Photosynthesis, Photorespiration and Related Aspects
PHOTOSYNTHESIS, PHOTORESPIRATION AND RELATED ASPECTS
OF C02 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
in the Department
of
Botany
accept this thesis as conforming to the required standard
THE UNIVERSITY OF BRITISH COLUMBIA
April, 1970 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and Study.
I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thes.is for financial gain shall not be allowed without my written permission.
Department
The University of British Columbia Vancouver 8, Canada ii
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 02 concentration on apparent photosynthesis of juvenile wheat and corn shoots were measured at different temperatures,
CO2 concentrations and light intensities using infra-red CO2 analysis and both open and closed gas-flow systems. The only condition in
which apparent photosynthesis of wheat was not inhibited by 02 was
in 20.8% 02 when the C02 concentration was saturating and the temperature was 30° C or less. The degree of inhibition increased
with increasing 02 concentration, increasing temperature, and decrea•
sing C02 concentration and was independent of the light intensity.
During some of the growing season in temperate regions, the effect of
atmospheric 02 on the photosynthetic productivity of wheat may be
negligible. The effect of 02 on wheat was ascribed to both a stimulation of photorespiration and an inhibition of photosynthesis
by 02.
In corn, which apparently lacks photorespiration, photosyn•
thesis was also inhibited by 99% 02- No inhibition occurred at
20.8% 02, however, and the stability and reversibility of the inhibi•
tion observed at 99% 02 differred from that found with wheat. These differences between wheat and corn are correlated with differences in leaf anatomy and photosynthetic carbon metabolism and with
differences in the response of apparent photosynthesis and the C02
compensation point to environmental conditions. Many of the gas iii exchange characteristics of corn and similar plants seem to be advantageous for the warm dry areas they often inhabit.
Initially high rates of CO2 production are exhibited by wheat immediately following illumination, and it has been suggested that this post-illumination CC>2 burst is an extension of photo• respiration into the dark period. In accord with this, CO2 concentration was found to influence both the post-illumination CO2 burst and the inhibition of apparent photosynthesis by O2 in a similar way. Except for Amaranthus edulis, plants which lack photorespiration also lack significant post-illumination CO2 bursts.
On the basis of its response to O2 concentration, however, the burst of Amaranthus edulis is concluded to be unrelated to photorespiration.
Measurements of "^C02 and ^C02 exchange were used to estimate the quantity of carbon in wheat and corn shoots which was in free exchange with atmospheric CO^. The free exchange pool size was found to be very small and it cannot be an important factor in the CO2 concentration response of photosynthesis or in the post-illumination
COo burst. iv
TABLE OF CONTENTS
Page
PREFACE . . . xiii
LITERATURE CITED • xv
CHAPTER I. The Effects of Temperature, CO2 Concentration and Light Intensity on the Oxygen Inhibition of Apparent Photosynthesis in 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 II. Some Comparative Aspects of the Physiology of CO2 Exchange by Wheat and Corn Shoots
INTRODUCTION 50 V
Page
MATERIALS AND METHODS 51
RESULTS AND DISCUSSION
1. The C02 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 III. Photorespiration and the Post-illumination C02 Burst in 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 02 and CO2 Concentrations on the Post-illumination CO2 Burst 91
3. Effect of 02 Concentration on the Post-illumination CO2 Burst of Amaranthus edulis 101
LITERATURE CITED .' . '. 104
CHAPTER IV Estimation of the C02 Free exchange Pool Size in Wheat and Corn Leaves
INTRODUCTION 106
MATERIALS AND METHODS 107
RESULTS AND DISCUSSION 114
LITERATURE CITED 118 vl
Page
CONCLUSIONS 120
APPENDIX I ^Calculation of the C02 Concentration in the Air Entering the IRGA during the Post-illumination
C02 Burst 122
APPENDIX II Calculation of the Absorption of C02 by 0.2 M Phosphate Buffers 124 vii
LIST OF TABLES
Table Page
II-l. Comparison of the Apparent Kinetic Constants of Photosynthetic CO2 Assimilation of Excised Wheat and Corn Shoots 65
IV-1 Magnitude of CO2 Free-Exchange Pools in 0.2 M Phosphate Buffer 115
IV-2 Magnitude of CO2 Free-Exchange Pools in Wheat and Corn Shoots . . . 116 viii
LIST OF FIGURES
Figure Page
1-1. Plant Chamber Used for Excised Shoots in Experiment I 3
1-2. Plant Chamber Used for Excised Shoots in Experiment II 4
1-3. Lamp Tower and Freezer Assembly Used for Control of Light Intensity and Temperature 6
1-4. Spectral Energy Distribution of Light from a General Electric "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 in Atmospheres Containing 300 ul./l. CO2
and 20.8% 02 or 3± 1% 02 14
1-8. Effects of Temperature on the CO2 Compensation Points of Excised Wheat Shoots in Atmospheres Containing 20.8% O2
or 3± 1% 02 16
1-9. Effects of CO2 Concentration and Temperature on the Apparent Rates of Photosynthesis of Excised Wheat Shoots in Atmospheres
Containing 20.8% 02 or 3± 1% 02 18
I-10. Effects of Temperature and C02 Concentration on the Per Cent Inhibition of Apparent Photosynthesis by 20.8% O2 in Excised Wheat Shoots 20
1-11. Effects of O2 Concentration and Temperature on the Apparent Rate of Photosynthesis
of Excised Wheat Shoots in 300 -ul./l. C02 .... 22
1-12. Effects of O2 Concentration and Temperature on the CO2 Compensation Point of Excised Wheat Shoots 24 ix
Figure Page
1-13. Effects of 02 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 02 Concentration and C02 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 Inhibition of Apparent Photosynthesis in Excised Wheat Shoots at 25° C 32
1-20. Effects of CO2 Concentration and Light Intensity on the Per Cent Inhibition of Apparent Photosynthesis in Excised Wheat
Shoots at 25° C and 20.8% 02 or 60.9% 02 33
1-21. The Carbon Deficit in Excised Wheat Shoots
Caused by 20.8% 02 at Different Temperatures and CO2 Concentrations (Experiment L) . 35
1-22. The Carbon Deficit in Excised Wheat Shoots
Caused by 20.8% 02 at Different Temperatures and CO2 Concentrations (Experiment II) 36
II-l. Open System Used to Generate Constant CO2 Concentrations in an Air Stream Containing Different O2 concentrations . 54 X
Figure Page
II-2. Time Course of CO2 Exchange by Excised Wheat Shoots in Atmospheres Containing
300 ul./l. C02 and Different 02 Concentrations 58
II-3. Time Course of C02 Exchange by Excised Corn': Shoots in Atmospheres Containing
300 ul./l. C02 and Different 02 Concentrations 59
II-4. Effects of 02 Concentration, Temperature,
C02 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 in Air Containing 20.8%
O2 and 335 ul./l. C02 63
II-6. Effects of Light Intensity on the Apparent Rates of Photosynthesis of Excised Wheat and Corn Shoots in Air Containing 20.8%
02 and 335 ul./l. C02 68
II-7. Effects of Exposure to High Light Intensity on the Apparent Rate of Photosynthesis of Excised Corn Shoots 69
III-l. Effects of Darkening on C02 Exchange of
Excised Wheat Shoots in .20.8% 02 87
III-2. Relative Response of the IRGA to Pulse Injections of CO2 into the Air Stream in the Plant Chamber and at the Entrance to the IRGA Sample Cylinder 90
III-3. Post-illumination CO2 Exchange'by Excised
Wheat Shoots in 1.8% O2 and 20.8% 02. The
C02 Concentration of the Air Entering the
Plant Chamber was 100 ul./l. C02 92
III-4.Post-illumination CO2 Exchange by Excised
Wheat Shoots in 1.8% 02 and 20.8% 02. The CO2 Concentration of the Air Entering the
Plant Chamber was 200 yl./l. C02 93
III-5. Post-illumination CO2 Exchange by Excised
Wheat Shoots in 1.8% 02 and 20.8% O2. The CO2 Concentration of the Air Entering the
Plant Chamber was 300 yl./l. C02 94 xi Figure Page
III-6. Post-illumination CO2 Exchange by Excised
Wheat Shoots in 1.8%-0, and 20.8% 02. The
C02 Concentration of the Air -Entering
the Plant Chamber was 400 ul./l. C02 95
III-7. Post-illumination C02 Exchange by Excised
Wheat Shoots in 1.8% 02 and 20.8% 02. The
C02 Concentration of the Air Entering the
Plant Chamber was 500 ul./l. C02 96
III-8. Effects of C02 Concentration on the Size of
the Post-illumination C02 Burst and the Magnitude of the Depression of Apparent
Photosynthesis by 20.8% 02 97
III-9. Effects of C02 Concentration on the Per Cent Inhibition of Apparent Photosynthesis by
20.8% 02 and on the Analogous Burst
Function % Ib 100
111-10. The Post-illumination C0.2 Burst of Amaranth us
edulis in 1.8% 02 and 20.8% 02 102
IV-1. Plant Chamber Used for Measurements of the
C02 Free-exchange Pool Size with Excised Wheat and Corn Shoots 108
12 IV-2. Gas Flow Systems Used for C02 Exchange
and -^C02 Exchange Measurements with Excised Wheat and Corn Shoots 110
12 IV-3. Gas Flow Systems Used for C02 Exchange
and 1^C02 Exchange Measurements with 0.2 M Phosphate Buffers 113 xii
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 helpful 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 is gratefully acknowledged.
Dr. G. R. Lister of Simon Fraser University kindly supplied the Gallenkamp gas mixing pumps and ISCO spectroradiometer used in these investigations. The use of the airflow planimeter of Dr.
D. P. Ormrod, formerly of the Department of Plant Science, is acknowledged.
The author was a recipient of a University of British Columbia
Scholarship in 1966-67, and of National Research Council of Canada
Scholarships during 1967-68 and 1968-69.
Finally, this thesis is dedicated to my wife Ann, who assisted in drafting many of the diagrams, and whose encouragement made this thesis possible. xiii
PREFACE
Two relatively 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 02 on plant C02 exchange (2, 3, 4, 10, 11). These
investigations culminated in the suggestion that many plants release
C02 during photosynthesis by a process called photorespiration. The
distinctive feature of photorespiration is that it is enhanced by 02
concentrations greater than 2% 02 while respiration by leaves in the
dark is not.
Meanwhile, in 1965 it was reported that malate and aspartate,
instead of the phosphorylated sugars typical of the Calvin cycle,
were the initial products of photosynthetic C02 fixation in sugar cane
(9). Hatch, Slack and their 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
"tropical" species which possessed the same type of photosynthetic
carbon metabolism (6, 7).
These two trends in 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•
piration, while plants which possess only the Calvin cycle can
rapidly photorespire (1).
This thesis includes experiments designed to indicate the
activity of photorespiration in different environmental conditions as
well as to indicate the differences in C02 exchange characteristics xiv of plants which possess and lack photorespiration. In addition, the relationship between photorespiration and post-illumination 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 - its relation 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 C02 fixation, 02 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 C02 - Gaswechsel wahrend die Licht-und Dunkelphase - Beitr. Biol. Pflanzen 42: 213-239.
Forrester, M. L., G. Krotkov and C. D. Nelson. 1966. Effect of oxygen on photosynthesis, photorespiration and respiration in 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 fixation in sugar-cane and its occurrence in other plant species. Biochem. J. 102: 417-422.
Johnson, H. S. and M. D. Hatch. 1968. Distribution of the C4-dicarboxylic acid pathway of photosynthesis and its occurrence in dicotyledonous plants. Phytochem.7: 375-380.
Johnson, H. S. and M. D. Hatch. 1969. The C^-dicarboxylic acid pathway of photosynthesis. Identification 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 fixation in sugarcane leaves. Plant Physiol. 40: 209-213.
Krotkov, G. 1963. Effect of light 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 in detached tobacco leaves. Physiol. Plantarum 19: 723-733. 1
Chapter I
THE EFFECTS OF TEMPERATURE, C02 CONCENTRATION AND
LIGHT INTENSITY ON THE OXYGEN INHIBITION OF
APPARENT PHOTOSYNTHESIS IN WHEAT
INTRODUCTION
The apparent rate of photosynthesis, whether it is measured in
terms of C02 assimilation or 02 evolution, is often found to be inhibited in the presence of molecular oxygen. This inhibition was first demonstrated by Warburg (46) and has come to be known as the
"Warburg effect". The effect has been observed in many photosynthetic organisms, including algae, mosses, liverworts, ferns, gymnosperms and angiosperms (31, 37, 43,'.44, 46).
It is known that the inhibitory effect of 02 is influenced by the environmental conditions, but information on the environmental
relations of the 02 effect is limited and often inconsistent. The
effect of 02 is reduced and may be absent at high C02 concentrations
(7, 40, 44), and is increased at C02 concentrations below atmospheric
levels (40) . In Chlorella at low C02 concentration, temperature did not affect the degree of inhibition 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 inhibition increases between 30° and 40° C
(23). Most studies have shown that the degree of inhibition is not affected by moderate to saturating light intensities (3, 31, 43, 47).
In several other cases, the degree of inhibition increased with 2 increasing light intensity (43, 47).
Numerous suggestions have been advanced concerning the
mechanism(s) by which 02 inhibits apparent photosynthesis (12, 43).
Both the light (3, 32, 34, 36) and dark (10, 40, 45) processes of
photosynthesis, as well as photorespiration (14, 17, 18, 41) have
been implicated as possible sites for the 02 effect.
In the present investigation, the effect of 02 concentration
on the apparent rate of photosynthesis of wheat shoots in different
conditions of temperature, C02 concentration and light intensity was
examined. The results permit us to assess the significance of the 02
effect on photosynthetic productivity, and add perspective to current
proposals on the mechanism of the 02 effect.
MATERIALS AND METHODS
Seeds of wheat (Triticum aestivum L. var. Spring Thatcher),
obtained from Buckerfield's Seed Co., Vancouver, were planted in flats
of vermiculite, watered daily, and grown in a ventilated room, at
21,600 lux of f lubres^eentrf and... incandescent! .light and 16 hr. photoperiod.
At the start 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 in 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 in Figures I-l and 1-2. Within these chambers, the cut ends of
the shoots were submerged in approximately 5 mm. of water. After the
shoots were enclosed in a chamber, they were allowed to adjust to the Figure I-l ant Chamber Used for Excised Shoots in Experiment air outlet /
shoot chamber-
water jacket
plant shoot
water trough
wing nuts and bolts
water r inlet 2 4 cm inlet and outlet FRONT VIEW SIDE VIEW Figure 1-2
Plant Chamber Used for Excised Shoots in 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
Electric "Cool Beam" 750 watt incandescent lamps. The lamp tower, shown in Figure 1-3 was used in experiments with excised shoots. The
chamber was placed on the freezer immediately below the lamp tower or, when more efficient cooling was required, within the freezer supporting
the lamp tower. Before the light was incident on the shoots, it was passed through 14 cm. of water to remove much of the infra-red
radiation. Figure 1-4 shows the spectral energy distribution of the un-
filtered 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 illumination of 32,300 lux (3000 ft-c) was equivalent to a
radiant intensity of 1.6 x 10^ erg./cm./sec. between 400 and 700 nm. when no water filter was used. The light 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 in 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 circulating 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 ectral Energy Distribution of Light from a General Electric Cool Beam" Lamp as Measured by a ISCO Spectroradiometer
8
flask, a Beckman IR 215 infra-red gas analyzer (IRGA), a Fisher
"Dynapump" air pump, and a Matheson R-2-15-B flowmeter, which were
linked in series by Tygon tubing. A Bausch and Lomb VOM-5 strip chart
recorder was connected to the IRGA and recorded the CC^ concentration
changes in the system.
Two studies were made of the effect of 0^ concentration on CO^ exchange by excised wheat shoots . Experiment I was carried out in
September 1967 to determine the effects of 20.8% (v./v.) and 3+ 1% 02 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 in Figure 1-1 was used, and the volume of the closed system was 860 ml. The air flow
rate in the closed system was maintained at 2 l./min., and the light intensity at the chamber surface was 10,760 lux. A Clark oxygen electrode, connected to a Beckman Oxygen Adapter and a millivoltmeter, was included in the system to measure the 0^ concentration in the air stream. Laboratory air or ^ from a compressed gas tank was flushed
through the system to adjust the 0^ concentration in the air stream.
Experiment II was carried out from February to May 1969 to in• 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 in
Figure i-2 was used. The volume of the closed system was 230 ml.,
and the air flow rate was 3 l./min. Except for the light intensity
study reported in Figure 1-15, the light intensity was 32,300 lux.
The 02 concentrations were established by flushing the closed system with laboratory air or with gas from compressed gas tanks. The 0 9 concentrations in the air stream were not measured during this experiment, but separate measurements with a Picker MS-10 mass spec•
trometer indicated that the 02 concentrations used were 1.8%, 20.8%,
60.9%, 78.6% and >99% 02. Between 11 and 24 measurements of the
apparent rate of photosynthesis were made at any one combination of 02
concentration, temperature, C02 concentration and light intensity used in Experiments I and II.
When intact plants were required, the wheat seeds were planted in a row so that a number of shoots could be sealed into the 135 ml. plexiglass chamber shown in Figure 1-5 by a rubber gasket coated with silicone grease. The environmental conditions during the growth of
these plants, and during the measurement of their C02 exchange were comparable to those described for Experiment I. When intact plants were used, the illumination system was similar to that described above except that the light was directed horizontally at the upright chamber.
The following sequence of operations was carried out during
the C02 exchange studies reported in this Chapter. After the system
was flushed to establish:.:the 02 concentration, it was closed. A hypodermic syringe was then used to inject less than 0.5 ml. of air
containing a high C02 concentration into the release flask to elevate
the C02 concentration in the system above 600 ul./l. The C02 concen•
tration in the system then decreased because of net C02 assimilation
by the enclosed shoots until the C02 compensation point was reached.
Thereafter, this sequence of operations was repeated until measurements with one sample of shoots was terminated. No sample of shoots was used for more than 3.5 hours following their enclosure in a chamber. Figure 1-5
Plant Chamber Used for Intact Shoots- water outlet
shoot chamber
water jacket
plant shoot
"wing nuts and bolts
vermiculite
O 4 cm
FRONT VIEW SIDE VIEW 11
Apparent rates of photosynthesis were calculated from the time required
for the shoots to reduce the CO2 concentration in the system by
50 yl./l. or by 25 yl./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 similar 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 indication 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 intact and excised shoots.
Figure 1-6 illustrates the similarity of the CO2 concentration res•
ponse of apparent photosynthesis by intact and excised shoots at high
and low temperatures. The vertical bars which extend above and below
the averages at each CO2 concentration indicate the 95 per cent
confidence limits about the sample means. Where similar vertical bars
occur on other figures in this thesis, they also show the 95 per cent
confidence limits about the sample means. If the excised shoots were well-supplied with water, their 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 is little 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 in CC>2 exchange (8, 28) or transpiration (33) when petioles or leaves are severed. Because their CO2 exchange behavior resembled that of intact shoots, and because of their convenience, excised shoots were used for the rest of the research reported in 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 initial 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 yl./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 C02 assimilation declined. It 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• sis was greatest between 20° and 26° C, but in 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 significantly less than at 20° C, while in 3± 1% O2, the rate was significantly greater at 34° C than at 20° C.
Therefore, a decrease in O2 concentration from 20.8% O2 to 3± 1% O2 appeared to cause an increase in the temperature optimum for apparent photosynthesis. In addition, it isnofceworthy that at 13° C or lower temperatures, there was no significant difference between the apparent rates of photosynthesis in 20.8% O2 and 3± 1% O2. At
higher temperatures, however, apparent photosynthesis in 20.8% 02 was greatly inhibited compared to the rates of CO2 assimilation observed
in 3± 1% O2. Thus, these results show that 02 concentration can alter 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 in Atmospheres Containing
300 yl./l. C02 and 20.8% 02 or;3± 1% 02
15
inhibitory effect of 02 on apparent photosynthesis is modified by the temperature.
(b) Effects of Low 02 Concentration and Temperature on the
C02 Compensation Point.
When plants are placed in a sealed chamber and illuminated,
C02 is assimilated until a C02 concentration is reached at which the
rate of C02 uptake is equal to the rate of C02 production. The C02
concentration at which this equilibrium occurs is known as the C02 compensation point (15). Figure 1-8 shows the effect of temperature
on the C02 compensation point of wheat shoots in 3± 1% and 20.8% 02 •
The C02 compensation point was always much lower in 3± 1% 02 than in
r 20.8% 02, but at both 02 concentrations the C02 compensation point increased with increasing temperature. At 32° C or less, the ratio
of the C02 compensation point in 3± 1% 02 to the C02 compensation
point in 20.8% 02 was constant. Also, this ratio was equivalent to
the ratio of the 02 concentrations used. Above 32°, however, the ratio increased sharply.
(c) Effects of Low 02 Concentration, Temperature, and C02 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 in 3± 1% O2
and 20.8% 02. At the same temperatures, however, Figure 1-8 indicated
that the C02 compensation point was proportional to the 02 concent• ration. Because of this -anomaly, a thorough study was made of the
effects of 3± 1% 02 and 20.8% 02 on apparent photosynthesis at
different temperatures and C02 concentrations-. Rates of C02 assimilation were measured at 4 temperatures from 13° C to 34° C and
at C02 concentrations between 500 yl./l. C02 and the C02 compensation Figure 1-8
Effects of Temperature on the CC>2 Compensation Points of Excised
Wheat Shoots in Atmospheres Containing 20.8% 02 or 3± 1% 02 point.
The results of this study are presented in 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 in CO2 concentration became less pronounced. Often, if the CO2 concentration was sufficiently high, the apparent rate of photosynthesis became insensitive'to changes in CO2 concentration.
When this occurred, apparent photosynthesis was saturated by CO2. It is 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 yl./l. Under all other conditions, apparent photosynthesis was saturated by 500 yl./l. CO2 or less, 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 in 20.8% O2 than in 3± 1% O2• The CO2 concentration required to eliminate the
inhibitory effect of 20.8% 02 was higher at 19.7° C than at 13° C and appears to correspond to the minimum CO2 concentration required to saturate apparent photosynthesis in 20.8% O2 in these cases. Under no condition of temperature'or CO2 concentration was the apparent rate
of photosynthesis significantly enhanced in the presence of 20.8% 02 compared to the rate in 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 in Atmospheres
. Containing 20.8% 02 or 3± 1% 02 18
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o o3±1 %02 13.CTC A * 20.8%0r 19.7°C 4= 4.0
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C02 CONCENTRATION (JJI/I) 19 points appeared to be less in 20.8% than ih 3± 1% O2.
Inspection of Figure 1-9 reveals that the inhibitory effects
of 20.8% 02 on apparent photosynthesis were greatest at high tempe•
ratures and low C02 concentrations. A useful quantitative index of the effect of O2 on apparent photosynthesis -is".-that of per cent
inhibition (% Ip) , which may be calculated (3, 18, A3) using the expression:
X p * P = l " P2 100 Pl (1)
in which P-^ and P2 represent the apparent rates of photosynthesis at a low (e.g. 3± 1% O2) and a high (e.g. 20.8% O2) concentration of O2 respectively. Examination of this formula shows that 100% inhibition must occur at the CO2 compensation point in the higher concen•
tration, since at that point P2 equals zero.
Figure 1-10 shows the per cent inhibition of apparent
photosynthesis by 20.8% O2 at different temperatures and C02 concentrations. These data are derived from the results presented in
Figure 1-9. It is clear that the per cent inhibition of apparent photosynthesis increased with increasing temperature. Except where the CO2 concentration approached the CO2 compensation point, the increase in degree of inhibition appeared to be linear with the same slope at all CO2 concentrations. In this linear portion of the
figure, the temperature at which a certain per cent inhibition o occurred appears to be related to the logarithm of the CO2 concen•
tration. At any one temperature, the per cent inhibition decreased in
a non-linear fashion with increasing CO2 concentration. Figure I-10
Effects of Temperature and CO2 Concentration on the Per Cent Inhibition of Apparent Photosynthesis by 20.8% O2 in Excised Wheat Shoots PER CENT INHIBITION 21
3. Further Studies on the Effects of 02 Concentration on C02
Exchange by Excised Wheat Shoots (Experiment II)
(a) Effects of 02 Concentration and Temperature on Apparent
Photosynthesis and the C02 Compensation Point.
A second series of tests was carried out to extend these
results to include higher 02 concentrations. Figure 1-11 is comparable to Figure 1-7 and shows the temperature response of apparent photo•
synthesis of wheat shoots in 300 yl./l. C02 and 02 concentrations
ranging from 1.8% 02 to >99% 02.
Throughout Experiment II, the apparent rates of photosynthesis were much higher than those obtained in Experiment I. This difference can be accounted for largely by the higher light intensity used in
Experiment II. In accord with the previous results, at low tempera-- tures there was no significant difference in the apparent rates of
photosynthesis in 20.8% 02 and 1.8% 02. In this case, however, the
apparent rates of photosynthesis at these 02 concentrations were similar at 20° C as well as at 13° C. At high temperatures apparent
photosynthesis in 20.8% 02 was again inhibited compared with the rates
observed at the low 02 concentration used. At all the temperatures
examined, apparent photosynthesis was inhibited by the presence of 02
concentrations from 60.9% 02 to >99% 02, and the degree of inhibition
was enhanced as the 02 concentration and temperature increased.
Although this time there was no significant difference in the optimum
temperature for apparent photosynthesis in the 20.8% 02 and 1.8% 02,
Figure-.IftlL.confirms, the earlier indication that the optimum
temperature tends to decrease as the 02 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
in 300 ul./l. C02 22 23
and 35° C when the 02 concentration was 1.8% 02 or 20.8% 02. When the
02 concentration was raised to >99% 02, however, the optimum occurred between 13° C and 25° C.
Figure 1-12 shows the effect of 02 concentrations from 1.8% 02
to >99% 02 on the C02 compensation point. Once again, the C02
compensation point was affected by the 02 concentration and temperature.
At any one temperature, the C02 compensation point was directly
proportional to the 02 concentration. The slope of the linear C02
compensation point-02 concentration relationship increased as the
temperature increased. If the data are extrapolated to zero 02
concentration, the C02 compensation points appear to become less than
15 yl./l. C02.
(b) Effects of 02 Concentration, Temperature, C02 Concen• tration 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 02 concentrations
from 1.8% 02 to >99% 02 on apparent photosynthesis in different
conditions of C02 concentration and temperature. The general pattern
of the results obtained at 1.8% 02 and 20.8% 02 was quite similar to the earlier results from Experiment I which were presented in
Figures 1-9 to 1-10. In Figures 1-13 to 1-18 it can be seen that at
higher 02 concentrations the trends observed between 1.8% 02 and 20.8%
02 were continued. At 60.9% 02 to >99% 02 the apparent rate of photosynthesis was significantly inhibited in all conditions except
at high C02 concentrations at 13° C and 60.9% 02. The degree of
inhibition increased as the 02 concentration and temperature increased
and the C02 concentration decreased. The slopes of the C02 concen• tration 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
C02 CONCENTRATION CLJI/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
o 25 C 4CN12. 0 o -Ol.8%02 O -A20.8°/oO2 (J A- ~A6Q9°/002 £ 1QC
0- -Q78.6°/O02 CO -•>99°/002 00 LU f 8.0 >- oo A _ _ O § 6.0 a /T /"
° 4.0
5 a/ or . , S*/ 2.0
LU m ol 00"c>-A' -L < 0 100 200 300 400 500 600
CONCENTRATION (ul/l) 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 O1.8%02
A20.8%02
A60.9%02
A E78.6%02
• >99°/002
A-— - / -f ° / _ B — /" - .•a A A' S 0/S / / 13
^2 "
100 200 300 400 500 600
C02 CONCENTRATION (ul/l) Figure 1-18
Effects of O2 Concentration and CC^ Concentration on the Apparent Rates of Photosynthesis of Excised Wheat Shoots at 40° C 30
14.0 40°C o—- —O1.8%02
A—— —A20.8%02 12.0
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10.0 D—- ^•>99%02
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C02 CONCENTRATION (pl/l) 31
were significantly decreased as the 02 concentration increased from
1.8% 02 to >99% 02. At 13° C and 78.6% 02, and at 20° C and 60.9% 02,
apparent photosynthesis was saturated by the highest C02 concentrations used but was still inhibited to some extent. Therefore it appears thatJ
saturating C02 concentrations may not be able to reverse the inhibitory
effects of very high 02 concentrations on apparent photosynthesis.
The data at 25° C were used for Figure 1-19 which shows the per cent inhibition of apparent photosynthesis by O2 concentrations
ranging from 1.8% 02 to >99% 02 at C02 concentrations from 100 yl./l.
e©2 to 500 yl./l. C02. At each C02 concentration, the degree of
inhibition increased with increasing 02 concentration. At low CO2 concentrations at least, this increase appears to be non-linear. When the results are extrapolated to zero inhibition, it appears that there
would be no inhibition of apparent photosynthesis in 300 ul./l. C02
below about 14% 02. Similarly, in 400 yl./l. C02 zero inhibition
would occur below about 22% 02, and in 500 yl./l. C02 below about
30% 02. This lack of inhibition by some 02 concentrations above 1.8%
02 is equivalent to that observed at high C02 concentrations in
Figures 1-9 and Figures 1-13 to 1-17.
Figure 1-20 summarizes other measurements which were made to test the influence of light intensity on. the per cent inhibition of apparent photosynthesis at 25° C. Although the data are somewhat scattered, light intensity had no apparent effect on the degree of
inhibition caused by 20.8% 02 or 60.9% 02. Changes in light intensity between 6460 lux and 107,640 lux affected the apparent rate of photosynthesis (see Chapter II), but the per cent inhibition was not
was altered because the ratio Pi/P2 constant over this range in light Figure 1-19
Effects of 02 Concentration and CO2 Concentration on the Per Cent Inhibition of Apparent Photosynthesis in 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 in Excised Wheat Shoots
at 25° C and 20.8% 09 or 60.9% 09
34 intensity. Figures 1-14 and IT-15 again show the decrease in per cent inhibition as the CO2 concentration increased.
4. Effect of C>2 on Photosynthetic Productivity
Per cent inhibition is a relative measure of the O2 effect and does not directly indicate the influence of O2 on the quantity of
Carbon assimilated in photosynthesis. The expression:
Carbon Deficit = 12 (Px - P2) (2) 44 can be used to calculate the decrease in photosynthetic productivity caused by the inhibitory effect of O2. The data from Experiment I
were used to calculate the carbon deficit produced by 20.8% 02 in different conditions of temperature and CO2 concentration, and these results are presented in Figure 1-21. It is apparent that there was a general increase in carbon deficit with each rise in temperature.
At 300 yl./l. CO2 or higher CO2 concentrations at 13° C and 450 ul./l.
CO2 or above at 19.6° C, the carbon deficit was zero. The absence of a carbon 'deficit under these conditions is related to the zero per
cent inhibition of apparent photosynthesis which was. noted in Figure
1-9. Also, according to Figure 1-21, the carbom:deficit tended to
decrease as the CO2 concentration approached 50 yl./l. CO2. As we have seen in the preceding sections, the degree of inhibition of apparent photosynthesis increases as the CO2 decreases. The carbon
deficit does not parallel this increase in per cent inhibition because
the apparent rate of photosynthesis is reduced by low CO2 concen•
trations .
The carbon deficit'values calculated from the results of
Experiment II are presented in Figure 1-22. Once again, the carbon Figure 1-21
The Carbon Deficit in Excised Wheat Shoots Caused by 20.8% 02 at Different Temperatures and CO2 Concentrations (Experiment I) CARBON DEFICIT (mg C/hr/g fr wt) o p o o o Figure 1-22
The Carbon Deficit in Excised Wheat Shoots Caused by 20.8% 02 at Different Temperature and CO2 Concentrations (Experiment II)
deficits were greatest at high temperatures and at CO2 concentrations
intermediate between the CO2 compensation point and saturating CO2
concentration. At the CC^ concentration to which plants are normally
exposed, about 300 yl./l. CO2, the carbon deficit ranged from zero
to 0.67 mg. C/hr./g. fr. wt., and there was no significant carbon
deficit at 20° C or less. The greatest carbon deficit observed in
these studies was 0.76 mg. C/hr./g. fr. wt. and this occurred at
40° Ccand 450 yl./l. CO2 in Experiment II. Therefore, the presence of
20.8% O2 in the atmosphere can greatly reduce the photosynthetic productivity of wheat. It is obvious from the results presented in
Figures 1-13 to 1-18 that the presence of higher O2 concentrations would reduce photosynthetic productivity even more. High CO2
concentrations and low temperatures, however, tend to reduce the
carbon deficit. 38
DISCUSSION
These results provide a comprehensive description of the
inhibitory effects of 02 on apparent photosynthesis by wheat under different environmental conditions. The degree of inhibition of
apparent photosynthesis by 02 was enhanced by high 02 concentrations,
high temperatures and low C02 concentrations, and under some
conditions the presence of 02 can cause significant reductions in photosynthetic productivity.
These results extend and are in general accord with the
findings of most previous studies on the effect of 02 on apparent
photosynthesis. The relationship between the 02 concentration and the per cent inhibition of apparent photosynthesis, shown in Figure 1-12, is similar to that obtained by other researchers using algae (40) and terrestrial plants (18). For the first time, except in algae (40), it
has been demonstrated that high C02 concentrations can eliminate the
inhibitory effects of 20.8% 02 on apparent photosynthesis. Some previous investigations have indicated that the degree of inhibition
of apparent photosynthesis by 02 is greatest at low C02 concentrations
(40, 43), but the details of the response of the 02 effect to C02 concentration are now clearly evident. The observation that light intensity does not affect the per cent inhibition of apparent photo•
synthesis by 02 coincides with most other reports (3, 31, 43, 47).
A few experiments with algae and mosses (43, 47) which found the per cent inhibition of apparent photosynthesis to increase with increasing light intensity do not agree with the present results. A conflict 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
inhibition of apparent photosynthesis by 02, but early investigations with Chlorella (40) and Funaria (43) revealed no such effect. Some
recent results with angiosperms, however, are in agreement with the
present observations (23). Whenever 21% 02 was found to inhibit
apparent photosynthesis, the degree of inhibition increased between
30° C and 40° C. There is little previous information on the effect
of 02 concentration on the optimum temperature for apparent photosyn• thesis. Contrary to the results in Experiment I but in agreement with
Experiment II, however, BjBrkman found no increase in the optimum
temperature when Marchantia polymorpha was placed in 2% 02 (4). The difference between the present observations and those of others
cannot now be resolved, but it is possible that some of the
discrepancies are due to the different species or different growing
conditions utilized in different investigations.
The effects of 02 on apparent photosynthesis are reflected in
the rates of plant growth at different 02 concentrations. The dry matter productivity of Phaseolus vulgaris, Mimulus cardinalis and
Marchantia polymorpha was found to be enhanced when the plants were
grown in 2.5 to 5% 02 compared with those grown in 21% 02 (5, 6).
This enhancement of growth at low 02 concentrations near the C0.2:cO;mpen-
sation point.was more than when the C02 concentration was 320 yl./l.
or 640 yl./l. C02. Therefore, the response of plant growth to 02 and
C02 concentrations corresponds well to, andmaybe explained by, the present observations on apparent photosynthesis. It is expected that
the present results may be useful in predicting the effect of atmos•
pheric 02 on the productivity of wheat in conditions where 02 40 concentration, temperature, CO2 concentration and light intensity are
limiting productivity through their effects on apparent photosynthesis.
The CO.2 compensation point results confirm earlier studies
which demonstrated that the C02 compensation point is directly
proportional to the O2 concentration (18, 41). This relationship is
now extended to include a wide range of temperatures, and the increase with increasing temperature in the slope of the linear response of the
C02 compensation point to O2 concentration is now apparent. In most
previous studies, the C02 compensation point has been found to extra• polate to zero at zero O2 concentration (18, 41). In the present
investigation, this was observed only at 25° C or lower temperatures.
Above 30° C, the extrapolated CO2 compensation point appears to be
significantly greater than zero at zero 02- Heath and Orchard (22) have reported that the extrapolated CO2 compensation points of
Pelargonium and Hydrangea were much higher than zero CO2 at zero O2 when the temperature was 27° C. It would be interesting to learn whether these plants are capable of attaining much lower CO2
compensation points in the absence of O2 at lower temperatures.
The linear relationship between the CO2 compensation point arid
O2 concentration and the finding that it extrapolates to zero at zero
O2 has previously been used as evidence that during photosynthesis,
dark respiration is replaced by a different process of CO2 production
called photorespiration (18, 41). In contrast to dark respiration,
which is saturated by about 2% 02 (2, 18, 48), photorespiration is
distinguished by a lower affinity for 02 and it continues to increase
in rate with increasing O2 concentration up to 100% O2 (18, 41). The
observations that the CO2 compensation point-02 concentration relationship sometimes extrapolates to greater than zero at zero 02, however, is consistent with the possibility that some CC^ production by dark respiration may occur during photosynthesis. It is possible
that during illumination the rate of dark respiration may appear to be
small because much of the CO2 produced is quickly reassimilated by
photosynthesis before it can escape from the leaf. At 25° C or less
the rate of C02 release by dark respiration during photosynthesis may be too low to cause a significant increase in the CO2 compensation v point in wheat. The operation of a classical tricarboxylic acid cycle has been demonstrated in the alga Scenedesmus obliquus during photosynthesis, but illumination did reduce the quantity of carbon
entering the cycle (29, 30). Therefore, CO2 production by dark
respiration may occur in illuminated photosynthetic tissue, but
probably at a reduced rate.
The CO2 compensation point and the apparent rate of photosyn•
thesis are both determined by the opposing processes of CO2 uptake
and CO2 production. It follows from this that the observed response
of CO2 exchange to O2 concentration could be due to an inhibition by
O2 of photosynthetic CO2 uptake, or to a stimulation by O2 of CO2 production, or to a combination of these two effects.
The results of recent investigations do not support earlier
suggestions (3, 43) that O2 inhibits apparent photosynthesis by acting
on photosynthetic electron transport in the chloroplast. For example,
a typical O2 effect has been observed in CO2 fixation by a chloro-
plast-free fraction from Euglena gracilis (16).
The activities 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 inhibited by These
enzymes could be the primary sites for the 02 effect, but the
significance of their inhibition by 02 in terms of the rate of photo• synthesis, and whether the inhibition occurs in vivo remains to be determined.
Direct evidence that photorespiration is a component of the
02 effect has been provided by estimates of the individual rates of
C02 uptake and production obtained from simultaneous measurements of 1U 12
C02 and C02 exchange by illuminated leaves. Bulley and Tregunna
(9) found that, at the C02 compensation point, about 80% of the total
effect of 21% 02 on apparent photosynthesis in soybean was due to
photorespiratory C02 production. The remainder was due to an inhibi•
tion of photosynthetic C02 uptake. In addition, D'Aoust and Canvin
(11) have reported that a large portion of the 02 effect in sunflower
was due to photorespiratory C02 production. It is interesting to note
that there was a noticeable inhibition at the saturating C02
concentration in Figure 1-13 at 80% 02 at 13° C and at 60% 02 at
20° C. This may correspond to the residual non-photorespiratory
portion of the 02 effect.
Further support for the correlation between photorespiration
and the 02 effect comes from studies of C02 production by illuminated
plants into a C02-free atmosphere. The effect of 02 concentration on
the rate of C02 production by illuminated barley leaves (48) is
similar to its effect on the per cent inhibition of apparent photosynthesis shown in Figure 1-19. Also, the large inhibitory
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 02 effect at high CO^ concentrations does not by itself prove the absence of photorespiration under those
conditions. It is conceivable that, at C02 saturation, photores• piration may not influence the apparent rate of photosynthesis. This situation would occur if photorespiration involves the oxidation of an
intermediate between the C02 fixation step and the limiting step in photosynthesis. Results which will be presented in Chapter III, however, demonstrate that the size of the post-illumination CC^ burst,
and therefore the rate of photorespiration, is decreased and may be
negligible at high C02 concentrations.
It is possible that the portion of the 02 effect which is not
due to photorespiratory CC^ production may be associated in another way with the photorespiratory process. For example, photorespiration
could depress the apparent rate of photosynthesis through competition
for the photosynthetic C02 acceptor or a precursor of it (19) as well
as by the production of C02. Of course, that component of the 02
effect which cannot be accounted for by photorespiratory C02 production
could be entirely unrelated to photorespiration.
It has been suggested that glycolic acid or a related metabolite is the substrate for photorespiration (13, 20, 49, 50, 51).
The present results are consistent with this hypothesis. The
excretion of glycolate by algae is greatest at low C02 concentrations
and high 02 concentrations (1, 38) and it is in these conditions that
the degree of inhibition of apparent photosynthesis by 02 is greatest.
Glycolate may be formed from intermediates of the Calvin cycle (1)
and its formation may inhibit photosynthesis by lowering the levels of photosynthetic intermediates as just described above.
An alternative to the glycolic acid hypothesis has recently
been advanced by Samish and Roller (39). They suggest that C>2 acts to
increase the leaf mesophyll resistance to C02 uptake. As a result,
increased quantities of the C02 produced within the leaf by dark
respiration are released from the leaf at high 02 concentrations instead of being reassimilated in photosynthesis. This is an intere• sting concept and can account for the present, data equally as well as
the glycolic acid hypothesis. Recent results on the behavior of C02
uptake and production rates at the C02 compensation point (9),
however, tend to support the hypothesis that the action of 02 is to
stimulate internal photorespiratory C02 production rather than to
directly inhibit photosynthetic C02 fixation.
At the present time, C02 production by photorespiration does not appear to be beneficial to the plant. Growth can be reduced by
this activity and no ATP or reduced pyridine nucleotide formation has
been associated with the C02 production. Goldsworthy (21) has
recently speculated that photorespiration has been inherited from primitive photosynthetic and non-photosynthetic microorganisms which
underwent symbiotic union early in the history of life. He has suggested that until recently photorespiration was not detrimental to
plants because the C02 concentration of the earth's atmosphere was
too high for an 02 effect to occur. Now, however, photorespiration is becoming disadvantageous, especially for plants growing in dense stands in the tropics.
There is much to be learned about photorespiration, however,
and I hesitate to condemn as entirely detrimental a plant activity which can involve a carbon flux similar to that involved in photosynthesis. Nevertheless, the function of photorespiration in the life of a plant is puzzling and requires further investigation. It should be recalled, however, that in air at temperatures of less than
13° C to 20° C, the apparent rate of photosynthesis in wheat was not
inhibited. Thus, photorespiration and the 02 effect may not be dis• advantageous to a plant like x^heat during some of its life cycle in temperate conditions. 46
LITERATURE CITED
1. Bassham, J. A. and M. Kirk. 1962. The effect of oxygen on the reduction of CO2 to glycolic acid and other products during photosynthesis by Chlorella. Biochem. Biophys. Res. Comm. 9: 376-380.
2. Beevers, H. 1960. Respiratory metabolism in plants. Row, Peterson and Co., White Plains, N.Y.
3. Bjorkman, 0. 1966. The effect of oxygen concentration on photo• synthesis in higher plants. Physiol. Plantarum 19: 618-633.
4. Bjorkman, 0. and E. Gauhl. 1969. Effect of temperature and oxygen concentration on photosynthesis in Marchantia poly• morph a. Carnegie Inst. Year Book 67: 479-482. Carnegie Inst, of Wash.
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7. Briggs, G. E. and C. P. Whittingham. 1952. Factors affecting the rate of photosynthesis of Chlorella at low concentrations of carbon dioxide and in 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 CO2 compensation point. Can. J. Botany, in press.
10. Coombs, J. and C. P. Whittingham. 1966. The mechanism of inhi• bition of photosynthesis by high partial pressures of oxygen in Chlorella. Proc. Roy. Soc. B. 164: 511-520.
11. D'Aoust, A. L. and D. T. Canvin. 1969. Effect of oxygen concen• tration on the CO2 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 in electron transport reactions of photosynthesis. Progr. in 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 CO2 fixation, 02 pressure and glycollate concentra• tion. 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- Ke^"ensatTc^pl"unktesi.Planta 43: 83- 97. V " ' " "'
16. Ellyard, P. W. and A. San Pietro. 1969. The Warburg effect in a chloroplast-free preparation from Euglena gracilis. 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. Beitr. Biol. Pflanzen 42: 213-239.
18. Forrester, M. L., G. Krotkov and C. D. Nelson. 1966. Effect of oxygen on photosynthesis, photorespiration and respiration in detached leaves. I. Soybean. Plant Physiol. 41: 422-427.
19. Gibbs, M., P. W. Ellyard and E. Latzko. 1968. Warburg effect: control of photosynthesis by oxygen. In: Shibata, K., Takamiya, A., Jagendorf, A. T. and Fuller, R. C. (editor). Comparative Biochem. and Biophysics of Photosynthesis. Uni• versity Park Press, Pennsylvania, pp. 387-399.
20. Goldsworthy, A. 1966. Experiments on the origin of C02 released by tobacco leaf segments in the light. 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 levels. II. The process of apparent assimilation. J. Exp. Bot. 19: 176-192.
23. Hesketh, J. 1967. Enhancement of photosynthetic C02 assimilation in 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 CO2 evolution in light 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 in light 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. Keller. 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. Jr., 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 light on the tricarboxylic acid cycle in 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 Hill reagents. Arch. Biochem. Biophys. 33: 65-77.
33. Meidner, H. 1965. Stomatal control of transpirational water loss. Symp. Soc. Exp. Biol. 19: 185-204.
34. Miyachi, S., S. Izawa and H. Tamiya. 1955. Effect of oxygen on the capacity of carbon dioxide fixation 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 in 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 in Chlorella cells. Biochem. Biophys. Acta 34: 202-210.;'
37. Poskuta, J. 1968. Photosynthesis, photorespiration and respira• tion of detached spruce twigs as influenced by oxygen concentration and light intensity. Physiol. Plantarum 21: 1129-1136.
38. Pritchard, G. G., W. J. Griffin and C. P. Whittingham. 1962. The effect of carbon dioxide concentration, light intensity and isonicotinyl hydrazide on the photosynthetic production of glycollic acid by Chlorella. J. Exp. Botany 13: 176- 184. 49
39. Samish, Y. and D. Koller. 1968. Estimation of photorespiration of green plants and of their mesophyll resistance to CO2 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 in detached tobacco leaves. Physiol. Plantarum 19: 723-733.
42. Turner, W. B. and R. G. S. Bidwell. 1965. Rates of photosyn• thesis in 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. Brittain. 1962. Oxygen as a factor in photosynthesis. Biol. Rev. 37: 130-170.
44. Turner, J. S., M. Todd and E. G. Brittain. 1956. The inhibition of photosynthesis by oxygen. I. Comparative physiology of the effect. Aust. J. Biol. Sci. 9: 494-510.
45. Turner, J. S., J. F. Turner, K. D. Shortman and J. E. King. 1958. The inhibition of photosynthesis by oxygen. II. The effect of oxygen on ^gly.ceraldehyde phosphate dehydrogenase from chloroplasts. Aust. J. Biol. Sci. II: 336-342.
46. Warburg, 0. 1920. Uber die Geschwindigkeit der photochemischen Kohlensaurezersetzung in lebenden Zellen. II. Biochem. Z. 103: 188-217.
47. Wassink, E. C., D. Vermeulen, G. H. Remen and E. Katz. 1938. On the relation between fluorescence and assimilation in photosynthesizing cells. Enzymologia 5: 100-109.
48. Yemm, E. W. 1969. Photorespiration and photosynthesis in young leaves of barley. Progr. in Photosynthesis Res. 1: 474-481.
49. Zelitch, I. 1958. The role of gly colic acid oxidase iii the respiration of leaves. J. Biol. Chem. 233: 1299-1303.
50. Zelitch, I. 1959. The relationship of glycolic acid to respira• tion and photosynthesis in tobacco leaves. J. Biol. Chem. 234: 3077-3081.
51. Zelitch, I. 1966. Increased rate of net photosynthetic carbon dioxide uptake caused by the inhibition of glycolate oxidase. Plant Physiol. 41: 1623-1631. 50
Chapter II
SOME COMPARATIVE ASPECTS OF THE PHYSIOLOGY OF C02 EXCHANGE
BY WHEAT AND CORN SHOOTS
INTRODUCTION
In recent years, certain plants have been distinguished from
others on the basis of their characteristics of C02 exchange, photosynthetic carbon metabolism, and leaf anatomy. The chloridoid- eVagrbstoidand some panicoid grasses, such as corn, as well as some members of the Cyperaceae, Amaranthaceae, Chenopodiaceae, Portulacaceae
and Euphorbiaceae exhibit C02 compensation points below 5 yl./l. C02
in the presence of 20.8% 02 (17, 66, 79, 80), and are capable of very rapid photosynthesis at warm temperatures and high light intensities
(11, 23, 24, 25, 35, 39, 41, 44, 67, 68). These plants possess the
C4-dicarboxylic acid pathway of photosynthesis (17, 36, 38, 51, 52) in
which C02 is initially combined with phosphoenolpyruvic acid by the enzyme phosphopyruvate carboxylase. In this thesis, these plants will be called C^ plants since the four-carbon acids oxaloacetate, malate
and aspartate are their initial products of photosynthetic C02 fixation (36, 54). The leaves of C4 plants contain a well-developed parenchyma bundle sheath surrounded by palisade mesophyll tissue.
The size, structure, and ability to accumulate starch differsnbetween chloroplasts of the bundle sheath and palisade mesophyll (5, 16, 17,
55, 56, 57) .
In contrast, wheat is representative of other plants which
exhibit C02 compensation points much above 5 yl./l. C02 in the 51 presence of 20.8% 02 (17), and have relatively low apparent rates of photosynthesis at warm temperatures and high light intensities (12,
23, 24, 40, 41, 42, 44, 67, 68). For convenience, plants like wheat
which carryco.ut photosynthetic C02 fixation by the Calvin cycle in which the three-carbon acid 3-phosphoglycerate is the initial product
(2) will hereafter be called C3 plants. In leaves of C3 plants, the parenchyma bundle sheath is usually poorly defined and the palisade mesophyll can be much more extensive. Chloroplasts in the palisade mesophyll and the parenchyma bundle sheath are not strikingly different in appearance (5).
The research presented here was carried out to survey the
differences in the C02 exchange characteristics of C3 and C^ plants, as exemplified by wheat and corn. Forrester e^t _al. have shown that
02 inhibits apparent photosynthesis in corn as well as in several C3 plants (28, 29). In the present study, further tests were made to
determine whether the inhibitory effects of 02 are similar in wheat
and corn. Measurements were made of C02 exchange by corn in different
02 concentrations, temperatures, C02 concentrations and light intensities. Additional measurements were made with wheat to supp• lement the information already given in Chapter I. Major differences
were observed in the pattern of C02 exchange by wheat and corn shoots.
The relationship of these differences to photorespiration and ecology offCg and C^ plants will 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 II in Chapter I. The chamber shown in Figure 1-2 was 52
used for all CC>2 exchange measurements reported in this Chapter. The
methods used to control and measure the light and temperature
environment of enclosed shoots were identical to those used in
Chapter I. Also, the closed system described in Chapter I was used
to obtain the CO2 compensation point and CO2 concentration results
presented in Figure;;11-4.
A simple open system was used for the temperature and light
intensity studies reported in Figures II-5, II-6 and II-7. Outside
air was drawn into this system and passed in series through the
reference cell of the IRGA, the plant chamber, the sample cell of
the IRGA, a Fisher "Dynapump" air pump, a Matheson R-2-15-B flowmeter,
and then out of the system. The IRGA was calibrated to giveuzero to
full-scale deflection between 250 yl./l. and 350 yl./l. CO2. As in
the closed system, the air 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 air flow rate and the CO2 concentration
differences between the reference and sample cells of the IRGA.
When measurements were made of the response of apparent
photosynthesis to temperature, the excised shoots were preconditioned
for an initial 30 minutes at 25° C and 32,300 lux. Then, the
temperature in 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
light intensity studies. When the light intensity was altered,
approximately 20 minutes elapsed before apparent photosynthesis
became stable, and measurements were made only after this time.
Throughout these temperature and light intensity studies, the CO2 concentration of the air entering the system was measured at about
15 minute intervals and was 335± 10 -ul./l. C©2«
A more complex open system, illustrated in Figure II-l, was used for the time course studies reported in Figures II-2 and II-3.
This system was developed to provide constant CO2 concentrations inya gas stream containing different O2 concentrations. Pure CO2 from a compressed gas tank was diluted with gas containing 1.8%, 20.8% or
100% 02, balance N2, by mixing these gases together using two
Gallenkamp gas mixing pumps and a Matheson gas proportioner in series
An IRGA measured the CO2 concentration which resulted from these dilutions. Once the desired composition of the mixed gas was established, the gas was passed in series through the reference cell of a second IRGA, the plant chamber, the sample cell of the second
IRGA, a Matheson R-2-15-B flowmeter, and out of the system. The second IRGA was calibrated to give a full scale deflection for a
100 yl./l. CO2 concentration difference between the reference and the sample cells. Once again, the apparent rate of photosynthesis was
calculated from the observed CO2 concentration difference between the reference and sample cells, and the gas flow rate, which was 1 l./min
In Figures II-2 to II-7, each point is the result of a single deter• mination of the apparent rate of photosynthesis. As in Chapter I, th apparent rates of photosynthesis are expressed in 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 II-l
Open System Used to Generate Constant,CO2 Concentrations in
an Air Stream Containing Different 02 Concentrations 54
ascarite CO2-absorbers
pressure release flasks
plant chamber lowmeter 55
RESULTS AND DISCUSSION
1. The CO2 Compensation Point
It has been reported that in air containing 21% O2, the C0£ compensation point of C^ plants is close to zero yl./l. CO2 (17, 29,
60, 64, 66). In Chapter I, it was observed that the CO2 compensation point of wheat was always greater than 5 yl./l. CO2 except at very low
O2 concentrations. Also, the CO2 compensation point of wheat was increased by increasing temperatures and O2 concentrations. Other C3 plants also exhibit high CO2 compensation points which respond to temperature and O2 concentration (17, 20, 28, 81, 89). To determine whether a high CO2 compensation point could be obtained with corn by modifying the temperature or O2 concentration, measurements were made with corn shoots at 32,300 lux, at temperatures from 13° to 40° C and at O2 concentrations from 1.8% to 100% 02- In all conditions, the CO2 compensation point of corn was found to be less than 5 yl./l. CO2,
which was indistinguishable from zero yl./l. C02 by techniques used.
Therefore, within the limits tested, the low CO2 compensation point of corn does not depend on the ambient temperature or O2 concentration.
This difference between the CO2 compensation points of C3 and
C4 plants is important since it is indicative of a difference in the rates of CO2 production by two types of plants during illumination.
The high CO2 compensation points of C3 plants are evidence that substantial amounts of CO2 are released by these plants by photores• piration, and that the relative rates of CO2 production and release are altered by the temperature and the O2 concentration. On the other hand, the low C0~ compensation points of corn and other C4 56
plants indicate a low rate of CO2 release during photosynthesis by
these plants. In fact, it has proven difficult to demonstrate that C4
plants release any CO2 at all during illumination. When the leaves of
C4 plants are exposed to light and placed in a stream of CC^-free air,
no detectable release of CO2 into the air stream is observed (25).
Irvine (47) recently carried out an experiment in which a ^C-labelled
and an unlabelled corn plant were enclosed together in a sealed chamber
and illuminated. After 1 hour, he was able to detect very small but 14
measurable quantities of C accumulating in the initially unlabelled
plant. He concluded that corn does release small amounts of CO2
during photosynthesis. The sensitivity of this CC>2 release process to
different concentrations of O2 was not tested, so it is not known whether the CO2 originated from photorespiration or some other source.
Meidner (60, 61) was able to obtain an elevated CO2 compensation
point with corn by placing illuminated leaves under water stress.
This CO2 compensation point, however was not responsive to changes in
O2 concentration above 2% O2, and therefore was not due to an increased
rate of photorespiration. Instead, it was attributed to a reduction
in the rate of photosynthetic CO2 fixation by the high water deficits.
Corn also exhibits a high CO2 compensation point when the light
intensity is very low (9, 18). Once again, it does not appear that
photorespiration is implicated in this result, which seems to be due
to a simple balance between the opposing processes of CO2 fixation
and dark respiration.
The CO2 compensation point studies therefore demonstrate a
major difference between C3 and C4 plants. During photosynthesis, C3
plants can release substantial quantities of CO2 by an 02-sensitive 57
photorespiratory process, while plants apparently lack photores•
piration.
2. Effecf-of ©2 Concentration on the Apparent Rate of Photosynthesis
Apparent photosynthesis may be inhibited by the presence of
O2 in both wheat and corn, but the characteristics of the inhibition
appear to differ between the two species. This difference is illus•
trated by Figures II-2 and II-3 which show the time course of apparent
photosynthesis of wheat and corn shoots which were exposed to
atmospheres containing 300 yl./l. C02; 1.8%, 20.8% or >"99% 02; balance N2. These measurements were carried out at 25° C and 32,300
lux. Figure II-2 shows that, as in Chapter I, apparent photosynthesis
in wheat was inhibited by O2 concentrations above.1.8% O2. At any
particular O2 concentration, however, the apparent rate of photosyn•
thesis of wheat was constant following the initial 30 minute period
of photosynthetic induction. Also, if the O2 concentration was changed
from 1.8% or >99% O2 to 20.8% O2 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 20.8% O2 all the time. Thus, the
effect of O2 on apparent photosynthesis of wheat is constant in time
and is reversible. As discussed in Chapter I, at least part of the
effect of O2 on apparent photosynthesis of wheat appears to be due to
the response of photorespiration to O2•
Figure II-3 shows that the apparent rate of photosynthesis of
corn was the same in 20.8% O2 as in 1.8% 02^ When the O2 concentration was 100%, however, CO2 assimilation by corn was greatly inhibited.
Also, the degree of inhibition was not constant but increased with the
time the corn shoots were exposed to >99% 02. When the corn shoots Figure II-2
Time Course of CO2 Exchange by Excised Wheat Shoo in Atmospheres Containing 300 ul./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 in Atmospheres Containing 300 yl./l. CO2 and Different O2 Concentrations
The Shoots were Excised at Time = 0 APPARENT RATE OF PHOTOSYNTHESIS (mg G02./hr/g fr wt) O —< row -N- oi o >j co vo p b b b b b b b b b o b 60
were returned to 20.8% 02 after one or two hours in >99% 02, the
reversal of the inhibition was much more gradual than with wheat.
Forrester et_ al. (29) have previously shown that high 02 concentrations
inhibit apparent photosynthesis in corn. The present results reveal
that the inhibitory effect of >99% 02 is time-dependent and only slowly
reversible. These results confirm the findings of several other
studies which have also indicated that photosynthesis of plants is
not enhanced when the 02 concentration is reduced from 20.8% 02 to 2%
02 or less (15, 29, 40).
Additional measurements were made to determine whether 20.8%
02 inhibits apparent photosynthesis of corn under other conditions of
temperature, C02 concentration or light intensity. Figure II-4 shows
that the apparent rates of photosynthesis in 1.8% 02 and 20.8% 02
were similar in all conditions tested. Therefore, 20.8% 02 does not
appear to be sufficient to affect apparent photosynthesis in corn.
This absence of an inhibitory effect with corn does not resemble that
found with wheat in Chapter I when the inhibition was absent only at 1
low temperaturessand high C02 concentrations.
Since the C02 compensation point of corn was always less than
5 yl./l. C02 in these studies, photorespiration does not appear to be
implicated in the response of apparent photosynthesis of corn to high
02 concentrations. It is possible that >99% 02 inhibited photosyn•
thesis in corn by causing stomatal closure. There is no explanation,
however, why stomata in wheat would not be similarly affected. It is
perhaps more likely that the results in Figure II-3 should be inter•
preted in terms of an inhibitory effect of >99% 02 on fixation itself.
Because photosynthesis involves many steps, there are numerous sites Figure II-4
Effects of O2 Concentration, Temperature, CO2 Concentration and Light Intensity on the Apparent Rate of Photosynthesis of Excised Corn Shoots 61
A A^O-O
O 1.8%02 12 40 C 32,300 lux * 20.8% 02
11
SZ CN1 0 O u
A A A A ®^ o o o o o o o o~ 25°C 32,300 lux CO LCOU X
>- CO o t- O 25UC 6,460 lux X D_ ^-9-6-4 •A—-A—/-v— o o 5 6 '9"
9 6 < cn 13 C 32,300 lux
LU cn D_ <
100 200 300 400 500 CO. CONCENTRATION Cul/I) which could be susceptible to inhibition by 02 (13, 83). In
plants, it is possible that high 02 concentrations may inhibit one or
several steps in the C^-dicarboxylic acid pathway, although little evidence is now available to specify the particular site(s) of action.
One enzyme of the C^-dicarboxylic acid pathway, pyruvate, phosphate
dikinase, deserves some investigation, however, since it is reversibly
inactivated in the presence of 02 in vitro (1).
3. Effect of Temperature on Apparent Photosynthesis
There are marked differences in the responses of C3 and plants to temperature. In air, C3 grasses generally have optimum
temperatures for apparent photosynthesis and for growth which are
distinctly lower than those of C4 grasses (11, 12, 21, 25, 26, 30, 31,
44, 62, 63, 67, 68). For example, Figure II-5 shows that, at 20.8% 02
and 335 ul./l. CO2, the optimum temperature for apparent photosyn•
thesis in wheat was about 25° C, while for corn it was almost 40° C.
At the optima, and at all 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 similar to thos of De Jager (12) who used the
C3 plants Lolium perenne and Lolium multiflorum, and the plant
Paspalum dilatatum.
The temperature curve for wheat in Figure II-5 resembles the
results of Experiment I in Chapter I but differs from the results of
Experiment II. The data from Experiment II were collected over a
period of 3 months while Experiment I was performed during a 3 week
period and the measurements in Figure II-5 were obtained on one day.
Some drift in the apparent rate of photosynthesis or in the temperature Figure II-5
Effects of Temperature on the Apparent Rates, of Photosynthesis of Excised Wheat and Corn Shoots in Air Containing
20.8% 02 and 335 ul./l. C02 response may account for the different results found in Experiment II.
Since they were obtained over a short period of time, the results in
Figure II-5 probably represent the best estimate of the within plant variation in apparent photosynthesis in response to changes in temperature.
Photorespiration may contribute to this difference in
temperature response between C3 and plants. In Chapter I it was observed that the optimum temperature for apparent photosynthesis in
wheat decreased as the 02 concentration increased. This decrease in
temperature optimum was noticeable in only one case in 20.8% 02, however, and was not rioted in recent studies with Marchantia poly• morph a (6). Therefore, conclusive evidence is not available to
indicate whether or not photorespiration is a major cause of the differences in temperature response between C3 and C4 plants in 20.8%
02.
Treharne and Cooper (82) have suggested that these differences may be the consequence of differences in the temperature responses of the major carboxylating enzymes of C3 and C^ plants. They observed
that the optimum temperature for the ribulose-1, 5-diphosphate
carboxylase of the C3 plants Lolium perenne and Avena sativa was 20° C
to 25° C. In contrast, the activity of the phosphoenolpyruvate
carboxylase of the C4 plants Zea mays and Cenchrus ciliarus was
greatest between 30° and 35° C. In a different study, the combined
activities 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
differential effect of temperature on C02 exchange by C3 and C4 plants. 65 4. Effect of CO2 Concentration on Apparent Photosynthesis
The effect of CO2 concentration on apparent photosynthesis of wheat was presented in Chapter I, Figures 1-9 and 1-13 to 1-18.
Corresponding results with corn are shown in Figure 1-4. The effects
of CC>2 concentration are generally similar except that the CO2
compensation point of wheat is higher than that of corn at O2
concentrations above 1.8% O2. As a result, corn can carry out net
CC>2 assimilation at much lower CO2 concentrations than can wheat.
The CC>2 concentration response of apparent photosynthesis of wheat and
corn appears to resemble the results obtained in studies of the effect
of substrate concentration on reaction rate in isolated enzyme systems. This similarity has led some investigators to apply methods
analogous to those used in the study of enzyme kinetics to the analysis of the CO2 concentration response of photosynthesis. In this
approach, the rate of CC>2 assimilation at CC>2 saturation is termed the
apparent Vmax of photosynthesis, and the CC>2 concentration at which
Table II-1
Comparison of the Apparent Kinetic Constants of Photosynthetic CO2 Assimilation of Excised Wheat and Corn Shoots
Plant Temperature Light "02' Cone. Apparent Apparent (°C) Intensity (%) Vmax Km
(lux) (mg. C02/hr/g fr wt) (yl 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 66 the rate of CC^ assimilation is one-half the apparent Vmax is called the apparent (32) . Table II-l and the results of Goldsworthy (32)
demonstrate that at low 02 concentrations the apparent Km for C02 assimilation by C3 plants is similar to that of C4 plants. In 20.8%
02, the apparent of wheat was much higher than it was at 1.8% 02,
while the apparent Km of corn remained low. The similar apparent 1^
values for C3 and plants at low 02 concentrations indicates that
under these conditions the affinity for C02 of the overall C02 assimilation systems of these two plant types is similar. In 20.8%
02, the apparent affinity of C3 plants for CO2 is much lower than in
1.8% 02 because of photorespiration and other components of the 02
effectr:. As before, 20.8% 02 did not reduce the apparent Vmax of wheat or corn.
Goldsworthy (32) has argued that the similar Km values at low
02 concentrations is evidence that the internal C02 fixation processes
in C3 and plants are similar in their affinity for C02. This
conclusion could be correct if the resistances to C02 diffusion into leaves of C3 and plants were equal. Unfortunately, several other
studies have indicated that there is a greater resistance to C02 diffusion into leaves of plants than into leaves of C3 plants (14,
44, 70). Because of this difference in C02 diffusion resistance, the apparent kinetic constants cannot in themselves be used to describe
the characteristics of the internal C02 fixation systems of C3 and
C4 plants.
Studies with isolated enzymes have indicated that phosphoeno•
lpyruvate carboxylase has a higher affinity for C02 than ribulose-1,
5-diphosphate carboxylase (50, 86). These in vitro results are diffi-v. cult to relate to the observations with leaves, however, since the
latter are the consequence of the activities of many other enzymes as well 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 light intensities. These measurements
were carried out at 25° C, 335 ul./l. C02 and 20.8% 02. In wheat,
apparent photosynthesis was close to light saturation at light intensities above about 32,000 lux. This result is in agreement with
other studies which have found that light saturation of apparent photosynthesis of.C^iplants often occurs in 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 light intensity was similar to that of wheat at
intensities below 36,000 lux. When the light intensity was increased
above that value, however, an unusual response was observed. Figure
II-7 shows that after a change in light intensity from 32,000 lux to
100,000 lux there was a temporary increase in the apparent rate of photosynthesis by corn followed by a gradual decline in the rate.
This decline continued after the rate became less than that observed
at the preceding lower light intensity. The temporary increase
in the apparent rate of photosynthesis after the increase in light
intensity may have been due to the accompanying increase in tempe•
rature from 25° C to 31.5° C. The inhibitory effect of high intensity
light in corn was not reversible since the apparent rate of photosyn•
thesis remained below the initial rate when the light intensity was
restored to 32,300 lux. The occurrence of this high light intensity Figure I1-6
Effects of Light Intensity on the Apparent Rates of Photosynthesis of Excised Wheat and Corn Shoots in Air Containing
20.8% 02 and 335 ul./l. C02 68 Figure II- 7
Effects of Exposure to High Light Intensity on the Apparent Rate of Photosynthesis of Excised Corn Shoots APPARENT RATE OF PHOTOSYNTHESIS (mg C02/hr/g fr wt) response of corn was verified with five different samples of corn
shoots.
The behavior seems to be a peculiarity of the plants used,
since the author has not encountered other descriptions of high light
intensity inhibition of corn photosynthesis. It is possible that the photosynthetic apparatus of these corn shoots was susceptible to
damage by high light intensity because the plants were grown at a
relatively low light intensity, 21,600 lux. In the plant sugar•
cane, however, leaves which develop at low light intensities are
still capable of photosynthesis at full efficiency in full sunlight
(85). Since the optimum temperature for photosynthesis of these corn
shoots was about 40° C, it seems unlikely that the inhibition of
photosynthesis was due to a high temperature lesion. Therefore,
there is no convenient explanation for this high light intensity
effect on the basis of the available evidence. All other experiments with corn shoots were performed at 32,300 lux or lower, where there was no noticeable inhibition of apparent photosynthesis by light.
The high light intensity results with corn were unexpected
since most studies with corn and other plants have shown that the
apparent rate of photosynthesis by individual leaves continues to
increase, although not linearly, up to light intensities of 60,000 lux
or more (10, 12, 21, 22, 35, 39, 41, 42, 67, 74, 84, 85). The dotted
line on Figure II-6 illustrates this typical response. Thus, most
investigations have indicated that plants are much more efficient
than Cg plants in utilizing high intensity light for photosynthesis.
This difference in efficiency 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
C4~dicarboxylie acid pathway (10, 37). If this pathway operates in series with the reductive pentose phosphate pathway in C4 plants (36,
52) , then these plants will have a higher ATP requirement:-'for photosynthesis than do C3 plants which possess only the second path• way. Thus, plants may require a high rate of photosynthetic phosphorylation during CO2 fixation. In accord with this, Chen et al.
(10) have recently demonstrated that chloroplasts of the C4 plant
Cynodon dactylon have a higher affinity for inorganic phosphate and
ADP than chloroplasts of the C3 plants which have been tested.
The leaf anatomy of C4 plants may also contribute to their efficiency at utilizing high intensity light. The high chlorophyll content of the bundle sheath of C4 plants may require higher light intensities for saturation of photosynthesis than are necessary with leaves of C3 plants in which the chlorophyll is more evenly distributed (5).
6. General Discussion
It is clear from the preceding sections that there are pronounced differences in the CO2 exchange characteristics of wheat and corn. The COp compensation point of corn was ordinarily indis• tinguishable from zero yl./l. CO2 and was not dependent on the O2
concentration and temperature as was the CO2 compensation point of wheat. Apparent photosynthesis of corn was not affected by 20.8% O2, but >99% O2 inhibited CO2 assimilation, and the degree of inhibition increased with time and was only slowly reversible. The inhibition of apparent photosynthesis of wheat by 20.8% O2 or by >99% O2 was
constant in time and was rapidly reversible. In 20.8% O2, corn was v 72 more efficient at carrying out CO2 assimilation at low CC>2 concentra• tions and high temperatures than was wheat, and there is evidence from other studies that corn is more efficient at utilizing high intensity light 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% O2, where photorespira• tion is virtually suppressed, wheat resembled.com in that it possessed a low CO2 compensation point, a high apparent rate of photosynthesis, and possibly an elevated optimum temperature for apparent photo• synthesis (Chapter I). It would be an oversimplification, however, to conclude that differences in photorespiration are the source of all the differences in CO2 exchange between wheat and corn. It is evident from the preceding sections that many of the differences in
^2' exchange can be related to differences in photosynthetic carbon metabolism or in leaf anatomy. For example, in Section II-3, it was suggested that the differential effect of temperature on apparent photosynthesis by wheat and corn may arise from differences in the
temperature response of the major enzymes responsible for C02 fixation in the two species.
Although corn and other C^ plants do not release appreciable quantities of CO2 during photosynthesis, they do seem to contain some components which may be involved in photorespiration.by C3 plants. It has been suggested'that photorespiratory CO2 production by C3 plants may arise from the oxidation of glycolic acid (18, 19, 61, 65, 87, 88,
89, 90). Glycolic acid oxidase occurs in corn (69, 75, 76), sugar cane and Amararithus edulis, although the activity is low in these species (78). Glycolic acid has been shown to accumulate when corn is treated with an inhibitor of glycolic acid oxidase (87). In addition,
C02 release is stimulated when glycolic acid is fed to corn leaves
(18). Peroxisomes, which may be associated with photorespiratory metabolism (53, 77, 78), have been detected in species (78).
Finally, mass spectrometric measurements have indicated that large
quantities of 02 are assimilated by illuminated corn plants (48).
This lastresult, however, may not be due-; to photorespiration, but may have another cause, such as the activity of phenol oxidase which is abundant in chloroplasts of the C^ plant sugar cane (33).
It is possible that C02 may be produced in significant quantities during photosynthesis in corn, but it may be prevented from
leaving the plant because of an efficient internal C02 recycling mechanism (24). Phosphoenolyruvate carboxylase has a high affinity fr
for C02 (86) and it is located in the mesophyll surrounding the paren•
chyma bundle sheath (4, 72). If CC>2 is produced by the photores• piration of compounds in the parenchyma bundle sheath of C^ plants, it may be refixed into C^ dicarboxylic acids in the mesophyll before it .
can escape from the leaf. Therefore,, although the C02 compensation point of C^ plants is ordinarily indistinguishable from zero, it does not necessarily follow that C^ plants lack the internal capability of
photorespiratory C02 production during photosynthesis. If photores• piration does occur in corn, however, it does not cause a carbon deficit.
The general geographical distribution of and plants is
correlated with their C02 exchange properties, photosynthetic 74
metabolism and leaf anatomy. C3 grasses, such as members of the
Festuceae, Aveneae and Agrosteae, are abundant in temperate regions.
The C4 Andropogoneae, Eragrosteae and Paniceae, however, are widespread
in the tropics (34). In the. dicotyledonous C4 groups, the Amaran-
thaceae are thought to be of tropical origin (58). Also, species of
Atriplex which possess the characteristic C^-type of leaf anatomy are
native to the hot and arid regions of Australia and the United States
(55). plants, therefore, thrive in locations which have warm
temperatures, high light intensities and often seasonal dry periods
(55).
The physiological characteristics of plants, particularly
their ability to utilize high intensity light and high temperatures
for rapid photosynthesis, would seem to be well suited for a warm well-illuminated environment. Their ability to assimilate CO2 at low
CO2 concentrations may also be advantageous in closely packed stands
on calm days when the CO2 concentration within the canopy may
decrease appreciably. There is also evidence that C^ plants are
efficient in their use of water. Results of Schantz and Piemeisel
(71) indicate that C4 plants use about one-half as much water during
the production of 1 g. of dry matter as do plants. Since C4 plants
transpire at lower rates than C3 plants (14, 44), this difference in
water requirement does not simply reflect differences in C02
assimilation rates between the two types of plants.
In temperate conditions, C4 characteristics may not be
favoured. In Figure II-5 it was seen that, at temperatures below 18°
C, the apparent rate of photosynthesis of wheat was higher than that
of corn. Also, chlorophyll accumulation is retarded in corn leaves if 75 the day temperatures are below 15° C (59). No information is available on whether this effect of low temperature occurs in other C4 species.
These correlations between some physiological characteristics and the geographical distribution of C3 and plants are not absolute.
The Bambuseae, for example, have C3 characteristics (17), but are abundant and grow rapidly in 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 in a locality. A plant which exhibits low apparent rates of photosynthesis per unit leaf area may still dominate if it produces a large leaf area.
Many plants are common weeds (7). In fact, of the ten species listed 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 C4 species would be of great value for weed control. Black ejt _al. (7) have suggested that the high competitive ability of many C^ species may be based on the CO2 exchange characteristics outlined in the preceding sections.
There appear to be additional differences between C3 and C4 plants which may be related to some characteristics already discussed.
Compared with C3 species, C^ plants are composed of carbon which is enriched in (3, 73) . This difference could arise from the difference in the photosynthetic carboxylation pathway or the difference in apparent photorespiratory activity between the two types of plants. Carbonic anhydrase activity is low in C^ plants (27)uarid this may be associated with the C^-dicarboxylic acid pathway. The acidic compounds produced by photosynthetic CO2 fixation in C^ plants may be responsible for the slightly greater acidity of sap expressed from plants compared with sap obtained from C3 plants (46). Also,
translocation of recent photosynthate may be more rapid in C4 plants
than in Cg plants (43). Whether or not these differences as well as
the others noted in this Chapter exist between all C3 and C4 plants
remains to be proven. Future investigations may discover additional
differences between these two plant groups and should prove helpful in
relating their physiological properties to their distribution and behavior in nature. 77
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Chapter III
PHOTORESPIRATION AND THE POST-ILLUMINATION C02 BURST IN
WHEAT AND AMARANTHUS EDULIS
INTRODUCTION
During the first minute of darkness following a period of
illumination, plants often exhibit high rates of C02 production into
air containing 21% 02. This post-illumination C02 burst is eliminated
when the 02 concentration is reduced to 2% and it is enhanced when the
02 concentration is raised to 100% (2, 3, 7, 8, 10, 12, 24). The size of:the burst also increases with increasing light intensity in the period preceding darkness (3, 6, 7, 8, 14, 22, 23, 24). Mainly
because of the sensitivity of the burst to 02 concentration, it has been suggested that the burst is a brief extension of photorespiration into the dark period (5, 6, 7, 8, 12, 19, 22, 23, 24). In accord with this interpretation is the observation that, with the exception of
Amaranthus edulis, those plants which photorespire have post-illumina•
tion C02 bursts, and those plants which apparently lack photorespira• tion also lack bursts (13, 23).
Amaranthus edulis, on the other hand, exhibits a distinct
post-illumination C02 burst even though it does not release C02 in the light (2, 9). Also, Heath and Orchard (14) have demonstrated that the
post-illumination C02 burst and the CO2 compensation point of wheat leaves respond differently to light intensity and temperature. They concluded from this that the burst was not associated with photores• piration. 85 The interpretation of post-illumination CO 2 exchange transients of plants is complex since changes in CO2 diffusion resistance, and in the rates of photosynthesis, photorespiration, and dark respiration can be involved. Stomata close in the dark (25, 26), and transpiration is reduced (10, 25), indicating that the gas diffusion resistance of leaves is increased during the post- illumination period. These changes, however, are relatively slow,
(25, 26), compared with the kinetics of the burst. Also, since the burst occurs in liverworts (7, 8), it cannot be the result of stomatal response. Photosynthetic CO2 fixation may continue briefly after illumination, for example falling to zero within 30 seconds after darkening Scenedesmus (1). Dark respiration may be inhibited in the light and increase after the onset of darkness (4, 15, 16, 17, 18, 20,
21).
In an effort to clarify the relationship between photores• piration and the post-illumination CO2 burst, the kinetics of the burst and the effects of different O2 and CO2 concentrations on the two processes were examined. The effect of O2 concentrations on the burst of Amaranthus edulis was also investigated.
MATERIALS AND METHODS
Wheat plants were grown in the same way as described for
Experiment I in Chapter I. Amaranthus edulis Speg. plants were grown in similar conditions except that soil, not vermiculite,was used. At the start of each experiment, 2.0 g. fresh weight of 9 to 11 day-old wheat shoots, or single, fully expanded Amaranthus edulis leaves from
25 to 28 day-old plants, were excised in the usual manner and enclosed 86 in the transparent chamber shown in Figure 1-2.
After enclosure, the plant material was conditioned for an initial 30 min. period at 32,300 lux (3000 ft.-c.) and 25± 1° C in air. Thereafter it was exposed to a series of light - 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 in less than 0.5 sec. by covering the chamber with an opaque black cloth and turning off the light. During illumination, the air temperature in the chamber was 25± 1° C, and in the dark it fell to
24± 1° C. An air flow rate of 1.0 l./min. was maintained while plant material was in the chamber.
The gas flow system used for these experiments was the same as that shown in Figure II-l. Measurement of the temperature within the plant chamber, the CO2 concentration in the air stream, and control of
the 02 and the CO2 concentration of the air entering the plant chamber were all carried out as described in ChaptersBl and II.
RESULTS AND DISCUSSION
1. Kinetics of the Post-illumination C02 Burst
The solid line in Figure III-l illustrates the data obtained-.' when illuminated wheat shoots were darkened. The air entering the plant chamber contained 100 yl./l. CO2, 20.8% O2, balance N2. During illumination, CO2 assimilation reduced the CO2 concentration of the air leaving the chamber to 74 yl./l. When the shoots were darkened, there was a 3 sec. delay before the CO2 concentration readings increased sharply. This lag can be attributed to the time required for CO2 concentration changes in the chamber to reach the CO2 analyzer. Figure III-l
Effects of Darkening on CO2 Exchange
Excised Wheat Shoots in 20.8% 02 87
40 60 TIME (sec) 88
Within 12 sec. of darkening the shoots, the CO2 analyzer.readings exceeded 100 yl./l. CO2, and a peak of 121.4 ul./l. CO2 was reached after 27 sec. After the peak, the readings declined and reached
111 yl./l. CO2 at 120 sec. Wheat, therefore, exhibites a post- illumination CO2 burst under these conditions.
The burst as it occurred within the plant chamber, however, must have differed in kinetics and magnitude from these observations.
Part of this difference may be ascribed to characteristics of the CO2 analyzer which operates by detecting the absorption of infra-red light across a 100 ml. sample cylinder. During the burst, the rapid variations of the CO2 concentration of the air stream generate CO2 concentration gradients in the sample cylinder and distort the results.
This effect could be eliminated by calculating the CO2 concentration
which must have^been entering the C02 analyzer to produce the observed results. The method of this calculation is described in Appendix I.
The dashed line in Figure III-l shows that when the results were corrected this way, the initial CO2 concentration increase was more abrupt, and the peak of the burst was about 4 sec. earlier and slightly higher than the measured result. The difference between the observed and corrected results, however, was not large, and all subsequent results in this chapter were left uncorrected.
An additional source of error arose from the separation of the
site of the burst from the site of its measurement. Before a C02 exchange event which had occurred within the plant chamber was detected by the CO^ analyzer, it was modified according to the air 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 in time, and the peak of the burst was delayed and reduced in size. This was confirmed by comparing the results, shown in Figure III-2, of pulse (less than 0.5 sec.) injections of 0.1 ml.
CO2 in air into the air stream either at the entrance to the CO2 analyzer or into the middle of the empty plant chamber. The complexity of the air flow characteristics of the experimental system prevented the exact assessment of their effects on the kinetics of the burst.
It is clear, however, that the true peak of the burst occurred earlier than 20 sec. after the initial CO2 analyzer response, and that the peak was higher and more abrupt than that recorded by the CO2 analyzer.
Bulley (3) recently used a highly simplified open system to measure the post-illumination CO2 burst of soybean leaves. He estimated that the true peak of the burst occurred between 7 and 12 F- sec. after the initial CO2 analyzer response, and calculated that it was about 150% as high as the recorded peak. In view of the above considerations, similar kinetics would not seem to be unreasonable for the post-illumination CO2 burst of wheat. The peak of the burst, therefore, occurs very soon after illumination is stopped, and it is highly susceptible to modification by measuring techniques.
Other experiments by Bulley (3) have indicated that the burst is not the result of processes commencing at the onset of darkness.
When the light intensity was reduced, but not extinguished, a dip in the rate of CO2 assimilation occurred, and this dip corresponded kinetically to the post-illumination CO2 burst. It was concluded that the burst was the resultant of two CO2 exchange processes which differed in rate of response to changes in light intensity. Figure III-2
Response of the IRGA to Pulse Injections of CO2 into the Air Stream- in the Plant Chamber and at the Entrance to the IRGA Sample Cylinder lOOr INJECTION AT IRGA ENTRANCE 90
80
70
60 LU i \ CO \ O i
LU i or
40 I- <
* 30 NJECTION \ INTO PLANT CHAMBER
20
10
0l 0 4 8 12 16 20 TIME AFTER INJECTION (sec) 91
A second broad peak of CO2 production usually occurring between 2 and 10 minutes after darkening was observed in this study and has frequently been noted before (7, 12, 13, 22, 23, 24). This peak was evident in both 20.8% O2 and 1.8% O2 and is therefore not related to photorespiration. It may be the result of a post- illumination overshoot of dark respiration. Comparable peaks of post-illumination CO2 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 O2 and CO2 Concentrations on the Post-illumination
C02 Burst
Figures III-3 to III-7 show the recorded post-illumination CO2 exchange transients of wheat shoots in atmospheres containing 20.8% or 1.8% O2 and CO2 concentrations between 100 ul./l. and 300 yl./l.
Each point on these curves is the average of 5 determinations. There was no burst in 1.8% O2 at any of the CO2 concentrations used. In
20.8% O2, there was a pronounced burst in 100 ul./l. CO2, and the peak height of the burst decreased as the CO2 concentration increased. Two minutes after the shoots were darkened, there was no significant
difference in the rates of C02 production between the 20.8% 02 and
1.8% O2 treatments.
It is apparent from the preceding discussion of burst kinetics that the observed peak height is affected by the experimental system, so comparisons based on this characteristic of the burst are hazardous.
Since the burst in wheat is dependent on O2, an appropriate index of the size of burst is the overall difference in post-illumination CO2 exchange between the 20.8% O2 and 1.8% O2 treatments. Using tthds index, Figure III-8 summarizes these results on the effect of CO2 Figure III-3
Post-illumination CO2 Exchange by Excised Wheat Shoots in
1.8% 02 and 20.8% 02. The C02 Concentration of the Air
Entering the Plant Chamber was 100 ul./l. C02 92
>15 min LIGHT DARK
TIME (sec) Figure III-4
Post-illumination CO2 Exchange by Excised Wheat Shoots in
1.8% 02 and 20.8% 02. The CO2 Concentration of the Air
Entering the Plant Chamber was 200 yl./l. C02 93
15 min LIGHT DARK TTTl 11111111111111111111 III111\
-G •5
o 1.8 %02
G 20.8 %02
40 60 100 120 TIME (sec) Figure III-5
Post-illumination CO2 Exchange by Excised Wheat Shoots in
1.8% 02 and 20.8% 02. The C02 Concentration of the Air
Entering the Plant Chamber was 300 ul./l. C02 94
>15,min LIGHT DARK 111111111111111111111111111111111111111111////A 320
-o-
°1.8%02
o 20.8 % 02
20 40 60 80 100 120 TIME (sec) Figure III-6
Post-illumination CO2 Exchange by Excised Wheat Shoots
1.8% 02. The C02 Concentration of the Air Entering th
Plant Chamber was 400 yl./l. C02 95
>15.min LIGHT DARK mniiniLLLaLUMmzn 420 -o -O- -o- -o -o-
-o1.8 %02
-G 20.8 % 02
JL_ ' • 40 60 80 100 120 TIME (sec) Figure III-7
Post-illumination CO2 Exchange by Excised Wheat Shoots in
1.8% 02 and 20.8% 02. The C02 Concentration of the Air
Entering the Plant Chamber was 500 yl./l. C02 ' 96
>15.min LIGHT DARK
520
o- >1.8%02
o- =20.8 % 02 480
O A jZ 440 < or o UJ O o u CM 400 o o (J • ©—J
U-6—-!
360
8 J 0 20 40 " ~Q~ ~^0-~- 1|)Q ' ^ TIME (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 size of the post-illumination CO2 burst. The burst was greatest at 100 yl./l. CO2 and 200 yl./l. CO2 and it decreased at higher CO2 concentrations. As will be shown in Chapter IV, the size of the burst was sufficiently large that it is improbable that the burst originated from a CO2 free-exchange pool.
In Chapter I it was concluded that the results of studies on
the inhibitory effect of 02 on apparent photosynthesis could not be
used to indicate the rate of photorespiration at high C02 concentra• tions. If the post-illumination CO2 burst is a manifestation of photorespiration, however, then the CO2 concentration response of photorespiration must resemble that of the burst size shown in Figure
III-8. The effect of C02 concentration on the burst can be correlated with its effect on the inhibition of apparent photosynthesis by 20.8%
O2. Figure III-8 shows that the burst size and the magnitude of the depression of apparent photosynthesis by 20.8% O2 respond similarly
to C02 concentration.
Fock et al. (11) have recently studied the effect of CO2
concentration on the post-illumination CO2 burst of the liverwort
Conocephalum conicum and have also noted this similarity. In their study, 50% O2 and 75% O2 were used and the size of the bursttwas
greatest at about 300 yl./l. CO2 and declined at lower and higher C02
concentrations. If Figure 1-13 is reexamined, it can be seen that the
depression of apparent photosynthesis by 60% 02 or 80% 02 is greatest
between 225 yl./l. C02 and 300 yl./l. C02 at 25° C. Thus, the results with Conocephalum appear to be in general agreement with the present
observations.
The correlation between the effect of 02 on apparent 99
photosynthesis and on the burst at different C02 concentrations can be illustrated in another/way. In Chapter I, it was seen that the per
cent inhibition of apparent photosynthesis by 02 was:
%IP " Pl-P2 —jTj-— X 100
As before, P^ and P2 are the apparent rates of photosynthesis at a low
and a higher 02 concentration respectively. An analogous expression
can be developed for the post-illumination C02 burst. Let Pb be the
average rate of C02 production during the burst, in mg. COg/hr./g. fr. wt., as calculated from the burst size and duration. Once again, the
burst size is the overall difference in post-illumination C02 exchange
between the 20.8% and 1.8% 02 treatments. The duration of the burst was taken .to be the length of time after darkening the shoots that the
C02 concentration readings in 20.8% 02 and 1.8% 02 were different at the 5% level of significance.
Then, if the effects of 20.8% 02 on apparent photosynthesis
and on the burst are due to the same cause or causes, (P2 -k P^) may be substituted into the above formula in place of P^. That is:
% Ib = (P2 + Pb) - P2 Pb
—± 2 £ x 100 X 100 p p p P2 + b 2 + b
and the response of this function to C02 concentration should resemble that of % Ip. The results presented in Figure III-9 show the
similarity of the C02 concentration responses of % Ip and % 1^. This
is further evidence linking the effects of 02 on apparent photosynthesis
with its effects on the post-illumination C02 burst.
In addition to the data already presented on kinetics and the Figure II1-9
Effects of CC"2 Concentration on the Per Cent Inhibition Apparent Photosynthesis by 20.8% 0£ and on the Analogous Burst Function % B
101
effects of 02 and CO2 concentrations on the burst, other results are
consistent with the hypothesis that the burst is related to photo•
respiration. The size of the burst and the depression of apparent
photosynthesis by 20.8% 02 are affected similarly by light intensity
(3). Also, the effect of wavelength of light on the apparent rates
of photosynthesis in 21% 02 or 2% O2 is the same as its effect on the size of the burst (3). These effects of preceding light conditions ?,
are further evidence that the burst is linked to processes occurring
during the light.
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 CO2 compensation point, the
rates of CO2 fixation and production are unaffected by light
intensity. Recent studies (3), however, indicate that at the C02
compensation point, increases in light intensity result in increases
in CO2 fixation. Therefore, their assumption was not valid, and their
reasoning cannot be. used to contradict the view that the post-
illumination CO2 burst in wheat and similar plants is an extension of
photorespiration into the dark period.
3. Effect of O2 Concentration on the Post-illumination CO2 Burst of
Amaranthus edulis
Figure 111-10 shows the results of consecutive determinations
of post-illumination C02 exchange by an Amaranthus edulis leaf in 20.8%
02 or 1.8% O2. These data are representative of the results of 16
determinations at 300 yl./l. CO2 and 32,300 lux. Although the rates
of CO2 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
in 1.8% 02 and 20.8% O2 102
>15min LIGHT DARK
320
-Q
-° 1.8 %02
-o 20.8 % 02
0 0 20 40 60 80 100 120 TIME (sec) 103 nevertheless apparent. Unlike wheat, Amaranthus edulis exhibited a
substantial peak in post-illumination CO2 production in 1.8% 02 as
well as in 20.8% 02. The height of the peak in 1.8% 02 was similar to
the height of the peak in 20.8% 02, although there were often
differences in the kinetics of C02 exchange subsequent to the peak.
The results of Bjbrkman (2) appear to be similar.
Since the peak is not sensitive to 02, the burst of Amaranthus edulis cannot be related to conventional photorespiration or to the burst in wheat. It is possible that the burst of Amaranthus edulis is
the result of an overshoot of dark respiration which is recovering
from inhibition during the light (2), or to some other cause
associated with C4 metabolism. There is insufficient evidence to
specify the cause of the differences in C02 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 CO2 uptake in higher plants. Carnegie Inst, of Wash. Year Book. 66: 220-228.
3. Bulley, N. R. 1969. Effects of light quality and intensity on photosynthesis and photorespiration in attached leaves. Ph.D. Thesis. Simonj Fraser University.
4. Calvin, M. and P. Massini. 1952. The path of carbon in photosynthesis. XX. The steady state. Experientia 8: 445-457.
5. Decker, J. P. 1955. A rapid post-illumination deceleration of respiration in green leaves. Plant Physiol. 30: 82-84.
6. Decker, J. P. 1959. Comparative responses of carbon dioxide outburst and uptake in tobacco. Plant Physiol. 34: 100-102.
7. Egle, K. and H. Fock. 1966. Light respiration - correlations between CO2 fixation, O2 pressure and glycollate concentra• tion. In: T. W. Goodwin (editor) Biochemistry of Chloro• plasts 2: 79-87.
8. Egle, K. and W. Troll. 1966. uber die "Lichtatmung" bei grunen Pflanzen. I. Die Wirkung von Sauerstoff und Kohlendioxyd aut den CO2 - Gaswechsel wahrend der Licht - und Dunkelphase. Beitr. Biol. 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 in liverworts and leaves. Progress in Photosynthesis Res. 1: 482-487.
12. Forrester, M. L., G. Krotkov, and C. D. Nelson. 1966. Effect of oxygen on photosynthesis, photorespiration and respiration in 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 in detached leaves. II. 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. II. 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 in der Blattzelle. Ber. Deut. Bot. Ges. 77: (l)-(3).
16. Heber, U. , K. A. Santarius, W. Urbach, and W. Ullrich. 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 in light and darkness. Crop Sci. 6: 351-354.
20. Owens, 0. V. H. and G. Hoch. 1963. Enhancement and de- enhancement effect in Anacystis nidulans. Biochim. Biophys. Acta 75: 183-186.
21. Ried, A. 1968. Interactions between photosynthesis and respiration in Chlorella. I. Types of transients of oxygen exchange after short light 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 light of different intensities. Can. J. Botany 39: 1945-1056.
23. Tregunna, E. B., G. Krotkov, and C. D. Nelson. 1964. Further evidence on the effects of light 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 in detached tobacco leaves. Physiol. Plant 19: 723-733.
25. Virgin, H. I. 1956. Light-induced stomatal movements in 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 C02 FREE-EXCHANGE POOL SIZE
IN WHEAT AND CORN LEAVES
INTRODUCTION
Onee consideration in the study of photosynthesis, photores•
piration and respiration is that C02 exchange by other processes should be insignificant or accounted for.
There is some evidence that plants may contain quantities of
carbon which can exchange freely with atmospheric C02. Many studies
have shown that C02 is released by plants when they are treated with
acid (1, 10, 13, 17, 18, 19). In addition, C02 is reversibly absorbed
by leaves when they are placed in a vacuum and then exposed to C02
(13). Illumination and chlorophyll are not required (19, 13), and
leaves which are dried and then rewetted also absorb C02 (14). The
quantity of C02 involved is substantial. For example, 0.64 to 1.19 ml of CO2 per g. fresh weight were absorbed by leaves of 13 species at
15° and 1 Atm. C02 (14). The relationship., of these observations to a possible CO2 free-exchange pool under ordinary conditions is not known.
A number of processes could contribute to the information of a
CO2 free-exchange pool. CO2 is absorbed by water, partly by physical solution and partly by chemical hydration (4, 6, 11):
CO2 (gas)^=^C02 (solution)
+ H20
H2C03^=^HC03 + H - . " CO3 + 2H
At alkaline pH, the formation of HCO3 and CO3 are favored, and the 107 reaction:
CO 2 + 0H~"*—JHCO3
also becomes important (9, 11). In.plant cells, 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, 12), may accelerate the other• wise slow hydration steps. The solution of CO2 in water is enhanced
in the presence of alkaline earth carbonic acid esters, and with
amines to form carbamates (11).
The following experiments were designed to estimate the size
of the CO2 free-exchange pools in wheat and corn leaves.
MATERIALS AND METHODS
Wheat (Triticum aestivum L.) and corn (Zea mays L.) plants were grown in the same conditions described for wheat in Chapter I.
About 2 g. fresh weight of ten to fourteen day-old shoots were excised
and transferred to a 20 x 3 x 0.5 cm plexiglass chamber shown in
Figure IV-1 and placed in the dark. The temperature in the chamber was measured by a thermistor placed behind some of the leaves. A
Beckman IR 215 infra-red CO2 analyzer, used differentially in the open
system, measured the CO2 concentration. The air flow rate was 1.6
1./min.for all measurements.
The acid-labile CO2 was measured after the addition of 20 ml.
of 5 M HCl in ethanol to 1 g. fresh weight of plant material. The CO2
evolved was trapped in 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•
2 changed CO2. The first, ^ C02 exchange, involved the comparison of Figure IV-1
Plant Chamber Used for Measurements of the CO2 Free-Exchange Pool Size with Excised Wheat and Corn Shoots FRONT VIEW SIDE VIEW dark respiration rates measured in an open gas flow system with those observed in a closed system. The apparatus used is represented in
Figure IV-2A. Air from compressed gas tanks was introduced into the open system, and the respiration rate was calculated from the increase in CO2 concentration after the air stream had crossed the chamber.
CC>2 concentrations used with the open system, with relative frequency of about 1:2:1, were 130 yL/1, 325 yl./l, and 390 yl./l. In the
0.29 1. closed systems, the respiration rate was calculated from the time required for the CO2 concentration to increase from 100 to
400 yl./l.. Open and closed system measurements were alternated.
The CO2 free-exchange pool, if present, should enlarge res• ponse to the 300 yl./l. increase in CO2 concentration in the closed system. The exchange pool should not enlarge appreciably in the open system where the CO2 concentration varied by less than 5 yl./l.
Since some of the CO2 produced by respiration in the closed system contributes to the increase in exchange pool size, the observed rate of respiration should be less in the closed system than in the open
system. The change in pool size after the C02 concentration had in• creased from 100 yl./l. to 400 yl./l. was calculated from:
Pool C02 Increase (yl/g.) = RQAt - VA [C02]
W
where RQ represents the respiration rate in the open system (yl. CO2/- min) , A [CO2] was the CO2 concentration increase (300 yl./l.) in 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, "^C02 exchange, the CO2 free- Figure IV-2
Gas Flow System Used for CO2 Exchange and CO2 Exchange Measurements with Excised Wheat and Corn Shoots 12 14 A C02 EXCHANGE B COo EXCHANGE
OUTLET INLET .A- PUMP TELETHERMOMETER FLOW METER I o 4 ©
A
PLANT
.« CHAMBER Y RECORDER
RECORDER
MgCIQ4 DRJR] I
SAMPLE REFERENCE
IRGA Ill exchange pool size was estimated by measuring the incorporation of
1^C02 into the pool in the dark. A 0.23 1. closed system, depicted in
Figure TV-2B, was used. The entire system was initially flushed with
(X>2 free-air to reduce the ^QQ^ concentration below 100 ul./l...
One yl. of -^C02 was generated in isolation by the addition of
1 14 excess 1 M H2SO4 to NaH ^C03 in the release flask. The C02 was then let 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 activity in the air stream. The initial observation of the "^C activity was made within 10 seconds of the introduction of the ^C02, and subsequent measurements were made at 6 second intervals until the CO2 concentra• tion had reached 400 yl./l...
A consistent decrease in the -^C activity of the air stream was observed when plants were not included in 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 this, an equivalent number of measurements were made with
and without plants.
It was also necessary to correct for the dark fixation of
1^002. When the CO2 concentration reached 400 yl./l.., the plants were
removed from the chamber, immersed in liquid nitrogen and ground to a
powder. One yl. per g. fresh weight of 1 M acetate buffer, pH 4.0, was added to remove "unbound" CO2 and the mixture was dried in a
dessiccator. Fixed was measured by spreading the dried material
on planchets, counting with a Geiger counter, and correcting for self-
absorption and counter efficiency. 112
On the basis of this information, the total size of the CO2
free-exchange pool was calculated from:
14 14 14 Pool C02 (ul./g.) = CD - ( Cp + Cp V[C02]
Co W
4 where "*"Cp and -^C were the gas phase -^G activities in the presence
or absence of plants; "*"4Cf was the amount of "*"C4 fixed; V was the
closed system volume (1.); [CO,-,] was the final C02 concentration
(400 ul./l.); and W was the fresh weight (g.) of plant material.
4 The "^C02 and "^ C02 exchange techniques are similar in that
they are both based on gas exchange measurements. They differ because
12rj02 exchange requires the formation of a chemical equilibrium while
i L4A 12 • C02 exchange involves an isotopic equilibrium. Also the C02
exchange technique can only detect changes in exchange pool size,
4 while the ^ C02 exchange technique allows the estimation of the total
pool size. The two methods were equally difficult.
12 14 To test the sensitivity of both the C02 and C02 technique, TO -1 /
the absorption of C02 or C02 by a wick of Whatman No. 1 filter
paper containing solution of 0.2 M phosphate buffers, pH 6.0 and
2 pH 7.75, was used. In the case of the -*-C02 absorption measurements,
the phosphate buffers had previously been stored for seven days over
1 M NaOH in a closed desiccator and were therefore C02-free. At the
-1 o
x start of the C02 absorption measurements, the phosphate buffers were
in equilibrium with normal air. 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 IV-3
1 12 Exchange and ^C02 Exchange Gas Flow System Used for C02 Measurements with 0.2 M Phosphate Buffers 12 A C02 EXCHANGE CONTROL B CQ2 EXCHANGE CONTROL
1 FLOW GEIGER PUMP METER TUBE
A
RELEASE RATEMETER £ >- FLASK RECORDER PL
WICK WITH BUFFER CHAMBER CHAMBER 1141
RESULTS AND DISCUSSION
Between 40 and 55 ul. of CO2 per g. fresh weight were released after wheat and corn shoots were acidified with 5 M HC1. Similar results were recently reported by Yemm (19). Smith (13) found that over 300 yl. of CO2 per g. fresh weight were released if plants were boiled in 4.4 M HC1 for 1 hour. If a substantial portion of this acid-labile CO2 were ordinarily in free and rapid exchange with the atmosphere, it could prove to be a complicating factor in studies of
CO2 exchange by plants.
Measurements of the absorption of CO2 by 0.2 M phosphate buffers are given in Table IV-1. The calculated values were derived from the solubility of CO2 in pure water (7), the apparent pK of car• bonic acid (2) and the Henderson-Hasselbach equation (16)*. At pH
7.75, the CO2 absorption measured by both techniques was slightly less than the calculated value. Failure to come to equilibrium, or diffe•
rences in the solubility of C02 or the pK of carbonic acid in 0.2 M phosphate may account for this underestimation. Nevertheless, it is
apparent that small C02 free-exchange pools can be detected, and their approximate sizes' determined, by both the CO2 and CO2 exchange techniques.
From Table IV-2 it can be seen that the CO2 free-exchange pools in both wheat and corn were found to be very small or non• existent by both techniques used. During these experiments, no significant effect of CO2 concentration on the rate of dark respirar:
tion was observed in the open system. Although the measurements were
*Appendix II Table IV-1
Magnitude of C0~ Free-exchange Pools in 0.2 M Phosphate Buffers
C02 CO? Free-exchange Pool- Size. " Method Temperature Concentration pH Measured Calculated H2O) (°C) (yl./l.) (yl.C02/ml. Buffer) (yl.C02/ml.
12 C02 Exchange 27 382 6.0 1.62* 1.01
27 358 7.75 5.90±0.76** 7.05
14 C02 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 limits of the mean. Table IV-2
Magnitude of C09 Free-exchange Pools in Wheat and Corn Shoots
No. of Average CO2 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*
•^^C02 exchange wheat 6 20 .5 -0 .78 + 1.52*
corn 6 21 .0 -0 .04 + 2.90*
* 95% confidence limits of the mean. 117 made with the shoots in darkness, it seems reasonable to expect that the pool would not be larger during photosynthetic utilization of
CC>2. If this is correct, the size of exchange pool is too small for it to be an important factor ih gas exchange transients such as the post-illimination CO2 burst (Chapter III, 15), or as a source of CO2 released by acid. Nor could a CO2 free-exchange pool of such small size significantly effect the CO2 response of photosynthesis or act as an important CO2 reservoir for photosynthesis.
The pH of juice expressed from wheat leaves is about 6.0, and that from corn leaves, consistently lower, about 5.5 (8). The for• mation of HCO3 from the hydration of CO2 is low in this pH range.
This is a further indication that a CO2 free-exchange pool, if it is dependant on the carbon dioxide-water equilibrium, ought to be small. 118
LITERATURE CITED
1. Berthelot, M. M. , and Andre. 1887. Recherches sur la vegetation. Sur les carbonates dans les plantes vivantes. Ann. Chem. Phys. 10: 85-107.
2. Edsall, 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. Distribution of carbonic anhydrase in relation 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. Biol. Chem. 238: 3502-3507.
5. Hansl, N., and E. R. Waygood. 1952. Kinetic 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. Biol. 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 in relation 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 CO2 in Chlorella cells. In: Comparative Biochemistry and Biophysics of Photosynthesis. K. Shib.ata, A. Takamiya, A. T. Jagendorf and R. C. Fuller (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 catalytic
effect of buffers on the reaction CO2 + 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 Institution of Washington. Yearbook No. 23: 132-133.
15. Tregunna, E. B., G. Krotkov, and C. D. Nelson. 1964. Further evidence on the effects of light 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 cell physiology. Interscience Publishers, New York.
18. Willstatter, R., and A. Stoll. 1918. Untersuchangen uber die Assimilation der Kohlensaure. Julius Springer, Berlin.
19. Yemm, E. W. 1968. Bound CC>2 in leaves - carbonic anhydrase. Research Conference on CO2 Uptake and Photorespiration. Case Western Reserve University, Cleveland. (unpublished) 120
CONCLUSIONS
The inhibitory effect of atmospheric 02 on apparent photosynthesis of wheat is at least partly due to photorespiration and is increased by:
increasing 02 concentration,
increasing temperature and
decreasing CO2 concentrations.
Moderate to very high light intensities do not affect the per cent inhibition of apparent photosynthesis of wheat by O2. The effects of varying more than one of these factors are additive.
At temperatures below 30° C in saturating CO2 concentrations, ar~j apparent photosynthesis of wheat is not inhibited by 20.8% O2.
During part of the growing season in temperate conditions, the
O2 present in the air may not cause a significant decrease in photosynthetic productivity in wheat.
The inhibitory effect of atmospheric O2 on photosynthesis of corn
is not due to photorespiration. It differs from the effect of 02 on wheat in that inhibition of photosynthesis occurs only at O2 concentrations greater than 20.8% O2, and the degree of inhibition is not constant but increases with time of exposure to >99% O2.
The CO2 exchange characteristics of wheat and corn also differ with respect to their CO2 compensation concentrations and the
effects of temperature, C02 concentration and light intensity on apparent photosynthesis. These differences are correlated with and may be the result of differences in photorespiration, photo• synthetic carbon metabolism and leaf anatomy. 121
5".' The post-illumination CO2 burst of wheat is an extension of photo•
respiration into the dark period following illumination.
6. The CC>2 concentration response of the post-illumination burst of
wheat indicates that photorespiration decreases with increasing
CO2 concentration in the same way that the size of the depression
of apparent photosynthesis by 20.8% O2 decreases with increasing
CO2 concentration.
7. The post-illumination CO2 burst of Amaranthus edulis is not the
result of an extension of photorespiration into the dark period
following illumination, but it has some other cause.
8. The quantity of carbon within wheat and corn leaves which can
exchange fully with atmospheric CO2 is too small to cause the
post-illumination CO2 burst or to significantly affect the CO2
concentration response of apparent photosynthesis. 122
Appendix I
CALCULATION OF THE CO2 CONCENTRATION IN THE AIR ENTERING
THE IRGA DURING THE POST-ILLUMINATION C02 BURST
This calculation was developed to avoid the error in C02
concentration measurement caused by rapid fluctuations in C02 concen• tration in the air stream passing through the IRGA sample cylinder.
For this calculation to be used, the initial C02 concentration in all parts of the sample cylinder must be known. In the present case, the
C02 concentration was initially similar throughout the sample cylinder and was equivalent to the IRGA reading/
Let the air stream entering the IRGA sample cylinder be divided into a series of 10 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 C02 concentration of an element just entering the sample cylinder. It is assumed that as the element passes through the sample cylinder, the relative response, r, of the
IRGA to C is the same as the IRGA response to a pulse injection of C02 into the air stream entering the sample cylinder which was given in
Figure III-2. Therefore the IRGA will detect the C02 contained in the element according to rC.
In Figure III-2,iit can be seen that an element will exert an appreciable influence (l%'."'or more of maximum response) on the IRGA up to 9.6 sec. after it began to enter the sample cylinder. Therefore, at any one time 9.6/0.6 = 16 elements will contribute to the IRGA reading. From this reasoning, the following equation was developed to
relate the IRGA reading to the C02 present in the elements which 12.3 entered the sample cylinder during the preceding 9.6 sec:
C + r C 0.9223 (IRGA) = r^ + *2 n-l • • • + l6 n-15 _
IRGAn represents the CO2 concentration detected by the IRGA. In this equation, the factor 0.9223 can be considered to be a correction for
uneven flow of air through the sample cylinder. When the pulse inje•
ction data of Figure III-2 is integrated with respect to time, the
integral is almost 8% less than it would be if the air flow rate was
equal inaall parts of the sample cylinder.
The' above equation can be rearranged to give directly the CO2
concentration in the element which just entered the IRGA. Since
T± = 1.0:
Cn = 9.223 (IRGAn) - (r^ + . . . + r^C^).
This equation is then used in a serial calculation to give Cn at
0.6 sec. intervals during the post-illumination CO2 burst. 124
Appendix II
CALCULATION OF THE ABSORPTION OF C02
BY 0.2 M PHOSPHATE BUFFERS
The absorption of C02 by pH 6.0 phosphate buffer at 27° C will be used as an example. Ideal gas laws, are assumed throughout, and the pK of carbonic acid will be taken as 6.37^. The concentration of CO^ is very small when the pH is less than 8, and may be neglected for these calculations.
The Bunsen solubility coefficient, which is the volume of C02,
reduced to 0° C, dissolved in unit volume of water at t° C with a C02 pressure of 1 atm., is 0.718 for 27° C . This solubility coefficient
includes all aqueous species of carbon as "dissolved C02". However, under the conditions in which the Bunsen coefficient is measured, the
concentrations of species other than true dissolved C02 are not appreciable.
Therefore, when the partial pressure of C02 is 0.000382 atm.,
the concentration of dissolved C02 is: ,
C02 = 0.718 x 0.000382 x 300 = 0.30 yl. C02/ml. H20 273
From the Henderson-Hasselback equation-^:
lEdsall, 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 edition. 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 = C02 x antilog (pH - pK)
Therefore, "Total CO2" =? C02 + HCO3
= C02 + C02 x antilog (pH - pK)
= 0.30 + 0.30 x antilog (6.0 - 6.37)
= 1.01 ul. C02/ml. H20
The absorption of C02 by 0.2 M phosphate buffers at pH 7.75 and at other temperatures were calculated similarly and reported in
Table IV-1.