidner, H. 1962. The minimum intercellular-space C0 2-concentration (T) of maize leaves and i t s influence on stomatal movements. J. Exp. Botany 13: 284-293. 61. Meidner, H. 1967. Further observations on the minimum i n t e r -cellular-space carbon-dioxide concentration (r) of maize leaves and the postulated roles of "photo-respiration" and glycollate metabolism. J. Exp. Botany 18: 177-185. 62. M i l l e r , V. J . 1960. Temperature effect on the rate of the apparent photosynthesis of seaside bent and bermudagrass. Amer. Soc. Hort. S c i . 75: 700-703. 63. M i t c h e l l , K. J. 1956. Growth of pasture species under controlled environment. I. Growth at various levels of constant temperature. N. A. J. Sc i . Tech. 38A: 203-215. 64. Moss, D. N. 1962. The l i m i t i n g carbon dioxide concentration for photosynthesis. Nature 193: 587. 82 65. Moss, D. N. 1968. Photorespiration and glycolate metabolism i n tobacco leaves. Crop S c i . 8: 71-76. 66. Moss, D. N., E. G. Krenzer J r . and W. A. Brun. 1969. Carbon dioxide compensation points i n related plant species. Science 164: 187-188. 67. Murata, Y. and J. Iyama. 1963. Studies on the photosynthesis of forage crops. I I . Influence of a i r temperature upon the photosynthesis of some forage and grain crops. Proc. Crop Sci. Soc. Japan 31: 315-322. 68. Murata, Y., J. Iyama and T. Honma. 1965. Studies on the photosynthesis of forage crops. IV. Influence of a i r -temperature upon the photosynthesis and respiration of a l f a l f a and several southern type forage crops. Proc. Crop Sci. Soc. Japan 34: 154-158. 69. N o l l , C. R. J r . and R. H. Burris. 1953. Nature and d i s t r i b u t i o n of g l y c o l i c acid oxidase i n plants. Plant Phys. 29: 261-265. 70. Osmond, C. B., J. H. Troughton and D. J. Goodchild. 1969. Physiological biochemical and st r u c t u r a l studies of photosynthesis and photorespiration i n two species of A t r i p l e x . Z. Pflanzenphysiol. 61: 218-237. 71. Schantz, H. L. and L. N. Piemeisel. 1927. The water requirements of plants at Akron, Colorado. J. Agric. Res. 34: 1093-1189. 72. Slack, C. R., M. D. Hatch and D. J. Goodchild. 1969. D i s t r i -bution of enzymes i n mesophyll and parenchyma-sheath chloroplasts of maize leaves i n r e l a t i o n to the C4~dicar-boxy l i e acid pathway of photosynthesis. Biochem. J. 114: 489-498. 73. Smithi B. N. and S. Epstein. 1969. l 3C/ 1 2C ratios as evidence for two pathways of carbon f i x a t i o n i h photosynthesis. XI. Botan. Congr., Seattle, pp. 203 (Abstract). 74. Stoy, V. 1965. Photosynthesis, respiration and carbohydrate accumulation i n spring wheat i n re l a t i o n to y i e l d . Physiol. Plantarum Suppl. IV. 1-125. 75. Tolbert, N. E. and R. H. Burris.' 1950. Light activation of the enzyme which oxidizes g l y c o l i c acid. J. B i o l . Chem. 186: 791-804. 76. Tolbert, N. E. and M. S. Cohen. 1952. Activation of g l y c o l i c acid i n plants. J. B i o l . Chem. 204: 639-648. 77. Tolbert, N. E., A. Oeser, T. K i s a k i , R. H. Hageman and R. K. Yamazaki. 1968. Peroxisomes from spinach leaves containing enzymes related to glycolate metabolism. J. B i o l . Chem. 243: 5179-5184. 83 78. Tolbert, N. E., A. Oeser, R. K. Yamazaki, R. H. Hageman and T. K i s a k i . 1969. A survey of plants for leaf peroxisomes. Plant Physiol. 44: 135-147. 79. Tregunna, E. B. and J. Downton. 1967. Carbon dioxide compensation i n members of the Amaranthaceae and some related families. Can. J. Botany 45: 2385-2387. 80. Tregunna, E. B., J. Downton and P. A. J o l l i f f e . 1969. Genetic and environmental control of photorespiration. Progress i n Photosynthesis Res. 1: 488-493. 81. Tregunna, E. B., G. Krotkov and C. D. Nelson. 1966. Effect of oxygen on the rate of photorespiration i n detached tobacco leaves. Physiol. P1antarum 19: 723-733. 82. Treharne, K. J . and J. P. Cooper. 1969. Effect of temperature on the a c t i v i t y of carboxylases i n t r o p i c a l and temperate Gramirieae. J. Exp. Botany 20: 170-175. 83. Turner, J.'S; and E. G. B r i t t a i n . 1962. Oxygen as a factor i n photosynthesis. B i o l . Rev. 37: 130-170. 84. Waggoner, P.-E., D. N. Moss and J . D . Hesketh. 1963. Radiation i n the plant environment and photosynthesis. • Agron. J. 55: 36-39. 85. Waldron, J. C , K. T. Glasziou and T. A. B u l l . 1967. The physiology of sugarcane. IX. Factors affecting photosynthesis and sugar storage. Aust. J. B i o l . S c i . 20: 1043-1052. 86. Walker, D. A. 1962. Pyruvate carboxylation i n plant metabolism. B i o l . Rev. 37: 215-256. 87. Z e l i t c h , I. 1958. The role of g l y c o l i c acid oxidase i n the respiration of leaves. J . B i o l . Chem. 233: 1299-1303. 88. Z e l i t c h , I. 1965. The r e l a t i o n of g l y c o l i c acid synthesis to the primary photosynthetic carboxylation reaction i n leaves. J . B i o l . Chem. 240: 1869-1876. 89. Z e l i t c h , I. 1966. Increased rate of net photosynthetic carbon . dioxide uptake caused by the i n h i b i t i o n of glycolate oxidase. Plant Physiol. 41: 1623-1631. 90. Z e l i t c h , I. 1969. Mechanisms of carbon f i x a t i o n and associated responses. In_: Easton, J. D. (editor). Physiological Aspects of Crop Y i e l d . Amer. Soc. Agron., Crop Sc i . Amer. Madison, Wise. pp. 20 7-226. 84 Chapter I I I PHOTORESPIRATION AND THE POST-ILLUMINATION C 0 2 BURST IN WHEAT AND AMARANTHUS EDULIS INTRODUCTION During the f i r s t minute of darkness following a period of illu m i n a t i o n , plants often exhibit high rates of C 0 2 production into a i r containing 2 1 % 0 2 . This post-illumination C 0 2 burst i s eliminated when the 0 2 concentration i s reduced to 2 % and i t i s enhanced when the 0 2 concentration i s raised to 1 0 0 % ( 2 , 3, 7 , 8 , 1 0 , 1 2 , 24). The size of:the burst also increases with increasing l i g h t intensity i n the period preceding darkness (3, 6 , 7 , 8 , 14, 2 2 , 23, 24). Mainly because of the s e n s i t i v i t y of the burst to 0 2 concentration, i t has been suggested that the burst i s a b r i e f extension of photorespiration into the dark period ( 5 , 6 , 7 , 8 , 1 2 , 19, 2 2 , 23, 24). In accord with this interpretation i s the observation that, with the exception of Amaranthus edulis, those plants which photorespire have post-illumina-tion C 0 2 bursts, and those plants which apparently lack photorespira-tion also lack bursts (13, 23). Amaranthus edulis, on the other hand, exhibits a d i s t i n c t post-illumination C 0 2 burst even though i t does not release C 0 2 i n the l i g h t ( 2 , 9). Also, Heath and Orchard (14) have demonstrated that the post-illumination C 0 2 burst and the C O 2 compensation point of wheat leaves respond d i f f e r e n t l y to l i g h t intensity and temperature. They concluded from this that the burst was not associated with photores-p i r a t i o n . 85 The interpretation of post-illumination CO 2 exchange transients of plants i s complex since changes i n C O 2 d i f f u s i o n resistance, and i n the rates of photosynthesis, photorespiration, and dark respiration can be involved. Stomata close i n the dark (25, 26), and transpiration i s reduced (10, 25), indicating that the gas d i f f u s i o n resistance of leaves i s increased during the post-illumination period. These changes, however, are r e l a t i v e l y slow, (25, 26), compared with the kinetics of the burst. Also, since the burst occurs i n liverworts (7, 8), i t cannot be the result of stomatal response. Photosynthetic C O 2 f i x a t i o n may continue b r i e f l y after i l l u m i n a t i o n , for example f a l l i n g to zero within 30 seconds after darkening Scenedesmus (1). Dark respiration may be i n h i b i t e d i n the l i g h t and increase after the onset of darkness (4, 15, 16, 17, 18, 20, 21). In an e f f o r t to c l a r i f y the relationship between photores-p i r a t i o n and the post-illumination C O 2 burst, the kinetics of the burst and the effects of different O 2 and C O 2 concentrations on the two processes were examined. The effect of O 2 concentrations on the burst of Amaranthus edulis was also investigated. MATERIALS AND METHODS Wheat plants were grown i n the same way as described for Experiment I i n Chapter I. Amaranthus edulis Speg. plants were grown i n s i m i l a r conditions except that s o i l , not vermiculite,was used. At the s t a r t of each experiment, 2.0 g. fresh weight of 9 to 11 day-old wheat shoots, or single, f u l l y expanded Amaranthus edulis leaves from 25 to 28 day-old plants, were excised i n the usual manner and enclosed 8 6 i n the transparent chamber shown i n Figure 1-2. After enclosure, the plant material was conditioned for an i n i t i a l 30 min. period at 32,300 lux (3000 ft.-c. ) and 25± 1° C i n a i r . Thereafter i t was exposed to a series of l i g h t - dark cycles, each cycle consisting of a minimum of 15 min. of illumination at 32,300 lux followed by 5± 0.5 min. of darkness. Darkening was carried out i n less than 0.5 sec. by covering the chamber with an opaque black cloth and turning off the l i g h t . During i l l u m i n a t i o n , the a i r temperature i n the chamber was 25± 1° C, and i n the dark i t f e l l to 24± 1° C. An a i r flow rate of 1.0 l./min. was maintained while plant material was i n the chamber. The gas flow system used for these experiments was the same as that shown i n Figure I I - l . Measurement of the temperature within the plant chamber, the C O 2 concentration i n the a i r stream, and control of the 0 2 and the C O 2 concentration of the a i r entering the plant chamber were a l l carried out as described i n ChaptersBl and I I . RESULTS AND DISCUSSION 1. Kinetics of the Post-illumination C 0 2 Burst The s o l i d l i n e i n Figure I I I - l i l l u s t r a t e s the data obtained-.' when illuminated wheat shoots were darkened. The a i r entering the plant chamber contained 1 0 0 y l . / l . C O 2 , 2 0 . 8 % O 2 , balance N 2 . During illu m i n a t i o n , C O 2 assimilation reduced the C O 2 concentration of the a i r leaving the chamber to 74 y l . / l . When the shoots were darkened, there was a 3 sec. delay before the C O 2 concentration readings increased sharply. This lag can be attributed to the time required for C O 2 concentration changes i n the chamber to reach the C O 2 analyzer. Figure I I I - l Effects of Darkening on C O 2 Exchange Excised Wheat Shoots i n 20.8% 0 2 87 40 60 T IME (sec) 88 Within 1 2 sec. of darkening the shoots, the C O 2 analyzer.readings exceeded 1 0 0 y l . / l . C O 2 , and a peak of 1 2 1 . 4 u l . / l . C O 2 was reached after 27 sec. After the peak, the readings declined and reached 1 1 1 y l . / l . C O 2 at 1 2 0 sec. Wheat, therefore, exhibites a post-illumination C O 2 burst under these conditions. The burst as i t occurred within the plant chamber, however, must have differed i n k i n e t i c s and magnitude from these observations. Part of this difference may be ascribed to characteristics of the C O 2 analyzer which operates by detecting the absorption of infra-red l i g h t across a 1 0 0 ml. sample cylinder. During the burst, the rapid variations of the C O 2 concentration of the a i r stream generate C O 2 concentration gradients i n the sample cylinder and d i s t o r t the results. This effect could be eliminated by calculating the C O 2 concentration which must have^been entering the C 0 2 analyzer to produce the observed results. The method of this calculation i s described i n Appendix I. The dashed l i n e i n Figure I I I - l shows that when the results were corrected this way, the i n i t i a l C O 2 concentration increase was more abrupt, and the peak of the burst was about 4 sec. e a r l i e r and s l i g h t l y higher than the measured res u l t . The difference between the observed and corrected r e s u l t s , however, was not large, and a l l subsequent results i n this chapter were l e f t uncorrected. An additional source of error arose from the separation of the s i t e of the burst from the s i t e of i t s measurement. Before a C 0 2 exchange event which had occurred within the plant chamber was detected by the CO^  analyzer, i t was modified according to the a i r flow characteristics of the plant chamber and the tubing connecting the chamber to the IRGA. As the result of this modification, the burst 89 was extended i n time, and the peak of the burst was delayed and reduced i n si z e . This was confirmed by comparing the r e s u l t s , shown i n Figure I I I - 2 , of pulse (less than 0.5 sec.) injections of 0.1 ml. C O 2 i n a i r into the a i r stream either at the entrance to the C O 2 analyzer or into the middle of the empty plant chamber. The complexity of the a i r flow characteristics of the experimental system prevented the exact assessment of t h e i r effects on the k i n e t i c s of the burst. It i s clear, however, that the true peak of the burst occurred e a r l i e r than 20 sec. after the i n i t i a l C O 2 analyzer response, and that the peak was higher and more abrupt than that recorded by the C O 2 analyzer. Bulley (3) recently used a highly s i m p l i f i e d open system to measure the post-illumination C O 2 burst of soybean leaves. He estimated that the true peak of the burst occurred between 7 and 12 F-sec. after the i n i t i a l C O 2 analyzer response, and calculated that i t was about 150% as high as the recorded peak. In view of the above considerations, s i m i l a r kinetics would not seem to be unreasonable for the post-illumination C O 2 burst of wheat. The peak of the burst, therefore, occurs very soon after illumination i s stopped, and i t i s highly susceptible to modification by measuring techniques. Other experiments by Bulley (3) have indicated that the burst i s not the result of processes commencing at the onset of darkness. When the l i g h t intensity was reduced, but not extinguished, a dip i n the rate of C O 2 assimilation occurred, and this dip corresponded k i n e t i c a l l y to the post-illumination C O 2 burst. I t was concluded that the burst was the resultant of two C O 2 exchange processes which differed i n rate of response to changes i n l i g h t i n t e n s i t y . Figure I I I - 2 Response of the IRGA to Pulse Injections of CO2 into the A i r Stream- i n the Plant Chamber and at the Entrance to the IRGA Sample Cylinder lOOr 90 INJECTION AT IRGA ENTRANCE 80 70 60 L U C O O L U or 40 I-< * 30 20 10 0l 0 i \ i \ i NJECTION \ INTO PLANT CHAMBER 4 8 12 16 20 T I M E A F T E R IN JECT ION (sec) 91 A second broad peak of C O 2 production usually occurring between 2 and 1 0 minutes after darkening was observed i n this study and has frequently been noted before ( 7 , 1 2 , 13, 2 2 , 23, 24). This peak was evident i n both 2 0 . 8 % O 2 and 1 . 8 % O 2 and i s therefore not related to photorespiration. I t may be the result of a post-illumination overshoot of dark respiration. Comparable peaks of post-illumination C O 2 production are exhibited by plants lacking photorespirations such as corn (13, 23')' and Amaranthus edulis ( 2 ) , as well as by Chlorella ( 7 , 8 ) . 2 . Effect of O 2 and C O 2 Concentrations on the Post-illumination C 0 2 Burst Figures II I - 3 to I I I - 7 show the recorded post-illumination C O 2 exchange transients of wheat shoots i n atmospheres containing 2 0 . 8 % or 1 . 8 % O 2 and C O 2 concentrations between 1 0 0 u l . / l . and 300 y l . / l . Each point on these curves i s the average of 5 determinations. There was no burst i n 1 . 8 % O 2 at any of the C O 2 concentrations used. In 2 0 . 8 % O 2 , there was a pronounced burst i n 1 0 0 u l . / l . C O 2 , and the peak height of the burst decreased as the C O 2 concentration increased. Two minutes after the shoots were darkened, there was no s i g n i f i c a n t difference i n the rates of C 0 2 production between the 2 0 . 8 % 0 2 and 1 . 8 % O 2 treatments. It i s apparent from the preceding discussion of burst kinetics that the observed peak height i s affected by the experimental system, so comparisons based on this characteristic of the burst are hazardous. Since the burst i n wheat i s dependent on O 2 , an appropriate index of the size of burst i s the o v e r a l l difference i n post-illumination C O 2 exchange between the 2 0 . 8 % O 2 and 1 . 8 % O 2 treatments. Using tthds index, Figure I I I - 8 summarizes these results on the effect of C O 2 Figure II I - 3 Post-illumination CO2 Exchange by Excised Wheat Shoots i n 1.8% 0 2 and 20.8% 0 2 . The C 0 2 Concentration of the A i r Entering the Plant Chamber was 100 u l . / l . C 0 2 92 >15 min LIGHT D A R K TIME (sec) Figure III-4 Post-illumination CO2 Exchange by Excised Wheat Shoots i n 1.8% 0 2 and 20.8% 0 2. The CO2 Concentration of the A i r Entering the Plant Chamber was 200 y l . / l . C0 2 93 15 min L IGHT D A R K  TTTl 11111111111111111111 III111\ -G •5 o 1.8 % 0 2 G 20.8 % 0 2 40 60 T IME (sec) 100 120 Figure III-5 Post-illumination CO2 Exchange by Excised Wheat Shoots i n 1.8% 0 2 and 20.8% 0 2 . The C 0 2 Concentration of the A i r Entering the Plant Chamber was 300 u l . / l . C 0 2 94 >15,min L IGHT DARK 320 111111111111111111111111111111111111111111////A 20 40 60 T I M E (sec) 80 -o-°1 .8%0 2 o 20.8 % 0 2 100 120 Figure III-6 Post-illumination CO2 Exchange by Excised Wheat Shoots 1.8% 0 2. The C0 2 Concentration of the A i r Entering th Plant Chamber was 400 y l . / l . C0 2 95 >15.min L IGHT D A R K mniiniLLLaLUMmzn 420 - O --o-J L _ ' • 40 60 80 T IME (sec) -o- -o -o -o1.8 %02 -G 20.8 % 0 2 100 120 Figure III-7 Post-illumination CO2 Exchange by Excised Wheat Shoots i n 1.8% 0 2 and 20.8% 0 2. The C0 2 Concentration of the A i r Entering the Plant Chamber was 500 y l . / l . C0 2 ' 96 520 480 O A jZ 440 < or U J O o u C M 400 o (J 360 >15.min LIGHT DARK • ©—J U-6—-! o o o-o-> 1 . 8 % 0 2 =20.8 % 0 2 0 20 40 " ~ Q ~ 8 ~ ^ 0 - ~ - J 1 | ) Q ' ^ T IME (sec) Figure III-8 Effects of CO2 Concentration on the Size of the Post-illumination CC>2 Burst and the Magnitude of the Depression of Apparent Photosynthesis by 20.8% O2 97 98 concentration on the s i z e of the post-illumination C O 2 burst. The burst was greatest at 1 0 0 y l . / l . C O 2 and 2 0 0 y l . / l . C O 2 and i t decreased at higher C O 2 concentrations. As w i l l be shown i n Chapter IV, the size of the burst was s u f f i c i e n t l y large that i t i s improbable that the burst originated from a C O 2 free-exchange pool. In Chapter I i t was concluded that the results of studies on the inhibitory effect of 0 2 on apparent photosynthesis could not be used to indicate the rate of photorespiration at high C 0 2 concentra-tions. I f the post-illumination C O 2 burst i s a manifestation of photorespiration, however, then the C O 2 concentration response of photorespiration must resemble that of the burst size shown i n Figure III-8. The effect of C 0 2 concentration on the burst can be correlated with i t s e f f e c t on the i n h i b i t i o n of apparent photosynthesis by 2 0 . 8 % O 2 . Figure III-8 shows that the burst size and the magnitude of the depression of apparent photosynthesis by 2 0 . 8 % O 2 respond s i m i l a r l y to C 0 2 concentration. Fock et a l . (11) have recently studied the effect of C O 2 concentration on the post-illumination C O 2 burst of the liverwort Conocephalum conicum and have also noted this s i m i l a r i t y . In t h e i r study, 50% O 2 and 75% O 2 were used and the size of the bursttwas greatest at about 300 y l . / l . C O 2 and declined at lower and higher C 0 2 concentrations. I f Figure 1-13 i s reexamined, i t can be seen that the depression of apparent photosynthesis by 60% 0 2 or 80% 0 2 i s greatest between 225 y l . / l . C 0 2 and 300 y l . / l . C 0 2 at 25° C. Thus, the results with Conocephalum appear to be i n general agreement with the present observations. The correlation between the effect of 0 2 on apparent 99 photosynthesis and on the burst at different C0 2 concentrations can be i l l u s t r a t e d i n another/way. In Chapter I , i t was seen that the per cent i n h i b i t i o n of apparent photosynthesis by 0 2 was: % I P " P l - P 2 — j T j - — X 100 As before, P^ and P 2 are the apparent rates of photosynthesis at a low and a higher 0 2 concentration respectively. An analogous expression can be developed for the post-illumination C0 2 burst. Let P b be the average rate of C0 2 production during the burst, i n mg. COg/hr./g. f r . wt., as calculated from the burst size and duration. Once again, the burst size i s the overall difference i n post-illumination C0 2 exchange between the 20.8% and 1.8% 0 2 treatments. The duration of the burst was taken .to be the length of time after darkening the shoots that the C0 2 concentration readings i n 20.8% 0 2 and 1.8% 0 2 were different at the 5% l e v e l of significance. Then, i f the effects of 20.8% 0 2 on apparent photosynthesis and on the burst are due to the same cause or causes, (P 2 -k P^) may be substituted into the above formula i n place of P^. That i s : % I b = (P 2 + P b) - P 2 P b — ± 2 £ x 100 X 100 P 2 + pb p2 + pb and the response of this function to C0 2 concentration should resemble that of % Ip. The results presented i n Figure III-9 show the s i m i l a r i t y of the C0 2 concentration responses of % Ip and % 1^. This i s further evidence l i n k i n g the effects of 0 2 on apparent photosynthesis with i t s effects on the post-illumination C0 2 burst. In addition to the data already presented on kinetics and the Figure II1-9 Effects of CC"2 Concentration on the Per Cent I n h i b i t i o n Apparent Photosynthesis by 20.8% 0£ and on the Analogous Burst Function % B 1 0 1 effects of 0 2 and C O 2 concentrations on the burst, other results are consistent with the hypothesis that the burst i s related to photo-respiration. The size of the burst and the depression of apparent photosynthesis by 2 0 . 8 % 0 2 are affected s i m i l a r l y by l i g h t intensity (3). Also, the effect of wavelength of l i g h t on the apparent rates of photosynthesis i n 2 1 % 0 2 or 2 % O 2 i s the same as i t s effect on the size of the burst (3). These effects of preceding l i g h t conditions ?, are further evidence that the burst i s linked to processes occurring during the l i g h t . Heath and Orchard (14) , on the other hand, concluded that the burst was not related to photorespiration. This conclusion was founded on the assumption that at the C O 2 compensation point, the rates of C O 2 f i x a t i o n and production are unaffected by l i g h t i n t e n s i t y . Recent studies (3), however, indicate that at the C 0 2 compensation point, increases i n l i g h t intensity result i n increases in C O 2 f i x a t i o n . Therefore, t h e i r assumption was not v a l i d , and thei r reasoning cannot be. used to contradict the view that the post-ill u m i n a t i o n C O 2 burst i n wheat and s i m i l a r plants i s an extension of photorespiration into the dark period. 3. Effect of O 2 Concentration on the Post-illumination C O 2 Burst of Amaranthus edulis Figure 1 1 1 - 1 0 shows the results of consecutive determinations of post-illumination C 0 2 exchange by an Amaranthus edulis leaf i n 2 0 . 8 % 0 2 or 1 . 8 % O 2 . These data are representative of the results of 16 determinations at 300 y l . / l . C O 2 and 32,300 lux. Although the rates of C O 2 exchange were found to be more variable from one determination to the next than with wheat, several important features are Figure III-19 The Post-illumination CO2 Burst of Amaranthus edulis i n 1.8% 0 2 and 20.8% O2 102 >15min LIGHT DARK 320 0 0 20 40 60 T IME (sec) 80 -Q -° 1.8 %02 -o 20.8 % 0 2 100 120 103 nevertheless apparent. Unlike wheat, Amaranthus edulis exhibited a substantial peak i n post-illumination CO2 production i n 1.8% 0 2 as w e l l as i n 20.8% 0 2. The height of the peak i n 1.8% 0 2 was similar to the height of the peak i n 20.8% 0 2, although there were often differences i n the kin e t i c s of C0 2 exchange subsequent to the peak. The results of Bjbrkman (2) appear to be s i m i l a r . Since the peak i s not sensitive to 0 2, the burst of Amaranthus  edulis cannot be related to conventional photorespiration or to the burst i n wheat. I t i s possible that the burst of Amaranthus edulis i s the result of an overshoot of dark respiration which i s recovering from i n h i b i t i o n during the l i g h t (2), or to some other cause associated with C4 metabolism. There i s i n s u f f i c i e n t evidence to specify the cause of the differences i n C0 2 exchange often observed after the peak of the burst of Amaranthus edulis. 104 LITERATURE CITED 1. Bassham, J. A., K. Shibata, K. Steenberg, J . Boudon and M. Calvin. 1956. The photosynthetic cycle and respiration: Light - dark transients. J. Amer. Chem1; Soc. 78: 4120-4124. 2. Bjbrkman, 0. 1968. Further studies of the effect of oxygen concentration on photosynthetic C O 2 uptake i n higher plants. Carnegie Inst, of Wash. Year Book. 66: 220-228. 3. Bulley, N. R. 1969. Effects of l i g h t quality and intensity on photosynthesis and photorespiration i n attached leaves. Ph.D. Thesis. Simonj Fraser University. 4. Calvin, M. and P. Massini. 1952. The path of carbon i n photosynthesis. XX. The steady state. Experientia 8: 445-457. 5. Decker, J . P. 1955. A rapid post-illumination deceleration of respiration i n green leaves. Plant Physiol. 30: 82-84. 6. Decker, J . P. 1959. Comparative responses of carbon dioxide outburst and uptake i n tobacco. Plant Physiol. 34: 100-102. 7. Egle, K. and H. Fock. 1966. Light respiration - correlations between C O 2 f i x a t i o n , O 2 pressure and glycollate concentra-t i o n . In: T. W. Goodwin (editor) Biochemistry of Chloro-plasts 2: 79-87. 8. Egle, K. and W. T r o l l . 1966. uber die "Lichtatmung" bei grunen Pflanzen. I. Die Wirkung von Sauerstoff und Kohlendioxyd aut den C O 2 - Gaswechsel wahrend der Licht - und Dunkelphase. B e i t r . B i o l . Pflanzen 42: 213-239. 9. El-Sharkawy, M. A., R. S. Loomis, and W. A. Williams. 1968. Photosynthetic and respiratory exchanges of carbon dioxide by leaves of the grain Amaranth. J . Appl. Ecol. 5: 243-251. 10. Fock, H. and K. Egle. 1967. Uber die Beziehungen zwischen den Glycolsaure - Gehalt und dem Photosynthese - Gaswechsel von Bohnenblattern. Z. Pflanzenphysiol. 57: 389-397. 11. Fock, H., G. Krotkov and D. T. Canvin. 1969. Photorespiration i n liverworts and leaves. Progress i n Photosynthesis Res. 1: 482-487. 12. Forrester, M. L., G. Krotkov, and C. D. Nelson. 1966. Effect of oxygen on photosynthesis, photorespiration and respiration i n detached leaves. I. Soybean. Plant Physiol. 41: 422-427. 13. Forrester, M. L., G. Krotkov, and C. D. Nelson. 1966. Effect of oxygen on photosynthesis, photorespiration, and respiration i n detached leaves. I I . Corn and other monocotyledons. Plant Physiol. 41: 428-431. ICS 14. Heath, 0. V. S. and B. Orchard. 1968. Carbon assimilation at low carbon dioxide levels. I I . The process of apparent assimilation. J. Exp. Botany 19: 176-192. 15. Heber, U. and K. A. Santarius. 1964. Zur Steuerung von Photosynthese und Atmung i n der B l a t t z e l l e . Ber. Deut. Bot. Ges. 77: ( l ) - ( 3 ) . 16. Heber, U. , K. A. Santarius, W. Urbach, and W. U l l r i c h . 1964. Photosynthese und Phosphathaushalt. Z. Naturforsch. 195: 576-587. 17. Hoch, G., 0. V. H. Owens, and B. Kok. 1963. Photosynthesis and respiration. Arch. Biochem. Biophys. 101: 171-180. 18. Kandler, 0. and I. Habersr-Liesen Kotter. 1963. Uber den Zusammsihang zwischen Phosphathaushalt und Photosynthese. Z. Naturforsch. 18b: 318-330. 19. Moss, D. N. 1966. Respiration of leaves i n l i g h t and darkness. Crop S c i . 6: 351-354. 20. Owens, 0. V. H. and G. Hoch. 1963. Enhancement and de-enhancement effect i n Anacystis nidulans. Biochim. Biophys. Acta 75: 183-186. 21. Ried, A. 1968. Interactions between photosynthesis and respiration i n Chlorella. I. Types of transients of oxygen exchange after short l i g h t exposures. Biochim. Biophys. Acta 153: 653-663. 22. Tregunna, E. B., G. Krotkov, and C. D. Nelson. 1961. Evolution of carbon dioxide by tobacco leaves during the dark period following illumination with l i g h t of different i n t e n s i t i e s . Can. J. Botany 39: 1945-1056. 23. Tregunna, E. B., G. Krotkov, and C. D. Nelson. 1964. Further evidence on the effects of l i g h t on respiration during photosynthesis. Can. J . Botany 42: 989-997. 24. Tregunna, E. B., G. Krotkov, and C. D. Nelson. 1966. Effect of oxygen on the rate of photorespiration i n detached tobacco leaves. Physiol. Plant 19: 723-733. 25. V i r g i n , H. I. 1956. Light-induced stomatal movements i n wheat leaves recorded as transpiration. Physiol. Plant 9: 280-303. 26. Williams, W. T. 1954. A new theory of the mechanism of stomatal movement. J . Exp. Botany 5: 343-352. Chapter IV 106 ESTIMATION OF THE C 0 2 FREE-EXCHANGE POOL SIZE IN WHEAT AND CORN LEAVES INTRODUCTION Onee consideration i n the study of photosynthesis, photores-p i r a t i o n and respiration i s that C 0 2 exchange by other processes should be i n s i g n i f i c a n t or accounted for. There i s some evidence that plants may contain quantities of carbon which can exchange freely with atmospheric C 0 2 . Many studies have shown that C 0 2 i s released by plants when they are treated with acid ( 1 , 1 0 , 13, 17, 18, 19). In addition, C 0 2 i s reversibly absorbed by leaves when they are placed i n a vacuum and then exposed to C 0 2 (13). Illumination and chlorophyll are not required (19, 13), and leaves which are dried and then rewetted also absorb C 0 2 (14). The quantity of C 0 2 involved i s substantial. For example, 0.64 to 1.19 ml of C O 2 per g. fresh weight were absorbed by leaves of 13 species at 15° and 1 Atm. C 0 2 (14). The relationship., of these observations to a possible C O 2 free-exchange pool under ordinary conditions i s not known. A number of processes could contribute to the information of a C O 2 free-exchange pool. C O 2 i s absorbed by water, partl y by physical solution and partly by chemical hydration (4, 6, 1 1 ) : C O 2 ( g a s ) ^ = ^ C 0 2 (solution) + H 2 0 H 2 C 0 3 ^ = ^ H C 0 3 + H - . " C O 3 + 2 H At alkaline pH, the formation of H C O 3 and C O 3 are favored, and the 107 reaction: CO 2 + 0 H ~ " * — J H C O 3 also becomes important ( 9 , 1 1 ) . In.plant c e l l s , the presence of the enzyme carbonic anhydrase (E.C. 4 . 2 . 1 . 1 . ) ( 3 , 5) or the anions of many acids, e.g. phosphate and acetate ( 4 , 1 2 ) , may accelerate the other-wise slow hydration steps. The solution of C O 2 i n water i s enhanced i n the presence of alkaline earth carbonic acid esters, and with amines to form carbamates ( 1 1 ) . The following experiments were designed to estimate the s i z e of the C O 2 free-exchange pools i n wheat and corn leaves. MATERIALS AND METHODS Wheat (Triticum aestivum L.) and corn (Zea mays L.) plants were grown i n the same conditions described for wheat i n Chapter I. About 2 g. fresh weight of ten to fourteen day-old shoots were excised and transferred to a 2 0 x 3 x 0.5 cm plexiglass chamber shown i n Figure I V - 1 and placed i n the dark. The temperature i n the chamber was measured by a thermistor placed behind some of the leaves. A Beckman IR 215 infra-red C O 2 analyzer, used d i f f e r e n t i a l l y i n the open system, measured the C O 2 concentration. The a i r flow rate was 1.6 1 ./min.for a l l measurements. The a c i d - l a b i l e C O 2 was measured after the addition of 2 0 ml. of 5 M HCl i n ethanol to 1 g. fresh weight of plant material. The C O 2 evolved was trapped i n 1 M NaOH and then released again into a closed system by the addition of excess 5 M aqueous HCl. Two techniques were used to measure the amount of freely ex-changed C O 2 . The f i r s t , ^ 2 C 0 2 exchange, involved the comparison of Figure IV-1 Plant Chamber Used for Measurements of the C O 2 Free-Exchange Pool Size with Excised Wheat and Corn Shoots F R O N T V I E W S I D E V I E W dark respiration rates measured i n an open gas flow system with those observed i n a closed system. The apparatus used i s represented i n Figure IV-2A. A i r from compressed gas tanks was introduced into the open system, and the respiration rate was calculated from the increase i n C O 2 concentration after the a i r stream had crossed the chamber. CC>2 concentrations used with the open system, with r e l a t i v e frequency of about 1:2:1, were 130 yL/1, 325 y l . / l , and 390 y l . / l . In the 0.29 1. closed systems, the respiration rate was calculated from the time required for the C O 2 concentration to increase from 100 to 400 y l . / l . . Open and closed system measurements were alternated. The C O 2 free-exchange pool, i f present, should enlarge res-ponse to the 300 y l . / l . increase i n C O 2 concentration i n the closed system. The exchange pool should not enlarge appreciably i n the open system where the C O 2 concentration varied by less than 5 y l . / l . Since some of the C O 2 produced by respiration i n the closed system contributes to the increase i n exchange pool s i z e , the observed rate of respiration should be less i n the closed system than i n the open system. The change i n pool size after the C0 2 concentration had i n -creased from 100 y l . / l . to 400 y l . / l . was calculated from: Pool C0 2 Increase (yl/g.) = R QAt - VA [C02] W where RQ represents the respiration rate i n the open system ( y l . C O 2 / -min) , A [ C O 2 ] was the C O 2 concentration increase (300 y l . / l . ) i n the closed system during At minutes; V was the closed system volume (1.); and W was the fresh weight of plant material (g.). For the second technique, " ^ C 0 2 exchange, the C O 2 free-Figure IV-2 Gas Flow System Used for CO2 Exchange and CO2 Exchange Measurements with Excised Wheat and Corn Shoots 12 A C 0 2 EXCHANGE B 14 COo EXCHANGE OUTLET A INLET . A -TELETHERMOMETER .« PLANT CHAMBER o 4 © MgCIQ4 D R J R ] I PUMP FLOW METER I Y RECORDER RECORDER SAMPLE REFERENCE IRGA I l l exchange pool size was estimated by measuring the incorporation of 1^C02 into the pool i n the dark. A 0.23 1. closed system, depicted i n Figure TV-2B, was used. The entire system was i n i t i a l l y flushed with (X>2 free-air to reduce the ^ QQ^ concentration below 100 u l . / l . . . One y l . of -^C02 was generated i n i s o l a t i o n by the addition of excess 1 M H 2 S O 4 to NaH1^C03 i n the release flask. The 1 4 C 0 2 was then l e t into the rest of the system. A Geiger tube, which was connected to a Nuclear Chicago "Labitron" ratemeter and a chart recorder, was placed at the entry to the plant chamber to detect the -^ C a c t i v i t y i n the a i r stream. The i n i t i a l observation of the "^ C a c t i v i t y was made within 10 seconds of the introduction of the ^ C 0 2 , and subsequent measurements were made at 6 second intervals u n t i l the C O 2 concentra-t i o n had reached 400 y l . / l . . . A consistent decrease i n the -^ C a c t i v i t y of the a i r stream was observed when plants were not included i n the system. Since no leakage of ^C02 was evident under these conditions, this loss was presumably due to absorption of "*"^ C02 on the walls of the system. Because of t h i s , an equivalent number of measurements were made with and without plants. I t was also necessary to correct for the dark f i x a t i o n of 1^002. When the C O 2 concentration reached 400 y l . / l . . , the plants were removed from the chamber, immersed i n l i q u i d nitrogen and ground to a powder. One y l . per g. fresh weight of 1 M acetate buffer, pH 4.0, was added to remove "unbound" C O 2 and the mixture was dried i n a dessiccator. Fixed was measured by spreading the dried material on planchets, counting with a Geiger counter, and correcting for s e l f -absorption and counter e f f i c i e n c y . 112 On the basis of this information, the t o t a l size of the CO2 free-exchange pool was calculated from: Pool C 0 2 (ul./g.) = 1 4 C D - ( 1 4C p + 1 4Cp V[C0 2] Co W where "*"4Cp and -^ C were the gas phase -^ G a c t i v i t i e s i n the presence or absence of plants; "*"4Cf was the amount of "*"4C fixed; V was the closed system volume (1.); [CO,-,] was the f i n a l C0 2 concentration (400 u l . / l . ) ; and W was the fresh weight (g.) of plant material. The "^C02 and "^4C02 exchange techniques are si m i l a r i n that they are both based on gas exchange measurements. They d i f f e r because 12rj02 exchange requires the formation of a chemical equilibrium while i A 12 • L 4C0 2 exchange involves an isotopic equilibrium. Also the C0 2 exchange technique can only detect changes i n exchange pool s i z e , while the ^ 4C0 2 exchange technique allows the estimation of the t o t a l pool si z e . The two methods were equally d i f f i c u l t . To test the s e n s i t i v i t y of both the 1 2 C 0 2 and 1 4 C 0 2 technique, TO -1 / the absorption of C0 2 or C0 2 by a wick of Whatman No. 1 f i l t e r paper containing solution of 0.2 M phosphate buffers, pH 6.0 and pH 7.75, was used. In the case of the -*-2C02 absorption measurements, the phosphate buffers had previously been stored for seven days over 1 M NaOH i n a closed desiccator and were therefore C0 2-free. At the -1 o s t a r t of the x C0 2 absorption measurements, the phosphate buffers were in equilibrium with normal a i r . These absorption measurements were terminated after 25 minutes, since that was the average duration of experiments with plants. Figures IV-3A and IV-3B indicate the appa-ratus used for the absorption measurements. Figure Gas Flow System Used for 1 2 C 0 2 Measurements with 0.2 IV-3 Exchange and 1^C0 2 Exchange M Phosphate Buffers A 1 2 C 0 2 EXCHANGE CONTROL B CQ 2 EXCHANGE CONTROL PUMP CHAMBER A 1 FLOW GEIGER METER TUBE WICK WITH BUFFER £ >-RELEASE FLASK RATEMETER RECORDER PL CHAMBER 1141 RESULTS AND DISCUSSION Between 40 and 5 5 u l . of C O 2 per g. fresh weight were released after wheat and corn shoots were a c i d i f i e d with 5 M H C 1 . Similar results were recently reported by Yemm (19). Smith (13) found that over 300 y l . of C O 2 per g. fresh weight were released i f plants were boiled i n 4.4 M H C 1 for 1 hour. I f a substantial portion of this a c i d - l a b i l e C O 2 were or d i n a r i l y i n free and rapid exchange with the atmosphere, i t could prove to be a complicating factor i n studies of C O 2 exchange by plants. Measurements of the absorption of C O 2 by 0 . 2 M phosphate buffers are given i n Table I V - 1 . The calculated values were derived from the s o l u b i l i t y of C O 2 i n pure water ( 7 ) , the apparent pK of car-bonic acid ( 2 ) and the Henderson-Hasselbach equation (16)*. At pH 7 . 7 5 , the C O 2 absorption measured by both techniques was s l i g h t l y less than the calculated value. Failure to come to equilibrium, or d i f f e -rences i n the s o l u b i l i t y of C 0 2 or the pK of carbonic acid i n 0 . 2 M phosphate may account for this underestimation. Nevertheless, i t i s apparent that small C 0 2 free-exchange pools can be detected, and thei r approximate sizes' determined, by both the C O 2 and C O 2 exchange techniques. From Table I V - 2 i t can be seen that the C O 2 free-exchange pools i n both wheat and corn were found to be very small or non-existent by both techniques used. During these experiments, no s i g n i f i c a n t effect of C O 2 concentration on the rate of dark respirar: tio n was observed i n the open system. Although the measurements were *Appendix I I Table IV-1 Magnitude of C0~ Free-exchange Pools i n 0.2 M Phosphate Buffers Method Temperature (°C) C0 2 Concentration ( y l . / l . ) pH CO? Free-exchange Measured (yl.C0 2/ml. Buffer) Pool- Size. " Calculated (yl.C0 2/ml. H 2 O ) 1 2 C 0 2 Exchange 27 382 6.0 1.62* 1.01 27 358 7.75 5.90±0.76** 7.05 1 4 C 0 2 Exchange 20 300 6.0 -0.85±3.12** 0.94 20 300 7.75 4.96±1.19** 7.05 * Average of 2 measurements. ** 95% confidence l i m i t s of the mean. Table IV-2 Magnitude of C 0 9 Free-exchange Pools i n Wheat and Corn Shoots No. of Average C O 2 Free-exchange Method Plant Measurements Temperature Pool Size (°C) (ul.CC-2/g. fresh weight) 12c02 exchange itfheat 27 21 .4 1 .69 + 2.48* corn 12 22 .9 0 .14 + 2.99* •^ C^02 exchange wheat 6 20 .5 -0 .78 + 1.52* corn 6 21 .0 -0 .04 + 2.90* * 95% confidence l i m i t s of the mean. 117 made with the shoots i n darkness, i t seems reasonable to expect that the pool would not be larger during photosynthetic u t i l i z a t i o n of CC>2. I f this i s correct, the size of exchange pool i s too small for i t to be an important factor i h gas exchange transients such as the p o s t - i l l i m i n a t i o n C O 2 burst (Chapter I I I , 15), or as a source of C O 2 released by acid. Nor could a C O 2 free-exchange pool of such small size s i g n i f i c a n t l y effect the C O 2 response of photosynthesis or act as an important C O 2 reservoir for photosynthesis. The pH of juice expressed from wheat leaves i s about 6.0, and that from corn leaves, consistently lower, about 5.5 (8). The for-mation of H C O 3 from the hydration of C O 2 i s low i n this pH range. This i s a further i n d i c a t i o n that a C O 2 free-exchange pool, i f i t i s dependant on the carbon dioxide-water equilibrium, ought to be small. 118 LITERATURE CITED 1. Berthelot, M. M. , and Andre. 1887. Recherches sur l a vegetation. Sur les carbonates dans les plantes vivantes. Ann. Chem. Phys. 10: 85-107. 2. E d s a l l , J. T., and J. Wyman. 1958. Biophysical chemistry. Vol. I . , Chapter 10. Academic Press, Inc., New York. 3. Everson, R. G., and C. R. Slack. 1968. D i s t r i b u t i o n of carbonic anhydrase i n r e l a t i o n to the C4 pathway of photosynthesis. Phytochem. 7: 581-584. 4. Gibbons, B. H.,aarid J. T. Edsall. 1963. Rate of hydration of carbon dioxide and dehydration of carbonic acid at 25°. J . B i o l . Chem. 238: 3502-3507. 5. Hansl, N., and E. R. Waygood. 1952. Kin e t i c studies of plant decarboxylases and carbonic anhydrase. Can. J. Botany 30: 306-317. 6. Ho, C., and J . M. Sturtevant. 1963. The kinetics of the hydration of carbon dioxide at 25°. J. B i o l . Chem. 238: 3499-3501. 7. Hodgman, C. D. (editor). 1954. Handbook of chemistry and physics. 36th edition. Chemical Rubber Co., Cleveland. 8. Hurd-Karrer, A. M. 1939. Hydrogen-ion concentration of leaf Juice i n r e l a t i o n to environment and plant species. Am. J. Botany 26: 834-846. 9. Kern, D. M. 1960. The hydration of carbon dioxide. J. Chem. Educ. 37: 14-23. 10. Miyachi, S., R. Kanai, and A. A. Benson. 1968. Aerobically bound C O 2 i n Chlorella c e l l s . In: Comparative Biochemistry and Biophysics of Photosynthesis. K. Shib.ata, A. Takamiya, A. T. Jagendorf and R. C. F u l l e r (editor). University Park Press, State College, Pennsylvania.a 11. Rabinowitch, E. I. 1945. Photosynthesis. Vol. I . , Chapter 8, Interscience Publishers, Inc., New York. 12. Roughton, F. J. W.,, and V. H. Booth. 1938. The c a t a l y t i c effect of buffers on the reaction C O 2 + H20^H2C03. Biochem. J. 32: 2049-2069. 13. Smith, J. H. C. 1940. The absorption of carbon.Idioxide by unilluminated leaves. Plant Physiol. 15':.'-183^224. 119 14. Spoehr, H. A., and J . M. McGee. 1924. Absorption of carbon dioxide by leaf material. Carnegie I n s t i t u t i o n of Washington. Yearbook No. 23: 132-133. 15. Tregunna, E. B., G. Krotkov, and C. D. Nelson. 1964. Further evidence on the effects of l i g h t on respiration during photosynthesis. Can. J. Botany 42: 989-997. 16. Umbreit, W. W. 1949. Carbon dioxide and bicarbonate. In: Manometric techniques and tissue metabolism. W. W. Umbreit (editor). Burgess Publishing Co., Minneapolis, pp. 21-29. 17. Warburg, 0. 1962. New methods of c e l l physiology. Interscience Publishers, New York. 18. W i l l s t a t t e r , R., and A. S t o l l . 1918. Untersuchangen uber die Assimilation der Kohlensaure. J u l i u s Springer, B e r l i n . 19. Yemm, E. W. 1968. Bound CC>2 i n leaves - carbonic anhydrase. Research Conference on CO2 Uptake and Photorespiration. Case Western Reserve University, Cleveland. (unpublished) 120 CONCLUSIONS The inhib i t o r y effect of atmospheric 0 2 on apparent photosynthesis of wheat i s at least partly due to photorespiration and i s increased by: increasing 0 2 concentration, increasing temperature and decreasing C O 2 concentrations. Moderate to very high l i g h t i n t e n s i t i e s do not affect the per cent i n h i b i t i o n of apparent photosynthesis of wheat by O 2 . The effects of varying more than one of these factors are additive. At temperatures below 30° C i n saturating C O 2 concentrations, ar~j apparent photosynthesis of wheat i s not inhib i t e d by 2 0 . 8 % O 2 . During part of the growing season i n temperate conditions, the O 2 present i n the a i r may not cause a s i g n i f i c a n t decrease i n photosynthetic productivity i n wheat. The in h i b i t o r y effect of atmospheric O 2 on photosynthesis of corn i s not due to photorespiration. I t d i f f e r s from the effect of 0 2 on wheat i n that i n h i b i t i o n of photosynthesis occurs only at O 2 concentrations greater than 2 0 . 8 % O 2 , and the degree of i n h i b i t i o n i s not constant but increases with time of exposure to > 9 9 % O 2 . The C O 2 exchange characteristics of wheat and corn also d i f f e r with respect to their C O 2 compensation concentrations and the effects of temperature, C 0 2 concentration and l i g h t i ntensity on apparent photosynthesis. These differences are correlated with and may be the result of differences i n photorespiration, photo-synthetic carbon metabolism and leaf anatomy. 121 5".' The post-illumination C O 2 burst of wheat i s an extension of photo-respiration into the dark period following il l u m i n a t i o n . 6 . The CC>2 concentration response of the post-illumination burst of wheat indicates that photorespiration decreases with increasing C O 2 concentration i n the same way that the size of the depression of apparent photosynthesis by 20.8% O 2 decreases with increasing C O 2 concentration. 7 . The post-illumination C O 2 burst of Amaranthus edulis i s not the result of an extension of photorespiration into the dark period following i l l u m i n a t i o n , but i t has some other cause. 8. The quantity of carbon within wheat and corn leaves which can exchange f u l l y with atmospheric C O 2 i s too small to cause the post-illumination C O 2 burst or to s i g n i f i c a n t l y affect the C O 2 concentration response of apparent photosynthesis. 122 Appendix I CALCULATION OF THE C O 2 CONCENTRATION IN THE AIR ENTERING THE IRGA DURING THE POST-ILLUMINATION C 0 2 BURST This calculation was developed to avoid the error i n C 0 2 concentration measurement caused by rapid fluctuations i n C 0 2 concen-t r a t i o n i n the a i r stream passing through the IRGA sample cylinder. For this calculation to be used, the i n i t i a l C 0 2 concentration i n a l l parts of the sample cylinder must be known. In the present case, the C 0 2 concentration was i n i t i a l l y s i m i l a r throughout the sample cylinder and was equivalent to the IRGA reading/ Let the a i r stream entering the IRGA sample cylinder be divided into a series of 1 0 ml. elements. At the /air flow rate of 1 . 0 l./min., 0.6 sec. are required for each element to enter the sample cylinder. Let C be the C 0 2 concentration of an element just entering the sample cylinder. I t i s assumed that as the element passes through the sample cylinder, the r e l a t i v e response, r, of the IRGA to C i s the same as the IRGA response to a pulse in j e c t i o n of C 0 2 into the a i r stream entering the sample cylinder which was given i n Figure I I I - 2 . Therefore the IRGA w i l l detect the C 0 2 contained i n the element according to rC. In Figure I I I - 2 , i i t can be seen that an element w i l l exert an appreciable influence (l%'."'or more of maximum response) on the IRGA up to 9.6 sec. after i t began to enter the sample cylinder. Therefore, at any one time 9.6 / 0.6 = 16 elements w i l l contribute to the IRGA reading. From this reasoning, the following equation was developed to relate the IRGA reading to the C 0 2 present i n the elements which 12.3 entered the sample cylinder during the preceding 9.6 s e c : 0.9223 (IRGA) = r ^ + *2Cn-l + • • • + rl6 Cn-15 _ IRGAn represents the C O 2 concentration detected by the IRGA. In this equation, the factor 0.9223 can be considered to be a correction for uneven flow of a i r through the sample cylinder. When the pulse i n j e -ction data of Figure III-2 i s integrated with respect to time, the inte g r a l i s almost 8% less than i t would be i f the a i r flow rate was equal i n a a l l parts of the sample cylinder. The' above equation can be rearranged to give d i r e c t l y the C O 2 concentration i n the element which j u s t entered the IRGA. Since T± = 1.0: C n = 9.223 (IRGAn) - ( r ^ + . . . + r ^ C ^ ) . This equation i s then used i n a s e r i a l calculation to give C n at 0.6 sec. intervals during the post-illumination C O 2 burst. 124 Appendix I I CALCULATION OF THE ABSORPTION OF C0 2 BY 0.2 M PHOSPHATE BUFFERS The absorption of C0 2 by pH 6.0 phosphate buffer at 27° C w i l l be used as an example. Ideal gas laws, are assumed throughout, and the pK of carbonic acid w i l l be taken as 6.37^. The concentration of CO^ i s very small when the pH i s less than 8, and may be neglected for these calculations. The Bunsen s o l u b i l i t y c o e f f i c i e n t , which i s the volume of C0 2, reduced to 0° C, dissolved i n unit volume of water at t° C with a C0 2 pressure of 1 atm., i s 0.718 for 27° C . This s o l u b i l i t y coefficient includes a l l aqueous species of carbon as "dissolved C02". However, under the conditions i n which the Bunsen coefficient i s measured, the concentrations of species other than true dissolved C0 2 are not appreciable. Therefore, when the p a r t i a l pressure of C0 2 i s 0.000382 atm., the concentration of dissolved C0 2 i s : , C0 2 = 0.718 x 0.000382 x 300 = 0.30 y l . C02/ml. H20 273 From the Henderson-Hasselback equation-^: l E d s a l l , J . T. and J. Wyman. 1958. Biophysical chemistry. Vol. I. Chapter 10. Academic Press, Inc., New York. 2Hodgman, C. D. (editor). 1954. Handbook of chemistry and physics. 36th e d i t i o n . Chemical Rubber Co., Cleveland. %mbreit, W. W. 1949. Carbon dioxide and bicarbonate. In Manometric Techniques and Tissue Metabolism. W. W. Umbreit (editor), Burgess Publishing Co., Minneapolis. 125 HCO'-jj = C 0 2 x antilog (pH - pK) Therefore, "Total C O 2 " =? C 0 2 + H C O 3 = C02 + C 0 2 x antilog (pH - pK) = 0.30 + 0.30 x antilog ( 6 . 0 - 6 . 3 7 ) = 1 . 0 1 u l . C 0 2 / m l . H 2 0 The absorption of C 0 2 by 0 . 2 M phosphate buffers at pH 7 . 7 5 and at other temperatures were calculated s i m i l a r l y and reported i n Table I V - 1 . 

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