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UBC Theses and Dissertations

Some effects of supplemental carbon dioxide on the physiology of plant growth and development Hicklenton, Peter R. 1978

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SOME EFFECTS OF SUPPLEMENTAL CARBON DIOXIDE ON THE PHYSIOLOGY OF PLANT GROWTH AND DEVELOPMENT by PETER ROBERT HICKLENTON B.Sc, University of Wales (Swansea) 1973 M.Sc, McGill University 1975 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Plant Science) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1978 © Peter Robert Hicklenton In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I ag r ee tha t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r ag ree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Depar tment o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depar tment o f ? w f t t » . T ^ c x E A t t t The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date Occet\te»- i?. v^iP ABSTRACT This research was concerned with some physiological effects of supra-normal CG^  concentrations on cucumber, tomato and Japanese Morning Glory (Pharbitis nil), and with measurement of CO^  levels in a commercial greenhouse. Measurements of CG^  concentrations in a cucumber greenhouse showed that, in the early stages of crop development, early morning CC^  levels reached 0.18% as a result of straw decomposition in the plant beds. Later in crop development, daytime levels were much lower and required gas combustion to restore high concentrations. Stomatal resistances in cucumber leaves were relatively insensitive to high greenhouse concentrations. Variation in stomatal resistance through the crop canopy was, however, detected. Generally, the two most recently developed leaves showed higher resistances than those of a slightly greater physiological age. Differences in leaf irradiance could not ful l y explain this effect, which may be related to the stage of leaf development. Subsequent experiments on greenhouse tomato crops showed that C^-enriched (0.09% CT^) plants flowered earlier and produced 30% more fru i t than those grown in normal air. Photosynthetic rates were inherently higher in apical and basal leaves developed under CO^  enrichment - 2 . -1 at irradiances above 50 m. s .Behavioral indicies of photosynthetic efficiency indicated an enhanced capacity to u t i l i z e C O 2 in enriched plants. Measurements of CO^  exchange in leaves of plants grown in chambers at 3 CG^  concentrations (0.03, 0.1 and 0.5%) confirmed the enhancement of inherent photosynthetic rates in young leaves of 0.1% grown plants. Reduced rates df photorespiration, total 0^ inhibition of photosynthesis, glycolate oxidase (GaO) activity, and an increased rate of ribulose-biphosphate-carboxylase (RuBP-case) activity, contributed to this enhancement. Maximum photosynthetic rates in young leaves developed at 0.5% CO^  were similar to those developed in 0.03% CO^. Growth rates of the 0.1% C02~grown plants were higher than the similar rates of plants from the 0.03 and 0.5% regimes. Apparently maximum benefit from CO2 enrichment is achieved by maintaining atmospheric CO2 concentrations close to 0.1%. At a later stage of development, however, GaO and RuBP-case activities were similar in the 0.03 and 0.1% C02-grown plants and photosynthetic rates did not differ between growth regimes. Observations on the effects of 0.03, 0.1, 1.0 and 5.0% C02 on development in the Short-Day Plant P h a r b i t i s n i l revealed that 1.0 and 5.0% CO2 modified normal flowering. These concentrations induced a weak flowering response in Long-Days and Short-Days and promoted stem elongation and leaf production under both photoperiods. These modifications were apparently unrelated to patterns of CO2 exchange which showed a relatively small increase above 0.5% CO2. These results are discussed in relation to possible mechanisms for the effects of supra-normal CO^- concentrations on development. The diversity of physiological effects mediated by CX^, and their relationship to one another are discussed. iv TABLE OF CONTENTS Page GENERAL INTRODUCTION 1 PART I Studies in a cucumber greenhouse: Atmospheric CO^  concentration and leaf stomatal resistance. INTRODUCTION 8 MATERIALS AND METHODS i) Conductimetric CO^  Analyser 12 i i ) Greenhouse Description and Crop Management Practices 17 i i i ) Measurements of Greenhouse CO^  Concentration . . 19 iv) Measurements of Stomatal Resistance 21 RESULTS AND DISCUSSION i) Greenhouse CO^  Concentrations (Experiment 1) . . 23 i i ) In situ Measurements of Stomatal Resistance (Experiment 1) 33 PART II The effect of atmoshperic CO^-enrichment on the growth, yield and gas-exchange physiology of the tomato plant. . . . . . . . INTRODUCTION 47 PART I I , SECTION 1 Experiments on Greenhouse Crops MATERIALS AND METHODS i) Selection of Suitable Greenhouses for a Comparative Study (Experiment 2) 52 V Page i i ) CC^- Enrichment Experiments (Experiments 3 and 4) 55 PART II, SECTION 1 RESULTS AND DISCUSSION i) Comparative Study Concerning Growth of Tomato Crops in Unenriched Greenhouses (Experiment 2). 64 i i ) C02 Concentrations in CO^-Enriched and Control (Normalair) Greenhouses 71 i i i ) Plant Growth and Yield (Experiment 3) 73 iv) Leaf Photosynthesis (Experiment 4) 78 v) Resistances to C02 Assimilation 86 vi) Behavioral Indicies of Photosynthetic Responses to C02 Enrichment 90 PART II, SECTION 2 Experiments on seedlings grown under controlled environment conditions MATERIALS AND METHODS i) Growth Chambers and Control Systems 96 i i ) Gas Exchange Measurement System 102 i i i ) Experimental Design 105 PART II, SECTION 2 RESULTS AND DISCUSSION i) Analysis of Plant Growth in Relation to C02 Concentration (Experiment 5) 113 Page i i ) Effects of Growth CO^  Concentration on Photosynthesis, Photorespiration and Enzyme Activity in Leaf 3 at LPI^^.O (Experiments 6 and 8) 122 i i i ) Comparison of the Effects of Growth CO^  Concentration on Photosynthesis, Photorespiration and Enzyme Activity in Leaf 3 at LPI^:7.5 and LPI3:2.0 (Experiments 6 and 8) 145 iv) Replacement Experiment (Experiment 7) 162 PART II DISCUSSION ' 169 PART III The influence .of supra-normal CO^  concentrations on photosynthesis, and vegetative and reproductive development of Pharbitis n i l plants in Long and Short Days INTRODUCTION 178 MATERIALS AND METHODS i) Growth System 183 i i ) Experiments a) Effects of Growth CO^  Concentration and Photoperiod on Development (Experiment 9) • 187 b) Effects of Growth CO2 Concentration and Photoperiod on Photosynthesis Rate and Stomatal Resistances (Experiment 10). . . . 190 v i i Page RESULTS i) Effects of CO^  Concentration on Flowering and Vegetative Growth in LD and SD (Experiment 9) . 192 i i ) Effects of 0.03 and 1.0% C02 During Growth in LD and SD on Subsequent Rates of Net PhotosynC • thesis and Stomatal Resistances (Experiment ". 10) 208 DISCUSSION 214 GENERAL DISCUSSION AND CONCLUSIONS 228 LITERATURE CITED 238 APPENDIX 1 261' APPENDIX 2 . 263 APPENDIX 3 265 v i i i LIST OF TABLES . Page I I - l Functions of plant dry weight and leaf area with time derived from multiple regression equations for tomato plants grown in experimental greenhouses A and B, without CC^ enrichment 65 II-2 Mean frui t yield from tomato plants grown in experimental greenhouses A and B, from March 16 to May 18, 1978, without CC^ enrichment 70 II-3 Time course of leaf development in tomato plants grown with or without CO^ enrichment 74 II-4 compensation points for leaves of tomato plants grown with br N without CO 2 enrichment 84 II-5 Behavioral indices: for photosynthetic exchange: ut i l i z a t i o n ef f iciency (E^), radiant energy ut i l i z a t i o n efficiency (E^ .)', and maximum photosynthetic C0 2 flux (F.^) 9 1 II-6 Comparison of observed rates of net photosynthesis with rates predicted by the model of Van Bavel, for a test C0 2 concentration of 340 y l l " 1 94 II-7 Experiment 6 - CO2 exchange of leaf 3 of tomato plants grown at 3 CO2 concentrations in chambers, at 2 stages of development: Summary of experimental design 110 i x Page II-8 E f f e c t s of CC^ concentration on net a s s i m i l a t i o n rate (NAR), r e l a t i v e growth rate (RGR) and le a f area r a t i o (LAR) i n tomato plants grown at 3 CC^ concentrations, i n chambers 120 II-9 compensation points of l e a f 3 of tomato plants grown at 3 CO2 concentrations, measured at 2 stages of development (Leaf Plastochron index, LPI) 132 11-10 Components of oxygen i n h i b i t i o n of photosynthesis i n l e a f 3 of tomato plants grown at 3 CO2 concentrations, at Leaf Plastochron Index: 2 . (LPI 3:2) 138 11-11 Components of oxygen, i n h i b i t i o n of photosynthesis i n l e a f 3 of tomato plants grown at 3 CO2 concentrations at Leaf Plastochron Index: 7.5 (LPI 3:7.5) 154 11-12 Rates of photorespiration (R^) and mesophyll resistance to CO2 transfer ( r m ) for le a f 3 of tomato plants grown at 0.03 or 0.1% CO^ before and a f t e r replacement i n a common 0.03% CO2 atmosphere* 164 I I I - l R elative growth rates (RGR) of Pharbitis plants and leaves exposed to photoperiod and CO2 treatments during a 14 day period 193 III-2 S i g n i f i c a n c e l e v e l s of terms i n Analysis of Variance on various parameters of growth and development i n Phavbitis plants subjected to d i f f e r e n t photoperiod and C0o treatments 194 Page III-3 Mean Plastochron Index of Pharbitis seedlings at end of 14 days in photoperiod and CC^  treatments . . . 195 III-4 Mean change in stem height (mm) of Pharbitis plants exposed to photoperiod and CG^  treatments during a 14 day period 197 III-5 Mean number of f l o r a l buds formed on Pharbitis plants after 14 days i n photoperiod and CC^ treatments, and 7 days after the end of treatments 198 III-6 Mean node of f i r s t f l o r a l bud formed on Pharbitis plants after 14 days in photoperiod and CC^  . . treatments . . .....,....«...,.., ««....<.... . ... 203 III-7 Combined leaf and air (boundary layer) resistances to CC^  transfer in the second leaf of Pharbitis plants grown under LD or SD photoperiods and 0.03 or 1.0% C02 . . . 211 x i LIST OF FIGURES Page 1-1 Flow diagram of conductimetric CG^  analyzer used in Experiment 1 13 1-2 Calibration curve for conductimetric CC^  analyzer. . . . 15 1-3 CG^  concentrations measured at 3 locations inside and outside the greenhouse on January 24, 1977 24 1-4 CO^  concentration measured inside the greenhouse on February 7, 1977 26 1-5 CC^  concentration measured inside the greenhouse on March 7, 1977. 30 1-6 Stomatal diffusion resistance of 4 leaves in the cucumber canopy, and greenhouse CG^  concentration on February 23, 1977. Figures in parentheses indicate irradiance at leaf level during resistance measurement -2 -1 (yE m s . 400 - 700 nm) 34 1-7 Stomatal diffusion resistance of 4 leaves in the cucumber canopy, and greenhouse CO2 concentration on March 7, 1977. Figures in parentheses indicate irradiance at leaf level during resistance measurement -2 -1 -(yE m s , 400 - 700 nm) .- , 36 1-8 Stomatal diffusion resistance and irradiance at leaf level of successive leaves in the cucumber canopy, at 4 times during the day on February 23, 1977. Times x i i Page indicate start of measurement sequence . . stomatal diffusion resistance ......_ quantum flux density 39 1-9 Stomatal diffusion resistance and irradiance at leaf level of successive leaves in the cucumber canopy, at 4 times during the day on March 7, 1977., Times indicate start of measurement sequence. . . stomatal diffusion resistance . quantum flux density 40 I I - l Spectral energy distribution of 100 W incandescent lamp and 300 W cool beam lamp used to illuminate assimilation chamber in Experiment 4 61 II-2 Time course of RGR derived from f i t t e d curves, for tomato crops' grown in experimental greenhouses A and B without CO^  enrichment. . 66 I1-3 Time course of NAR derived from fit t e d curves, for tomato crops grown in. experimental greenhouses A and B without CO^ enrichment 67 II-4 Time course of CGR derived from values of NAR and LAI, for tomato crops grown in experimental greenhouses A and B without CO^ enrichment 68 II-5 Atmospheric CO^ concentrations in C02~enriched and normal air (unenriched) greenhouses 72 x i i i Page II-6 Truss development. Open bars: CO^-enriched growth regime. Cross-hatched bars: normal a i r growth regime-(cross-hatching superimposed). A: Number of trusses developed with f r u i t set on at least one flowerhead. B: Number of trusses developed with open flowerheads. C: Number of trusses developed with closed flowerheads. NOTE: Data are means for 17 replicate tomato plants per treatment. At each date, A + B + C are significantly different between treatments according to Witney-Mann test for non-parametric data (P < 0.001) 7 5 II-7 Cumulative f r u i t yield for plants grown in CO2 enriched and normal a i r growth regimes. NOTE: Data are means of 17 tomato plants per treatment. Differences at any date after May 24 are significant according to t-test (P < 0.05) . . 77 II-8 Rates of apparent photosynthesis in basal leaf 5 of plants grown in normal air =(._ ..) and (, ,) and C02~enriched growth regimes. NOTE: Data are means of ,2 tomato plants per "treatment.- Points marked with the -same letter xiv Page are not significantly different (P > 0.05) according to SNK multiple range test 79 II-9 Rates of apparent photosynthesis in f i r s t unrolled apical leaf of plants grown in normal air (. ,) and C09-enriched (, ,) growth regimes. NOTE: Data are means of 2 tomato plants per treatment. Points marked with the same letter are not significantly different (P > 0.05) according to SNK multiple range test 80 11-10 Differences in apparent photosynthesis rates beweeen tomato plants grown in CO^-enriched and normal a i r growth regimes (APs), in relation to (A) C02 concentration and .(B). quantum flux density. Points represent mean APs for a fixed level of one factor over a l l tested levels of the other. Apical leaves: A A; Basal leaves: A k 81 11-11 Leaf resistances to C02 transfer. NOTE: Data for r (stomatal resistance) are means of 2 s determinations per treatment. r represents m mesophyll resistance calculated as described i n the text. C02 represents test C02 concentration. Bars show one standard deviation either side of the mean for test C02 concentration = 340 y l 1 1 (standard deviations for other r means were of s similar magnitude) 87 X V Page 11-12 Diagram of 8-chamber growth system used for Experiments 5, 6, 7, and 8 97 11-13 Diagram.of one chamber of the system shown in Figure 11-12 ( f o i l facing removed to show internal detail). . 98 11-14 Spectral energy distribution of mixed fluorescent and incandescent lamps used in the system shown in Figure 11-12. 100 11-15 Diagram of assimilation chamber used in Experiments 6 and 7 .104 11-16 Plastochron development of tomato plants grown at 3 C0„ concentrations as a function of time. NOTE: Points are means of measurements on a total of 6 plants, in two chambers shown ± one standard deviation 114 11-17 Growth in length of successive leaves of tomato plants grown at 0.03% C02 as a function of LPI . •<•..-.. NOTE: Points are means of measurments on a total of six plants in two chambers 115 11-18 Growth in length of successive leaves of tomato plants grown at 0.1% CO2 as a function of LPI. NOTE: Points as for Figure 11-17 116 11-19 Growth in length of successive leaves of tomato plants grown at 0.5% CO2 as a function of LPI-NOTE: Points as for Figure 11-17 117 xvi Page 11-20 Apparent rates of photosynthesis in relation to quantum flux density in leaf 3 of tomato plants grown at 3 concentrations. Test C0 2 concentration: 330 y l l " 1 Leaf temperature: 30 ± 2°C LPI: 2 ± 0.5 NOTE: Points bearing the same letter at any irradiance are not significantly different according to SNK multiple range test (P > 0.05) 1 2 3 11-21 Stomatal resistance to CO2 transfer i n relation to quantum -flux density in leaf 3 of tomato plants grown at 3 CO2 concentrations. Test C0 2 concentration: 330 y l 1 _ 1 Leaf temperature: 30 ± 2°C LPI: 2 ± 0.5 NOTE: Mean resistances at each irradiance are not significantly different (P > 0.05) according to SNK multiple range test. ...... . 127 11-22 Apparent rates of photosynthesis in relation to intercellular CX^ concentration in leaf 3 of tomato plants grown at 3 CO2 concentrations. -2 -1 Radiant flux density: 520 yE m s , 400-700 nm Leaf temperature 30 ± 2°C LP.I: 2 ± 0.5 . . 129 x v i i Page 11-23 Rates nf phntorespir^tion (R^), and mesophyll resistance (r ) for leaf 3 of tomato plants grown m at 3 C0? concentrations and measured at LPI: 2 ± 0.5 NOTE: Values of RT or r represented by bars L m bearing the same letter are not significantly different (P > 0.05) according to SNK multiple range test. Rates of R^  and r^ derived from regressions relating apparent photosynthesis to intercellular CO^ concentration, as described in the text 130 11-24 Effect of CO2 concentration on the percentage total oxygen inhibition of photosynthesis in leaf 3 of tomato plants grown at 3 CO 2 concentrations. -2 -1 quantum flux density: 520 yE m s , 400-700 nm Leaf temperature: 30 ± 2°C LPI: 2 ± 0.5 136 11-25 Activities of RuBP carboxylase and glycolate oxidase extracted from leaves of tomato plants grown at 3 CO2 concentrations. PI: 5 ± 0.5 NOTE: Values are means of 3 replicate samples per CO2 regime shown ± one standard deviation. . . 142 x v i i i Page 11-26 Apparent rates of photosynthesis in relation to quantum flux density in leaf 3 of tomato plants grown at 3 CO^  concentrations. Test C02 concentration: 330 y l 1 _ 1 Leaf temperature: 30 ± 2°C LPI: 7.5 ± 0.5 NOTE: Points at any irradiance are not significantly different from one another (P > 0.05) according to SNK multiple range test 146 11-27 Apparent rates of photosynthesis in relation to intercellular CO^  concentration in leaf 3 of tomato plants grown at 3 CO^  concentrations. -2 -1 quantum flux density: 520 yE m s , 400 - 700 nm Leaf temperature: 30 ± 2°C LPI: 7.5 ± 0.5 149 11-28 Rates of photorespiration (R^) and mesophyll resistance (r m) for leaf 3 of tomato plants grown at 3 CO concentrations and measured at LPI: 7.5 ± 0.5. NOTE: Values of R^  or represented by bars bearing the same letter are not significantly different (P > 0.05) according to SNK multiple range test . .Rates.of R_.and.r. „ „ L m xix Page derived from regressions relating apparent photosynthesis to intercellular CC^ concentration, as described in the text. . . . 151 11-29 Stomatal resistance to CO^  transfer in relation to quantum flux density in leaf 3 of tomato plants grown at 3 CO^  concentrations. Test CC>2 concentration: 330 y l 1 _ 1 Leaf temperature: 30 ± 2°C LPI: 7.5 ± 0.5 NOTE: Mean resistances at each irradiance are not significantly different (P > 0.05) according to SNK multiple range test • 152 11-30 Effect of C02 concentration on the total oxygen inhibition of photosynthesis in leaf 3 of tomato plants grown at 3 G02 concentrations. -2 -1 quantum flux density: 520 yE m s , 400-700 nm Leaf temperature: 30 ± 2°C LPI: 7.5 ± 0.5 155 11-31 Activities of RuBP carboxylase and glycolate oxidase extracted from leaves of tomato plants grown at 3 CO concentrations. Plastochron Index: 10.5±0.5. NOTE: Values are means of 3 replicate samples per treatment shown ±-one standard deviation . . . 157 XX Page 11-32 Apparent rates of photosynthesis in relation to intercellular CO^  concentration in leaf 3 of tomato plants grown at 0.03% or 0.1% CO^  before placement in common 0.03% atmosphere. -2 -1 quantum flux density: 520 liE m s , 400-700 nm Leaf temperature: 30 ± 2°C LEI: 2 ± 0.5 163 11-33 Apparent rates of photosynthesis in relation to intercellular GO^  concentration in leaf 3 of tomato plants grown at 0.03% or 0.1%/CO , 14 -days after placement in common 0.03% CO^  atmosphere. -2 -1 quantum flux density: 520 pE m s Leaf temperature: 30 ± 2°C LPI: 12 ± 0.5 166 I I I - l Diagram of gas flow system used in Experiments 9 and 10 . . III-2 Pharbitis plants A and B. A exposed to 14 SD cycles and 0.1% C02; B exposed to 14 SD cycles and 1% C02. Plants photographed 7 days after return to uniform LD conditions (0.03% CO^ 199 III-3 P h a r b i t i s plants C and D. C exposed to 14 SD cycles and 0.03% C02; D exposed to 14 LD cycles and 5% C02. Note the flower formed.on Plant C. Plants photographed 7 days after return to uniform LD conditions (0.03% C02) 200 xx i Page III-4 F i r s t f l o r a l bud at node 6 on plant D (see Figure III-3) exposed to 14 LD cycles and 5% CCy Photograph at same time as Figure III-3. . . 201 III-5 Detail of developing f l o r a l buds and one flower on plant C (see Figure III-3) exposed to 14 SD cycles and 0.03% CO. Photograph at same time as Figure III-3 204 III-6 Third f l o r a l bud at node 6 on plant A (see Figure III-2) exposed to 14 SD cycles and 0.1% C02. Photograph at same time as Figure III-2 205 III-7 Detail of plant A showing terminal f l o r a l bud (see also Figure III-2). Plant exposed to 14 SD cycles and 0.1% (X>2. Photograph at same time as Figure III-2 . . 206 III-8 D e t a i l of f i r s t f l o r a l bud at node 7 on plant B (see Figure III-2) exposed to 14 SD cycles and 1% C02. Photograph at same time as Figure III-2 207 III-9 Apparent rates of photosynthesis i n r e l a t i o n to C02 concentration i n Phavbitis seedlings grown i n 0.03% or 1.0% C02 in Long Days 209 111-10 Apparent rates of photosynthesis i n r e l a t i o n to C02 concentration i n Phavbitis seedlings grown i n 0.03% or 1.0% C02 i n Short Days 210 x x i i Page A3-1 RuBP-case activity in relation to substrate concentration at pH 7.8, 30°C 2 6 7 A3-2 RuBP-case activity in relation to pH at saturating substrate concentration 268 A3-3 GaO activity in relation to pH at saturating substrate concentration, 27°C 269 x x i i i ACKNOWLEDGEMENTS I wish to take t h i s opportunity to thank the many people who have assi s t e d me throughout the course of t h i s research. I would e s p e c i a l l y l i k e to express my appreciation of the help and f r i e n d l y advice of my research supervisor, Dr. Peter J o l l i f f e , the technical assistance of Mr. Ilmars Derics, and the s k i l l and patience of my t y p i s t Ms. Joyce Hollands. Thanks are also due to Mr. Albert Van Marrewyk for permission to conduct experiments i n his greenhouse i n P i t t Meadows, B.C., and to Mr. R. D. B u l l i v a n t f or the basic design of the a s s i m i l a t i o n chamber used i n Part I I , Section 2 and Part I I I . F i n a l l y , to my wife Joyce, my deepest gratitude for the love and support which have helped me i n so many ways during the course of t h i s work. ABBREVIATIONS USED IN THE TEXT Abbreviations A ANOVA CGR C. 1 EDTA E c E I F FIM GaO GSH I P IRGA LAR LD LDP Meaning Leaf area Analysis of variance Crop growth rate(s) Intercellular (inside leaf) CO2 concentration Ethylenediaminetetracetic acid Efficiency of CO^  u t i l i z a t i o n (see Appendix 2) Efficiency of radiant energy ut i l i z a t i o n (see Appendix 2) Flux density of CC^  Maximum possible CC^  flux (see Appendix 2) Glycolate oxidase Glutathione Quantum flux density (400 - 700 (see Appendix 2) Infra-Red Gas Analyzer Leaf-area ratio Long-Day Long-Day Plant(s) XXV Abbreviations LPI.:: j 1 J NAR PhAR PI % I tot Ps 21 •ab Lad m RGR \ RuBP Meaning Leaf plastochron index f o r le a f i = j Net a s s i m i l a t i o n rate(s) Photosynthetically a c t i v e r a d i a t i o n (400 - 700 nm) Plastochron Index Total percentage i n h i b i t i o n of apparent photosynthesis by 21% oxygen Rate of photosynthesis i n 0 - 1% oxygen Rate of apparent photosynthesis i n 21% oxygen Stomatal resistance measured on abaxial l e a f surface Stomatal resistance measured on adaxial l e a f surface Mesophyll resistance to CO^ transfer T o t a l stomatal resistance to CO^ transfer Relative growth rate(s) Rate of photorespiration D-Ribulose-l , 5-biphophate xxvi Abbreviations Meaning RuBP-case Ribulose-1,5-biphosphate carboxylase SD Short-Day SDP Short-Day Plant(s) SNK Student-Newman-Keuls multiple range test t Time W Plant dry weight APs Difference in photosynthesis rate between tomato plants grown in CCv, enriched and normal air greenhouses (Part I I , Section 1) T CC^  compensation point yE Micro-Einstein GENERAL INTRODUCTION The role of carbon dioxide as a primary reactant in the process of photosynthesis is fundamental to l i f e on earth since i t represents the sole inorganic carbon source available to green plants for the production of carbohydrate. The concentration of CO^  in the atmosphere is maintained by the respiration of plants, animals, and micro-organisms, geothermal activity and, in recent times by the extensive combustion of f o s s i l fuels. Due to the low, natural concentrations of the gas (approximately0.033% by volume), extremely large quantities of air are required by plants to produce relatively small amounts of dry matter. For example, Norman (219) has calculated that the production of 5500 pounds (2500 kg) of organic carbon in a corn crop required 20,000 pounds (9091 kg) of CX^  drawn from some 21000 tons (19091 metric tons) of a i r . Current rates of plant photosynthesis are sustained by a complex series of reactions (the carbon cycle) which result in efficient CO,, replenishment on a global scale. Probably the largest single contribution to the atmospheric pool of CO^ is the microbial decay of organic matter in the s o i l , oceans, lakes and rivers. The large water masses of the earth represent an important buffering system in the regulation of atmospheric CO2 concentration but recent work has suggested that the world's forests are an even more important reservoir in the carbon cycle (300). C0„ content of the atmosphere 2 changes significantly with season, with highest concentrations occurring in late winter and the lowest in late summer (33) . The most striking global variation in CC^ concentration, however, i s the steady year to year increase (300). Until very recently this increase (which has raised the atmospheric C0^ level from approximately 0.029% in 1850 to 0.033% in 1977) was attributed to the: increased combustion of f o s s i l fuels. It now seems l i k e l y that an even more important cause is the clearing of forested land which accelerates the breakdown of tree biomass and s o i l humus and removes a potentially large carbon reservoir from the biosphere (300). Despite the recent increases i n concentration, the atmosphere contains considerably lower CO^  levels today than were present in primordial times. The most l i k e l y estimates of the content of the early atmosphere on earth range between 0.3 and 3% (229). In most present day land plants using the system of CO2 assimilation, rates of photosynthesis:.increase very l i t t l e above 0.15 - 0.2% CO2 under optimal conditions of irradiance (60, 103, 134, 252). The a b i l i t y of plants to u t i l i z e concentrations in excess of the ambient atmospheric level of 0.03% may be a remnant, of evolutionary development during a period when the atmosphere contained much greater quantities of the gas. The pressures of natural selection may gradually reduce the concentration required for photosynthetic saturation since i t seems l i k e l y that, in previous ages, plants could u t i l i z e even higher levels of CO2 than are shown today. 3 The promotive effect of increasing CO^ concentration on photosynthetic fate was f i r s t noted in the late nineteenth century (166) and was confirmed several years later by the work of Blackman and Smith (27) on the aquatic plants Elodea and Fontinalis... These early studies provided the background for further research into the effects of supra-normal CO^  concentrations on plant growth and development. At f i r s t , studies concentrated on plant responses to increased CC^  concentrations which seemed to be directly correlated with enhanced photosynthesis. Demoussy (74) showed that dry weight increases of 158% could be obtained in plants grown with additional CO^ , and in Europe and North America, experiments were soon undertaken to determine the potential benefits of CC^-enrichment for greenhouse crops (69, 70). The commercial application of greenhouse CO^-enrichment in North America was not f u l l y substantiated, however, u n t i l the results of a series of experiments on several crops were collated in 1964 (298). At about that time, Krotkov and co-workers (279) presented the results of their studies on light stimulated respiration (photorespiration) in tobacco. Further work established that rates of photorespiration increased with oxygen concentration (280). The inhibition of apparent photosynthesis by oxygen in plants had f i r s t been demonstrated by Warburg in 1920( 287), but i t was not u n t i l 1966 that the physiological components of this inhibition were f u l l y recognized (96, 280). Those studies established, not only that photorespiration was increased by high oxygen concentration, but that 4 oxygen also had a direct Inhibitory effect on photosythesis. A few years later further work showed that high levels of CG^  could markedly reduce the oxygen inhibition of apparent photosynthesis (150). This finding put into perspective the results of Mortimer (207) who showed that the production of serine and glycine (two intermediates of photorespiration) was reduced at CO^  concentrations of 2.5%. It became evident that supra-normal levels of CO^  exerted their effects on plant growth, not merely through increased true photosynthetic rates but also via a reduction in photorespiration. This was further supported by subsequent studies which showed that the.synthesis of glycolate, the probable substrate for photorespiration, i s inhibited at high C02 concentrations (35, 83, 172,, 239, 258). While much research into the physiological role of C02 in higher plants has been concerned with photosynthesis and photorespiratory metabolism, a large amount of information has also accumulated on the effects of C02 on stomatal movement. Linsbauer (174) f i r s t noted that C02 free a i r causes maximum stomatal opening in both light and darkness. This early observation was confirmed by a number of subsequent studies (119, 120, 122, 157). • I t . i s presently unclear exactly how CI^ concentration affects stomatal action although a number of detailed hypotheses have been put forward (173;, 235) . In addition to i t s effects on stomatal movement and photosynthetic physiology, CO appears to affect a number of other 5 processes involved in plant growth and development. The growth of root tissue (58, 260) and coleoptile sections (39, 80, 90) are influenced by increasing CO^  concentrations over a range of 0 to 0.5%. Other studies have shown that CO2 i s required for the effective inhibition of flowering in Xanthium pennsylvanicum during a single red light break in the middle of an inductive dark period, and for seed germination following red illumination in lettuce (13). At higher concentrations (1% and above) C02 affects budset in Douglas f i r (247) , and flowering in a number of SD* and LD plants (51, 234). CO^  may also affect plant survival under adverse environmental conditions as exemplified by the increased salt tolerance of tomatoes grown at 0.8% as compared with those grown at 0.035% C02 (85). The wide variety of the effects of CO^  on the physiology of plants provided the background for the present studies. While considerable attention has previously been paid to the effects of C02 enrichment on yield in greenhouse crops, there remains a lack of information on two important aspects of this practice. In the f i r s t case, many growers and horticulturists are acutely aware of the need to assess ambient C02 concentrations within greenhouses under enriched and non-enriched conditions. Secondly, very few studies have been concerned with the physiological adjustments which may occur when plants are grown at supra-normal C02 concentrations. *Abbreviations used here and throughout the thesis are explained and listed on p.xxiv 6 The f i r s t part of the research in this thesis (Part I) con-sisted of experiments designed to provide information on CO^  levels within a commercial greenhouse at various stages in the development of a cucumber crop. In addition, measurements were conducted during the course of the work to assess the influence of varying ambient CO^  concentrations on stomatal resistance in this crop. The scope of these studies were limited, however, since i t was impractical to conduct a more detailed investigation of other physiological responses of these plants to enrichment. The studies were extended by further experiments designed to investigate growth and photosynthetic metabolism of tomato plants grown under CG^-enriched conditions in a greenhouse (Part I I , Section 1). The results of these experiments prompted further investigation into the physiological mechanisms underlying observed changes in photosynthetic capacity i n the greenhouse-grown plants. Subsequent studies on photosynthesis, photorespiration, and enzyme activities associated with these processes were, therefore, conducted on plants grown at 3 levels of CC^  in controlled environment chambers (Part I I , Section 2). Previous studies on plant response to CO^  have suggested that the gas may mediate physiological changes in systems other than those directly involved in photosynthetic metabolism. To investigate such effects, the f i n a l part of this research (Part III) examined the relationship between CO^  enrichment during growth and vegetative and reproductive development in Pharbitis nil seedlings. The particular interest in these responses originated in the results of earlier studies which had demonstrated marked effects of high CO^ levels on flowering in Pharbitis and other photoperiodically sensitive plants (51, 234). PART I Studies in a cucumber greenhouse: Atmospheric concentration and leaf stomatal resistance. 8 INTRODUCTION Over the past 15 years, CO^-enrichment has become widely adopted by commercial growers engaged i n greenhouse vegetable crop production throughout Europe and North America. The introduction of the procedure i n the early part of the 1960's and i t s rapid acceptance by the greenhouse industry represented an outstanding example of cooperation between research workers and the growers to improve crop production techniques. The beneficial effects of supplemental during crop growth, in terms of increased earliness, yield and quality have been recognized since the early part of this century (32, 69, 74, 255). In more recent years i t has been demonstrated that the yields of a wide range of greenhouse crops show an increase with CO^-enrichment to a concentration of approximately 1000 y l 1 (0.1.%), although the most extensive literature refers to responses of lettuce (85, 295, 298), tomato (47, 49, 50, 163, 165, 189, 273, 298), and cucumber (72, 85, 135, 245, 298). Of these three crops, cucumbers have received the least attention with regard to the effects of C^-enrichment. This seems to be due, primarily, to the fact that for many years a r t i f i c i a l means of supplementing supply in cucumber greenhouses (by combustion or injection of pure gas) were thought to be unnecessary. CO2 concentrations in these situations attained naturally high levels as a result of decomposition of organic matter (straw and manure) traditionally used in preparation of the cropping beds (245, 162). Recently, the highly encouraging results obtained by tomato growers using CO^-enrichment in their greenhouses throughout the growth of the crop, have prompted many British Columbia cucumber growers to i n s t a l l CO^-enrichment equipment to supplement production from the soil-straw growing medium (W. Gates, personal communication). From the lower Fraser Valley region of British Columbia (where this research project was conducted) there are, as yet, few data to suggest whether the new CO^-enrichment practices have proved beneficial in terms of increased f r u i t yields. The introduction of a new cultural practice in crop production always raises important questions, not only with regard to effectiveness in yield improvement but also concerning possible detrimental side effects. Some of the questions may be answered in experiments conducted under controlled environmental conditions in growth chambers. The answers to other questions must rely on results from experiments carried out under f i e l d conditions. This is especially true of C02~-enrichment, since the constant concentration conditions of most growth chamber experiments do not reflect the situation in greenhouses where co2 levels often fluctuate widely and rapidly. In early experiments on CO^-enrichment, Brown and Escombe (41) found that C02 concentrations of 1100 ]il 1 1 inhibited flowering and caused bud abortion in a number of plants grown in closed containers. A few years later, Demoussy (74) pointed out that their results were almost certainly due to toxic impurities in the gas 10 supplied to the plants, and since that time considerable care has been taken to purify gas used in laboratory experiments. The potential problems of toxicity are, however, often present i n greenhouse situations. Sulphur dioxide (114) and nitrogen oxides (54, 55) at concentrations sufficient to inhibit tomato growth are sometimes present i n greenhouses using hydrocarbon burners for enrichment. Incomplete combustion of natural gas may give rise to detrimental concentrations of ethylene in greenhouse atmospheres (17). Whenever CO2 enrichment i s introduced as a new procedure, and particularly when i t i s practiced on a new crop, careful attention should be paid to the method of enrichment and the composition of the resulting gas. Even in the absence of toxic impurities some crops respond unfavourably to very high concentrations of CO2. Cucumbers seem particularly susceptible in this respect. Two studies have indicated that atmospheric enrichment to 2000 or 3000 y l 1 1 can result i n severe leaf necrosis, whereas a constant concentration of 1000 u l 1 produces beneficial effects on growth and f r u i t yield (72, 295). The research which is the subject of Part I of the thesis was prompted by the decision of a cucumber grower in the lower Fraser Valley to i n s t a l l natural gas burning, CO^ enrichment equipment in his greenhouse. Prior to the installation, supplementary CO2 had been provided solely from decomposition of straw bales covered with s o i l in the cropping beds. No information was available on CO^ levels attained in the house atmosphere using these cultural procedures. In view of the considerations mentioned above, there was a clear need to determine ambient concentrations achieved with arid without supplementary CC^ and, also, the composition of the gas produced by the burners. The present study was concerned, primarily, with the f i r s t of these objectives. The generally high cost, inaccuracy or inconvenience of most C02 measuring equipment currently available for use in greenhouses (3) prompted the development of a new system for this study. It was, also, of interest to measure the stomatal resistance of leaves within the developing cucumber crop under naturally fluctuating levels of C02 and irradiance. Although C02 concentrations in excess of those normally present in the atmosphere have often been shown to result in stomatal closure and a reduced potential for photosynthesis (123, 152) in a number of plants, no data were available for cucumbers grown as a crop.„ The following studies were, therefore, carried out to establish the levels of C02 present in one cucumber greenhouse at various stages of crop growth and with different methods of C02 supply. Some measurements were also made to determine the variation of stomatal resistance in the crop canopy under different environmental and developmental conditions. The applicability of the present findings to other greenhouses and other crops awaits further investigation. It is hoped, however, that the information provided here, and the design of the CC>2 measuring equipment developed for these studies, w i l l prove useful in other studies on greenhouse CO - enrichment. 12 MATERIALS AND METHODS i) Conductime trio CO„ Analyzer The requirement for a portable analyzer to monitor ambient concentrations of CC^  was met with a conductimetriee measurement system based on a design principle f i r s t described by James (145). The system i s shown diagramatically in Figure 1-1. Deionized water was induced to flow through the system by means of a small electric pump. Water passed vertically through a glass column (length: 40cm, internal diameter: 2 cm) incorporating a lower side arm to which a one-way valve was f i t t e d . This formed the point of entry for the sample a i r stream. At the top of the bubble column the glass was extruded into an atrium with an upper port for exhaust of the air stream and a side arm from where the water flowed, through plastic tubing, into the conductivity c e l l . From the conductivity c e l l water returned directly to the pump and was thus driven through a cylindrical deionizing column (length: 15 cm, internal diameter: 6 cm) packed with approximately 450 g of Amberlite MB-3 ion exchange resin (Mallinckrodt Inc.). During operation, water flow rate was maintained at a constant 100 ml min 1 and a sample air stream was introduced into the bubble column at a rate of 90 ml min 1. The air supply was maintained by a small diaphragm pump (Universal Electric Co., Owosso, MI) and i t passed through a rotameter type flowmeter fi t t e d with a precision needle 12a FIGURE 1-1: Flow diagram of conductimetric CC^ analyzer used i n Experiment 1. 13 a i r /N outlet J, upper atrium waterTTeveT conductivity transmitter bubble column thermi stor (for temperature compensation) water pump air inlet deionizing column L E G E N D -» direction of water flow :-- electrical connections valve (Matheson Ltd; tube number 610) before entering the column. As the air bubbles rose with the water, some of the CT^  contained in the sample became dissolved resulting in a change in conductivity (according to the equilibrium between CT^  in solution, and carbonic acid i n i t s associated and dissociated forms). The conductivity of water in the system was sensed by a flow-through c e l l with wall-embedded electrodes (Electronic Switchgear, London Ltd.: Model EFA/01/NPT). The c e l l was used in conjunction with a model TX2/2P conductivity transmitter, and conductivity could be measured on any one of three ranges (0-1, 0-10 and 0-100 micromhos/cm Changes i n temperature have a marked effect on conductivity. In this system the effects of temperature changes over a wide range (between 0 and 100°C) were automatically corrected by means of a temperature sensor connected to the conductivity transmitter. Temperature changes also affect water flow rate through a change in density, but this effect i s small (16) and was considered insignificant in the operation of the system. Calibration of the instrument was achieved by bubbling gases of known CO^  concentration into the water column and recording relative conductivity readings. A zero point was set by bubbling a sample of nitrogen gas through the system. A calibration curve for the analyzer is shown in Figure 1-2. Conductivity over the range 0 :to 1650 y l 1 1 changed quadratically although the deviation from a linear relationship was not great. A linear change was assumed in subsequent calculation FIGURE 1-2: C a l i b r a t i o n curve for conductimetric C0„ analyzer. 180Ch 1600H 1 2 3 4 5 6 RECORDER READING 16 of a i r sample CO^ concentration, since only r e l a t i v e l y gross assessments of ambient greenhouse l e v e l s were required. 17 ii) Greenhouse Description and Crop Management Practices The study was concerned with a greenhouse located in P i t t Meadows, British Columbia.' It was typical of many modern cucumber-producing houses in the lower Fraser Valley, with a 2 total area under glass of approximately 2250 m giving enough space for a crop of 2800 plants. In early January 1977 seed of Cuaumis sativus L. cv. Farbio was sowm in 15 cm square pots (1 seed per pot) containing a mica peat mix supplemented with Magamp and dolomitic limestone. Germination was evident in 95% of the pots after two days at a temperature of 24°C. This temperature was 2 maintained i n a section of the greenhouse (approximately 200 m ) throughout the propagation period of three and a half weeks. During this time, preparation of the plant beds was carried out by the greenhouse staff. 230 straw bales were laid in shallow trenches and thoroughly moistened before the addition of a potassium nitrate/lime mix at a rate of approximately 1 kg per 50 kg straw. The straw was allowed to decompose for several days before i t was covered with 15-20 cm of loam. The cucumber seedlings were transplanted into the beds in the f i r s t week of February. Each plant had formed at least two true leaves by this date. A seven-day period had elapsed since i n i t i a l moistening and f e r t i l i z e r addition to the straw, thus ensuring adequate decomposition of this substrate and dissipation of gaseous ammonia. During transplanting, the seedlings were planted i n double rows 18 at a spacing sufficient to allow 0.5 m of ground area per plant. Subsequently standard management practices were used on the crop, and growth was vigorous throughout the course of this study (January to March, 1977). Daily irrigation and sprinkling of plants were carried out automatically. Superphosphate was supplied as a s o i l dressing and applications were repeated as necessary during growth of the crop. Other nutrients, including ammonium, calcium and potassium nitrates, were supplied via the irrigation system. Rates of nutrient application varied somewhat throughout the study period but broadly conformed to that recommended in the 1976-77 B.C. Greenhouse Tomato and Cucumber Production Guide (4). Approximately three weeks after the seedlings were transplanted, two natural gas-burning CO^  generators (Priva Ltd., Netherlands) were installed in the house. Each burner incorporated a fan to disperse the CO2 and, according to the manufacturers specificiations, was capable 2 of enriching a covered area of 12000 to 16000 m . Unfortunately no details of the rate of CO^  production were;provided.by the manufacturer. During the f i r s t four weeks after transplanting the burners were not available to provide CO2- enrichment within the greenhouse. Enrichment was commenced on a daily basis at the beginning of March. The installation and operation of the CO2 enrichment equipment and a l l crop management procedures were undertaken by the greenhouse staff. 19 iii) Measurement of Greenhouse CO Concentrations CC>2 concentrations inside the greenhouse were measured throughout the daylight period on four occasions (January 24, February 7, 23 and March 7, 1977) during the early stages of crop growth and development. At the f i r s t sampling date the seedlings had not been transplanted and were enclosed i n the propagation area which was closed off from the main greenhouse. • Within the remainder of the house the beds had been partially prepared for transplanting (straw bales had been l a i d and moistened but were not covered with s o i l ) . Sampling of the atmosphere was commenced in the early morning and consisted of half-hourly periods during which air was drawn into the analyzer alternately from the seedling enclosure and a randomly selected sampling site in the main part of the greenhouse. Upon changing from one sampling location to another, spot checks were made on ambient external CC^  levels by drawing an air sample from the atmosphere above the greenhouse roof. A l l measurements within the greenhouse were based on air samples drawn from a height of 2 m above the ground surface. Within the seedling enclosure, the open end of the sample tube was positioned directly above the cucumber seedlings which were arranged in rows on slatted benches. In the main greenhouse the open end of the sample tube was arranged to face upwards above the cropping beds. Measurements of the greenhouse atmosphere on the other dates (February 7 and 23, and March 7) were carried out on air samples from a similar height. On these occasions the air samples were drawn continuously into the analyzer from randomly selected sites within the greenhouse. The output from the conductivity transmitter was recorded on a strip chart recorder (Yellow Spring Instrument Co. Model 80A) and the CO^  concentrations were integrated over successive fifteen minute periods throughout the day. 21 iv) Measurements of Stomatal Resistance Stomatal resistance was determined on leaves of one plant i n the crop on February 23, and March 7, 1977. On each date, plant selection was at random but care was taken to exclude any plant growing on the end of a row where i t might be subjected to different light and temperature conditions, and other edge effects. At each date stomatal resistance measurements were conducted on successive leaves in the canopy on four occasions during the daylight period (measurements starting at 0820, 1030, 1300 and 1630 h). A diffusion resistance porometer (Lambda Instrument. Co. Inc., Lincoln, Nebraska; Model LI-60) was used for the measurements. On February 23 the f u l l complement of 13 leaves was used for stomatal resistance determinations. On March 7, when each plant had in excess of 20 leaves, only the f i r s t thirteen counting from the newly expanded leaf at the top of the canopy, were used. Throughout a l l measurements a specific sequence of operations was followed for each leaf, whereby the porometer was clasped to the abaxial leaf surface, leaf temperature was recorded (by means of a thermistor incorporated in the porometer sensor), and a resistance measurement was taken. The sequence was repeated for the adaxial surface. PhAR incident upon each leaf was measured concurrently with the resistance measurements using a Lambda Instrument Co. Model LI 190S quantum sensor with a LI 185 meter., 22 Calibration of the porometer was carried out in the laboratory<as recommended by the manufacturer (Lambda Instrument Model LI 60 operation manual). Total leaf resistance was calculated as: 2 / r s = 1 / r a d + 1 / rab where r = total leaf resistance s .r . •= resistance of adaxial leaf surface ad r , = resistance of abaxial leaf surface (156) ab Measurements on a l l thirteen leaves occupied approximately one hour so that there was considerable temporal as well as spatial var i a b i l i t y in the radiation and, possibly, the stomatal resistance data. For descriptive convenience, i n Figures 1-8 and 1-9 and throughout the text, measurement times are referred to by the time at which the series of measurements commenced. In Figures 1-6,and 1-7. resistance values are plotted at the actual time that they were taken. 23 RESULTS AND DISCUSSION i) Greenhouse CO Concentrations. (Experiment 1) * Measurements of CC^  levels in the greenhouse atmosphere indicated that considerable increases in ambient CC^ concentrations can be achieved simply as a result of straw decomposition within the plant beds. Figure 1-3 illustrates the diurnal pattern of CC^  concentration within the greenhouse during seedling propagation. Spot checks on external atmosphere concentrations showed l i t t l e variation with concentrations remaining between 320 and 330 y l 1 1 throughout the daylight period. In the bulk atmosphere within the greenhouse, CO^  concentrations were also relatively stable and were some 130 y l 1 1 higher than external air values. Within the propagation area, CO^  concentrations in the early morning (0830 - 0845 h) were approximately 420 y l 1 _ 1, but by the next sampling period at 0930 h levels had declined to 385 y l 1 1 and thereafter remained quite steady u n t i l measurements were terminated at 1600 h. Evidently, the elevated concentrations inside the greenhouse were due to production within the beds where straw bales had been allowed to settle and undergo some decomposition for a week prior to the January 24. measurements. In the early morning, levels i n the enclosed propagation area were similar to those in the bulk greenhouse atmosphere, but by mid-morning concentrations had been depleted to a value intermediate between ^Experiments are numbered sequentially throughout the thesis and are listed in Appendix 1. FIGURE 1-3: CC^  concentrations measured at 3 locations inside and outside the greenhouse on January 24, 1977. 500 400-3001 200 bulk greenhouse atmosphere inside seedling enclosure external atmosphere n r 1 1 1 1 i i 0800 0900 1000 1100 1200 1300 U 0 0 1500 1600 TIME OF DAY 25 the greenhouse and external air levels. Ventilators were not opened in the propagation area on this day and the CO^ depletion was apparently due to photosynthetic uptake by the young plants. The most complete information on ambient CC^ levels in a cucumber house in which beds were prepared with a sub-soil straw layer i s provided by Klougart (162). He reported concentrations of approximately 4500 y l 1 1 within a 20-metre greenhouse during the Spring pre-planting period suggesting that decomposition was already proceeding at a fast rate. The present results show that CO^  levels in the house used in this study were an order of magnitude lower than Klougart's (162) data. It is l i k e l y that straw decomposition had not reached maximum rate during the week preceding the January 24 measurements and the young seedlings were probably CO^-limited during this early stage of growth. The high CO^  concentrations often described i n cucumber houses well supplied with organic matter (162, 298) have been assumed to be more than adequate for seedling propagation. The present data, however, indicate that CO^  levels could be usefully raised (by alternate methods of CO^  production) for at least a week after bed preparation in operations where young plants are raised in the main greenhouse. Figure 1-4 shows the diurnal trend of greenhouse CX^ concentration on February 7. Plants had developed 5 leaves by this date and had already been transplanted into the beds. CO 25a FIGURE 1-4: CO^  concentration measured inside the greenhouse on February 7, 1977. 27 levels i n the early morning were over 5 times higher than normal atmospheric concentrations, and i t was apparent that straw decomposition was contributing significantly to CO^  production within the house. It i s noticeable, however, that concentrations as high as 1800 y l 1 1 (measured at 0800 h) were quickly depleted during the morning and by 1030 h had fallen to a constant -1 daytime level of 600 y l 1 . Similar decrements i n house CO^ concentration during the f i r s t few hours after dawn have been reported in previous studies (162), and may be due to a number of factors. Even in late winter some ventilation i s frequently necessary to control greenhouse temperature and this occurred at intervals during the sunny morning of February 7. The opening of roof vents quickly reduces internal CX^  concentration (298) and this effect i s enhanced by strong wind (17, 253). In addition, CO^  consumption by the developing crop undoubtedly contributed to the drop in ambient concentration during the early morning on this day. Despite the rapid decline in CO^ levels between 0800 and 1030 h, greenhouse concentrations remained close to 600 y l 1 1 as a result of the continued production of CO^  from the soil-straw substrate. Considerable benefit i n terms of increased photosynthesis and growth might be obtained at this stage of crop development by the injection of additional CO^  into the house atmosphere. It i s evident, however, that even without CO^  generating equipment the replenishment of lost by crop consumption and ventilation is 28 quite efficient in cucumber houses prepared with a sub-soil straw layer. Morris et at. (206) calculated that 10 air changes per hour were necessary to produce a non-diminished growth rate of tomato plants in a conventional greenhouse where no supplementary CO^  source was provided. This value is clearly dependent upon season, stage of crop development and cultural conditions (45, 206). The present results suggest that ventilation of many cucumber houses i s unnecessary for CO^  replenishment, at least during the f i r s t 2-3 weeks of crop development. Frequent ventilation might, in fact, be expected to reduce internal concentration close to that of the external atmosphere. In recent years, various management d i f f i c u l t i e s commonly associated with growing greenhouse crops in s o i l (e.g. control of pests and levels of essential nutrients) have prompted some growers in the Lower Fraser Valley to change to a soilless-culture for grow-ing cucumbers (W. Gates, personal communication). One of the disadvantages of this method of production w i l l be a lack of supplementary CO^  produced from the traditional manure/straw and s o i l cropping beds. For some growers this has been an important reason fo^- installing equipment to provide an alternate means of CO2~ enrichment. Since l i t t l e accurate information has been available on CO2 levels in commercial greenhouses, previous recommendations have tended to underestimate the importance of efficient f a c i l i t i e s for CO - enrichment in cucumber houses (245). It i s clear from the profiles of diurnal CO2 concentrations presented above that the addition of C09 from a r t i f i c i a l sources may be of benefit even i n houses where t r a d i t i o n a l cropping systems are used. The r e s u l t s obtained from measurements of i n t e r n a l CO^ concentrations on March 7 are shown i n Figure 1-5. On t h i s date the natural gas burners were used from mid-morning u n t i l l a t e afternoon to replenish CO^ supply i n the greenhouse atmosphere. A s i m i l a r pattern of C ^ — d e p l e t i o n to that observed on. .February 7 was evident i n the early morning before the burners were turned on, although lower concentrations at 0800 h probably r e f l e c t e d the diminished contribution of organic substrate decomposition to house CO l e v e l s i n early March. When the i n t e r n a l concentration had f a l l e n to approximately 550 U l 1 1 the burners were turned on and the concentration was restored to between 1100 and 1200 l i l 1 1 within 2 hours. Enrichment was discontinued at 1530 h. A l l operations were performed by the greenhouse s t a f f without reference to the output from the conductimetric analyzer, : and enrichment procedures were s i m i l a r to those c a r r i e d out each day when house v e n t i l a t i o n was minimal. On March 7 outside temperatures were low and there was l i t t l e need for v e n t i l a t i o n . Burners would have been turned off for periods on other days when r i s i n g house temperatures necessitated vent opening. The enrichment period on March 7 appeared to be quite e f f i c i e n t i n maintaining house l e v e l s near optimum concentrations for crop growth. Each plant had formed at l e a s t 20 leaves by t h i s time and had set several FIGURE 1-5: CC^ concentration measured inside the greenhouse on March 7, 1977. T I M E O F D A Y 31 f r u i t . It is at this stage of development that serious CO^  limitations may occur inside closed greenhouses. These clearly cannot be remedied by ventilation or by natural CC^ production processes even in the presence of abundant organic matter in the cropping beds.' Nevertheless, the use of burners in the early morning was evidently unnecessary in a house of this type since CC^ concentrations were high, presumably as a result of night respiration by the extensive crop biomass and residual production, from the plant beds. For most growers who lack the instruments necessary to monitor CO^  levels in their greenhouses on a daily basis, the operation of enrichment equipment is dictated by the recommendations of agricultural bulletins and manuals, although few recommendations relating to cucumber culture are available. The usual recommendation is that CO^  addition should be commenced soon after dawn and continued un t i l 1 1/2 hours before sunset, when light levels become severely limiting to photosynthesis (e.g. 3, 257). Based upon the present results, i t appears that these recommendations may be legitimately applied to Spring-grown cucumber crops, although some economic benefit might be derived by slightly delaying the onset of enrichment in the morning. In this study, however, i t is evident that enrichment could have been usefully commenced at least an hour earlier on March 7, and i t is doubtful that the financial saving in fuel costs by this delay is very significant'. Calvert and Slack (50) , in their studies on tomatoes, found that any reduction i n the d a i l y enrichment period, extending from sunrise to sunset,had a marked detrimental e f f e c t on f i n a l y i e l d s which was not o f f s e t by savings i n the cost of enrichment. Their findings advise caution i n delaying morning C0 0-enrichment i n cucumber houses. 33 ii) In situ •Measurements of Stomatal Resistance (Experiment 1) Stomatal closure during CC^  enrichment has been the subject of several studies, which have disagreed on the importance of this effect in limiting photosynthesis and growth (138, 152, 225). It now seems probable that the magnitude of stomatal response to increasing CO^  concentration is species-specific, with tomatoes apparently showing a rather low sensitivity (138, 225). There are, apparently, no previous reports dealing with the response of stomates .in cucumber leaves to elevated CO^  concentrations, despite the fact that CO^-enrichment by organic matter fermentation or gas combustion i s frequently practiced in cucumber greenhouses. Figure 1-6 shows the stomatal resistance of four leaves from the top, middle and bottom of the crop canopy throughout the daylight period on February 23. Also shown on this figure is the pattern of internal C02 concentration on this day, during which no additional CO2 was supplied to the atmosphere from the gas burners. Stomatal resistances in leaves 4, 8, and 13 tended to increase throughout the day, reaching a maximum i n the late afternoon between 1630 and 1700 h. Radiation incident on individual leaves (figures in parentheses) was variable on this ^predominantly sunny day, with only leaf 13 (at the bottom of the canopy) receiving relatively low irradiance at each measurement time. The progression of stomatal resistance from low to high values in leaves 4, 8, and 13 during the day was not obviously correlated 33a FIGURE 1-6: Stomatal d i f f u s i o n resistance of 4 leaves i n the cucumber canopy, and greenhouse CC^ concentration on February 23, 1977. Figures i n parentheses in d i c a t e irradiance at l e a f l e v e l during resistance measurement (yE m s » 400 - 700 nm). 35 with decreasing levels of radiation or increasing CO^  concentration. In the early morning resistances in a l l leaves were at their lowest daily value, when atmospheric CC^  concentration was highest. Leaf 2 exhibited considerably higher stomatal resistance than the other leaves except i n the late afternoon (1630 h) and did not show the same gradual increase in this parameter. A similar trend of increasing resistance through the day was shown by leaves.4, 8, and 13 on March 7 (Figure 1-7), although resistance values were generally higher than on February 23. In contrast to that day, March 7 was predominantly overcast, as indicated by the relatively low values of leaf irradiance, which may have accounted for :the observed differences. Resistances were higher during the CO^r-enrichment period (from 1030 to 1530 h) than in the early morning, but there was no indication of a decrease during the f i n a l measurements, roughly an hour after the CO^  burners were turned off. The similar trends in resistance throughout the daylight period in leaves 4, 8, and 13 on March 7 (when (X^-enrichment was practiced) and February 23 (without enrichment) do not suggest that this parameter was strongly affected by ambient CO^  concentration. The pattern of gradually increasing resistance on both days may be related to a diurnal rhythm of stomatal opening potential which has been shown to result in maximum aperture in mid-morning followed by gradual closure through the afternoon and early evening (155, 197, 261). 35a FIGURE 1-7: Stomatal diffusion resistance of 4 leaves i n the cucumber canopy, and greenhouse CC^ concentration on March 7, 1977. Figures in parentheses indicate irradiance at leaf level during resistance —2 —1 measurement (yE m s , 400 - 700 nm) . T I M E O F D A Y 37 It i s worthwhile to mention at this point, some shortcomings of these experiments. F i r s t l y , on both dates that stomatal measurements were taken no adequate controls were available against which to compare the responses of the selected leaves to the greenhouse environment. In each case the results are purely descriptive and conclusions must, therefore, remain tentative. Secondly, measurements of radiation incident on the leaves were instantaneous values and may not have provided a true assessment of general irradiance conditions at that time of the day. This problem was probably greatest on February 23, since ambient light levels below the canopy tended to fluctuate rapidly, during alternate periods of bright sun, and cloud. On March 7 the sub-canopy radiation environment was much more constant. A third problem was the confounding of the effects of varying CO^  concentration and irradiance on stomatal response, which was unavoidable in the greenhouse. The apparent lack of an effect of C02 concentrations around 1000 y l 1 1 (present in the greenhouse atmosphere during the enrichment period on March 7 and at the 0830 h measurement time on February 23) i s not consistent with the results..of Heath and Russell (123). They showed that the diffusive resistance of illuminated stomata increased markedly between 170 and 1840 y l 1 i i n wheat leaves. The results of the present study are, however, similar to those of Hurd (138) i n studies on a greenhouse tomato 38 crop. He,itoo, discovered a pattern of v a r i a t i o n i n stomatal resistance of mid-canopy leaves throughout the daylight period which was unaffected by CO^ -enrichment to 1000 y l 1 X . High l e v e l s of CO^ i n greenhouse atmospheres may, therefore, be of small consequence i n determining the use e f f i c i e n c y of the gas i n cucumber and tomato crops. Confirmation of t h i s suggestion requires further work p a r t i c u l a r l y with cucumber plants, tested under co n t r o l l e d conditions of CO^ concentration and i r r a d i a n c e . A clearer assessment of the pattern of d i u r n a l and s p a t i a l v a r i a t i o n i n stomatal resistance w i t h i n the crop canopy can be made from the data presented i n Figures 1-8 and 1-9. These show stomatal resistances of the f i r s t 13 leaves of cucumber plants measured on 4 occasions during the day on February 23 and March 7. At most measurement times, minimal resistance was found to occur i n leaves 3 and 4, situated j u s t below the top of the canopy. In very few cases were these low values found to correspond with the highest irradiance i n the canopy. Sometimes r a d i a t i o n incident on these leaves at the time of measurement, was lower than that on leaves of higher and/or lower i n s e r t i o n (e.g. February 23 and March 7, 1030 h). These observations corresponded reasonably w e l l with other in situ measurements on a tomato crop (138). In that case, minimal resistances were found i n leaves 8 and 9. In tomato, however, i t was not possible to determine whether l i g h t played a r o l e i n these e f f e c t s , since 38a FIGURE 1-8: Stomatal diffusion resistance and irradiance at leaf level of successive leaves in the cucumber canopy, at 4 times during the day on February 23, 1977. Times indicate start of measurement sequence. . . stomatal diffusion resistance •• •* quantum flux density or U J CO 1 • 2 3 i U \\ 7-8H 9-10-1H 12-13H 0 8 3 0 h top of c a n o p y 1 0 3 0 h b o t t o m of 1 c a n o p y r 0 1 3 0 0 h t o p of c a n o p y 1630h r g (s c m " 1 ) 2 0 0 400 600 800 1000 1200 200 400 600 Q U A N T U M F L U X D E N S I T Y (uE m" 2 s"UoO-700nm) 8 0 0 1000 1200 I D 39a FIGURE 1-9: Stomatal diffusion resistance and irradiance at leaf level of successive leaves in the cucumber canopy, at 4 times during the day on March 7, 1977.• Times indicate start of measurement sequence. • • stomatal diffusion resistance • - quantum flux density LEAF NUMBER LEAF NUMBER 41 irradiance within the canopy was not measured. The present results are also in agreement with the work of Fischer (93) who demonstrated that stomata i n epidermal strips from leaf 5 to 7 of tobacco showed greater opening a b i l i t y than those from younger (numbers 1 to 4) and older (> 5) leaves. Thus, maximum aperture under test conditions was found in the newest f u l l y expanded leaves (93). The results obtained in the present study seem to indicate a similar effect of leaf age on stomatal resistance to that shown by Fischer's work. In view of the uncertainty that instantaneous measurements of irradiance on individual leaves were truly representative of effective, longer term radiation conditions, the possibility exists that minimal stomatal resistancesin leaves 3 and 4 were mediated by higher irradiances than those experienced by leaves at other levels. This seems to be a somewhat unlikely explanation, however, since the shading effects of the upper leaves and neighbouring plants were considerable at most times during the day and the measurements showed no consistent trend of higher irradiance just below the top of the canopy. On most occasions during the present study, the highest stomatal resistances were shown by the oldest leaves at low levels in the crop canopy (Figures 1-8 and 1-9: leaves 11-13). These high values were, for the most part, correlated with the lowest irradiances within the canopy. The results may be 42 consistent with those of Turner (785) who showed that increases in stomatal resistance at progressively lower levels in canopies of corn, poplar and dogwood could be explained entirely in terms of reduced irradiance, u n t i l senescence induced leaf chlorosis. Davis et al. (73) have pointed out that the effect of irradiance on stomatal resistance has two components. These consist of a conditioning effect whereby leaves adapt physiologically and structurally to ambient radiation conditions, and an instantaneous effect by which stomates are influenced by short term fluctuations in radiant flux density. The physiological age of individual leaves was found to exert an influence on stomatal resistance which was exclusive of both components. Results obtained in the present experiment at 1630 h on February 23 and March 7 (Figures 1-8 and 1-9) suggested that low irradiance during measurement had a more pronounced effect on stomatal resistance in bottom leaves than in those higher in the canopy. In addition, the increase in stomatal resistance below leaf 7 at 1300 h on March 7 (Figure 1-9) did not correlate with irradiance level which remained relatively constant with leaf position. These results may be due to the f i r s t component of the irradiance effect mentioned previously, or to the greater age of leaves at progressively lower insertions. Unfortunately, the two factors were confounded in these experiments. Some support for the involvement of leaf age as a determinant of stomatal resistance in this 43 crop comes from the work of Hurd (138) who showed that leaf resistances in exposed tomato plants (at the edge of greenhouse rows) were higher in old leaves from the low canopy than in uppermost, young leaves. It appears from the data presented in Figures 1-8 and 1-9 that high stomatal resistance in leaves 1 and 2 i s a relatively constant feature of these greenhouse cucumber plants. On several occasions on February 23 and March 7 resistances i n these two leaves were higher than in those at the bottom of the canopy (e.g. 0830 and 1030 h, Figure 1-8), and at practically every measurement time they exceeded values for leaves 3 and 4. These observations are again in accord with those of Hurd (138) who found that leaves i n the uppermost part of a greenhouse tomato canopy sometimes showed the highest resistances of a l l leaves on the plant. This effect may have been due to dehydration of the upper leaves which were often out of the range of the mid-canopy sprinkling system. In this case i t i s , however, d i f f i c u l t to explain the much lower resistances generally shown by leaves 3 and 4, which were also situated well above the sprinkling system. Another possibility i s that the higher resistances of leaves 1 and 2 are due to a closing response induced by high levels of irradiance, sometimes experienced at the top of the canopy, although such responses are more prevalent i n old than in young leaves (182). The appeal of this explanation i s diminished by the observation that relatively high resistances occurred i n these leaves in the late afternoon (1630 h; Figure 1-8) when almost uniform irradiance conditions prevailed at successive leaf insertions. It seems more l i k e l y that the effects were related to the developmental stage of the leaves and possibly, a reduced stomatal density in comparison with leaves of a slightly greater physiological age (265). In summary, the profile of stomatal resistance through the cucumber canopy displays a distinct pattern quite similar to that of a greenhouse tomato crop (138). As in that case, stomatal resistance of leaves from a l l levels in the canopy did not show a clear response to CCX,-enrichment. Resistances varied considerably according to the position of the leaf and level of irradiance, and i t was apparent that the lowest stomatal resistances occurred in leaves just below the top of the canopy. Measurements of CC^  concentration using a conductimetric analyzer showed that greenhouse CC^  concentrations were normally maintained at a f a i r l y high level throughout the day in the early stages of crop growth. This effect probably resulted from CC^  production by decomposition of straw and organic material within the cropping beds. It was evident, however, that by the time the crop canopy had developed appreciably, daytime CO^  levels became depleted below those necessary to maintain maximum rates of photosynthesis under optimal irradiance. CO^  production from natural gas burners was effective in restoring CO^ concentrations to optimal daytime levels. In recommending suitable enrichment procedures for cucumber crops, i t i s worth bearing in mind that supplementary CG^ (from combustion or other a r t i f i c i a l sources) may be of benefit f o r c e r t a i n periods immediately a f t e r the crop i s transplanted. As w i l l be shown i n Part II of t h i s t h e s i s , the b e n e f i c i a l e f f e c t s of CC^-enrichment to approximately 1000 y l 1 ^ i n another crop (tomato) may be enhanced by commencing an e f f e c t i v e enrichment program as early i n the growing period as possible. In cucumbers t h i s may mean gradually increasing the proportion of supplied by a r t i f i c i a l means, as the rate of decomposition i n the straw-soil cropping bed slows. Previous to t h i s study there has been l i t t l e a v a i l a b l e information on the e f f e c t i v e l e v e l s of C0? i n commercial cucumber houses u t i l i z i n g t r a d i t i o n a l cropping methods. There was considerable uncertainty amongst growers as to whether a d d i t i o n a l CO2~ enrichment equipment, s i m i l a r to that used for other greenhouse crops, would be b e n e f i c i a l to th e i r operations. Using a r e l a t i v e l y simple apparatus, i t has been possible to demonstrate that ambient concentrations i n one house may be i n s u f f i c i e n t to promote maximum crop growth without the i n j e c t i o n of a d d i t i o n a l CO2 into the atmosphere. I t i s hoped that t h i s system can be used elsewhere i n cucumber greenhouses to e s t a b l i s h ambient CO2 concentrations i n d i f f e r e n t s i t u a t i o n s . Such surveys should serve to provide basic information on the a d v i s a b i l i t y of i n s t a l l i n g CO2-enrichment equipment. 46 PART II The effect of atmospheric CO^ enrichment on the growth, yield and gas exchange -physiology of the tomato plant. PART II - INTRODUCTION Tomato crops have been the subject of a large number of studies concerned with plant growth and yield responses to CC^-enrichment. This considerable interest seems to have arisen for several reasons. Probably the most important of these i s the crop's very great economic importance in the greenhouse industry. In an early study, Bolas and Melville (32) showed that yield could be increased between 14 and 24% in tomato crops grown in air enriched with to approximately 850 y l 1 X. Later work has established that yield increases of 40% over normal air-grown tomato plants are possible with CX^-enrichment of crops grown in the Spring (298). The plants also respond to enhanced C02 concentrations by the earlier production of ripe f r u i t and a reduction of the harvest period (115). As a result of these findings, CO^-enrichment in tomato greenhouses has been enthusiastically adopted throughout Europe and North America (295) . The result's of early studies on tomato response to CO2"" enrichment also provided a strong impetus for similar research in a number of other greenhouse crops. Yet, despite numerous studies on the direct effects of supplemental CO2 on yield and growth under various conditions of "light, temperature and nutrient status (47, 52) very l i t t l e research has been concerned with the physiological basis for these effects (190). Yield increases due to CO^-enrichment of greenhouse atmospheres may be due to increased rates of photosynthesis as a result of a steepened CG^  diffusion gradient from the bulk atmosphere to the sites of fixation at the chloroplasts (103, 104). It i s , however, doubtful that this represents the only important factor. Reduction in light compensation point (121), lower rates of glycolate" production and photorespiration (310), and the suppression of oxygen inhibition of photosynthesis (149, 150, 167) may also result from high concentrations and contribute to higher net rates of photosynthesis. It is also worthwhile to note that the growth of plants and plant parts i s the result of interactions between many factors, and the link between yield and photosynthetic rate is often complex. The information available concerning the effects of C^ -enrichment during plant growth on subsequent rates of photosynthesis is very limited. Three studies have indicated that some persistent changes may occur in the photosynthetic response of enriched plants (21, 102, 185). In each case, the physiological basis for these change was not investigated. Recently, however, studies have been carried out which may help to determine the basis for the persistent changes i n photosynthetic metabolism observed in other work. CO^ - enrichment has been found to increase the activity of a number of enzymes associated with CO2 fixation in a variety of plants (59, 92, 112), and to decrease activities of others important in photorespiration (92). Thes results suggest that some changes in photosynthetic activity associated with C0„-enrichment may be due to a modification of enzyme systems in the leaves of enriched plants. Other studies have indicated significant increases in carbohydrate levels in plants grown under COy-enriched conditions (184, 186). In several cases, the high leaf starch content resulted in chloroplast deformation (186) which seemed to be correlated with a decrease in apparent photosynthesis (143, 185, 190). The present study consists of 2 sections, the f i r s t dealing with the effects of supplementary CO^  on greenhouse-grown tomato plants, and the second with the effects of 3 different levels of CO ~enrichment on tomato seedlings raised in growth chambers. The f i r s t section of the study was prompted by the lack of information on the relative photosynthetic responses of greenhouse crop plants grown under C02~enriched and normal air conditions. The primary objective was to compare the growth and gas exchange physiology of two greenhouse tomato crops grown with and without CO2-enrichment. Two greenhouses were available for this part of the project, so preliminary experiments were required to determine the f e a s i b i l i t y of conducting a comparative study where supplementary CO^  would be introduced into only one house. Once this was established, a further series of experiments to elucidate the effects of CO — enrichment on growth, development, and CO^  exchange were begun. The approach taken was f i r s t l y to measure rates of leaf and flower formation, and yield in each crop. Secondly rates of CO^  and water vapour exchange were monitored to assess photosynthetic potential and diffusion resistances in leaves developed under CO^—enriched and normal air atmospheres. Of particular interest at the outset of this research was the possibility that supplementary CO^  during growth might induce persistent changes in the photosynthetic system. Accordingly, due attention was paid to this possibility and to whether such changes might be influenced by leaf age. The greenhouse experiments established that, in fact, distinct differences in development and changes in photosynthetic physiology were evident in plants grown under CO^-enriched conditions. Upon completion of this part of the study the physiological basis for these effects was s t i l l not fu l l y clear. It was decided, therefore, to investigate this problem in greater depth. Conventional greenhouse environments were unsuitable for this second study since they did not permit maintenance of constant CO2 concentration. Therefore, a series of laboratory experiments was undertaken using small growth chambers within which ambient CO^  levels could be closely controlled and monitored. The greenhouse study clearly demonstrated an increase in inherent photosynthetic capacity of leaves developed at a CO2 concentration of approximately 0.09%y I t was not evident whether similar effects would be shown at higher growth concentrations which may be encountered in greenhouses lacking control systems on CO2-enrichment equipment. The scope of the laboratory study was expanded to investigate the growth and physiology of tomato plants at 0.5% in addition to 0.1% and 0.03%''.CO . An attempt was.made to determine whether the observed differences in photosynthetic response between CO2—enriched and non-enriched plants would persist after return to a common, normal air environment. The results of these studies provided considerable additional information on the response of tomato plants to supplemental C0 2 during growth, in terms of observable physiological changes and the underlying mechanisms for these changes. \ 52 PART ii, SECTION:.! EXPERIMENTS ON GREENHOUSE CROPS  MATERIALS AND METHODS i) Selection of Suitable Greenhouses for a Comparative Study (Experiment 2) During the Spring of 1976, an experiment was conducted to compare the growth and fr u i t yield of two tomato crops grown under similar temperature and s o i l conditions in adjacent greenhouses of glass construction. Each house had a floor area 2 3 of 58 m and air volume of 182 m . They w i l l be referred to as House A and B. Seeds of tomato {Lycopersi con esculentum L.) cv. Vendor, (from Stokes Seeds Ltd., St. Catharines, Ont.) were germinated in moist s o i l in flats during February 1976. At the beginning of March, 160 seedlings were transplanted into buckets containing 9 l i t r e s of steril i z e d s o i l with an admixture of 3 g;l 1 Osmocote 14-14-14 slow release f e r t i l i z e r (Sierra Chemical Co., Milpitos, Ca.). Eighty seedlings were placed in rows i n each house with 2 approximately 0.3 m of bench space provided per plant. Hand watering was carried out daily and the plants were subject to occasional pest control procedures (monthly fumigation with Plant Fume 103, Plant Products Co., Bramalea, Ont.). Daytime temperatures in both houses were maintained at 24°C in the early season, and roof ventilators were set to open at this temperature. Night temperatures varied between 15 and 18°C. Three weeks after transplanting, four plants were harvested from each house to determine total leaf area and plant dry weight. Three similar harvests took place at 21 day intervals thereafter. Relative Growth Rate (RGR) and Net Assimilation Rate (NAR) were calculated by means of functions derived from fitted-curves of ^he primary values (leaf area (A) and plant dry weight (W)) against time (t) (286) . A multiple regression program was used to derive suitable curves using the U.B.C. IBM 370 d i g i t a l computer. Input to the program consisted of the dependent variable (W or A) and two independent variables (t and the second degree polynomial, 2 t ). Parabolic functions of W and A against time were calculated for the two crops. Analysis of Variance of each regression showed a l l the relationships to be highly significant (P < 0.01). 1 dW 1 dW Using accepted definitions of RGR = — and NAR = — — the following revised formulae were established in terms of multiple regression functions X(t) = W and Y(t) = A. RGR dt X(t) N A R = dX£tl . _1_ N A K dt Y(t) Mean Leaf Area Index (LAI) was estimated at several dates after transplanting from the regression of A against t, and the bench 54 area occupied by each plant in the respective houses. Crop Growth Rate (CGR) was derived from the product of LAI and NAR at selected dates (169,.). Plants in both houses began to set f r u i t during the second week in A p r i l . Fruit yield was determined by picking and weighing marketable f r u i t from plants as i t became ripe. Picking continued un t i l May 18, 1976. 55 ii) CO,, Enrichment Experiments (Experiments 3 and 4) Cl During January 1977, tomato seeds of the same cultivar as used in the preliminary experiments were germinated in moist mica-peat. Upon emergence in late January, seedlings were planted in mica-peat in 40 small pots, each containing one plant. The pots were randomly divided into two groups of 20 and placed into the two greenhouses used in the preliminary study. When the f i r s t two true leaves were developed, in mid-February, 1977, the seedlings were transplanted into separate plastic bags which contained c_a. 9 1 of Douglas Fir (Pseudotsuga menziesii (Mirb.) Franco) sawdust. The bulk sawdust contained an admixture of 2.55 g 1 1 of 19.5% superphosphate, 4.18 g 1 1 of dolomitic limestone and 1.0 ml 1 1 of minor element solution (183). During growth, nutrient solution containing 5.8 g 1 1 KNO^  plus 0.15 or 0.27 or 0.40 g 1 1 NH^ NO^  (different concentrations associated with different stages of development as specified by Maas and Adamson (183)) was supplied via separate plastic feeding tubes to each bag. The rate of supply of the nutrient solution to each bag was increased in stages from 450 ml day 1 in February to 2500 ml day-1 in mid-April. This nutrient regime conformed to that recommended by Maas and Adamson (183) except that iron was supplied in the minor element solution as FeEDTA (ferrous ethylenediaminetetraacetic acid containing 5 g 1 1 available iron) at the rate of 0.51 ml per 1 of sawdust. 56 Temperature regimes in each greenhouse were similar to those used in the 1976 study although daytime temperatures in both houses rose to 27°C or above during many warm sunny days in April and May. In general, CC^—enrichment procedures were similar to those in use in the local greenhouse industry. The house in which CO-enrichment was applied in 1977 was that which had produced the quantitatively lower yield in the 1976 study. C02-enrichment was begun at the time of seedling emergence and continued u n t i l A p r i l . Each day CO^  was added between 30 min before sunrise and 30 min after sunset. The source was a propane burner (No. 1332, Johnson Gas Appliance Co., Cedar Rapids, Iowa) which produced C0 2 at a constant daytime rate. The burner was thermostatically controlled to switch off i f the house temperature was above 24°C in order to prevent the addition of C02 at times when the roof vents were open. After late A p r i l , continued C02—enrichment was not feasible because the roof ventilators remained open virtual l y a l l day. At two week intervals during the experiment, C02 concentrations in each house were checked over a 24 h period using the conducti-metric analysis system described in Part I of this thesis. During early growth, leaf development of plants in the enriched and normal air regimes (as the greenhouse treatments w i l l be referred to throughout this text) :was assessed using the plastochron index. (87). A reference length of 30 mm was chosen and leaf lengths were measured 57 on 3 replicate plants in each regime using a di a l micrometer. These measurements were discontinued after March..18 because the i n i t i a t i o n of flowering in some plants could have produced non-linearity in the relationship between plastochron index and time (87). After March 18, plant development was assessed in terms of flowering and truss development. At approximately 3-day intervals each plant was assigned a grade on the basis of the number of trusses developed with closed flowers, the number with open flowers, and the number with one fr u i t set on at least one flowerhead. These data were collected u n t i l the study ended on July 9. Fruit yield was determined as described previously with harvests taken at irregular intervals ranging from 3 to 7 days apart. Due to the death from Fusarium wilt of 3 plants in the normal air growth regime in late May, the experimental design became unbalanced. This problem was remedied by randomly discarding f r u i t yield values for 3 plants from the enriched regime. Cumulative yields are therefore expressed as means of 17 replicate plants per growth regime from the beginning of the harvest period. Analysis of flowering, leaf development and yield in the two crops constituted experiment 3 of the thesis research. Experiment 4 was concerned with simultaneous determinations of CC>2 and water vapour exchange by attached leaves carried out during a 3 week period from mid-April to early May. Measurements 58 were conducted on true leaf 5 ("basal" leaf), counting from the f i r s t above the cotyledons, and on the most recently unrolled apical leaf on the main axis of the same plant. Gas exchange determinations were made in situ on two replicate plants per CO^  regime by enclosing the terminal leaflet portion of a compound leaf in a 43 ml, trap type Plexiglas chamber. The leaf chamber was connected in an open gas flow system, and the inlet and outlet ports of the chamber were arranged to create turbulence in the gas stream passing by the enclosed leaf. Gas of known CC^  concentration was supplied to the chamber by mixing CO^-free compressed ai r with air containing 1.0% using a Matheson 7321 gas proportioner. Ingoing concentrations were quite stable and were checked frequently with a Beckman IR 215 CO2 analyzer (IRGA). During measurements, the gas supply to the leaf chamber was maintained at 825 ml min x. The IRGA was used in i t s differential mode to determine the change in CO2 concentration as the gas passed through the leaf chamber. The CO2 concentration differential across the chamber was never greater than 40 ul 1 _ 1 and was- usually less than 20 y l 1 i.. The ingoing air was humidified to a relative humidity between 65 and 75%, and an electric hygrometer (Model 880, EG & G Ltd., Waltham, Mass.) was used to measure the dew point of the gas before and after the leaf chamber. The returning air stream was dried by passing through a calcium sulphate drying column before entering the IRGA. 59 Rates of gas exchange were calculated from the gas flow rate and the changes in CO^ or water vapour concentration as the gas stream traversed the chamber. After a leaflet was used for a series of measurements i t s outline was traced onto coarse f i l t e r paper. The outlined leaf model was cut from the paper, moistened, and used to determine air boundary layer resistance to gas transfer. Stomatal resistances were calculated from rates of transpiration, boundary layer resistances and water vapour pressure within the leaf chamber. The logarithmic relationship discussed in yiarvis and S a t s k y ' (147) was used to estimate mean CG^  and water vapour levels in the chamber atmosphere. Intercellular space CG^  concentrations were calculated as indicated by Moss and Rawlins (210). Me'sophyll resistances ( r m ) t o GO^  uptake w e r e calculated from t h e reciprocal of the slope of the regression relating intercellular space concentrations (Ci) minus CO^ compensation concentration (17) to the flux of C02 in net photosynthesis (F) (146, 177, 282). Mesophyll resistances were not calcuated for the apical leaves of plants grown in the normal air regime since the relationship between CO^ flux and internal CO^  concentration was not significantly linear in that case. Rates of gas exchange were measured under a l l combinations of 4 levels of photosynthetically active radiation (PhAR; 50, 150, -2 -1 250, and 750 yE m s , 400 to 700 nm) and 4 concentrations of C00 (240, 340, 440 and 540 y l 1 _ 1 ) . The enclosed leaf was allowed 60 to equilibrate for 20 to30 minutes after each change of test conditions. Leaf and a i r temperatures were monitored continuously using 0.2 mm diameter copper-constantan thermocouples pressed to the abaxial surface of the leaf and positioned i n the a i r stream adjacent to the gas i n l e t ports. Light was provided by either a 100 W incandescent lamp situated d i r e c t l y above the chamber or by a 300 W cool beam lamp (General E l e c t r i c Ltd.) 1 m -2 -1 above the chamber (for 750 yE m s only). Figure I I - l shows the spectral energy d i s t r i b u t i o n of radiation from each lamp as measured by an ISCO model SR spectroradiometer. PhAR was determined by a Lambda Instruments Inc. (Lincoln, Nebraska) LI 190 S Quantum Sensor with a LI 185 meter. Several layers of cheesecloth interposed between the lamps and the chamber were used to adjust the PhAR l e v e l s . Leaf temperature in the chamber was maintained at 25 ± 2°C by c i r c u l a t i n g water through a 3 cm deep jacket attached to the top of the chamber. Rates of dark respiration and CO^ compensation points were determined for apical and basal leaves on three randomly selected plants from each growth regime. Compensation points for irradiance were calculated by interpolation between the CO2 flux of the lowest l e v e l of PhAR and the relevant mean dark respiration rate. These measurements involved a simple closed gas analysis system incorporating an a i r pump, the leaf chamber and the CO2 analyzer. Leaf areas were determined using a photoelectric planimeter (Hayashi Denko Co. Ltd., Japan). 66a FIGURE I I - l : Spectral energy distribution of 100 W incandescent lamp and 300 W cool beam lamp used to illuminate assimilation chamber in Experiment 4. 62 Certain behavioral indices of photosynthesis were calculated for apical and basal leaves grown under CG^-enrichment, and for basal leaves grown in normal a i r . The indices were derived from a model for leaf CO^  assimilation described by Van Bavel (14). The model allows the flux of CC^  in photosynthesis to be separated into two elements depending upon: (a) the radiant flux density and associated photochemical u t i l i z a t i o n efficiency of incident radiation, and (b) the internal CC^ concentration and associated CC^  u t i l i z a t i o n efficiency. The model yields two parameters, E and E respectively defined as the efficiencies of radiant energy and CO^  u t i l i z a t i o n . The model can also be used to estimate the theoretical maximum flux of C0„, FTl,, which would 2 IM occur at fu l l y saturating levels of PhAR and CG^  concentration. The calculation of each parameter was carried out as described by Van Bavel (14), and i s reviewed in Appendix 2'.... S t a t i s t i c a l analysis of the gas exchange results was carried out .by multiway analysis of variance (ANOVA) on an IBM 370 di g i t a l computer. In order to elucidate differences i n photosynthesis rates between growth regimes, separate ANOVA models were used for basal leaves and apical leaves. This approach provided an alternative to pooling the data for different ages which preliminary tests had shown to be significantly (P < 0.05) different. A two stage multiple range test (Student-Newman-Keuls, a = 0.05) was included in the analysis to provide a method of mean separation. 63 S t a t i s t i c a l tests applied to other data i n the study are i d e n t i f i e d , wherever appropriate, i n the text and i n fi g u r e and table headings. 64 PART II, SECTION 1  RESULTS AND DISCUSSION i) Comparative Study Concerning Growth of Tomato Crops in  Unenriched Greenhouses (Experiment 2) The functions derived from multiple regression analysis of the primary growth parameters A and W against t are list e d in Table II—1. These functions were subsequently used to calculate values for RGR, NAR and CGR by differentiation at various dates after transplanting and to produce the curves shown in Figures II-2 to II-4. The forms of the expressions used to estimate the respective growth parameters are shown in the corner of each figure. Mean RGR and NAR as well as CGR were considerably higher i n house A as compared with house B. Specific reasons for the discrepancies in vegetative growth between the two crops were not investigated. Temperature regimes and cultural conditions were unlikely sources of the variation between the two houses. It i s more probable that the differences were due to higher incident light levels throughout the growing period in house A, and this unfortunately represented an important uncontrolled variable throughout these and subsequent experiments. The importance of irradiance in determining growth rates and NAR for plants and crops is well established. In general, RGR, CGR, and NAR decline proportionally with a reduction in incident radiation (28, 289). TABLE I I - l : Functions of plant dry weight and leaf area with-time derived from multiple regression equations for tomato plants grown i n experimental greenhouses A and B, without CO^  enrichment. Coefficient: Leaf Area (A) HOUSE B Coefficient: Dry Weight (W) Const, 2065.2 A Const, 10.0 w 119.4 W 1.6 0.1 c -0.002 W HOUSE A Coefficient: Leaf Area (A) Coefficient: Dry Weight (W) Const, 5215.8 A Const, 16.6 W 169.2 W 1.4 -2.1 c-, -0.004 W Function for W = Consty + b w ( t ) + cy(t ) with time Function for A = Const A + t>A(t) + c A ( t ) with time Where t = time in days from transplanting 65a FIGURE II-2: Time course of RGR derived from f i t t e d curves, f o r tomato crops grown i n experimental greenhouses A and B without CO -enrichment. D A Y S F R O M T R A N S P L A N T I N G 66a FIGURE II-3: Time course of NAR derived from f i t t e d curves, f o r tomato crops grown i n experimental greenhouses A and B without C0„ enrichment. DAYS FROM T R A N S P L A N T I N G 67a FIGURE II-4: Time course of CGR derived from values of NAR and LAI, f o r tomato crops grown i n experimental greenhouses A and B without C0o enrichment. DAYS FROM TRANSPLANTING The clearcut differences i n rates of vegetative growth and dry matter a s s i m i l a t i o n contrasted sharply with s i m i l a r mean f r u i t y i e l d s on a per plant basis obtained i n each greenhouse (Table II-2). Evidently, the environmental factor or factors which caused the growth discrepancies did not a f f e c t f r u i t p r o d u c t i v i t y on the plants i n the two houses. Largely on the basis of t h i s close s i m i l a r i t y , i t was decided to proceed with the CO - enrichment experiments i n these l o c a t i o n s . The unavoidable confounding of greenhouse and treatment (C02~enrichment versus normal a i r ) was ameliorated by supplying a d d i t i o n a l CO2 i n the house which produced lower growth and net a s s i m i l a t i o n rates and q u a n t i t a t i v e l y lower f r u i t y i e l d ( i . e . house B), i n the preliminary study. I t seemed reasonable to assume that any demonstration of increased f r u i t y i e l d , rate of vegetative or reproductive development by a crop grown i n t h i s house would constitute strong evidence f o r the effectiveness of the enrichment regime. TABLE II-2: Mean fr u i t yield from tomato plants grown in experimental greenhouses A and B, from March 16 to May 18, 1976C without CO -enrichment. House A House B Mean frui t fresh weight (g per 606.7* 598.8 plant) * d i f f e r e n c e i n y i e l d between houses not s i g n i f i c a n t (P > 0.05) by t - t e s t . 71 ii) CO ^ Concentrations in CO -Enriched and Control (Normal-Air)  Greenhouses The results in Figure II-5 are representative of the house CO^  concentration measurements taken during early and late stages of the second part of the greenhouse experiment. On March 12, roof vents opened infrequently and daytime concentrations remained between 800 arid 1000 y l 1 X. On warmer days-(as exemplified by data for April 23), vent opening was more frequent and CO2 levels in the enriched house ranged between 550 and 850 y l 1 x. Daytime maximum and minimum CO2 levels recorded i n the unenriched house were often well below 300 y l 1 x. The lower concentrations on April 24 compared to March 13 occurred despite more frequent vent opening and may be correlated with an increase i n foliage within the house, between these two dates. 71a FIGURE I I - 5 : Atmospheric CO^ concentrations i n C O 2 -enriched and normal a i r (unenriched) greenhouses. \C0 2 enriched house A March 12 C0 2 enriched house / \ April 23 0700 0900 1100 1300 1500 1700 1900 2100 0700 0900 1100 1300 1500 1700 1900 2100 T I M E O F D A Y K) 73 iii) Plant Growth and Held (Experiment 3) Early leaf production by plants grown in the CO^-enriched house was quantitatively higher than production by plants grown in normal air (Table II-3) but this difference was not s t a t i s t i c a l l y significant. This observation corresponds closely with those of previous studies where CG^-enrichment to 1000 y l 1 1 has been shown to have negligible or very small effects upon the rate of leaf development (137, 215). In this respect the effects of CX^ -enrichment differ from those of other environmental parameters known to enhance tomato growth such as high light intensities and temperatures. In several cases these parameters have been shown to increase leaf production (46). If should be noted, however, that since leaf production in the present study was recorded only in terms of the plastochron index, i t i s possible that unobserved aspects of leaf production, such as area or dry weight accumulation, might have been influenced by regime during growth. Wittwer and Robb (29,8), for example, noted an increase of ca. 25% in total dry weight of lettuce leaves grown under 1000 y l 1 1 compared to leaves produced under 400 y l 1 1-CO . The impact on vegetative growth of similar levels of CO^—enrichment has also been demonstrated i n terms of increased relative growth rates and net assimilation rates (273). In the present study, CO — enrichment caused a distinct advancement of truss development (Figure II-6). Flowers opened ca. 3 TABLE II-3: Time course of le a f development i n tomato plants grown with or without CO enrichment. Plastochron Index Date (1976) A i r Regime CO -Enriched Regime Feb. 28 5.30±0.89* 5.99±0.37 Mar. 2 5.91±0.62 6.69±0.44 Mar. 8 8.73±1.31 9.16±0.47 Mar. 12 9.72±1.32 10.9'3+0.53 Mar. 15 11.11±1.17 12.31+0.67 Mar. 18 11.58±1.60 13.43±0.58 *Values shown are means ± 1 standard deviation from 3 r e p l i c a t e plants per regime. Differences between regimes at any date were not s i g n i f i c a n t (P > 0.05) by t - t e s t . 74a FIGURE II-6: Truss development. Open bars: C02~enriched growth regime. Cross-hatched bars: normal a i r growth regime (cross-hatching superimposed): A: Number of trusses developed with f r u i t set on at l e a s t one flowerhead. B: Number of trusses developed with open flowerheads. C: Number of trusses developed with closed flowerheads. NOTE: Data are means for 17 r e p l i c a t e tomato plants per treatment. At each date, A + B + C are s i g n i f i c a n t l y d i f f e r e n t between treatments according to Whitney-Mann test for non-parametric data (P < 0.001). 7.0 6 0 5 0 -4 0-3 0 2-0-1 0 -FRUIT SET TRUSSES WITH OPEN FLOWERS TRUSSES WITH CLOSED FLOWERS 25-3 28-3 2 - 4 5-413 - 4 18-4 26-429-4 3-5 9-5 13-516-519-5 24-530-5 2-6 6-6 DATE (day-month) 76 days earlier in the enriched plants, beginning at about 6 weeks after seedling emergence, and the number of trusses bearing f r u i t after April 13 was always significantly higher under the enriched treatment. These results are in agreement with most other studies in which flowering and f r u i t set in a number of different tomato cultivars has been shown to be increased by 3 to 5 days as a result of C02-enrichment to 1000 y l 1 _ 1 (49, 116, 298). They do not correspond with the results of Knecht and O'Leary (163) who found no significant difference between numbers of open flowers sixty-four days after seeding on tomato plants grown at 400, 800, and 1200 y l 1 X Those plants, however, were cultivated i n growth chambers under a r t i f i c i a l light i n contrast to the other, greenhouse experiments, which may explain some of the differences between them. Plants exposed to the higher CO^ levels also showed greater frui t yields throughout the harvest period (Figure II-7) and differences in cumulative fru i t yield were accentuated toward the end of the experiment. Yield increases in response to CO^-enrichment in tomatoes have been widely reported (165, 189, 297., 298), the magnitude of the increase over normal air grown plants depending on other cultural conditions, notably light and temperature .regimes (116). The percentage increase (ca. 30%) in marketable f r u i t yield as a .result of CO-enrichment in the present experiments was consistent with that obtained in many commercial and experimental greenhouses for Spring-grown tomato crops (165). J 7 6 a FIGURE II-7: Cumulative f r u i t yield for plants grown in CO2-enriched and normal air growth regimes. NOTE: Data are means of 17 tomato plants per treatment. Differences at any date after May 24 are significant according to t-test (P < 0.05). 77 78 iv) Leaf Photosynthesis (Experiment 4) It i s often assumed that growth and yield increases resulting from CO^-enrichment are brought about by increased rates of photosynthesis. There i s , however,- very l i t t l e information relating to the photosynthetic responses of plants which have been grown at supra-normal CO^  concentrations (185). In order to elucidate the basis for the differences in yield and plant development between CO^-enriched and normal air grown plants i t was decided to evaluate some components of photosynthetic CO^  exchange in plants from each regime. Under comparable test conditions of PhAR and CO^  concentration, the net rates of photosynthesis of basal (Fig. II-8) or apical (Fig. II-9) leaves were generally greater in leaves developed under CO - enrichment. The overall pattern of the responses to variations i n PhAR and test CO2 concentration, however, was similar for leaves grown i n the enriched and normal air regimes. Sta t i s t i c a l analysis of these results showed significant (P <0.05) interactions between growth regime, PhAR, and CO^  concentration. A more direct impression of the effect of growth regime on leaf CO^ exchange is given by Fig. 11-10 which shows the difference in net photosynthesis rate (APs) between leaves grown in the two regimes. In both basal and apical leaves, APs tended to increase with increasing C09 concentration (Fig. II-10A). Increasing PhAR 78a FIGURE: II-8: Rates of apparent photosynthesis in basal leaf 5 of plants grown in normal air (- --•• ) and CC^-enriched (•———•) growth regimes. NOTE: Data are means of 2 tomato plants per treatment. Points marked with the same letter are not significantly different (P > 0.05) according to SNK multiple range test. 79a FIGURE I I - 9 : Rates of apparent photosynthesis in f i r s t unrolled apical leaf of plants grown in normal a i r (- •*) and C02~enriched (• • ) growth regimes. NOTE: Data are means of 2 tomato plants per treatment. Points marked with the same letter are not significantly different (P > 0.05) according to SNK multiple range test. APPARENT > oo o 80a FIGURE 11-10: Differences in apparent photosynthesis rates between tomato plants grown in C02~enriched and normal air growth regimes (APs), in relation to (A) concentration and (B) quantum flux density. Points represent mean APs for a fixed level of one factor over a l l tested levels of the other. Apical leaves: A A;. Basal leaves: A A. g apical leaves basal leaves 1 1 1 1 200 400 600 800 QUANTUM FLUX DENSITY (JJE m ' 2 s"1. 400-700nm) 82 also increased APs in basal leaves, but no consistent trend was evident with apical leaves (Fig. II-10B). The data suggest that APs was more responsive to changes in test CC^  concentrations than changes'in PhAR. Thus, in basal leaves, a 2.25-fold increase in test CC^  concentration caused a 1.8-fold increase in APs, while a 15-fold increase in PhAR resulted in a 4.5-fold increase in APs. It i s worth pointing out, however, that over the range of test levels of PhAR used in these experiments, leaf photosynthesis undoubtedly varies between a state of irradiance limitation to near irradiance saturation. At the highest test CG^  concentration, on the other hand,, photosynthesis is probably proceeding at less than half i t s maximum rate (103). These differences probably hold important consequences for the relative effects of varying PhAR and CO2 concentration on APs. The tendency of APs to increase at progressively higher levels of PhAR and CO2 concentration is in contrast to the results of:some other studies. Bishop and Whittingham (21) found persistent increases in photosynthesis rate of tomato seedlings grown in 1000 y l 1 CO2 following transfer to normal a i r at low light intensity, but not at high intensity. More recently, i t was found (102) that the transfer of cucumber seedlings from a growth concentration of 1500 y l 1 ' _ . CO2 to normal air resulted in lower photosynthesis rates -2 at an irradiance of 98 W m than in seedlings cultivated in normal air . At approximately half that irradiance, there was no difference 83 between the two regimes. It has been suggested (21) that the modification of photosynthesis rates following CC^-enrichment may be related to changes in pool sizes of photosynthetic intermediates. There are many- other possible explanations and some recent work has suggested that after growth of plants:in CO^-enriched conditions changes in activities of enzymes involved in CC^  exchange may be important (92). The results shown in Table II-4 indicate that compensation points were reduced following growth under CO^ - enrichment. The magnitude of the CO^  compensation point often reflects the rate of photorespiration (7$, 196;, 278) . The present results, therefore, raised the possibility that changes in photo-respiratory activity may contribute to the alterations in net photosynthetic rates. The CC^  compensation point differences prompted further study of photorespiration and the activity of enzymes involved in CC^  exchange in CG^-enriched plants. The results of these studies w i l l be reviewed i n Part I I , Section 2 of this thesis. It is well known that the photosynthetic capacity of leaves changes with age (6, 97, 148, 161, 223, 248, 256). Generally rates of photosynthesis rise to a maximum shortly after leaf emergence and thereafter decline. In accord with this, net photosynthesis rates in apical leaves tended to be about double the rates of older basal leaves (Figures II-8 and II-9). The results also indicate a different response to growth under C0„-TABLE II-4: compensation points for leaves of tomato plants grown with or without C0„-enrichment. CO2 Compensation Point (yl 1 ^ ) Air Regime C0 2-Enriched Regime Apical leaves 55 * 46 b a Basal leaves (5th) 55 44 b a *Values are means of 3 replicate determinations. Values designated with the same letter are not significantly different (P > 0.05) by • AN OVA and SNK test. enrichment between the apical and basal leaves. In apical leaves, values of APs tended to be much more sensitive to changes i n test CC>2 concentrations (Fig. II-10A), and possibly less sensitive to changes in PhAR (Fig. II-10B), than in basal leaves. CO^  compensation points, however, were not significantly affected by leaf age (Table II-4). The differences in effects of CO^ -enrichment between apical and basal leaves could be due to a number of factors. Apical leaves clearly emerged much later than basal leaves and were therefore exposed to the aerial environment for a shorter time period. I t i s possible that different times of exposure to CO^-enriched conditions or differential effects of elevated CO^  concentration on the photosynthetic system of young and older leaves were responsible for the observed differences. 86 v) Resistances to C0„ Assimilation Stomatal behavior was also influenced by growth regime and by conditions during the gas exchange measurements (Fig. 11-11). In plants grown in normal a i r , stomatal resistances under comparable test conditions were lower in apical leaves than in basal leaves. Similar differences were not obvious with leaves from the CG^-enriched plants, and this may indicate a need to consider apsects of leaf development and growing conditions when undertaking studies on stomatal physiology. Stomatal resistances tended to decrease with increasing PhAR irrespective of growth regime, although this tendency was not strong in apical leaves from the normal air regime which consistently had low resistances. Overall, the effect of test concentration on stomatal resistance was not s t a t i s t i c a l l y significant (P < 0.05). Other investigations have often observed increased stomatal resistances at higher than normal CO^  concentrations in various plant species (99, 123, 152). • The relative stomatal insensitivity-to test ' C02 concentration found in the present study, however, coincides with the observations of Pallas (225) who examined tomato leaves over a wide range of CO2 concentrations. Mesophyll resistance was always considerably greater than stomatal resistance at equivalent levels of PhAR (Fig. 11-11), and i t was therefore the dominant resistance to CO 2 assimilation under the conditions tested in this study. Because of the procedure used for i t s calculation, mesophyll resistance is in turn composed of elements related to liquid phase transport of CO2 and to carboxyla-tion at the photosynthetic sites within leaf cells (146, 171)-; Since FIGURE 11-11: Leaf resistances to C0 2 transfer. NOTE: Data for r (stomatal resistance) are means s of 2 determinations per treatment, r m represents mesophyll resistance calculated as described in the text. C0 2 represents test C0 2 concentration. Bars show one standard deviation either side of the mean for test C0 2 concentration = 340 y l 1 1 (standard deviations for other r means were s of similar magnitude). 87 — i 1 1 I ' 1 1 1 1 200 400 600 800 200 400 600 800 Q U A N T U M F L U X D E N S I T Y (uE m" 2 s\400-700nm) 88 carboxylation i s dependent on photochemical events, mesophyll resistance can be strongly affected by PhAR (e.g. 19, 291). The contribution of carboxylation resistance to mesophyll resistance i s very large at low PhAR levels and is minimum at light saturation. Also, Jarvis (146) and Whiteman and Roller(292)have noted that mesophyll resistance calculated as described in this study i s overestimated unless the gas exchange determinations are made using conditions of light saturation and CO^  limitation. Clearly (Figs. II-8 and II-9), many of the conditions used in the present study do not satisfy those c r i t e r i a . The values for mesophyll resistances found in these experiments (Fig. 11-11) are considerably larger than those reported for some other plant species. Gaastra (103) reported mesophyll resistances between 2 and 10 s cm 1 in turnip leaves, and Bierhuizen and Slatyer (19) found similar values for cotton leaves. The values calculated from the present data are, however, of similar magnitude to those reported by Ku et al. (167) for potato leaves. Mesophyll resistances tended to be higher in basal leaves (Fig. 11a) than in apical leaves (Fig. lib) on CO^-enriched plants. These differences were particularly evident at low (50 yE m~2 s"1) and high(/50 yE m~2 s _ 1) levels of PhAR. In comparing basal leaves from the two growth regimes, similar mesophyll resistances were obtained at the higher PhAR levels -2 -1 used, but at low levels of PhAR (50 and 150 yE m s ) these 89 resistances were much lower in leaves from the CO^-enriched regime. As discussed above, individual contributions of the components of mesophyll resistance to these differences could not be assessed, and i t is possible that they were due either to induced changes in liquid phase resistance to CC^ transfer in the mesophyll, or to changes in carboxylation efficiency dependent upon enzyme activity (RuBP carboxylase) or upon photochemical reactions. More than one of these factors may, of course, be implicated, but the differential effects of low and high irradiance strongly suggest the involvement of changes in photochemical-dependent fixation reactions. 90 vi) Behavioral Indiaies of Photosynthetic Responses to CO ^ -Enrichment The confounding of components within mesophyll resistance described above has made i t desirable to model photosynthesis without reference to internal inseparable resistances. The behavioral model of Van Bavel (14) which describes leaf assimilation i n terms of radiant flux density, internal C02 concentration and associated ut i l i z a t i o n efficiencies (Ej and E^), is particularly relevant to the present study. Values of E„, E T, and FT>;r calculated from the present C I IM data are summarized in Table II-5. E^ , was significantly higher in basal leaves of CO^-enriched plants than in comparable leaves of those grown in normal air. However, values of E^ did not differ between those treatments. In the CO -enriched treatment E was 2. t» somewhat lower in apical than in basal leaves but E^. was almost twice as great. Clearly, the relatively high photosynthesis rates and F ^  value for these apical leaves was associated with a higher efficiency of PhAR util i z a t i o n . This is consistent with the results of McCree (181) who found that the highest quantum yield (rate of photosynthesis per unit rate of quanta absorption) was obtained for sections of the youngest leaves of corn plants. In the terms of the Van Bavel model, E^ . is the maximum possible quantum yield although in these calculations, this parameter i s based on incident rather than TABLE II-5: Behavioral indices f o r photosynthetic C O 2 exchange: C 0 2 u t i l i z a t i o n e f f i c i e n c y ( E c ) , radiant energy u t i l i z a t i o n e f f i c i e n c y ( E T ) , and maximum photosynthetic CO f l u x ( F I M ) . Leaf Growth Regime E r E I _ i _ 5 (g y E i n s t e i n x 10 ) 2 > g m ^ s x x 10--5 Basal A i r 2.8±0.5* 0.27±0.07 0.85±0.20 C0 2~Enriched 10.9+0.6 0.27±0.02 1.00±0.20 Apic a l A i r — — — CO -Enriched 7.5±0.5 0.50±0.03 1.50±0.21 *Values shown are means ± 1 standard deviation. 92 absorbed quanta. In view of the low reflectance and trans-mittance relative to the absorbance of leaves over the waveband 400-700 nm, i t is doubtful that the difference between incident and absorbed PhAR results in more than a small, constant error in values of E^ .. The value of E for basal leaves from the normal air regime is of similar magnitude to that reported previously for the 12th leaf of a sunflower plant (14). The values obtained for the other leaves in these experiments are considerably higher than this, suggesting that E^ may be very sensitive to supplemental during growth and leaf age. Values for E^ . and F are about an order of magnitude lower than those reported for the sunflower leaf, and this may account for the relatively low rates of net photosynthesis in tomato compared with those reported for sunflower (14). Although i t i s clear that E^ , and E^ are influenced by growth regime and/or leaf age, the physiological changes underlying these responses are obscure. Modifications in leaf structure, chloroplast photophysiology, and the activities of photosynthetic enzymes may be involved, but the relative importance of such modifications i s not evident from the present data. In studies of the aging of bean leaves, Fraser and Bidwell (97) observed a repetitive pattern of changes in photosynthetic activity in . successive leaves. They suggested that this pattern may be controlled by intrinsic factors. The data i n Table 5 seem to 93 indicate that aging is accompanied by changes in efficiencies of both radiation and possibly carbon dioxide u t i l i z a t i o n . The validity of the behavioral model (14) used in these calculations i s supported by the accuracy of prediction of net CC^  assimilation rates from values of E^, E^ ., and (Table II-6). Calculated rates (only those for a test CC^  concentration of 340 pi. .1 have been shown here) were closely correlated with observed rates over the range of CO^  concentrations and light intensities used in these experiments. The present study thus demonstrated the applicability of the model to an additional plant species grown under various environmental conditions. This research has, therefore, indicated that the increases in growth and yield which result from C02_enrichment may not simply be due to an increased concentration gradient driving the diffusion of CO2 from the greenhouse air to the chloroplasts. Physiological modifications occurred in plants grown at high CO2 levels which increased inherjentt rates of C0 2 assimilation and decreased CO2 compensation points relative to those plants grown in normal a i r . Also,the responses to CO2- enrichment during growth differed in leaves of different physiological ages. It i s apparent that increased tomato yields following CO2-enrichment are at least partially due to these changes. The experiments so far performed were designed to provide general descriptive information on the physiology of tomato plants grown under C0?-enriched and normal air conditions. No special TABLE II-6: Comparison of observed rates of net photosynthesis with rates predicted by the model of Van Bavel, for a test CO. concentration of 340 y l 1 ±. Leaf Growth Regime Incident Radiation yE m~2 s ~ x (400-700 nm) Rate of Observed Net (g/m" Photosynthesis "2 s- ]xl0 4) Predicted Basal Air 50 0.42 0.29 150 1.13 1.29 250 1.74 1.91 750 2.04 3.05 c o 2 - 50 0.61 0.62 Enriched 150 1.70 2.33 250 2.42 3.09 750 3.02 4.88 Apical c o 2 - 50 1.20 1.30 Enriched 150 2.14 3.20 250 3.45 3.90 750 6.07 6.90 emphasis was placed on determining underlying mechanisms for the observed e f f e c t s of CO^-enrichment on the physiology of CO^ exchange. The following section w i l l describe a seri e s of experiments which were devised to focus more attention on these mechanisms. 96 PART II, SECTION 2  EXPERIMENTS ON SEEDLINGS GROWN UNDER  CONTROLLED ENVIRONMENT CONDITIONS MATERIALS AND METHODS i) Growth Chambers and Control Systems An integrated system of 8 plant chambers (Fig. 11-12) was designed to satisfy the specific needs of the laboratory experiments. The skeleton of the system was as previously described (234) but the chambers and control systems they used were considerably modified for use in the present study. Each chamber was a 30 x 30 x 46 cm plexiglas box surmounted by a 6 cm deep water bath (Fig. 11-13). Separate inlet ports were provided for a i r and CO^  supply to each chamber, and the internally mounted fan facilitated mixing of the atmosphere. Different concentrations of CO^  were maintained i n different chambers by supplying compressed air from a central source and pure, compressed CO^  at different flow rates. The flow rates needed to achieve a given atmospheric concentration varied only slightly from chamber to chamber. Where normal air concentrations (0.03%) were required the compressed air flow rate was maintained at 4.5 1 min ^  and no additional CO2 was added. Gas flow to each chamber was controlled by a pair of rotameter flowmeters incorporating needle values (Matheson tube 600 or 610 96a FIGURE 11-12: Diagram of 8-chamber growth system used for Experiments 5, 6, 7, and 8. FIGURE 11-13: Diagram of one chamber of the system shown Figure 11-12 ( f o i l facing removed to show i n t e r n a l d e t a i l ) . 98 99 for CC^j and tube 603 for a i r ) . Each pair was connected to one chamber by means of 0.6 cm internal diameter Tygon plastic tubing. Gas outlet ports, near the top of each chamber, were connected to three-way solenoid values which normally exhausted the chamber ai r . Monitoring of the outlet gas stream from a single chamber atmosphere was possible by activating the appropriate solenoid valve, thus directing gas flow from the chamber through a manifold, a calcium sulphate drying column,and into the sample c e l l of an IRGA (Beckman Instruments Ltd., Model 864). Illumination of the chambers was by two banks of mixed i i fluorescent and incandescent lights. Each bank (illuminating four chambers) consisted of 20 high output fluorescent tubes (135 W) and 15 incandescent bulbs (40 W). The lamps were positioned at a height above the chambers sufficient to provide an irradiance of -2 -1 150 UE m s (400 - 700 nm) (approximately 12,000 lux) at plant level. The radiation was filt e r e d through 6 cm of cooled water in the chamber bath before i t was incident on the plants. The radiant spectral energy distribution did not vary between the chambers, and a representative plot based on data obtained with an ISCO spectroradiometer on several occasions during the four month course of the experiments, is shown in Figure 11-14. In these experiments, photoperiod was kept constant at 12 hours light and 12 hours darkness; the lamps were set to switch on and off automatically at 0700 h and 1900 h each day. The sides 99a FIGURE 11-14: Spectral energy distribution of mixed fluorescent and incandescent lamps used in the system shown in Figure 11-12. 100 o *x *x "x * x x (N cn ^ i n ro * -w u UD S^OM ri A1ISN3Q " I V H I O B d S 101 and bottom of the chambers were shielded by facing with aluminum f o i l , and, during the dark period, a screen of black cloth moved automatically over the chamber tops to exclude extraneous room light. Air temperature was not controlled but was continously monitored by means of a shielded copper-constantan thermocouple placed in each chamber. Temperatures were quite consistent between chambers during the light and dark periods, a differential of 4°C being the maximum observed under any conditions. Excessive heat was removed by means of the flowing water bath located on each chamber top (Figures 11-12, 11-13). Chamber air temperatures varied between 30 and 35°C during the light period whereas during darkness they were consistent at approximately 25°C. Chamber humidity varied between 80 and 90% relative humidity throughout the experiments. Throughout the experiments plants within each chamber werf. watered two times per week. On each occassion the nutrient solution of Epstein (86) was used except that a 1 M stock solution of potassium dihydrogen phosphate was used in place of ammonium dihydrogen phosphate, and no potassium nitrate was added. 102 ii) Gas Exchange Measurement System The system used to measure rates of CO^  and water vapour exchange in these laboratory studies was similar to the portable system used in the greenhouse study. The fixed location of the apparatus, however, allowed certain improvements to be made to some of the components, which w i l l be described here. The system was, once again,of an open flow design. The IRGA (Beckman Instruments Inc. Model 1^215) was used to establish the appropriate concentration in the air stream supplied to the chamber and to measure the CO2 differential of ingoing and outgoing streams. Throughout measurements of gas exchange at normal (21%) oxygen concentrations, C02~free air and 1% CO2 in air were mixed to give a required CO2 concentration as described previously (Part I I , Section 1: Materials and Methods). For measurements at low oxygen concentration, nitrogen and 3% CO2 in nitrogen were similarly mixed, and the composition of the resulting gas was checked by means of the IRGA and a Servo.mex model OA. 184 oxygen analyzer. Chamber inlet air was humidified to between 65 and 75% relative humidity as measured by the EG'&G Ltd. Model 880 electric hygrometer, and flow rate was monitored by means of a Datametrics model 800 VTP mass flow meter and sensor. Throughout a l l gas exchange measurements, gas flow was maintained at a sufficiently high velocity to limit the C0o differential across the chamber to < 40 y l 1 x. For most measurements, this was achieved by maintaining gas flow at 800 ml min x. Details of the assimilation chamber used in these studies are shown in Figure 11-15. The fan mounted in the bottom of the chamber maintained conditions of turbulence in the air about the leaf. Air .and leaf temperatures were measured using 0.2 mm diameter copper-constantan thermocouples placed near the air inlet ports and against the abaxial leaf surface, respectively. Light was provided to the chamber by two 300 W cool beam lamps (General Electric Ltd.) vand was filte r e d through 5 cm of water before i t was incident on the chamber. Irradiance was varied by means of cheesecloth f i l t e r s and by varying the number of lamps used. PhAR was measured as described previously (Part I I , Section 1 Materials and Methods). The spectral energy distribution of the lamps was similar to that shown for the 300 W lamp i n Figure I I -During a l l experiments leaf temperature was maintained between 30 and 33°C by circulating water through the 2 cm deep bath mounted on the top plate of the chamber. FIGURE 11-15: Diagram of assimilation chamber used in Experiments 6 and 7. 104 SIDE VIEW 105 iii) Experimental Design i i The laboratory study consisted of four experiments. The f i r s t to be described here (Experiment 5), occupied approximately 6 weeks commencing i n early April 1978. It was designed to provide information on vegetative growth and development of plants exposed to different CO2 concentrations during growth. Seeds of Lycopersicon esculentum L. (cv. Vendor) were germinated as described in Part I I , Section 1: Materials and Methods^at three day intervals and allowed to develop under a 12 hour photoperiod (14000 lux;180 yE m~2 s _ 1 (400 - 700 nm)) for 14 days from sowing on a greenhouse bench. Daytime and nightime temperatures were maintained at 25°C and 18°C respectively. At the end of the 14 day period, six uniformly developed seedlings were transplanted into 10 cm square pots (one seedling per pot) which were transferred to one of the plexiglas growth chambers. The CO^  concentration within the chamber was maintained at 0.03% as described previously. At three day intervals oth°r seedlings were similarly transplanted and transferred to other chambers supplied with air containing 0.1 and 0.5% C02, respectively. Other growth conditions were maintained as described above. A total of six chambers were used in this study, providing two replicate locations for each CO treatment (0.03, 0.1 and 0.5%). Before placement in the chambers, three of the six plants were randomly selected for subsequent harvest. The others were 106 labelled for identification, and the lengths of a l l unrolled leaves on these plants were measured. The leaf measurements were then repeated at three day intervals for the next three weeks. The mean developmental stage of plants at each measurement date in the three growth regimes, was calculated in terms of Plastochrons from the leaf length data (Part II, Section 1: Materials and Methods). At the end of the f i r s t week in the growth regimes (three weeks from germination) the previously selected plants were harvested to determine leaf area and total plant and leaf dry weight. Three weeks later, the rest of the plants were similarly harvested. The data obtained were used to calculate RGR, NAR and Leaf Area Ratio.(LAR) of plants in each chamber. The growth analysis formulae given by Kvet et al. (169) were used in each case. Single factor ANOVA was used in conjunction with a two-stage SNK test (a = 0.05) to distinguish between the mean values for growth analysis parameters in each growth regime. Experiment 6 occupied a period of three months from January to March 1978 and was designed to study effects of CO^  concentration during growth on subsequent CO2 exchange response to irradiance and.CO2 concentration in atmospheres containing normal (21%) and low (0 - 1%) oxygen. Tomato plants were raised from seed as described for Experiment 5. Six plants were placed in each chamber at three day intervals. CO^  concentrations and growth conditions were the same as in Experiment 5. The course of plant development was 107 followed by monitoring Plastochron Index (PI). When a majority of plants within any one chamber had attained a PI value of 5, one of the majority was randomly selected for use in measurements of leaf gas exchange. Upon completion of the measurements the plant was returned to the same chamber to permit continued development under the same growth conditions as before. A second series of measurements were carried out on the same leaf when the plant had attained a PI value of 10.5.. These gas exchange measurements were carried out on leaf 3 (counting from the f i r s t true leaf above the cotyledons). Each measurement series was begun by measuring photosynthesis rate at 500 -2 -ls-or520 yE m s (400 — 700 nm, irradiance under normal atmospheric conditions, and this was followed by measurements at -2 -1 4 lower irradiances (^ 00, 200, 130, 32 yE m s ) and complete darkness. Leaf irradiance was then readjusted to a level -2 -1 sufficient to saturate photosynthesis (520 yE m s in each case), prior to a series of measurements at 5 different CO^  concentrations (540, 440, 340, 240, 140 u l 1 _ 1 ) . After each change of irradiance and/or CO 2 concentration, the leaf was allowed to adjust to the new chamber conditions for at least 30 minutes. Measurements under conditions of saturating irradiance and the same five CO2 concentrations were subsequently repeated in atmospheres containing 0 - 1% 0^. The CO 2 compensation point for the leaf was then determined using a simple closed gas exchange system as 108 described i n Part II, Section 1: Materials and Methods. Finally the; leaf was removed from the chamber and carefully photocopied. Area was determined from the leaf outline using a photoelectric planimeter (Hayashi Denko Co. Ltd., Japan). In these and the subsequent replacement experiments, rates of gas exchange were again calculated from gas flow rates and measured CO2 and water vapour concentration differences between the leaf chamber ingoing and outgoing gas streams. Intercellular space CO2 concentrations, and boundary layer and stomatal resistances were also calculated as described in the greenhouse study. Mesophyll resistance to C0^ uptake was calculated from the reciprocal of the regression slope relating the intercellular space C0 2 concentration (C^) plus CO2 compensation concentration (P) to rate of net photosynthesis (F), so that estimates of photorespiration rate (R ) could be obtained by extrapolating JLi each regression to zero F (246). From the data obtained relating F to test CO2 concentration at 21% and 0 - 1% O2, the percentage inhibition of photosynthesis (% I ) • In air was calculated from the following formula (22, 283) (Ps - Ps ) = ^ — " 100 tot PSQ where PSQ represents the rate of net photosynthesis in 0 - 1% O2 and Ps„, represents the rate in 21% 0 . Experiment 6 had a randomized block design in which a complete series of gas exchange measurements on plants from each growth regime constituted a single block. Each series was replicated three times to provide three complete blocks. The experimental design is summarized i n Table II-7. S t a t i s t i c a l analysis of the calculated values of and r^ was performed by means of covariance analysis in combination with a Student-Newman-Keuls multiple range test (a = 0.05) (303). Tests were applied to the regression relationships relating C\ to F. Experiment 7 (Replacement Experiment) was designed to investigate whether permanent changes occurred i n the gas exchange physiology of leaf 3 after a period of growth under supra-normal CO^  concentrations. Plants were grown, as described above, for approximately two weeks under 0.03% or 0.1% CO,,. At PI = 5, two plants were removed from each growth regime. Measurements df CT^  and watp.r vapour exchange at 5 Ct^ concentrations (540, 440, 340, 240 and 140 y l 1 x) at saturating irradiance were conducted on leaf 3. The plants were removed to a greenhouse bench and l e f t for a two week period under conditions of light and temperature similar to those used for early propagation of the seedlings. Then, f i n a l l y , the plants were returned to the laboratory and the same series of measurements was repeated on leaf 3. Experiment 8 .'investigated activities of the enzymes RuBP-case and GaO in leaves 2, 3, 4, and 5 of plants grown at TABLE I I - 7 : Experiment 6 - C O 2 exchange of leaf 3 of tomato plants grown at 3 C O 2 concentrations in chambers, at 2 stages of development: Summary of experimental design. Number of Factor Levels Blocks (B) 3 Growth CC^ concen- 3 tration (C02) B x C02 9 Plastochron index 2 (PI) PI x C0o 6 Definition Replicate measurements: 1 block representing 1 complete set of the other factors, randomized in space (chambers) and staggered in time 0.03, 0.1, 0.5% C02 Error term PI = 5; PI = 10.5 Interaction of leaf age (defined by PI), and growth C0„ concentration Residual error Error term I l l 0.03, 0.1 and 0.5% C^. 500 mg of leaf material was cut into pieces to exclude the petiole and major veins. Homogenization was carried out using a mortar and pestle at 4°C in 5 ml of a medium containing 0.5 mM potassium phosphate buffer (pH 7.3), 0.2 yM EDTA and 1 mg solid sodium glycolate. The homogenate was decanted and centrifuged at 12,000 g for 15 minutes. GaO activity was determined spectrophotometrically in the unpurified extract by the rate of phenylhydrazine oxidation to phenylhydra'zone, using the reaction mixture and procedures described by Baker and Tolber.t (10). A Gilford 2400S spectro-photometer was used in a l l determinations. The assay procedure for RuBP-case was similar to that of Wishnick and Lane (294). Enzyme activity was determined from 14 the amount of C radioactivity fixed i n 0.1 ml aliquots of the unpurified leaf extract reaction mixture incubated for 10 minutes in a water bath at 39°C. Each 0.1 ml aliquot was dried in a forced air draft in a fume hood for 60 minutes and then resolubilized in 10 ml of aquasol (Nuclear Chicago Ltd.) before counting on a Nuclear Chicago Mark 1 liquid s c i n t i l l a t i o n counter. The reaction mixtures for each enzyme assay are summarized in Appendix 3" at the end of this thesis. Before commencing Experiment 8,trials were conducted with each assay procedure to determine the pH optimum and saturating substrate concentration for GaO and RuBP-case. The 112 details of these preliminary experiments are also presented in Appendix 3. Final pH of reaction mixtures in experiment 8 were adjusted to 8.3 and 7.8 for GaO and RuBP-case, respectively. A l l pH adjustments were carried out by dropwise addition of 1 M Tris-HCl buffer. Activity assays were replicated three times using plants grown in different chambers. With the exception of the f i n a l replicate, plants for assay were selected from those which were not used for gas exchange determinations, two plants being harvested at PI = 5 and another pair at PI = 10. Thus the enzyme assays were conducted, for the most part, at the same time as the gas exchange, determinations on plants from each growth regime. The f i n a l replication was done without concurrent measurements of gas exchange but otherwise growth conditions were identical to those.described above. 113 PART II, SECTION 2  RESULTS AND DISCUSSION i) Analysis of Plant Growth jv> Relation to CO„ Concentration (Experiment 5) The effects of greenhouse CO ^ - enrichment on tomato leaf development in terms of plastochron index have been reviewed in Part I I , Section 1. Analysis of the results of the present experiment confirmed the earlier finding that supplemental CO^  had l i t t l e effect on this aspect of vegetative development:. A close similarity between the i n i t i a l slopes of the lines in Figure 11-16 indicated that plastochron index increased linearly with time u n t i l a value of between 8 and 9 was attained. After this point there was some indication of a decreasing rate of development in plants grown in 0.1 and 0.5%, but not in 0.03% C09. Rogan and Smith (240) noted a transition to a lower rate of leaf i n i t i a t i o n and emergence between the appearance of leaves 6 and 7 i n Agropyron: repens, which seems to correspond reasonably well with the present data. However, in these experiments the trend at any CO2 concentration was not marked and may be attributable to measurement vari a b i l i t y . Figures T.I-17 to 11-19 show the development of leaf length in the f i r s t five leaves of tomato seedlings exposed t<~> 0.03, 0.1 and 0.5% C0?, in relation to Leaf Plastochron Index (LPI). At each CO2 concentration, the rate of length increase i n the early exponential growth phase appeared to be similar irrespective of leaf number, although measurements were not available for leaves 1 and 2 at negative values of LPI. This confirms the results of FIGURE 11-16: Plastochron development of tomato plants grown at 3 CG^  concentrations as a function of time. NOTE: Points are means of measurements on a total of 6 plants, in two chambers shown f one standard deviation. 10H 1 0 11 -1 1 T 1 1 f 12 1 3 U 1 5 1 6 17 DAYS FROM I N I T I A L M E A S U R E M E N T tdayO is arbitrary for each growth regime) 1 1 4 a FIGURE 11-17: Growth in length of successive leaves of tomato plants grown at 0.03% CO^  as a function of LEI. NOTE: Points are means of measurements on a total of six plants in two chambers. L E A F P L A S T O C H R O N I N D E X 115a FIGURE 11-18: Growth i n length of successive leaves of tomato plants grown at 0.1% C0 2 as a function of LPI. NOTE: Points as for Figure 11-17. L E A F PLASTOCHRON INDEX FIGURE 11-19: Growth i n length of successive leaves of tomato plants grown at 0.5% as a function of LPI. NOTE: Points as for Figure 11-17. 117 250-1 230H L E A F P L A S T O C H R O N I N D E X 118 Coleman and Greyson (64) who noted, in addition, that the LPI at which a single tomato leaf l e f t the exponential growth phase depended on i t s serial number on the main shoot of the plant. This observation also corresponded with the present data where leaves 1 and 2 had attained maximum length at an LPI of between 3 and 4 in contrast to higher leaves which showed exponential growth u n t i l they had attained an LPI of between 4 and 6. These transitions from phase 1 (exponential) growth to phase 2 (mature) growth (64) were apparently independent of CO^  concentration although the mean lengths of individual leaves at the transition point were CO^-dependent. In general, leaves which developed at a growth concentration of 0-5% were shorter at the growth curve asymptote than those developed at 0.03%. This difference was particularly marked above leaf 2. At the start of phase 2, the lengths of leaves 1 and 2 which were developed in 0.1% CO^  were greater than at the other two concentrations. This difference was not evident between the 0.03 and 0.1% CO^~grown plants at higher leaf numbers. The lack of effect of either 0.1% or 0.5% CT^ during growth on leaf production i s consistent with results from the previous greenhouse study (Part I I , Section 1). It i s , nevertheless, interesting to note that lengthening of leaves 1 and 2 was respectively inhibited and promoted in plants grown at 0.5% and 0.1% CX^. To my knowledge there are no previous reports on the effect of atmospheric CO,, concentrations on the development of leaf length in tomato or other plants. 119 An i n t e r e s t i n g comparison may be made between these observations and the values of LAR for plants exposed to the three growth CO^ concentrations (Table II-8). LAR was s i g n i f i c a n t l y decreased by 0.1 and 0.5% C0 2 r e l a t i v e to the 0.03% treatment i n accordance with several previous studies (95, 136, 140, 273). The differences i n l e a f length at the s t a r t of phase 2 growth were not r e f l e c t e d i n d i f f e r e n t values for LAR between plants grown i n 0.1 and 0.5% CO^. This may suggest that high growth C0 2 concentrations exert morphogenetic influences on the leaves (length reduction at 0.5% C0 2 and increase at 0-1%) which are compensated by d i f f e r e n t i a l production and/or p a r t i t i o n i n g of dry matter wi t h i n the plant. In a l a t e r section (Part III) data w i l l be reviewed which ind i c a t e that high C0 2 concentrations greatly influence o v e r a l l morphological development i n Pharbitis nil. Compared with 0.03% C0 2, RGR were increased i n plants grown at 0.1% C0 2 > but at 0.5% C0 2 > RGR was not changed (Table I I - 8 ) . Photosynthetic e f f i c i e n c y , as indicated by NAR, was highest at 0.1% and lowest at 0.03% C0 2. NAR of plants grown under 0.5% C0 2 was intermediate between the othe*" two values. I t may be noted that the increase i n NAR i n the 0.5% COp-grown plants was not s u f f i c i e n t to increase RGR over plants grown i n 0.03% C0 2, due to the reduced LAR at the higher C0 2 concentration. RGR and NAR have previously been shown to increase with ambient C0 o concentration i n a number nf species (25, 139, 140, 273). 120 TABLE II-8: Effects of CO2 concentration on net assimilation rate (NAR), r e l a t i v e growth rate (RGR) and leaf area r a t i o (LAR) i n tomato plants grown at 3 C0^ concentrations, i n chambers. Growth CO2 NAR RGR LAR* Concentration (%) (mg cm - 2 day"--*-) (mg g~l day -^) cm2 mg~ 0.03 0.40a** 229.40a 0.55a 0.10 0.65b 234.90b 0.35b 0.50 0.59c 215.60a 0.36b *LAR determined 22 days after placing plants i n growth regimes. *Mean values i n any column designated with the same l e t t e r are not s i g n i f i c a n t l y different (P > 0.05) according to SNK multiple range test. 121 Basing their conclusions on detailed mathematical analysis, Thornley and Hurd (271) have suggested that the main effect of CO^ " enrichment in tomatoes is upon NAR, with other changes in RGR and LAR resulting directly from these effects. The present data are not entirely inconsistent with this view although they are more easily explained by direct effects of CO^  on NAR and LAR which thus determine RGR. Support for this explanation is provided by the data of Monsi et al. (202) concerning the effect of another environmental factor, light intensity, upon these three growth parameters. Monsi at al. (202) showed that, in mung bean, a reduction in light intensity decreased NAR but concurrently increased LAR so that RGR remained constant. It is important to emphasize that the increase in NAR i n the 0.5% CO2 grown plants was not sufficient to promote the rate of dry matter production. NAR represents net^photosynthetic gain by the plant. Differences in NAR between plants may be a result of differences in rates of respiration or photosynthesis (290) . The contribution of changes in either of these processes to the reduction in NAR in tomato plants grown at 0.5% CO2 relative to those grown at 0.1% CO2 is unclear from the present data. The results described later in this Section w i l l serve partly to c l a r i f y the relationships between NAR and growth CO2 concentration. 122 Effects of Growth C0„ Concentration on Photosynthesis„ Photorespiration and Enzyme Activity in Leaf 3 at LPI : 2.0 (Experiment 6&8) In Part II, Section 1 of this thesis i t has been shown that tomato plants grown at ambient CO^  concentrations of approximately 900 y l 1 x (0.09%) exhibit higher rates of net photosynthesis and higher efficiencies of CO^  u t i l i z a t i o n than those grown in normal a i r . In order to determine i f these effects were evident at higher concentrations experiments were conducted to investigate the effects of 0.5, 0.1, and 0.03% during growth on subsequent rates of photosynthesis in relation to irradiance and concentration. At LPI3:2 net rates of photosynthesis were highest i n -2 -1 plants grown at 0.1% CO2 at irradiances of 130 yE m s and above, when test CO^  concentration was 330 y l 1 ^ (Figure 11-20) . This confirmed earlier observations and indicated a similar response of young leaves to growth CO^ concentration under greenhouse conditions and in small controlled environment chambers. At 0.5% growth CO2, however, a different response pattern was observed. -2 -1 At irradiances above 200 yE m s , and a test concentration of 330 y l 1 x, net photosynthetic rates were similar to those of leaf 3 on plants grown at 0.03% CC^' At lower irradiances, leaf rates in both 0.03 and 0.1% CO^ grown plants were significantly higher than in those from the 0.5% CO2 regime. It was clear that the application of supplementary C09 during growth at concentrations 122a FIGURE 11-20: Apparent rates of photosynthesis in relation to quantum flux density in leaf 3 of tomato plants grown at 3 concentrations. Test concentration: 330 y l 1 x Leaf temperature: 30 ± 2°C LPI: 2 ± 0.5 NOTE: Points bearing the same letter at any irradiance are not significantly different according to SNK multiple range test (P > 0.05). 124 5 times the normal commercial enrichment level of 1000 y l 1 (0.1%) resulted in depressed leaf photosynthetic rates at low irradiances when plants were returned to a normal (0.03%) CO^ atmosphere. This effect partly resembles that described for cucumber seedlings returned to 320 y l 1 CO^ after growth under CO^-enriched conditions (102). In that case, the photosynthetic rates in three newly emerged leaves were lower when plants grown at 1500 y l 1 ^ CO^ were measured at 320 y l 1 ^  at an irradiance -2 of 98 W m . Yet, there are many differences between the present results and those obtained in that study. Notably, the discrepancies in photosynthetic rates in cucumber plants were not evident at low irradiances, and the growth CO^  concentration of 1500 y l 1 ^  was more similar to the 0.1% treatment used in the present study. It i s not possible to resolve these differences at present, but the lack of uniformity between the two studies, in terms of growth conditions and plant type,may be important. The results showing net photosynthetic rate differences at low irradiances (Figure 11-20) may be due to differences i n rates of dark respiration. In plants grown at 0.5% CO^ , dark respiratory rates were higher than in those grown at either 0.1 or 0.03% CO^. Furthermore, compensation point for irradiance (estimated from the intercept of the curve relating CO^ exchange to irradiance, with the abscissa) was much higher in plants from the 0.5% C0? regime 125 than in those grown at 0.3 or 0.1% CO^ - The reasons for the higher rates of dark respiration in the 0.5% C02-grown plants are not clear from the present data. Madsen (184) has noted that the starch content of plants grown at CO^ concentrations of 0.22 to 0.5% may be as high as 13% of total plant dry matter as compared with 2 to 6% in plants grown in normal air or 0.1% CO^ . It seems possible that such large accumulations of starch may result in an increase in mitochondrial respiration through increased availability of respiratory substrates (266). Similar increases in dark respiration rates of young tomato leaves grown in C02- enriched air have been described by Ito (143). It has often been observed that dark respiration in plant leaves continues under conditions of low irradiance (126, 132) although the rate of production is inhibited (193), apparently as the result of direct inhibition of carbohydrate breakdown (132). Inhibition of dark respiration increases with increasing light intensity, and, in plants u t i l i z i n g only the pathway of photosynthesis,is replaced by peroxisomal photorespiration -2 -1 (144). At irradiances below 200 yE m s in the present experiments, i t seems lik e l y that net photosynthetic rate in leaves of plants grown at 0.5% CO^  is reduced relative to those grown at 0.03 or 0.1% C0 2 because of greater production of C02 by mitochondrial respiration. There are alternative explanations for the observed effects. It might be suggested, for example, that 126 higher quantum yields in the 0.03 and 0.1% CO^- grown plants could cause the relatively greater photosynthetic rates in those cases. However, the close similarity between the i n i t i a l slopes of the irradiance response curves in Figure 11-20 tends to refute this hypothesis. Another possible source of the differences in photosynthetic rates is that stomatal resistances to CO^ transfer were inherently higher at low irradiances in the 0.5% CO^-grown plants, but this is not borne out by the data shown in Figure 11-21. Variability in stomatal resistance within plants from any one growth regime was high at each test irradiance. Resistance values for leaf 3 of plants grown at 0.5% CO^ were quantitatively lower than for those from the 0.03% regime at low irradiance, but analysis revealed no s t a t i s t i c a l differences between growth regimes. It may be concluded that the particular CO^- enrichment regime under which tomato plants were grown did not influence stomatal resistance. The lack of stomatal acclimatization to increased CX^ concentration during growth has been well demonstrated for lettuce and Xanthium (152). It has also been shown with two lettuce varieties, that instantaneous rates of photosynthesis may be depressed at CO^  concentrations of 1700 y l 1 \ an effect which i s unrelated to stomatal closure (238). The present data suggest that variations in photosynthetic rates in plants grown under different CO concentrations are not related to differences in stomatal resistance. 1 2 6 a FIGURE 11-21: Stomatal resistance to CO^  transfer in relation to quantum flux density in leaf 3 of tomato plants grown at 3 CO^  concentrations. Test C02 concentration: 330 y l 1 _ 1 Leaf temperature: 30 ± 2°C LPI: 2 ± 0.5 NOTE: Mean resistances at each irradiance are not significantly different (P > 0.05) according to SNK multiple range test. 2 0 n 18-16 e o tu H -o ? 12-t -£ 10-I/) LU oc 8 _J < 6 2 < o r -CO 100 0 03% C 0 2 growth regime 0 1% CO, •0-5%CO, Line: y=10 52-1 58 ln(x) [all points] - r 200 300 ^00 500 Q U A N T U M F L U X D E N S I T Y (uE ir f 2s"!400-700nm) 128 Some results discussed in Part I I , Section 1 suggested that rates of photorespiration and magnitudes of mesophyll resistance at low irradiances were reduced in leaves of tomato plants grown at approximately 900 y l 1 1 CO . In order to assess the importance of these factors, two tests involving the effects of and 0^  concentration on photosynthesis at saturating irradiance were undertaken. Figure 11-22 illustrates the relationship between CO^  concentration in the intercellular spaces within the leaf and apparent rate of photosynthesis. The enhancement of photosynthesis at each value of in leaf 3 of plants grown at 0.1% CX^ is clearly shown by these data. The relationship also provides some insight to the mechanisms responsible for this enhancement, and also the lack of effect of higher CO^  concentrations (0.5%) on inherent rates of net photosynthesis. The steep slope of the regression line for the 0.1% CO^-grown plants i s reflected i n a lower value for mesophyll resistance as compared to leaves of plants f rom the 0.03 or 0.5% CO^ growth regimes (Figure 11-23). In addition, values of photorespiration were significantly lower in leaf 3 developed under 0.1% C02. It was apparent that both factors played important roles in determining the maximum photosynthetic rate i n the leaf at this developmental stage. 128a FIGURE 11-22: Apparent rates of photosynthesis in relation to intercellular CO,, concentration in leaf 3 of tomato plants grown at 3 CO concentrations. -2 -1 quantum flux density: 520 yE m s , 400 - 700 nm Leaf temperature: 30 ± 2°C LPI: 2 ± 0.5 NOTE: Extrapolation to zero CO^  concentration to estimate rate of photorespiration. 129a FIGURE 11-23: Rates of photorespiration (R^ , and mesophyll resistance (r ) for l e a f 3 of tomato plants m grown at 3 CO^ concentrations and measured at LPI: 2 ± 0.5. NOTE: Values of L or r represented by bars L m bearing the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (P > 0.05) according to SNK multiple range t e s t . Rates of R_ and r L m derived from regressions r e l a t i n g apparent photosynthesis to i n t e r c e l l u l a r CO2 concentration, as described i n the text. 130 131 The rates of photorespiration in leaf 3 of plants grown at 0.03, 0.1, and 0.5% CO^ correspond closely with the relative magnitudes of the observed CO^  compensation points (Table II-9) . Reduction in photorespiratory activity can result in substantial enhancement of photosynthesis in plant species u t i l i z i n g only the photosynthetic system (62, 170). The possibility of effectively decreasing photorespiration by supplying additional CX^ to the atmosphere during plant growth has been discussed (110). Short-term 14 exposure to 0.1% C09 has been shown to reduce C incorporation into the photorespiratory intermediates serine and glycine in tomato leaves (172) but not in leaves of Atriplex (224) or tobacco (258). Until now the relationship between CO^-enrichment and photorespiration has not, however, been well understood. The present results clearly indicate that after a period of growth at 0.1% CO^ , photorespiration i s inhibited relative to that of normal air grown plants. Yet, an increase of growth CX^ concentration to 0.5% resulted in similar photorespiratory rates to those observed in leaves developed in normal a i r . There is considerable evidence that the substrate for photorespiration in plants is glycolate or a related metabolite (79, 81, 144, 276, 308). An increase in ambient concentration from 0.03 to 0.1% has been shown to result in a marked reduction in glycolate synthesis in isolated chloroplasts (83, 239), tobacco leaf discs (310), and in algae (35). A possible explanation for the 132 TABLE II-9: C02 compensation points of leaf 3 of tomato plants grown at 3 CO2 concentrations, measured at two stages of development (Leaf Plastochron Index, LPI). CO2 Compensation CO2 Compensation Growth C02 Point at LPl3:2.0±0.5 Point at LPl3:7 ,5±0.5 Concentration (%) (g m~3 (ul l - 1 ) ) (g m-3 (ul I - 1 ) ) 0.03 0.095 (54)a* 0.069 (39)a 0.10 0.065 (37)b 0.076 (43)a 0.50 0.090 (51)a 0.083 (47)a ^Values are means of 6 determinations per treatment. Values for each LPI designated with the same letter are not significantly different (P > 0.05) by ANOVA and SNK test. 133 results reported here is that, i n plants grown under 0.1% CO^ the biochemical capacity for glycolate production is decreased relative to those grown under 0.03% . In this case the availability of photorespiratory substrate would be reduced leading to an inhibition of the overall process. A relatively large number of mechanisms have been proposed for the synthesis of glycolate in green leaves (9, 37, 65, 276, 308). There is currently f a i r l y wide support for two of these viz. the oxidation of the transketoluse-glycoaldehyde complex by hydrogen peroxide (produced by reoxidation of a photosynthetically produced reductant - the Mehler reaction) (65, 249) and the direct oxidation of RuBP to phosphoglycolate in a reaction catalyzed by RuBP carboxylase/oxygenase (37, 176, 276). Both mechanisms may operate in some cells as is suggested by some recent work on the alga Hydrodiatyon afvioanwn (107, 108). There are, however, a number of serious criticisms of the Mehler reaction pathway proposed by Coombs and Whittingham (65) and i t seems that, in most c e l l s , the relative activities of RuBP-carboxylase and oxygenase determine the rate of glycolate production and hence the rate of photorespiration. Later in this section i t w i l l be shown that, in plants grown in 0.1% CO2, the carboxylation activity of this enzyme is significantly increased relative to normal air-grown plants, thus supporting the hypothesis of a reduced capacity for glycolate production after growth at this concentration. 134 It i s possible that an increase of CO concentration above a c e r t a i n optimal l e v e l brings about furt h e r changes i n photorespiratory mechanisms, which may u l t i m a t e l y r e s u l t i n higher rates than exhibifed by plants grown i n normal a i r . This i s suggested from experiments on Hydrangea i n which i t was observed that an increase i n ambient CO^ concentration to 1% resulted i n 25% more re s p i r a t o r y CC^ release i n the l i g h t as compared to plants tested i n 0.03% CO^ (216). I t i s , however, important to point out that those experiments were concerned e n t i r e l y with instantaneous rates of photorespiration and not with the long term e f f e c t s of high concentrations during plant growth. The r e s u l t s presented here have shown that c h a r a c t e r i s t i c s of l e a f CO^ exchange are determined, not only by the current gaseous environment, but also by CO^ conditions during a previous period of growth. That the growth conditions a f f e c t e d other aspects of l e a f physiology i s evident from the lower values of mesophyll resistance i n the young l e a f 3 of plants grown at 0.1% (Figure 11-23). It i s worth r e c a l l i n g that mesophyll resistance as c a l c u l a t e d i n t h i s study incorporates two separate components, one r e s u l t i n g from the p h y s i c a l resistance to CO2 t r a n s f e r i n the l i q u i d phase through the mesophyll, and another a t t r i b u t a b l e to the photochemical and biochemical resistances to CO2 f i x a t i o n at the c h l o r o p l a s t s . I t i s not p o s s i b l e , a t present,to completely separate these 135 components since even under conditions of saturating irradiance, the slope of the line relating internal CO^  concentration to net photosynthesis w i l l depend on both physical transport processes across the mesophyll and the rate of dark carboxylation reactions (146). Hesketh (125) has stated that inherent differences in net photosynthetic rates between species are principally due to variations in CO^  diffusion resistance across the mesophyll and the kinetics of the dark reactions of photosynthesis. It i s possible that both factors play a role in determining the magnitude of r^ in plants grown in 0.03 and 0.1% CO^ . Other data to be presented shortly, however, have provided strong support for the idea that differences in the carboxylation efficiency of RuBP largely account for the observed differences in r . m In leaf 3 at L?Iy2, there were certain close similarities between the effects of CO^~enrichment during growth on photorespiration and mesophyll resistance. Just as photorespiration was suppressed by growth at 0.1% but not at 0.5% CO2, mesophyll resistance was significantly decreased at 0.1% but, again, not at 0.5% CO2 (Figure 11-23). The similar effects of supra-normal CO2 concentrations suggested a close correlation between these parameters which warranted careful examination. Figure 11-24 shows the percent inhibition of photosynthesis by 21% O2. Inhibition was highest at low test concentrations of CO^ (140 y l 1 x) in plants from each growth regime, a finding which 135a FIGURE 11-24: Effect of CO^  concentration on the percentage total oxygen inhibition of photosynthesis in leaf 3 of tomato plants grown at 3 CO^  concentrations. -2 -1 quantum flux density: 520 yE m s , 400 - 700 nm Leaf temperature: 30 ± 2°C LPI: 2 ± 0.5 136 137 corresponds with observations on oxygen inhibition of photo-synthesis in wheat (151), soybean (96, 170), and sunflower (179). Total oxygen inhibition of photosynthesis in air among plants appears to be of variable magnitude. Experiments on soybean and sunflower have indicated values between 40 and 45% (96, 179) whereas similar determinations for wheat have shown between 10 and 15% inhibition (149). The present data for total inhibition of photosynthesis in leaves of tomato plants grown at 0.03 and 0.5% C02 f a l l between these values (Table 11-10). The 10% inhibition shown by plants grown at 0.1% CO^ is less than half the values for plants from the other regimes. The total inhibition of photosynthesis by 21% 0^ comprises two components, a stimulation of photorespiration and a direct 0^ inhibition of photosynthesis (96, 280). Different studies have produced differing estimates of the relative contributions of these components to overall inhibition. In soybean, for example, Curtis et al. (71) found that photorespiration accounted for slightly more than 50^ of total inhibition of photosynthesis whereas a later study by Laing et al. (170) showed that photorespiration was responsible for only 33%, the remaining 67% being contributed by direct oxygen inhibition. Other studies on sunflower (178, 179) support the findings of Laing et al. (170). An explanation for the differing results probably l i e s in the methods used by different workers to determine photorespiratory "Total" refers to a l l components of oxygen inhibition of photosynthesis. TABLE 11-10: Components of oxygen i n h i b i t i o n of photosynthesis i n l e a f 3 of tomato plants grown at 3 CO concentrations, at l e a f plastochron index: 2 (LPI :2). Growth C O 2 Concentration (%) Mean Irradiance Saturated Rate of Photosynthesis i n Normal A i r (20.8% 09, 0.03% C0 2) (g m ,-1 xlO 4) Rate of Photorespiration (g m"2 s - i x l O 4 ) I n h i b i t i o n of Photosynthesis by Photorespiration i n A i r Dire c t I n h i b i t i o n of Photosynthesis by Oxygen i n A i r % Total Oxygen I n h i b i t i o n of Photosynthesis i n A i r 0.03 2.00 0.37 18.5 6.8 25.3 0.1 2.55 0.24 9.4 0.6 10.0 0.5 1.80 0.33 18.3 5.2 23.5 CO 139 rates. Ludwig and Convin (179) and Laing et al. (170) both measured photorespiration directly by methods involving the differential uptake of C02 and CO^  (180), and the measurement of CO^  efflux into CO^—free a i r , respectively. Curtis et al. (71), on the other hand, calculated soybean photorespiration from a simple relationship between net photosynthetic rate and the C02 compensation point. The data obtained from graphically determined rates of photorespiration in the present study (246) , indicate that the major part of the total oxygen inhibition of photosynthesis was accounted for by photorespiratory C02 production. It i s noteworthy that i n plants grown in 0.1% C02 both photorespiratory and direct oxygen inhibition of photosynthesis were reduced relative to those from the 0.03 or 0.5% C02 regimes. In the case of 0.1% grown plants, some 94% of total inhibition was accounted for by photorespiration alone. These observations help to c l a r i f y the close relationship between reduced photorespiratory rate and mesophyll resistance i n leaves of 0.1% grown plants, discussed above. Direct oxygen inhibition of photosynthesis may be a result of depleted supplies of the C02 acceptor RuBP (65, 83, 230, 239, 249), a depressed rate of pyridine nucleotide oxidation (284), or the competitive inhibition of RuBP-case by oxygen with respect to C02 (36, 221) which reduces the availability of the carboxylating enzyme for photosynthetic C09 fixation. If the latter mechanism i s correct, 140 as is strongly suggested by evidence implicating the RuBP carboxylase/oxygenase enzyme system in the production of glycolate, then any increase in carboxylase activity w i l l reduce biochemical carboxylation resistance. This in turn, would lead to a reduction in total mesophyll resistance as observed for plants grown at 0 . 1 % CG^  in these experiments. A l l the available evidence thus points towards a closely integrated system of photosynthesis and photorespiration whereby an increase in RuBP-case activity leads to a stimulation of photosynthesis and concomitant inhibition of photorespiration. This does not discount the possibility that other systems may be involved in increasing the photosynthetic potential of leaves developed i n 0 . 1 % C^. Increased carbonic anhydrase activity at this C O 2 concentration has been demonstrated in young cotton plants ( 5 9 ) and may be an important determinant of maximum photosynthetic rate. The enhancement of photosynthesis in leaf 3 at L71^:2 of tomato plants grown at 0 . 1 % CO^  may thus be a result of a number of changes in leaf physiology. Experiment 8 , which was conducted to determine act i v i t i e s of two enzymes in relation to growth CO^  concentration, provided further support for the hypothesis that ultimate control of potential net photosynthetic rate was through RuBP-case. In leaves 2 and 3 of tomato plants grown at 0 . 1 % C O 2 at a PI of 5 , activity of RuBP-case was well over twice that of the same leaves 141 developed in normal air (Figure 11-25). In leaves 4 and 5 the differences were not as great, but a similar pattern was evident. In contrast to the effect of the 0.1% CX^  growth regime, the data obtained from leaves developed in 0.5% CO^  showed lower activities in a l l four tested leaves, as compared with those from the normal air-grown plants. These data do not correspond precisely with a previous study using barley, in which growth of plants at supra-normal concentrations resulted i n increased RuBP-case activity in the f i r s t leaf on the main stem (92). In this case increased activities were noted after growth for two days in an atmosphere containing between 1 and 5% CO^ . It i s possible that after a longer growth period under C^ -enriched conditions, reduced RuBP-case activities would have been evident in the experiments with barley. Treatment with high (1 - 5%) concentrations for a short period may e l i c i t similar effects to the longer term 0.1% exposures used in the present experiments, where plants were subjected to each growth regime for at least 14 days. Enhancement of RuBP-case activity in leaf 3 of plants grown at 0.1% CO^ is consistent with the concept that increased carboxylation results in decreased photorespiration and oxygen inhibition of photosynthesis. The similar rates of photorespiration shown by the same leaf on plants grown in normal air a"d 0.5% CO^  are not, however, reflected i n similar RuBP-case activities (Figure 11-25). It is apparent that a growth concentration 1 4 1 a FIGURE 11-25: Activites of RuBP carboxylase and glycolate oxidase extracted from leaves of tomato plants grown at 3 CO^  concentrations. PI: 5 ± 0.5 NOTE: Values are means of 3 replicate samples per CO^  regime shown ± one standard deviation. GLYCOLATE OXIDASE ACTIVITY (umoles glyoxylate formed gfw^mirJ) m > T I c W m 3J • • • o o o en o o o o h o K J o CO o o KJ - i o CD 3 (0 m o m D m > a; m o o p p o o p _ » cn _ l_ ro co _ i i _ __i I W-»-H I—•—I I » 1 RuBP CARBOXYLASE ACTIVITY (umoles RuBP converted g f w~1 min"1) O O O O —' —' —' Co <ln -vj t]o —» co ai i I I I I L_ CO A cn ro N) M N> NJ *^ co cn -sj io ' ' I L _ - _ - l -•-I 143 of 0.5% CO2 i s inhibitory to carboxylation activity of RuBP-case which may explain reduced assimilation rates and growth in some plants subjected to this and higher concentrations (11). Since the oxygenase activity of the enzyme was not measured in these experiments, i t can only be speculated that the competitive advantage of the RuBP-oxygenase system, which in the absence of high rates of carboxylation would be expected to increase glycolate production and photorespiration, is not realized due to an overall suppression of enzyme activity by high ambient (X^ concentrations during growth. The differential effects of growing plants at 0.1% as compared with 0.03% CO2 upon RuBP-case activity, are similar to those noted between "sun" and "shade" grown plants, where enzyme activities are higher in plants developed under high light intensities (29, 67). Such plants also show higher rates of net photosynthesis when measured at high irradiances (30). Clearly, growth conditions which favour high instantaneous rates of photosynthesis seem to be closely correlated with higher RuBP-case activities and higher inherent rates of photosynthesis. The pathway of photorespiration in higher plants and some algae after the synthesis of glycolate apparently proceeds through the enzymic oxidation of this substrate by glycolate oxidase (98, 274, 275). A reduced availability of glycolate, which seems to be a l i k e l y result of the 0.1% growth C09 treatment in these 144 experiments, might be expected to result in a reduced activity of glycolate oxidase relative to the 0.03% CO^-grown plants. This is confirmed by the data shown in Figure 11-25. In leaf 3, GaO activity was approximately halved in the 0.1% CO^  grown plants. A similar reduction in activity of a number of enzymes associated with photorespiratory metabolism including glycolate oxidase, resulted from the two day exposure of barley plants to 1-5% CO^  (92). In the present experiments, growth at 0.5% CO^  once again resulted in suppression of enzyme activity relative to both the 0.03-and 0.1%- grown plants. The poor correlation between glycolate oxidase activites in leaf 3 of plants from the 0.03 and 0.5% CO^  growth regimes is d i f f i c u l t to reconcile with their similar photorespiratory rates (Figure 11-23). It i s , however, important to recall that the enzyme is involved in other aspects of carbon flow within the leaf including the oxidation of glyoxylate to oxalate, a reaction involved in the proposed glycolate-glyoxylate shuttle (237, 274, 277). This oxidation does not contribute directly to the main stream photorespiratory pathway resulting i n the production of CO2 via the decarboxylation of glycine (7, 160). The lack of substrate specificity demonstrated by glycolate oxidase, and i t s consequent involvement in other reactions within the peroxisome, may account for the poor correlation observed between i t s activity in extracts and measured rates of photo-respiration or C0? compensation points (91). 145 iii) Comparison of the Effects of Growth CO„ Concentration on Photosynthesis, Photorespiration and Enzyme Activity in Leaf S  at LPI :7.5 and LPI : 2,0 (Experiments 6&8) The effect of age on net assimilation rate in plant leaves is d i f f i c u l t to predict. Patterns of behavior tend to differ }even within a single species (6) and are mediated by external factors such as light intensity (18) and the sequence of reproductive and vegetative development within the plant (97, 228). Figure 11-26 shows the results of a second series of measurements of net CC^  exchange in relation to irradiance in leaf 3 of tomato plants grown at 0.03, 0.1 and 0.5% CO^ . At the time of this experiment, the leaves had attained a LPI of 7.5 and the plants had been exposed to the treatment regimes for approximately five weeks. Photosynthetic rates at saturating irradiance were reduced relative to rates at LPI^:2 in leaves of plants from each growth regime (cf. Figure 11-20). In the plants subjected to 0.03 -4 or 0.5% CO2 this reduction corresponded to approximately 0.9 x 10 -2 -1 g CO^  m s . In the case of the 0.1%-grown plants, however, -4 -2 -1 the reduction was greater, being 1.1 x 10 g CX^ m s . The resulting maximum rates of net photosynthesis at this later stage of leaf development were, therefore, similar irrespective of growth regime. A similar relationship was observed over the entire range of irradiances tested. Lower rates of dark respiration in 145a FIGURE 11-26: Apparent rates of photosynthesis in relation to quantum flux density in leaf 3 of tomato plants grown at 3 CO^  concentrations. Test C02 concentration: 330 y l l " 1 Leaf temperature: 30 ± 2°C LPI: 7.5 ± 0.5 NOTE: Points at any irradiance are not significantly different from one another (P > 0.05) according to SNK multiple range test. 147 leaf 3 or the 0.5% C^-grown plants at this stage apparently resulted in higher rates of net assimilation at low irradiance -2 -1 (30 yE m s ) as compared with the response at LPI^:2 (Figure 11-20) . These results are not consistent with those obtained from the experiments on greenhouse grown plants (Part I I , Section 1), where differences between rates of net assimilation in plants grown under enriched or non-enriched conditions were evident in young and old leaves on the main stem. To my knowledge, there are no other reports dealing with the variation of net assimilation rate with age in leaves developed under different CO 2 concentrations. Some data, however,are available on such variations in plants grown under different irradiances. Osman and Milthorpe (223) found that leaves of plants grown at high irradiance show inherently higher maximum rates of photosynthesis early i n development followed by a rapid decline during aging to a rate equal to or lower than those grown at lower irradiance. The high irradiance leaves had a shorter l i f e span and senesced earlier. Thus, conditions resulting in a high rate of net assimilation induced an early decline in photosynthetic capacity,, an observation which may bear upon results of the present experiments. At LPI^:7.5, leaf 3 of plants grown at 0.1% CO^  could be at a more advanced stage of senescence than a leaf at the same developmental stage i n 0.03% CO . The data obtained in this study do not allow further 148 interpretation of the situation, but i t should be borne in mind that senescence is a complex phas^ of development during which leaves pass through several changes of metabolism each of which is susceptible to environmental modification (251, 301). Physiological differences between plants grown in controlled environments and in f i e l d situations present a constant source of d i f f i c u l t y in the interpretation of experiments using a r t i f i c i a l growth regimes (88). The differences between results obtained in the experiments discussed here and those involving greenhouse grown plants may be directly related to the more restrictive growth conditions encountered in the controlled environment chambers. An earlier onset of senescence i n the growth chambers would be li k e l y to mask differences in inherent photo-synthetic rates discernable in the older basal leaves i n the greenhouse experiment. The particular susceptibility of the tomato plant to continuous a r t i f i c i a l light (5, 128) may indicate a special d i f f i c u l t y in growing these plants in controlled conditions, although the 12-hour photoperiod used i n these experiments was selected to circumvent problems of early leaf senescence which can be induced by unusual light-dark cycles (2, 127). The close similarities between photosynthetic rates in leaf 3 (LPI^:7.5) among plants from each growth regime are also exhibited in relation to leaf response to intercellular CO^  concentration (Figure 11-27). Mesophyll resistance to CO^  transfer 148a FIGURE 11-27: Apparent rates of photosynthesis in relation to intercellular CO^  concentration in leaf 3 of tomato plants grown at 3 CO^  concentrations. -2 -1 quantum flux density: 520 yE m s , 400 - 700 nm Leaf temperature: 30 ± 2°C LPI: 7.5 ± 0.5 NOTE: Extrapolation to zero CO^  concentration to estimate rate of photorespiration. I—1 150 and rate of photorespiration, as calculated from the regression relationship, were independent of growth regime (Figure 11-28). Mesophyll resistances showed a significant increase over the LPI^:2 stage of leaf development in a l l plants, the average change being 13 s cm x in the 0.03 and 0.5% CO^-grown plants, and 12 s cm x in those from the 0.1% regime. Increases in internal resistance with leaf age a"d nutrient deficiency have bee" shown to contribute significantly to an overall decline in photosynthetic activity in a number of species (223, 242). In others this resistance has been shown to remain quite constant with age (218). The decrease in maximum rates of photosynthesis with age in leaf 3 of tomato plants in this study seemed to be closely connected to an increase in mesophyll resistance. This resistance was, once again, dominant in the transfer catena at a l l irradiances, since values were always higher than stomatal resistances even when the latter were measured at low irradiance (Figure 11-29). Stomatal resistances were generally higher than at the LPI^:2 stage of development (cf. Figure 11-21). The increase of this resistance with age is in accord with a number of previous studies (73, 133, 254), but since i t was smaller than mesophyll resistance, the possibility that this represented a major determinant of photosynthetic capacity seems unlikely (24). In a l l plants except those grown under 0.1% 00^, rates of photorespiration were depressed at the LPI^:7.5 stage of development 150a FIGURE 11-28: Rates of photorespiration (R^) and mesophyll resistance (r ) for leaf 3 of tomato plants m r grown at 3 CO^  concentrations and measured at LPI: 7.5 ± 0.5. NOTE: Values of RT or r represented by bars L m bearing the same letter are not significantly different (P > 0.05) according to SNK multiple range test. Rates of R_ and r L m derived from regressions relating apparent photosynthesis to intercellular CO^  concentration, as described in the text. 151 C M E < cr CL CO LU OC o r -o X Q_ U. O LU I — < OC 0 Al 0-3 H 0 - 2 H 0-1-1 r A 0 h-20 -10 R L P m 0 0 3 0-1 0 5 % C 0 2 GROWTH REGIME E o h30 i LU O < h-co • — i LU cr >-x CL O CO LU 151a FIGURE 11-29: Stomatal resistance to CO^  transfer in relation to quantum flux density in leaf 3 of tomato plants grown at 3 CO^  concentrations. Test CO^  concentration: 330 y.1 1 ^ Leaf Temperature: 30 ± 2°C LPI: 7.5 ± 0.5 NOTE: Mean resistances at each irradiance are not significantly different (P > 0.05) according to SNK multiple range test. 20-, — I 1 ! ' ' 100 200 300 A00 500 Q U A N T U M F L U X DENSITY (uE rrf z s ,400-700nm) U l 153 (Figure 11-28). In many plants photorespiratory activity changes with leaf age, although frequently the pattern of change i s dependent upon leaf type, developmental events taking place in other parts of the plant (9 7) and on previous growth conditions (84). Data obtained from the present experiments are inconsistent with some previous work on leaves of tobacco and some citrus varieties, showing an increase in photorespiration with leaf age (243). ^ock and Krotkov (94), however, found an increase in photorespiratory activity in young, expanding leaves of kidney bean, which paralleled an increase in net photosynthesis. At a later stage of leaf development both photosynthesis and photorespiration declined, a finding which is comparable to the present results and to some previous observations on wheat (97). Clearly, the decline in net photosynthetic rate with leaf age shown in these experiments i s not attributable to increased photorespiration. It i s , however, interesting to note that rates of photorespiration in leaf 3 of plants grown under 0.1% CO2 were unaffected by leaf age; but the advantage of those leaves in terms of net carbon gain at the LPI^:2 developmental stage was lost at LPI^:7.5. This may be due, in part, to a marked increase in the total oxygen inhibition of photosynthesis which, at LPI^:7.5 was of similar magnitude to total inhibition in the 0.03 a™d 0.5% CO^-grown plants (Table 11-11, Figure 11-30). The increase i " the direct oxygen inhibition component largely accounts TABLE 11-11: Components of oxygen inhibition of photosynthesis in leaf 3 of tomato plants grown at 3 CC>2 concentrations, at leaf plastochron index: 7.5 (LPI3:7.5) . Growth C02 Concentration Mean Irradiance Saturated Rate of Photosynthesis in Normal Air (20.8% 0 2, 0.03% C02) (g m-2 s - 1 x l 0 4 ) Rate of Photorespiration (g m~2 s-^xlO 4) Inhibition of Photosynthesis by Photorespiration in Air Direct Inhibition of Photosynthesis by Oxygen in Air % Total Oxygen Inhibition of Photosynthesis in Air 0.03 1.33 0.19 14.3 16.0 30.3 0.1 1.45 0.19 13.1 14.2 27.3 0.5 1.20 0.18 15.0 10.0 25.0 154a FIGURE 11-30: Effect of CO,, concentration on the total oxygen inhibition of photosynthesis in leaf 3 of tomato plants grown at 3 CO2 concentrations. -2 -1 quantum flux density: 520 yE m s , 400 - 700 nm Leaf temperature: 30 ± 2°C LPI: 7.5 ± 0.5 1 5 6 for the higher total inhibition of photosynthesis at LPI^:7.5 in the 0.1% CO^ grown plants (Table 11-11). It was notable that, total inhibition in leaves of plants grown at 0.03 and 0.5% C02 showed l i t t l e change from the LPI^:2.0 developmental stage. A review of the present data yields a number of interesting points. The decline in net photosynthesis in leaf 3 of plants grown in the 0.03 or 0.5% C02 regimes from LPI 3:2 to LPI3:7.5, appears to be directly related to an overall decline in C02 exchange activity. Both photosynthesis and photorespiration are decreased at the later developmental stage, while total oxygen inhibition of photosynthesis remains relatively unchanged. In the 0.1% C02-grown plants, however, the lower photosynthetic rates exhibited at LPI^:7.5 are partly due to an enhanced total oxygen inhibition of photosynthesis, including a greatly increased direct oxygen inhibition component (Table 11-11). The results provide further evidence to suggest that leaves developed under 0.1% C02 show a degree of physiological adjustment resulting in different C02 exchange characteristics at both early and late stages of development, compared with leaves of 0.03 or 0.5% C02~grown plants. Lower maximum rates of photosynthesis at LPI^:7.5 as compared to LPI^:2 in plants from each growth regime are generally correlated with lower RuBP-case activities (Figure IT-31). In leaves of the 0.5% C00-grown plants, activity was quite similar 156a FIGURE 11-31: Activities of RuBP carboxylase and glycolate oxidase extracted from leaves of tomato plants grown at 3 CO^  concentrations. Plastochron index: 10.5 ± 0.5 NOTE: Values are means of 3 replicate samples per treatment shown ± one standard deviation. GLYCOLATE OXIDASE ACTIVITY (umoles gloxylate formed gfw"1mirT1) • • •. o o o cn vP CO o o o o ° NJ rO O O e g 3 to m > (D m 30 m o m O o O O O o O O — * — * ~ * ~ * | s J U ) * ~ - c n O ^ - v l O O * i D O — » N ) C 0 * ~ -I I L- I 1 1 1 1 1 M jl k i l l » CO A I I • 1 1 r m p i ' l -i r RuBP CARBOXYLASE ACTIVITY (umoles RuBP converted g fw"1 min"1) o o o p o p p o - ^ u> t~- cn cn -si 6o ib o ro I r->U 1 1 L 1 1 | » ' • ! i 1 m > 0D m co 4 * - 4 cn -* ro to J I 1 — l M — W — I I • 1 h 11 i 1 1 r =4] H • i 1 i 1 r Z.ST 158 to that obtained at LPI^:2, and, as before, was considerably lower than values for equivalent leaves of plants from the 0.1 or 0.03% growth regimes. The general decline in RuBP-case activity with leaf age is consistent with data obtained with a number of other plants (77, 124, 195, 256, 288). Maximum carboxylation activity has been found to occur during the period of leaf expansion. Thereafter, RuBP-case activity rapidly declines to a level which remains relatively constant u n t i l long after expansion has ceased (262, 264). In the present experiments, leaf 3 of plants from each growth regime was fu l l y expanded at LPI^:7.5 (equivalent to PI:10.5, Figure 11-31), and the reduction in RuBP-case activity at this later developmental stage therefore corresponded with those previous studies. The increases in mesophyll resistance at LPI3:7.5 in comparison with LPI^:2, may be correlated with a reduction in RuBP-case activity resulting in an increase in carboxylation resistance. Indirect support for this idea is provided by a recent study which showed that the transfer component of mesophyll resistance in bean leaves actually decreased with the -onset of leaf senescence, while the biochemical and/or photochemical components showed a marked increase (272). In several plants, variations in in vitro RuBP-case activity parallel changes in maximum photosynthetic rate (23, 38, 124, 256, 263, 264, 281, 288). Results obtained for leaf 3 of 159 plants from the 0.03 and 0.1% CO^  growth regimes seem to be consistent with those findings, but the close similarities between activities at PI 5 and PI 10.5 i n the 0.5% C^-grown plants were unexpected. In addition, similar rates of net photosynthesis in leaf 3 of plants grown under 0.5% and leaves from the 0.03 or 0.1% CO2 growth regimes were not reflected in similar RuBP-case act i v i t i e s . Such results lend some support to the previously discussed hypothesis that growth of tomato plants at very high '^ (-0.-05% and above) ambient CO2 concentrations results in an overall suppression of RuBP carboxylase/oxygenase activity. Although close correlations can exist between photosynthetic rate and RuBP-case (214), inconsistencies have also been reported (38). It i s evident from the present results that factors other than activity of the carboxylating enzyme are responsible for determining maximum photosynthetic rates in plants grown under 0.5% CO2. The light intensity under which plants are grown, has been shown to play an important role in determining the relationship between RuBP-case activity and photosynthesis (38). Perhaps a similar importance may be ascribed to ambient CO2 concentrations during growth. GaO activities at PI:10.5 did not show a consistent trend from leaf to leaf between the three growth regimes. The results were quite variable at this developmental stage, but indicated that there was l i t t l e difference in GaO activity among the leaves studied. This i s consistent with the similar rates of photorespiration 160 observed in leaf 3 from each growth regime (Figure 11-28), but represents a quite different situation to that described for the PI:5 stage of development. GaO activity in leaves of plants grown under 0.5% CO2 no longer showed lower values than leaves grown at other CO^ concentrations. In the 0.03 and 0.1% CO2- grown plants GaO activities i n leaves 2-to 5 were reduced relative to the PI:5 stage of development, but not in those developed under 0.5% CO2. A decrease in GaO activity with leaf age has been described in kidney bean (94). Salin and Homann (243), on the other hand found lowest GaO activities in the youngest leaves of tobacco and some citrus plants. In the latter study, variation in GaO activity corresponded closely with photorespiratory rate in a l l plants tested, but in kidney bean the decline in activity was much greater than the accompanying decrease in photorespiration. The results of the present study indicate that the relationship between GaO (and RuBP-case) activity, and leaf age may be altered by CO2 conditions during growth. It i s probable that many of the differences between the other studies cited above are related to different growth conditions or possibly to differences in vegetative or reproductive plant development (228). The results of experiments performed at the PI:10.5 stage of development (LPI^:7.5) have indicated physiological differences between plants grown under 0.03, 0.1 and 0.5% CO^. The patterns of response to the various measurement conditions were quite different to those at the PI:5 (LPI3:2) stage. Rates of net photosynthesis and photorespiration were similar in a l l of the plants at LPI3;7.5. The relative changes in these paramete with respect to increasing leaf age, however, differed markedly between growth regimes. 162 iv) Replacement Experiment', (Experiment 7) The growth of tomato plants at CG^  concentrations of 0.1% in air results in changes in the photosynthetic and photorespiratory metabolism of young leaves. The previous sections have shown that these changes are affected by leaf age, and that the effects are more marked in chamber-grown plants. None of the experiments described so far have indicated whether the physiological modifications induced by growth at 0.1% are retained after a subsequent period of growth in a non-enriched (0.03% C^) atmosphere. The results shown in Figures 11-32 and 11-33 provide some information on this question. Figure 11-32 illustrates the relationship between C:.. and apparent photosynthesis in leaf 3 of tomato plants at LPI : 2 after a 14 day growth period in 0.03 or 0.1% CO^ . The measurements were analogous to those described previously, and the results corroborate those shown in Figure 11-22. Mesophyll resistances and rates of photorespiration derived from the regression relationships shown i n Figure 11-32 are listed in Table 11-12. Plants exposed to 0.1% CO^  during the early stages of growth showed lower rates of photorespiration and mesophyll resistance than those grown in normal air (0.03% CO^) • Magnitudes of each of these parameters were similar to those determined in previous experiments (Figure 11-23). After a further two week growth period, during which plants were placed in a normal air atmosphere in the greenhouse, the 162a FIGURE 11-32: Apparent rates of photosynthesis in relation to intercellular CG^  concentration in leaf 3 of tomato plants grown at 0.03% or 0.1% CO^  before placement in common 0.03% atmosphere. -2 -1 quantum flux density: 520 yE m s , 400 - 700 nm Leaf temperature: 30 ± 2°C LPI: 2 ± 0.5 NOTE: Extrapolation to zero CO^  concentration to estimate rate of photorespiration. to , £9T TABLE 11-12: Rates of photorespiration (R^ and mesophyll resistance to CO2 transfer (r m) for leaf 3 of tomato plants grown at 0.03 or 0.1% CO2 before and after replacement in a common 0.03% CO2 atmosphere. Before Replacement 14 Days After Replacement Growth C02 RL_ 4 r m _2 RL 4 r m _ 1 Concentration (%) (g m s ixl0 ) (s cm (g m s ^xlO ) (s cm ) 0.03 0.30a* 20a 0.20c 22a 0.1 " 0.22b 15b 0.22cb . 20a ^Values of RL or r m bearing the same letter are not significantly different (P > 0.05) according to the SNK multiple range test. 165 relationship between CL and apparent photosynthesis was similar in plants from each growth regime (Figure 11-33). Mesophyll resistance was increased relative to the previous measurements in leaf 3 of plants grown i n i t i a l l y under 0.1% C02, although i t remained constant in the 0.03% CO2~grown plants (Table 11-12). Rates of photorespiration were decreased in plants from the 0.03% C0 2 growth regime but not in those grown i n i t i a l l y under 0.1% C02-These results are in broad agreement with those obtained at the LPI 3:7.5 stage of leaf development for plants maintained i n growth chambers throughout development (Figure 11-28). Interpretation of the results of these experiments is complicated by the confounding of effects of leaf age and replacement to a normal air atmosphere. Moreover, i t does not seem possible to separate these effects by any other experimental design, and the conclusions that can be drawn from the present data are, therefore, tentative. The results do suggest that the relative changes in mesophyll resistance and rates of photorespiration in plants grown under 0.03 and 0.1% C02 are determined by increasing leaf age and not by prolonged exposure to the particular C02 concentration in the growth chamber. Changes in photosynthetic or photorespiratory processes which occur in the young leaf in response to growth at 0.1% C02 are modified as the leaf ages. Plant replacement to an atmosphere containing normal levels of C0o has no effect on this modification. FIGURE 11-33: Apparent rates of photosynthesis i n r e l a t i o n to i n t e r c e l l u l a r concentration i n l e a f 3 of tomato plants grown at 0.03% or 0.1% C02,,14 days a f t e r placement i n common 0.03% C0 2 atmosphere. -2 -1 quantum f l u x density: 520 yE m s , 400 - 700 Leaf temperature: 30 ± 2°C LPI: 12 ± 0.5 NOTE: Extrapolation to zero C0 2 concentration to estimate rate of photorespiration. 167 The results are again in contrast to those obtained in the greenhouse study where mature basal leaves developed under CO^-enriched conditions showed inherently higher rates of photosynthesis than those developed in normal air. The i n i t i a l growth environment is clearly of great importance in determining the effect of aging on plants grown at 0.1% CO^ . It is unfortunate that these experiments have provided no definite answer to the question of whether changes induced in young leaves in response to CO^-enrichment to 0.1% are retained for an extended period upon replacement in normal air. Bishop and Wittingham (21) have suggested that inherently higher rates of photosynthesis in tomato plants grown in low light and 0.1% CO^ , upon transfer to a 0.03% C^*-atmosphere, are related to a greater pool size of certain photosynthetic intermediates. As they are careful to point out, the validity of this assumption depends upon the demonstration that differences in photosynthetic rates between 0.03 and 0.1%-grown plants are rapidly eliminated after replacement in a common, 0.03% CO atmoshpere. In general, studies on the effects of other environmental variables (notably light intensity and temperature) have indicated a rapid and reversible adaptation of plants to a change in growth conditions. An increase in light intensity resulted in a concomitant increase in maximum photosynthetic rate in shade grown maize and Amaranthus, which was complete after 6 days (118). An even faster adaptation (24 hours) was evident in Encelia califovnica and Polygonum bistortoides grown at cool temperatures and transferred to a 30°C growth chamber (204). The question of whether physiological adaptations:" induced by growth under 0.1% CO2 follow a similar pattern of rapid adjustment when plants are returned to a 0.03% CO2 atmosphere, remains to be answered. The results of experiments 4 and 6, which investigated CO2 exchange rates in relation to irradiance and CO2 concentration over a period of hours, seem to indicate, however, that very short term changes in physiology, which could be related to metabolic pool sizes, do not occur. 169 PART II - DISCUSSION The results of these experiments have focussed attention on the physiological adaptations of tomato plants to growth under supra-normal CG^  concentrations in greenhouses and controlled environment chambers. Results of experiments performed on chamber-grown plants have provided a comprehensive description of the physiological modifications which can take place under CO^-enrichment. They have answered many of the questions relating to the underlying basis for the changes observed in plants grown under supplementary CO^  in the greenhouse (Part I I , Section 1). It is clear, for example, that the increased efficiency of CC^  u t i l i z a t i o n (E^ ,) (14) in CC^-enriched plants was due, at least partly, to changes in photorespiration and the activities of two enzymes (GaO and RuBP-case). The discovery of these mechanisms extended the basic observations of the study that photosynthetic capacitywas improved as a result of CG^  enrichment to 0.1% during growth, and that this concentration was apparently optimal for maximizing inherent rates of photosynthesis. Further increases in concentration (to 0.5%) exerted different effects on the photosynthetic system in C02~enriched plants which may have masked or counteracted the enhancement due to growth at 0.1% 00^. These findings corresponded with previous studies on tomato which showed that highest rates of photosynthesis occurred in plants grown at 0.15% CO^ and that increasing growth concentrations above this level resulted in significant 170 rate depressions (185). The present studies have demonstrated, furthermore, that the enhancement of photosynthetic capacity apparently depends on the physiological age of the leaves since photosynthetic potential of older leaves of chamber-grown plants was independent of CO2 concentration. The results and observations of these studies may be considered on a number of levels. F i r s t l y , i t has been demonstrated that tomato plants can adapt photosynthetically to ambient CO2 concentrations. The relationships between this adaptation process and the more extensively investigated adaptations to irradiance and temperature are worth examining. Secondly, the physiological changes that take place are complex and require careful review to elucidate the response of plants to growth under CX^-enriched conditions. Finally, the relationship between growth CO2 concentration and photosynthetic capacity should be considered in relation to the agricultural practice of greenhouse enrichment. It may be asked, for example, how production techniques may be modified to maximize the use efficiency of (X^-enrichment f a c i l i t i e s . The present discussion is concerned with each of these points, and, in addition, serves to connect the growth analysis observations (Experiment 5) with the results of the other experiments performed on chamber-grown plants. The change in photosynthetic capacity in response to growth at 0.1% (X>2 shows many parallels with the response patterns of plants to growth under different light and temperature regimes. The higher 171 maximum rates of photosynthesis shown by plants grown at 0.1% CO^  as compared with 0.03% CO^  in these experiments, correspond to similar differences between plants grown under high and low ambient light intensities (24, 38, 44, 67, 76, 111, 233, 299). Similar photosynthetic adaptation has "also been shown i n relation to growth temperature. Plants from various environments tend to show higher maximum photosynthetic rates after growth under warm conditions (204, 205) although rates of arctic and alpine plants may be reduced by this treatment (20) . Previously, plant adaptation to different light and temperature regimes has recieved much more attention than the physiological changes resulting from growth at supra-normal CO^  concentrations. This emphasis is probably due to the greater variability in f i e l d light and temperature conditions as compared to atmospheric CO2 concentration (203). The widespread manipulation of CO^  levels in greenhouses has recently provided a strong incentive to investigate physiological adaptation to high CO2 concentrations. The present results also allow some comparisons to be made between the underlying mechanisms involved in adaptive responses to different environmental variables. The importance of changes in RuBP-case activity, photorespiration and the degree of oxygen inhibition in the regulation of photosynthesis i n plants grown at 0.1% CO2 as compared with those grown at 0.03% CO, has been emphasized in the preceding sections. As already discussed, variations in RuBP-case activity also play an important role in the photosynthetic adaptation of plants to high irradiance. On the other hand, increases in maximum 172 rates of photosynthesis in response to increasing irradiance during growth cannot be attributed to changes in photorespiration in at least one plant species (Atriplex; 24). The physiological basis for adaptation to different temperature conditions has been the subject of some controversy (26, 57, 204), but recent studies have suggested that changes in RuBP-case activity, dark respiration rates and thermo-stability of photosystem 2 activity are important factors in Atriplex lentiformis (226,' 227). It is evident, therefore, that adaptation to a growth CT^  concentration of 0.1% involves some physiological changes which are common to other adaptation processes (changes in RuBP-case activity) and others which are distinctive (changes in photorespiration and degree of oxygen inhibition of photosynthesis). It has been shown in the present experiments that the photosynthetic adaptation to supra-normal concentrations depends on the level of CO^-enrichment. Some of the mechanisms underlying the enhancement of photosynthesis at a growth concentration of 0.1% have been characterized in this study, but the reasons for the absence of a similar response when the concentration was raised to 0.5% requires further consideration. It seems l i k e l y that an overall suppression of enzyme activity (particularly RuBP-case) may be partly responsible, although the reasons for such an inhibition are not clear. Some explanation may be provided by previous studies on the relationship between CO^  concentration, leaf starch content and photosynthesis in tomato plants. At a growth C0o concentration of 0.5% tomato leaves tend to 173 accumulate excess quantities of starch (184, 186) and become rolled and mishapen (188). Madsen (190) postulated that these effects may result in chloroplast deformation and could explain his previous results which showed a reduction in photosynthetic rates at growth concentrations above 0.15% (185). Another explanation may be that high leaf starch contents cause a reduction in photosynthesis possibly through a feedback control on carboxylating enzyme activity. Reduction in leaf photosynthesis rate as a result of large accumulations of starch have been demonstrated in a number of species (75, 130, 143). In others, however, photosynthesis is unaffected by starch content (63) and there i s , as yet, no general support for a link between leaf photosynthesis rate and level of accumulated assimilate (213). The possibility that other regulatory mechanisms contribute to the effects discussed above cannot be discounted. It would be unwise to conclude from the present data that the mechanisms involved in the photosynthetic adaptation of tomato plants to growth at 0.1% CO^  have been f u l l y characterized. Undoubtedly increased RuBP-case activity plays an important role in this adaptation. The relatively lower mesophyll resistances at low irradiance in greenhouse C^-enriched plants (Figure 11-11) may not, however, be directly correlated with enhanced carboxylation by RuBP-case (a dark reaction). Elevated concentrations during growth may cause other changes in photochemical fixation reactions in the chloroplasts which \ relieve inhibition of fixation at low irradiances. 174 The finding that the photosynthetic capacity of tomato plants changes with growth CO^  concentration holds important implications for the use of supplemental CC^  in the production of commercial greenhouse crops. The data presented in Figure II-9a clearly indicate that photosynthesis in leaves of plants grown at approximately 0.1% (X^ in the greenhouse are more responsive to subsequent increases in ambient CO^  concentration, than leaves of unenriched plants. This i s confirmed by the relationship between intercellular CO^  concentration and net photosynthesis for young leaves of chamber-grown plants (Figure 11-22). These observations suggest that maximum benefit in terms of photosynthetic enhancement w i l l be obtained by exposing young tomato seedlings to supplemental as early as possible. General recommendations state that CO^-enrichment may be commenced at the time of "pricking out" the young plants (3, 48). The present data indicate that this practice results, not only in immediately increased rates of photosynthesis, but also in higher rates at a slightly later stage of development than i f the onset of CO2-enrichment i s delayed. The growth chamber experiments indicated that the enhancement of photosynthesis as a result of growth at 0.1% CO2 was increased at higher irradiances and was maximal under conditions of saturating irradiance (Figure 11-20). This was not fu l l y supported by the results of the greenhouse experiments, although there were indications that increasing irradiance enhanced differences between enriched and non-enriched plants (Figure 9b). If subsequent work verifies this effect, then the benefits obtained from 175 CO^-enrichment of greenhouses crops may be even greater than previously imagined. Not only are inherent rates of photosynthesis enhanced by growth at 0.1% CO^ , but this enhancement may be increased by seasonally increasing light levels. The beneficial effects of supplementary CO^ in greenhouse atmospheres are well established and require no further experimental verification (297). The results of the present studies have indicated, however, that the control of C02~enrichment levels at around 0.1% may be more important than has been realized. To i l l u s t r a t e this point i t is worthwhile to consider the results of growth analysis (Experiment 5) and i t s relationship to other experiments on chamber-grown plants. The higher NAR and RGR of 0.1% C02 versus 0.03% C02~ grown plants (Table II-8) are readily explained in terms of enhanced leaf photosynthesis at 0.1% C02. This enhancement is accentuated by an upward shift in photosynthetic capacity. The higher NAR in the 0.5% C02-grown plants as compared with those grown at 0.03% also resulted from an increased photosynthetic rate in direct response to the increased C02 content of the air. NAR of the 0.5% C02-grown plants was, nevertheless, lower than of those grown at 0.1% C02- This difference undoubtedly reflects the higher inherent photosynthetic capcity of the latter plants. It is worth repeating that the mutually compensating effects of decreased LAR and increased NAR in the 0.5% C02 plants resulted in similar RGR to those grown at 0.03% C0 2 > Plants grown at 0.1% C02, on the other hand, showed sufficient increase in NAR to produce a significant increase in RGR as compared to the 0.03% C02-grown controls. It is clear that increasing the CO content of a tomato 176 greenhouse atmosphere to concentrations greatly in excess of 0.1% is unwise. Enrichment to 0.5% is not effective in increasing NAR over values obtained at 0.1% CO^ . It may, in fact, result in a similar rate of vegetative development to that shown by plants grown without supplementary CO^. These effects may be of special concern in other crops, particularly greenhouse cucumbers which are often subjected to very high levels of CO in the early stages of growth (Part I; 162). It i s , perhaps, surprising in view of the considerable research that has been undertaken on the subject of CO^-enrichment, that the phenomenon of photosynthetic adaptation to high CO^ concentrations has only recently been recognized (190). The results of these studies have determined the responses of tomato plants to different CO^  concentrations in excess of normal atmospheric levels and, for the f i r s t time, have established physiological mechanisms for these responses. The question of whether similar adaptations are shown by other species opens a large and intriguing area for future research. PART III The -influence of supra-normal CO^ concentrations on photosynthesis, and vegetative and '•productive development of Pharbitis nil Ghoisy. plants Long and Short Bays . 178 INTRODUCTION In the early work of Garner and Allard (105, 106), the phenomenon of photoperiodism was clearly established as the basic controlling mechanism for flowering in at least some plant species. Since then some research has been concerned with characterizing other environmental variables, including light intensity, temperature, and carbon dioxide, which may substitute for, or modify the effect of the timing of light/dark periods. The importance of light intensity was established in early work which found that for f l o r a l induction to occur in obligate SDP, the long (longer than c r i t i c a l ) dark period must be followed by a suitable period of high intensity white light (34, 113, 191, 259). This light requirement may be replaced in some plants by the a r t i f i c i a l application of sucrose and some tricarboxylic acid cycle intermediates to the leaves or stems of plants maintained in continuous darkness (56, 159, 17/5, 19.8). Such studies strongly suggested that high intensity light preceding or following an inductive dark period was concerned with the photosynthetic production of respiratory substrates necessary for complete f l o r a l development. This explanation for the light intensity effects was supported by earlier work (34, 117) which established that atmospheric CO^  was required.during the inductive light period in Bi l o x i soybean and Kalanehoe'.. It has since been corroborated by studies which have demonstrated that flowering may be completely inhibited by the application of the photosynthetic inhibitor DCMU during a normally inductive photoperiod (15). 179 Temperature treatments have also been found to affect photoperiodic behavior, but the interpretation of these effects has not proven as straight forward in the case of light intensity effects. For example, De Zeeuw (304) showed that the SDP Xanthium pennsylvanianm/was induced to flower under non-inductive LD conditions i f the plants were maintained at 4°C for part of the long light period. Similar results were obtained with Xanthium pennsylvanioum by Nitsch and Went (217) and with Pharbitis nil by Ogawa (220). Some indication of the complicated nature of these temperature effects was provided by the finding that in Pharbitis, the cold treatment must be continuous throughout the light period (141), while in Xanthium, temperatures of 4°C must follow or precede a 25°C treatment for flowering to occur. Temperature has also been shown to exert negative effects on flowering. In Pharbitis, the normally inductive effects of a long dark period were n u l l i f i e d by low temperature treatment, the effect being most pronounced when low temperatures were applied near the middle of the dark period (212). • The mechanisms by which temperature affects flowering are not well understood. Nitsch and Went (217) explained their results with Xanthium by suggesting that low temperatures inhibited the light-mediated destruction of a labile flowering stimulus, formed in the dark. In LDP i t has been shown that some gibberellins are effective in promoting flowering in SD (201) and are produced under cold conditions. Gibberellins do not, however, substitute for SD in SDP in various temperature regimes (244). 180 It seems equally l i k e l y that temperature influences the flowering process by altering the rate of certain metabolic reactions (199), but the true mechanism probably involves effects on gibberellin production, reaction rates as well as other biochemical systems. Atmospheric carbon dioxide concentration has also been found to modify photoperiodic responses. In a recent, study, high concentrations of (1% and above) were shown to. inhibit flower formation in two SDP {Pharbitis and Xanthiim), and to promote flowering in the LDP SiV&ne armeria when a l l were maintained under SD conditions (234). In another study, 1% CO2 was found to inhibit flowering under normally inductive conditions in the SDP Lemna paucieostata (perpusilla) (232). In each case i t was suggested that the effect of the high (X^ concentration was due to increased accumulation of photosynthetic products which directly or indirectly prevented flowering. Inhibitory effects of high CO2 concentrations are not restricted to SDP. An earlier study with the LDP Lemna gibba (153), indicated that 3.5% GO2 suppressed flowering, and a subsequent review attributed this inhibition to an indirect effect of enhanced photosynthesis (154). Some research has also considered the effects of high CO2 concentrations on photoperiodically sensitive plants grown in non-inductive photoperiods. Studies on Sitene armeria (234) and Xanthiim (51), however, have indicated that at least some long and short day species may be induced to flower by the provision of 181 supplementary CO^  during a photoperiod normally ineffective for f l o r a l induction. The study of the effects of secondary factors such as light intensity, temperature, and CC^  on photoperiodism have, in the past, yielded important information on the mechanisms involved in f l o r a l induction. It has been shown that normal flowering can be controlled and modified by changing environmental conditions exclusive of the length of the light/dark period. Such modifications may hold important implications for the control of flowering in commercially important crops. At this time the potential of CO^  enrichment for this type of control is unknown and, in fact, the link between photoperiodic behaviour and CX^ concentration is not firmly established. It •seemed desirable therefore,,to carry out further investigations into the role of CO^  in f l o r a l induction. In addition, the interpretation that very high CO2 concentrations affected flowering via the enhancement of photosynthesis seemed inadequate. Many plants attain CO2 saturation of the photosynthetic system at concentrations between 0.1 and 0.2% (103), yet concentrations as low as this were ineffective in modifying the photoperiodic flowering response in Xanthium (51). Furthermore, very l i t t l e information was available on the effects of high levels of CO2 on vegetative development in photoperiodically sensitive plants. The general objective of the research described in this part of the thesis was to assess the influence of low (0.1%) and high 182 (1.0 - 5.0%), supplementary concentrations on the developmental physiology of Pharbitis nil seedlings. Investigations were carried out to obtain....information on both vegetative and reproductive development under different conditions of photoperiod and concentration (Experiment 9). The relationship of the effect of growth concentration on development, and photosynthetic activity was studied by measuring the gas exchange characteristics of plants at C00 concentrations up to 2.5% (Experiment 10). 183 MATERIALS AND METHODS i) Growth System Due to the unsuitability of conventional growth chambers for work requiring the maintenance of constant supra-normal CC^  concentrations i t was necessary to construct a new growth system for these experiments. The maintenance of appropriate conditions of irradiance, temperature, photoperiod and CC^  concentration in the plant environment were the general requirements of the experimental system, which was developed for use in conjunction with four growth chambers. A flow plan i s shown in Figure I I I - l . The central feature of the system was a group of four plywood cabinets lined with thin sheets of PVC. Each cabinet was divided in two by another sheet of PVC to give a total of 8 individual treatment boxes of internal dimensions 55 x 60 x 25 cm. Each of these was completely self-contained and sealed from i t s neighbour and the external atmosphere with silicone rubber wherever PVC sheets abutted. The top and bottom walls of each treatment box were of clear Plexiglas and access was provided by a 15 x 35 cm opening, which in normal operation was sealed with a Plexiglas plate tightly pressed to rubber gaskets in the front of the treatment box. During system construction, two treatment boxes (numbers 4 and 7, Figure I I I - l ) were randomly selected for subsequent use as normal air controls. These two were fitted with bottom plates perforated with 2.5 cm diameter holes to ensure adequate 183a FIGURE I I I - l : Diagram of gas flow system used in Experiments 9 and 10. LEGEND F: Flow meter MF: Variable connection to mass flow sensor (1 line may be connected at one time) C: Flow control clamp TB: Treatment box nc: normally closed no: normally open /: clamps for optional closure of individual gas lines air pump C1<> C2C ) C 3 < > C 4 c > C 5 < > C 6 0 \' v v v " " no/ F2F3 185 ventilation and exchange of air with the external atmosphere. Mixing of air within every treatment box was facilitated by a small electric fan, mounted close to the top of the front wall. Each of the gas-tight boxes was provided with one outlet and two inlet ports for the input and through-flow of air and CO gas streams. Tygon plastic tubing was used throughout for the gas supply lines. During the course of the experiments, air was supplied continuously to the treatment boxes from a single gas pump (Doerr Electronics Ltd.). The air flow rate was maintained at 0.25, 1.29 or 4.65 1 min"1 (in the 5.0, 1.0 and 0.1 % C0 2 treatments, respectively). Flow control was by means of screw-mounted tubing clamps which allowed variable constriction of the plastic tubing. Just after the flow control clamps, each supply line was divided into two with one branch terminating in an open-ended tube and the other passing directly to a treatment box. Air flow rates were monitored by completely closing the latter branch and connecting a mass flow sensor (Datametrics Inc. - see Part II, Section 2: Materials and Methods) to the open-ended tube. Whenever this was done an uninterrupted flow to the treatment box was maintained by completing the circ u i t , with the mass flow sensor in place through another plastic supply line, following the mass flow sensor. In normal operation, these auxilliary lines were completely closed by appropriately placed tubing clamps (Figure I I I - l ) . 186 Supplementary CG^ was supplied from a cylinder of pure, compressed CC^ and the flow of gas to each treatment box was metered through needle valves and rotameter flow meters (Matheson 610A). Flow rates were varied between 10 and 40 ml min depending on the required CO^  concentration. The treatment box CO^  inlet ports were located directly behind the fans, and the inlets for the air . stream, 10 cm to the l e f t . This arrangement allowed efficient mixing of the incoming gases. Concentrations of CO^  in the outgoing air streams were measured with a Beckman model 864 IRGA which had a range of 0 - 2.5% C0 2. In the case of the 5% C02 treatment, the outgoing air stream was mixed with nitrogen gas in a 1:10 ratio by means of a gas mixing pump (H. Wosthoff, BRD) before entering the IRGA. For the experiments, two treatment boxes were placed in each of 4 separate growth chambers (Sherer model 4005). The growth chamber controls were programmed to provide a photoperiod of 16 hours light and 8 hours darkness (LD treatment) on four of the treatment boxes, 8 hours light and 16 hours darkness (SD treatment) on the other four boxes. Light intensity at plant level inside a l l treatment chambers was between 3000 and 4000 lux (approximately 50 - 70 liE m s ., 400 - 700 nm), and air temperatures throughout the experiments were maintained at 25° ± 1°C. 187 ii) Experiments a. Effects of growth C0„ concentration and photoperiod on  development (Experiment 9) Two replicate t r i a l s were conducted to investigate the effects of CO^  concentration and photoperiod on vegetative and reproductive growth of Pharbitis nil seedlings. Experiment 9 was designed in a 2-way factorial arrangement with 4 levels of CC>2 concentration (0.03, 0.1, 1.0 and 5.0% by volume) and 2 photoperiods (LD and SD as specified previously). The objective was to evaluate parameters of vegetative plant development (relative growth rates of whole plants and leaves, leaf plastochron index and change in stem height over the treatment period), and flower development (number of flower buds formed and the serial number of the primary flower bearing node). Pharbitis nil seeds (cv. Imperialis Japanese from Stokes Seeds Ltd., St. Catharines, Ont.) were scarified and sown in moist mica peat. Germination took place in three to four days in a growth chamber at a constant temperature of 25°C. Immediately after the cotyledons emerged, seedlings which were uniform in height and development were individually transplanted to 10 cm square plastic pots containing mica-peat which were then replaced in the growth chamber. After transplanting, 72 seedlings were maintained for two weeks 188 under a photoperiod consisting of 16 hours light and 8 hours darkness. The light intensity was 4000 lux (ca. 70 yE m"2 s"1,400-700nm) and consisted of a mixture of fluorescent and incandescent illumination. Temperature was maintained at 25°C both day and night. At the end of the 14 day preconditioning period, a l l plants had formed two to three true leaves and were quite similar in terms of vegetative development. At this stage 6 seedlings were placed in each of the eight treatment boxes, 4 of which were exposed to SD conditions and 4 to LD conditions under the range of CG^  concentrations previously described. Leaf and total plant dry weights were determined for each of the remaining twenty four seedlings (Harvest 1). Also, at this time, the stem height (distance between the peat surface and apical shoot tip) of 3 randomly selected plants in each treatment box was measured. Before placing seedlings in the treatment boxes each pot was watered to f i e l d capacity with a nutrient solution (see Part I I , Section 2: Materials and Methods), and in most cases further watering during the course of the experiment was unnecessary due to the high humidity maintained within the treatment boxes. Watering of plants in those boxes open to the growth chamber atmosphere was required at approximately 4 day intervals. In these cases the treatment box front plate was removed and s o i l moisture was 189 readjusted to f i e l d capacity using tap water. The CC^ treatment period of each t r i a l was 14 days during which the gaseous concentrations within the chambers were constantly monitored and maintained at the desired l e v e l s . At the end of t h i s period three plants were harvested i n each treatment (harvest 2) for the determination of dry weights. Relative growth rates for whole plants and leaves were calculated from data obtained at harvests 1 and 2 using the formulae previously c i t e d (Part I I , Section 2: Materials and Methods). For the remaining three plants in each treatment box, the stem heights were remeasured and the number of flower buds and t h e i r nodal pos i t i o n s were noted. An a d d i t i o n a l assessment of vegetative development was made by determining the plastochron index of each plant based upon a l e a f reference length of 30 mm (Part I I , Section 1: Materials and Methods). Upon completion of a l l measurements, these remaining plants were returned to a growth chamber under i d e n t i c a l conditions to those used during the preconditioning period (LD, 25°C and 0.03% C^) . Seven days l a t e r , the number of flower buds per plant was reassessed. Data were analyzed i n accord with the f a c t o r i a l design using analysis of variance (P = 0.05). Mean separation was c a r r i e d out by the two-stage ..' •„ (SNK) multiple range test, (a = 0.05). 190 b. Effects of growth CO„ concentration and photoperiod on photosynthesis rate and stomatal resistances (Experiment 10) Sixteen plants for use in gas exchange t r i a l s were raised from seed and preconditioned as described above. After preconditioning, two plants were placed in daily sequence in each of the eight treatment boxes. The allocation of plants to chambers was at random except that plants assigned to the same chamber were inserted on consecutive days so that interruption of the gas flush treatments was minimized. In four of the treatment boxes, CO^  concentration was controlled at 1.0% with two of the boxes under SD conditions and two under LD conditions. The control treatment consisted of 0.03% CO2 (maintained by the flushing of growth chamber a i r ) 3 with two treatment boxes at each photoperiod. Each plant was subjected to fourteen consecutive 24 hour treatment cycles after which i t was removed for the gas exchange determinations. These determinations were conducted on the second true leaf and consisted of two complete sets of measurements performed daily between 0900 and 1500 h. The f i r s t set utilized a simple closed gas exchange system for the determination of photosynthetic rates at concentrations between 2.5% and 0.1% CO^ . The design of the system was identical to that described in Part II of this thesis (Section 2: Materials and Methods), except for the use of a Beckman Instruments Ltd. Model 864 191 IRGA. An open flow gas system incorporating a water-bath immersed bubbler, flow,meter, dew-point hygrometer and leaf chamber (Part I I , Section 2: Materials and Methods) was used in the second measurement set to determine stomatal resistances in relation to CO^  concentrations between 0.03% and 2.5%. In this case . CG^  concentrations were established by mixing CO^-free air and 3% CO^  in nitrogen from compressed gas sources, and verified prior to passing through the leaf chamber, by means of the IRGA. The air stream was humidified to between 70 and 80% relative humidity and rates of trans-piration of individual leaves were calculated from chamber inlet and outlet dew point temperatures, air temperature and flow rate. From these data a combined estimate of stomatal and boundary layer resistance was calculated (146). Upon completion of a l l measurements the leaf was excised and i t s area determined as described previously (Part I I , Section 1: Materials and Methods).. 1 192 RESULTS i) Effects of COr. Concentration on Flowering and Vegetative Growth  in LB and SD (Experiment 9) Mean relative growth rates of whole plants and leaves of Pharbitis seedlings grown in LD and SD and at 0.03, 0.1, 1.0, and 5.0% CO^ , are shown in Table I I I - l . In LD treatments, rates were increased at 0.1% CO2 relative to the 0.03% CO^  control, while 5% CO2 resulted in suppression. Overall, whole plant growth rates were higher in LD than in SD at 0.03 and 0.1%, although at higher concentrations (1 and 5%) increasing daylength had no effect on this index of dry matter accumulation. A similar pattern was shown by relative leaf growth rates in LD, with the exception that at 1% C02, rates were significantly higher than at the same CO2 concentration in SD. Further information was provided by consideration of a number of parameters of vegetative and reproductive development. In each case, the data indicated a significant interactive effect of CO2 concentration and photoperiod (Table III-2) . At the end of the 14 cycle, LD treatment, plants grown at 0.03% and 0.1% CO2 showed a slightly more advanced stage of plastochron development than those grown under SD (Table III-3). At 1 and 5% C0 2, however, the mean, f i n a l index was similar for the two photoperiods and was significantly higher than in those plants grown under the lower C0o concentrations. TABLE I I I - l : Relative growth rates (RGR) of Pharbitis plants and leaves exposed to photo-period and CO treatments during a 14 day period. SD* LD 0.03 oTl lTo 5T0 0.03 0.1- 1.0 5.0 v RGR whole plant 0.1643*** 0.159* 0.165* 0.171* 0.179b 0;.19.IC 0.177ab 0.164* (g g - 1 day - 1) RGR leaves 0.185* 0.181* 0.188* 0.189* 0.195b 0.202° 0.194b 0.185a (g g - 1 day - 1) *SD: Short Day treatment (8 hours light, 16 hours dark). LD: Long Day treatment (16 hours light, 8 hours dark). **Percentages of CO2 in treatments. ***Means in either row of the table designated with the same letter are not significantly different according to SNK multiple range test (P > 0.05). TABLE III-2: Significance l e v e l s of terms i n analysis of variance on various parameters of growth and development i n Pharbitis plants subjected to d i f f e r e n t photoperiod and C0„ treatments. Source of Var i a t i o n Number of F l . Buds at End of 2 Week Treatment Number of F l . buds 1 Week After End of Treatments Change i n Plant Height Over Treatment Period F i n a l Plastochron Index F i n a l Lf Area Per Plant F i n a l Lf Dry Wt Per Plant Photoperiod AA AA AA A AA AA CO concen-t r a t i o n AA • * AA AA AA AA Photoperiod a. j . X AA AA AA AA AA C0„ con-centration *Difference s i g n i f i c a n t at 95% l e v e l . **Difference s i g n i f i c a n t at 99% l e v e l . TABLE III-3: Mean plastochron index of Pharbitis.plants at end of 14 days in photoperiod and CO treatments. SD* LD 0.03** 0.1 1.0." 5.0 0.03 0.1 1.0" 5.0 5.63a*** 5.69a 7.47° 7.81° 6.27b 6.26b 7.44° 7.54° *SD: Short Day treatment (8 hours lig h t , 16 hours dark). ...LD: Long Day treatment (16 hours light, 8 hours dark). **Percentages of CO^  in treatments. ***Means designated with the same letter are not signficantly different according to SNK multiple range test (P > 0.05). 196 Under SD conditions and either 0.03 or 0.1% C02, plants tended to remain small with l i t t l e elongation of the main stem. Once again, the response to 1 and 5% CO^  during growth was quite different. Under those conditions the main axis of the plants showed a marked tendency to elongate. This effect i s clearly indicated by the values for mean change in stem length (Table III-4) and by the comparisons shown in Figure III-2. Elongation of the main stem in plants grown at the two highest CO^  concentrations in SD was approximately twice that of the normal air controls. In Long Days a l l plants showed greater stem elongation but again, the effect was most marked in those grown under 1 and 5% C02 (Figure III-3, Table III-4). It is interesting to note in the present context, that the stem elongation response exhibited at the two highest C02 concentrations was not accompanied by an increase in relative growth rate of the plants (Table I I I - l ) . The normal effects of photoperiod on flowering in Pharbitis were also modified by 1 and 5% C02 during growth. Table III-5 l i s t s the number of flower buds produced at the end of the 14 day C02 treatment period, and after an additional week, during which the plants were maintained under LD conditions. In normally non-inductive long photoperiods, flower bud formation occurred on some plants exposed to 1 and 5% C02 (Table III-5, Figure III-4). This response was not always exhibited in this experiment. In the f i r s t t r i a l two plants, and in the second only one out of three, showed flower bud development under LD and 1 or 5% C0„ TABLE III-4: Mean change in stem height (mm) of Pharbitis plants exposed to photoperiod and C02 treat-ments during a 14 day period. SD* * LD 0.03** 0.16 1.0 5.0 0.03 0.16 1.0 5.0 339.5a*** 497.0a 700.0b° 660.0b 810.3° 717.5b° 1214.0d 1135.0 *SD: Short Day treatment (8 hours light, 16 hours dark). LD: Long Day treatment (16 hours light, 8 hours dark) **Percentages of CO^ in treatments. ***Means designated with the same letter are not significantly different according to SNK multiple range test (P > 0.05). TABLE III-5: Mean number of f l o r a l buds formed on Pharbitis plants after 14 days in photoperiod and C0„ treatments, and 7 days after the end of treatments. SD* LD 0.03** 0.1 1.0 5.0 0.03 0.1, 1.0 5.0 End of treatments cb 1.5*** 2.75° 2.75C i.ooab o.ooa o.ooa 2.75° 2.0 C b End + 1 week 4.75b 3.50b 4.5b 3.00b o.ooa o.ooa 3.25b 4.00b (All plants replaced to LD conditions) *SD: Short Day treatment (8 hours light, 16 hours dark). LD: Long Day treatment (L6 hours light, 8 hours dark)., **Percentages of CO in treatments. ***Means in either row of the table designated with the same letter are not significantly different according to SNK multiple range test (P > 0.05). 198a FIGURE III-2: P h a r b i t i s plants A and B. A exposed to 14 SD cycles and 0.1% CO^ ; B exposed to 14 SD cycles and 1% CO^ . Plants photographed 7 days after return to uniform LD conditions (0.03% CO ) . 1'99a FIGURE III-3: Pharbitis plants C and D. C exposed to 14 SD cycles and 0.03% CO^; D exposed to 14 LD cycles and 5% CO^. Note the flower formed on plant C. Plants photographed 7 days a f t e r return to uniform LD conditions (0.03% C0 o). 2 0 0 200a FIGURE III-4: First f l o r a l bud at node 6 on plant D (see Figure III-3) exposed to 14 LD cycles and 5% CO . Photograph at same time as Figure III-3. 201 202 conditions. No flower buds were observed on plants maintained under 0.03 or 0.1% C02 in LD at the end of the C02 treatment period, or 1 week later. Higher than normal C02 concentrations had no significant effect on the total number of flower buds formed in SD. However, growth at 1 and 5% C02 in SD did have a marked effect on the position of the f i r s t f l o r a l bud upon the main stem, and on the overall morphology of flowering in these plants (Table III-6, Figures III-2 and III-5 to III-8). Plants grown at 0.03 or 0.1% C02 in SD formed their f i r s t f l o r a l buds in the axils of leaves 3 or 4 (Table III-6). Development proceeded with f l o r a l buds being formed at each subsequent node on the main stem axis (Figures III-5, III-6), and in many cases a terminal flower bud was formed (Figure III-7). On the other hand, at 1 and 5% C02, the f i r s t flower buds were formed at nodes 6, 7, or 8.(Figure III-8, Table III-6) and the cessation of stem elongation by induction of a terminal flower bud was never observed. It is interesting to note that when flower buds were formed under LD conditions at high C02 concentrations, the f i r s t ones also appeared at nodes 6, 7, or 8 (Figures III-4, Table III-6), with no f l o r a l bud development below this level on the stem. Visual observation of plants exposed to 1.0 and 5.0% C02 in LD or SD revealed a less vigorous flowering response in comparison to those grown at 0.03 or 0.1% C02 in SD (cf. Figures III-4 and III-5; III-6 and III-8). TABLE III-6: Mean node of f i r s t f l o r a l bud formed on Pharbitis plants after 14 days in photoperiod and CC^  treat-ments . SD* LD 0.03** 0.1 1.0 5.0 0.03 0.1. 1.0 5.0 3.5**** 3.25a 7.0° 6.75C nf b nf b 7.25° 7.75C nf: No f l o r a l buds formed. *SD: "."Short Day treatment (8 hours light, 16 hours dark). **LD: Long Day treatment (16 hours light, .8 hours dark).. ***Means designated with the same letter are not significantly/ different according to SNK multiple range test (P < 0.05). 203a FIGURE III-5: Detail of developing f l o r a l buds and one flower on plant C (see Figure III-3) exposed to 14 SD cycles and 0.03% C02. Photograph at same time as Figure III-3. 204a FIGURE III-6: Third f l o r a l bud at node 6 on plant A (see Figure III-2) exposed to 14 SD cycles and 0.1% CO^. Photograph at same time as Figure III-2. 205 205a FIGURE III-7: Detail of plant A showing terminal f l o r a l bud '(see also Figure III-2) . Plant exposed to 14 SD cycles and 0.1% CO^. Photograph,at same time as Figure III-2. 206 206a FIGURE III-8: D e t a i l of f i r s t f l o r a l bud at node 7 on plant B (see Figure III-2) exposed to 14 SD cycles and 1% CO2. Photograph at same time as Figure III-2. 208 ii) Effects of 0.03 and 1.0% CO During Growth in LD and SD on Subsequent Rates of Net Photosynthesis and Stomatal Resistances  (Experiment 10) Figures III-9 and 111-10 depict the effects of C02 concentration on net photosynthesis in the second leaf of Pharbitis seedlings, grown under four combinations of C02 and photoperiod. Plants grown for 14 days at a continuous concentration of 1% C02 showed lower inherent rates of net photosynthesis at test concentrations above 0.5% as compared to those subjected to normal air (0.03% C0 2). These differences were independent of photoperiod, except at a test concentration of 0.5% C02 where rates were significantly different between plants grown in LD and those grown in SD. An explanation for the rate reductions in the high C02-grown plants was sought in the relative magnitudes of leaf resistances at test C02 concentrations of 1.0% and 2.5% (Table III-7). It was apparent that.increased stomatal resistance was not a contributory factor, since, with values of 9.50 and 6.78 s cm x, mean resistances for leaves of the 1%-grown plants were quantitatively, but not significantly, lower at 2.5% than for those of plants grown at 0.03%.. C02. Similarly, at a test concentration of 1.0% C02 no significant differences were found between plants subjected to different growth C02 concentrations. An increase in test C02 concentration from 0.03 to ' 2.5% caused a marked increase in stomatal resistance (Table III-7). The magnitude of the increase was somewhat variable depending 208a FIGURE III-9: Apparent rates of photosynthesis in relation to CO^  concentration in Pharbitis -seedlings grown in 0.03% or 1.0% CO in Long Days. O 209a FIGURE 111-10: Apparent rates of photosynthesis in relation to CO^  concentration in Pharbitis seedlings grown in 0.03% or 1.0% CO in Short Days. to f—1 o TABLE III-7: Combined leaf and air (boundary layer) resistances to CO-2 transfer in the second leaf of Pharbitis plants grown under LD or SD photoperiods and 0.03 or 1.0% C0„. Test CO Concentration SD LD (8" hours.'.light (8 hours dark 16 hours dark) 16 hours light) 0.03% C02 1.0% C02 0.03% CO 1.0% C02 0.03% 6.33* 6.87 6.09 5.35 0.1% 7.34 6.56 6.59 5.93 1.0% 8.13 9.21 8.91 6.45 2.5% 10.58 9.50 9.14 6.78 *Leaf + air (boundary layer) resistances in s cm . Values in any row of the table are not significantly different according to analysis of variance (P > 0.05). 212 upon the particular combination of growth photoperiod and CO^  concentration, but averaged 2.8 s cm ^ between the lowest and highest test concentration. With one exception (1.0% growth CO^  under SD photoperiod), resistance increments were also noted between 0.03 and 0.1, and 0.1 and 1.0% test C02. At the lower concentrations these increments corresponded closely with those noted in a number of previous studies (e.g. 123). Increased stomatal resistance at high CO^  concentrations apparently imposed l i t t l e restriction on photosynthetic activity. In plants grown at 0.03 or 1% C02 in LD or SD, photosynthesis approached saturation between 0.1 and 0.5% CO^  (Figures III-9 and 111-10) . The gradual levelling off of the C02 response curves above 0.5% C02 was probably due more to an inability of the photosynthetic apparatus to u t i l i z e additional CX^  than to the effects of stomatal closure. This view is supported by other studies which have shown that increased CO concentrations around the leaves of corn and sunflower resulted in increased intercellular concentrations and enhanced photosynthesis despite an increase in stomatal closure (291). In plants grown at 1.0% CO^ , rates of net photosynthesis tended to level off at somewhat lower test C02 concentrations, compared to those grown in 0.03% C^. The response of plants subjected to SD photoperiods and 1.0% CX^  to test concentrations above 0.5% was quite erratic, and rates at 1.0% and 2.5% (2.68 and 213 2 . 4 5 x 1 0 g C O 2 m s respectively) were of s i m i l a r magnitude to those at 0 . 1 % ( 2 . 6 6 x 1 0 ~ 5 g C 0 2 m~2 s _ 1 ) (Figure 1 1 1 - 1 0 ) . Plants grown at 1 . 0 % CO^ i n LD showed a more consistent response (Figure I I I - 9 ) . Nevertheless, l i t t l e increase i n photosynthetic rate was observed above 0 . 5 % C^. In summary, the r e s u l t s presented i n Figures I I I - 9 and 1 1 1 - 1 0 show that net photosynthetic rates are inherently lower at test C 0 2 concentrations above 1% i n plants grown at 1% C 0 2 > as compared to those grown i n normal a i r ( 0.03%). The lower rates cannot be explained i n terms of increased stomatal resistance but appear to depend on photosynthetic reactions i n s i d e the leaves. It i s also c l e a r from these r e s u l t s that the photosynthetic f i x a t i o n of C 0 2 i s e s s e n t i a l l y saturated at concentrations between 0 . 1 and 0 . 5 % C 0 2 i n a l l plants. Only a slow, and sometimes e r r a t i c , increase of photosynthesis with increasing test C 0 2 concentration was evident above 0 . 5 % CO.. 214 DISCUSSION The results obtained in these experiments indicate that very high CC^  concentrations (1 and 5%) have distinctive effects on the vegetative and reproductive growth of Pharbitis nil under different photoperiods. The findings have provided more support for the hypothesis, established by a number of previous workers (51, 234, 247), that these CO^  concentrations are effective in modifying plant growth and development. , Further understanding of the developmental role of CG^  required close examination of the effects of very high CO^  concentration on vegetative and reproductive growth and how they relate to one another. It may be asked, for example, i f the effects share common physiological mechanisms, and which ones may be involved? The following discussion i s concerned with these questions. The relationship between CO^  concentration during growth under LD or SD photoperiods and vegetative development i s well illustrated by the data shown in Tables I I I - l , III-3, and III-4. The results showing increased stem length and leaf production, but a similar RGR in plants grown at 1 . 0 or 5.0% CO^  as compared with those grown at 0.03 or 0.1% C O 2 , suggested that very high CO^  concentrations induced changes in dry matter distribution within the plants. In a previous study Purohit and Tregunna (234) observed that Pharbitis seedlings grown in SD and 1.5% C O 2 had fewer leaves per plant and were shorter than those grown at 0.1% C 0 9 . The discrepancies between 215 their results and those of the present study may have arisen as a result of the different methods employed in the control of gas concentrations, or because of differences in irradiance supplied during growth. The conflicting results may also be due to an erratic morphogenetic behaviour of plants to very high CO^ concentrations, corresponding with the photosynthetic response of some species to concentrations above 0.2% (207). It is apparent that the increases in stem length and leaf production shown by Pharbitis seedlings in the present study are not simply, related to increased photosynthesis and growth caused by higher CO^  concentrations during development. A number of studies on the effects of 00 - enrichment in other plant species have shown that vegetative development is more vigorous at C O 2 concentrations above 0.03%. In tomato plants, stem height increases with concentration up to approximately 0.22% (188) and similar responses have been shown by roses (250) Chrysanthemum (8) and Poinsettia (109). In each case, increasing GO^  concentration from 0.03 to 0.1% also produced significant effects on dry matter accumulation and plant vigor, presumably through increased photosynthetic production. This is clearly in contrast to the present results which indicated no significant changes in stem morphology by CO^-enrichment to 0.1%. On the other hand, a further increase in growth C O 2 concentration to 1% produced certain changes in morphological development which were apparently independent of relative growth rate. The observations on f l o r a l bud formation (Tables III-5 and III-6) provide equally strong evidence for an effect of 1.0 or 5.0% on flowering in Pharbitis in both LD and SD. As an aid in recognizing these effects i t i s important to understand the normal course of development in Pharbitis under different photoperiods It has been known for many years that Pharbitis seedlings become photoperiodically sensitive with the unfolding of the cotyledons two to three days after germiantion (168). At this, or a later, stage of development, a dark period of 16 hours in a single 24 hour cycle w i l l induce flowering (142). Several repetitions of the cycle cause the development of a terminal flower and the complete cessation of vertical stem growth (269). Under weaker stimulation, for example, when plants are subjected to dark periods of only 13 or 14 hours in 3 or 4, 24 hour cycles, the apex continues to grow vegetatively and flower Buds are only developed in the axils of the leaves. The c r i t i c a l dark period in most seedlings appears to be between 9 and 10 hours (142) and plants maintained under 24 hour cycles each incorporating a 16 hour light period show only vegetative growth for ah almost indefinite period (194). Since Pharbitis seedlings w i l l i n i t i a t e flowering after a single SD, the 14 cycles each incorporating a 16 hour dark period given in the present experiments constituted an extremely strong stimulus to flower. This was shown by 217 the f l o r a l morphology of plants grown at 0.03% or 0.1% C02 in SD. In these cases flower buds were formed in the axils of leaves low on the stem (Table III-6) and frequently at the stem apex (Figure III-7). The effect of 1.0 or 5.0% C02 was to inhibit flowering in SD, since in plants maintained under these conditions flower buds were formed at much higher nodes than in the other C02 treatments (Table III-6), and the stem apex remained vegetative in a l l cases. This interpretation is in line with the discussion by Hillman (129) which established that, under two sets of treatment conditions, plants which have developed fewer leaves before the development of flower buds are considered to be at a more advanced flowering stage. Under non-inductive LD conditions, flowering was stimulated in a number of cases by growth at 1 or 5% C02 (Table III-5). It i s important to note, however, that in these cases morphology was similar to that shown by plants maintained at the same C02 concentrations in SD (cf. Figures III-2 and III-3). The results showing inhibition of flowering in Pharbitis seedlings kept in SD at 1 or 5% C02 (Table III-6, cf. Figures III-6 and III-8) confirm the results of Purohit and Tregunna (234). The present study has also considerably extended the work of those authors by demonstrating that these very high C02 concentrations can promote flowering in this species under normally non-inductive LD 218 conditions. Generalizing, i t may be stated that very high CO^  concentrations tend to oppose the photoperiodic influences on flowering in Pharbitis. Similar effects have previously been shown by studies on Xanthium pennsylvanicum (51, 234) in which i t was observed that CC^  concentrations in excess of 1% inhibit flowering under inductive SD conditions and promote i t in LD. It is worthwhile at this point, to compare the effects of supra-normal CO^  concentrations on vegetative and reproductive growth in Pharbitis in order to determine i f a relationship could exist between them. Under both LD and SD conditions an increase in CO^  concentration from 0.03 to 0 . 1 % had apparently l i t t l e effect on vegetative growth or flowering. The modifying effects on both types of development occurred only when the CO^  concentration was raised to 1 or 5%. In addition, the vegetative and f l o r a l morphology were affected by these concentrations under both photoperiods. These observations tend to suggest that the effects of 1 or 5% C O 2 on vegetative and reproductive growth have a similar basis. I f , on the other hand, i t is considered that flowering is inhibited by 1 or 5% C O 2 in SD but promoted by the same concentrations in LD, the conclusions that are drawn from these results may be quite different. In this case, i t is evident that conditions which inhibit flowering (LD and 0.03 or 0 . 1 % C O 2 ; SD and 1 or 5% C O 2 ) produce, respectively, the smallest and largest increases in stem length (Table III-4). This suggests, conversely, a different basis for the C 0 ? effects on reproductive 219 and vegetative development. This suggestion may be considerably strengthened i f flower formation and vegetative growth in Pharbitis can be shown to be governed by separate physiological mechanisms, as is the case in certain rosette plants (306). The difference between the two viewpoints rests on the question of whether very high concentrations influence development which is s t i l l primarily under photoperiodic control or whether they act by releasing development from this control. Evidence in favour of the latter mechanism would provide support for a common basis for the effect of C0^ on vegetative and reproductive growth. This question w i l l be addressed in a later part of this discussion. The present results do not indicate the nature of the physiolog-i c a l mechanisms which may be important in the control of development in Pharbitis by 1 or 5% C O 2 . They do, however, focus attention on the interpretation of some previous results which have also demonstrated the regulatory effects of C02 on flowering in a number of species (51, 153, 232, 234). In 1971, Posner (232) noted that the inhibitory effects of exogenously applied sucrose on flowering in Lemna paucicostata (perpusilla) were^slmilar to the effects of 1% (X^. He postulated that the correlation was due to an increase in photosynthesis, giving rise to greater sucrose production, in the presence of high CO^  concentrations. Similar observa-tions and conclusions were made by Kandeler (153, 154), working with Lemna gibba. In a more recent study, Purohit and Tregunna (234) suggested that the inhibitory effects of 1% C02 on flowering in Pharbitis might be due to the increased production of a photosynthetic product which is required in minimum quantities for f l o r a l induction. In the 220 present study, however, i t was observed that, in plants grown at -1% CO^ in SD, rates of net photosynthesis were quite erratic at test C O 2 concentrations between 0 . 1 and 2.5% (Figure 1 1 1 - 1 0 ) , the range over which large differences in development had been observed in Experiment 9 (e.g. Tables III-5 and III-6). In contrast, when test CO^ concentration was raised from 0.03 to 0 . 1 % in these plants, photosynthesis rates showed a clear increase from 1.81 to 2.66 x 1 0 ~ * g -2 - 1 m s (Figure . 1 1 1 - 1 0 ) . Yet, a similar increase in growth CO^ concentration was ineffective in modifying development in the plants tested. These results did not support the interpretation of previous workers that the developmental effects of high C O 2 concentrations are mediated by increases in photosynthetic rate. In fact, plants grown at 1% C O 2 in SD tended to have lower inherent photosynthetic rates than those grown at 0.03% C0 2 (Figure 1 1 1 - 1 0 ) . In plants grown in LD at 1% t n e response of net photosynthesis was less erratic at high iGO^ . concentrations (Figure III-9). In this case, the gradual increase in photosynthetic rate between test concentrations of 0 . 1 and 1% C O 2 in both 0.03 and 1% C O 2 grown plants, might be connected with the differences in development over this range. As with plants grown in SD, however, i t is d i f f i c u l t to explain the lack of developmental response to an increase in test G O 2 concentration from 0.03 to 0 . 1 % , which results i n a relatively large increase in net photosynthesis. It is important to distinguish between the apparent lack of correlation between photosynthetic rate and the very high C 0 0 221 concentration effects shown in this study, and the involvement of photosynthesis in flowering. Several studies, showing a requirement for high irradiance and atmospheric concentrations of CO^  during inductive light periods (15, 31, 34, 42, 100) have established a link between photosynthesis and flowering in both LDP and SDP. The precise role of photosynthesis in the flowering process, however, is not well understood. Some workers have suggested that adequate rates of photosynthesis alone are not sufficient for the induction of flowering. Cumming (68) has presented evidence in support of a dual requirement for flower induction in Chenopodium vubrum based on photoperiodic stimulation of the phytochrome system in combination with sugar substrates produced by photosynthesis during the light period. On the other hand, induction of flowering took place in dark-grown Pharbitis seedlings following two short red irradiations in a 24 hour dark period, even though they showed no net photosynthesis (101). These studies clearly demonstrate the complexity of the relationship between photosynthesis and flowering. It seems li k e l y that the major changes in physiology which are associated with flowering may require substrates provided by net assimilation. There are, however, many other modifying factors which are apparently independent of photosynthesis. Bassi et at. (13) found that C02 removal during a short light break in the middle of an inductive dark period in Xanthium pennsylvanicum reversed the inhibitory effects of this treatment on flowering. In a later study (12) i t was 222 demonstrated that CO exchange during similar light breaks was not correlated with flower induction. The results of the present study have demonstrated that flowering may also be modified by continuous treatment with very high CO^ concentrations, a factor which, once again, does not appear to be related to photosynthesis. The diversity of the effects of CO on flowering suggests that this gas may be involved through a number of physiological mechanisms. The present data permit only a speculative approach to the question of which ones may underlie the modification of flowering and/or vegetative development by 1 or 5% CO^ . The problem i s complex since i t is uncertain at present, whether similar mechanisms are involved in the response of plants exposed to LD and SD conditions, or whether the mechanisms depend on photoperiod. Each of these possibilities w i l l be considered in the following discussion. The effects of CCv, concentration on respiration represent a possible control point for the action of 1 and 5% CO^ . Kidd (158) noted that 1% CO^ could significantly suppress rates of dark respiration. Such an effect might be expected to reduce energy supply during an inductive dark period, which may be required for the synthesis of.a f l o r a l stimulus (306) and thereby inhibit flowering under SD conditions. Indirect support for this hypothesis is provided by the work of Nakaymama (212) who showed that flowering in Pharbitis depends on respiration during a 16 hour dark period, since induction could be prevented by the application of various respiratory poisons. Some SDP show an increase in respiratory rate after exposure to an 223 inductive photoperiod„(82)? which may be essential for flowering. In others, respiration follows a distinctive pattern with an increase in rate during the last 7 hours of a long dark period which differs from the continual respiratory decline shown during a short, non-inductive night (211). This explanation for the suppressive effect of 1 or 5% CC^  on flowering of Pharbitis in SD seems, therefore, plausible. It i s d i f f i c u l t to imagine, however, how the same mechanism relates to the flower inducing effects of these CO^  concentrations under LD conditions. Campbell (51) postulated that the inhibitory effects of 10% CO^  on flowering in Xanthium pennsylvanieum in SD and the promotion of flowering by the same concentration in LD, were mediated through separate mechanisms. Under LD conditions he suggested that CO^  may interact with some system to produce a f l o r a l stimulus or, alternatively, that CO^  acts in some way to prevent the destruction of a labile stimulus. I t is unfortunate that since the time that those hypotheses were advanced, no other studies have provided evidence to prove or disprove them. Another possible mode of action for the effects of 1 or 5% CO^  on development in Pharbitis is via the competitive inhibition of ethylene activity (1, 43). Ethylene can promote flowering in a number of plants (1). but-Siige (267) has shown that high concentrations -1 (100 y l 1 ) can completely inhibit flowering in Pharbitis under normally inductive conditions. The antagonism between CO^  and ethylene action represents a possible mechanism for the induction of flowering 224 in Pharbitis in LD. This requires that ethylene is produced under LD conditions and is normally effective in inhibiting flowering, whereas under inductive photoperiods production is eliminated or maintained at a low level. Although this is a somewhat complex explanation, the role of ethylene in flowering is not well understood and its'.possibler.involvement with very high CC^  concentrations i s worthy of careful consideration. Clearly, i t i s d i f f i c u l t to propose a mechanism which adequately explains the effects of 1 and 5% CO^  on floweringiin Pharbitis under both inductive and non-inductive photoperiods. It is interesting to note, however, that there is a close correlation between the responses shown in these experiments, and those caused by low temperature treatments. In SD, low temperatures applied during the inductive dark period inhibit flowering (212), whereas in LD similar temperatures given during the light period cause flower induction (220, 268, 270). Campbell (51) showed that the effectiveness of C02 flushes (10%) in the control of flowering in Xanthium pennsytvanicum depended on whether the gas was supplied during the light or dark period in either LD or SD. Application times for flower promoting or inhibitory effects were found to coincide precisely with those which were effective for low temperature treatments. A similar finding for Pharbitis would . largely substantiate the correlation between these factors. Possibly the effects of very high CO^  concentration and low temperature share a common physiological 225 mechanism, or alternatively, they may operate by altering the rate of reactions involved in flower induction. In an earlier part of this discussion, a hypothesis was introduced which stated that 1 and 5% CCXj may act by' releasing development in Pharbitis from photoperiodic control. Such an effect i s worth consideration since i t could provide a satisfactory explanation for the observed flowering responses under LD and SD conditions, and may also be related to patterns of vegetative growth. It has been shown for a number of SDP and LDP that when seedlings are grown in continuous darkness they are capable of producing flower primordia, providing that adequate reserve materials are available (222, 305). It i s , therefore, clearly possible to separate f l o r a l induction from photoperiodism. There are, at present, no available data to suggest any mechanism through which very high concentrations could exert these effects. The appeal of" this explanation i s , however, enhanced by the results which showed that flower development and vegetative morphology were very similar under 1 or 5% CO^  and both photoperiods. As mentioned previously, i f this theory is proven correct i t w i l l provide evidence in favour of a common basis for the CO^  effects on vegetative and reproductive growth. It w i l l also, however, raise other questions related to the interaction between these two aspects of development. The results obtained in the present experiments suggest that flowering may not be easily controlled on a commercial scale 226 by atmospheric CO^-enrichment. At present, the development of many photoperiodically sensitive flower crops, notably Chrysanthemum, is regulated by black-out screening of supplementary illumination depending on the time of year and the type of development that is required (231). Due to the considerable cost of illuminating plants during the winter months, there has been some interest in developing new, cheaper methods of flowering control. The practicality of gaseous treatments to maintain plants in the vegetative condition has been demonstrated using low concentrations of ethylene (241), but .these treatments may also lead fco?a reduction in growth. Although CC^-enrichment to 0 . 1 % has been used successfully to promote yield of many flower crops in commercial greenhouses (131), this study has shown that plant development at very '- -high C O 2 concentrations in SD may not resemble that shown in LD at normal CO^  concentrations. Further work i s necessary to determine whether other flower crops show similar responses to those observed in the present study. Ultimately, an assessment of the potential of C^-enrichment as a control on flower development requires a thorough understanding of the effective concentrations, and the time and duration of their application. Although plant response to very high C O 2 concentrations may hold l i t t l e f l o r i c u l t u r a l significance, the present study has provided important insight into the physiological role of C O 2 in plant development. The results have confirmed the modifying effects of 1 and 5 % C O 2 on flowering and vegetative development which have also 227 been shown to oppose the normal responses to photoperiod in Xanthium pennsylvanioum (51, 234), and Pseudotsuga menziesii (247). For the f i r s t time since the early work of Campbell (51) on Xanthium, i t has been established that very high CO^  concentrations can affect flowering in another SDP under both inductive and non-inductive photoperiods. Clearly such effects are important in several species in which development is under photoperiodic control. The pattern of plant response to 1 and 5% COv, established in this and previous studies, has provided a strong basis for future research to discover the physiological mechanisms which are involved and to assess the significance of these C0o effects under natural conditions. GENERAL. DISCUSSION AND CONCLUSIONS The experiments described in this thesis have dealt with a wide range of CO^ effects on the physiology of plant growth and development. Information has been provided on atmospheric CC^ concentrations within a commercial greenhouse, thus providing a factual basis for some decisions on the most effective use of natural and a r t i f i c i a l CO^-enrichment systems. It has been demonstrated that stomates in leaves of a cucumber crop are relatively insensitive to greenhouse CO^ concentrations as high as 1600 y l 1 ^ (0.16%), but that stomatal resistance varies with leaf age and/or position in the canopy. In tomato, the benefits of greenhouse CO^-enrichment to ca. 0.09% i n terms of reduced time to flowering, and f r u i t yield have been confirmed. It is clear from the results of these studies that the supply of supplementary CO^ i s a worthwhile cultural practice for commercial tomato production in Bri t i s h Columbia. Investigations of CO^  exchange rates in leaves of tomato plants grown at 0.09 - 0.1% C02, and in normal air (0.03%) have established that plants grown with supplementary CO^  show higher inherent rates of net photosynthesis. This finding has been confirmed using both greenhouse and growth chamber-grown plants. Laboratory experiments have indicated that these differences in photosynthetic physiology are at least partly due to changes in photorespiration and activities of the enzymes GaO and RuBP-case. It has also been shown, 229 however, that different physiological changes occur when tomato plants are grown at higher CO^ concentrations (0.5%). These changes do not result in higher rates of photosynthesis or lower rates of photorespiration as compared with normal air-grown plants. Moreover, RGR of plants grown in 0.5% C O 2 correspond with those of plants grown in normal a i r , and contrast with the higher values shown by plants grown in 0.1% CO^ - The relationship between inherent net photosynthetic rate and growth C O 2 treatment has been shown to change with leaf age, in chamber-grown plants. The effects of very high C O 2 concentrations (1 'and 5%) on vegetative and reproductive growth in Pharbitis nil were also investigated. Seedlings grown in LD or SD for 14 days in continuous 1 or 5% C O 2 showed greater stem elongation and leaf development than those grown, in 0.03 or 0.1% C O 2 under the same photoperiods. Flowering was induced by 1 and 5% C O 2 under normally non-inductive LD conditions, whereas in SD the normal flowering response was less vigorous at these concentrations. It is clear from the results obtained in each of these studies that the effects of C O 2 concentration on the physiology of plant growth and development are diverse. Until quite recently, plant physiological studies concerned with C O 2 have strongly emphasized i t s connection with photosynthetic metabolism. The great majority of previous investigations dealt only with changes in photosynthetic or photo-respiratory rate in response to a short term change in C O 2 concentration. The present studies have shown, however, that photosynthetic and 230 photorespiratory metabolism represent only part of the true physiological role of CO^ • Data obtained from the experiments on tomato and Pharbitis have established two effects of concentration on growth and development which have previously received l i t t l e attention. F i r s t l y , the results presented in Part II have indicated that photosynthetic adaptation to growth at supra-normal C0^ concentrations can occur in at least one plant species. The generality of this response in the plant kingdom requires further investigation, but the apparent ubiquity of the C 3 CO assimilation system leads me to speculate that similar adaptations may be shown by many other plants. Secondly, the experiments concerned with the effects of supplementary CO^  on the growth of Pharbitis seedlings have demonstrated significant effects of the gas at concentrations of 1-5%, on vegetative and reproductive development. It is important to note that these effects do not appear to be mediated by photosynthesis and are, therefore, quite separate to those discussed in Part I I . The mechanisms involved in the adaptation response of tomato plants to supplementary CO^  during growth have, to some extent, been characterized. Future research w i l l undoubtedly reveal other physiological modifications which occur during this adaptation, but the changes induced.! in photorespiration and enzyme activities seem to be basic to the overall process. On the other hand, insufficient data areavailable at present to determine the.mechanisms which may be involved in the very high CO^  concentration effects on development. The 231 apparent lack of correlation between CO^ effects on photosynthesis, and the developmental changes observed in Pharbitis should not be surprising in view of the number of non-photosynthetic effects of CG"2 on plant growth and development which have been reported in the literature. It does, however, raise the question of whether correlations exist between those effects and the response of Pharbitis seedlings to very high CO^  concentrations. Unlike the effects oh flowering and vegetative morphology in Pharbitis, many of the previously reported developmental responses to CO^  have been shown to occur at relatively low concentrations. For example, i t has been demonstrated that 0.03% CO 2 stimulates the elongation of Avena sativa coleoptiles in darkness (40) and root growth in tomatoes (260). Similar CO^  concentrations were found to be necessary to i n i t i a t e germination in lettuce seeds exposed to red ii g h t , and for maximum effectiveness of a light break in inhibiting flowering in Xanthium pennsylvanioum (13). A number of mechanisms have been proposed to explain the action of In these responses. Evans (89) postulated that the elongation of dark grown coleoptile sections in CO^-saturated water may be a response to lowered pH. Some later work, however, clearly separated the growth stimulating effects of CO^  and pH (236). More recently i t has been proposed that dark fixation reactions may play an important role in the response of etiolated coleoptiles to (39, .40-; ,80)'. . 232 The competitive inhibition of ethylene action by CO^  (1, 43) has received some attention as a possible mechanism for several non-photosynthetic effects of CO^ on plant development. For example, Zeevart (306) has suggested that interactive effects of CO^  and ethylene may be responsible for the CO^ dependency of the light break phenomenon in the inhibition of flowering in Xanthium (13). Ethylene has also been shown to inhibit root growth ( 58) in direct opposition to the stimulatory action of CX^ (260). It i s regrettable that no investigation has been carried out, to date, to assess the importance of ethylene in other CO^-mediated plant responses. Clearly, more than one of these mechanisms may be responsible for a single effect. For example, both dark fixation reactions (260) and the inhibition of ethylene action (58) could account for the stimulatory effect of atmospheric CC^ levels on root growth. The relationships between the known effects of C O 2 on plant development have received l i t t l e attention, however, and i t i s d i f f i c u l t to determine at present whether or not they share common mechanisms. From the data that are available, there appears to be l i t t l e support for a relationship between the effects of 1 and 5% CO^ on flowering and vegetative growth in Pharbitis, and other plant responses reported in the literature. Most non-photosynthetic effects of CO^  develop at atmospheric concentrations (0.03%) or lower. The effects on flowering and vegetative development in Pharbitis, however, are shown at much higher concentrations (1 - 5%) and normal development takes place under 0.03% C0„ in LD and SD. 233 It is tempting to suggest that there are at least 4 classes of CG^  effects viz: (1) those associated with the requirement for C02 as a substrate for photosynthesis (e.g. the instantaneous response of photosynthetic rate to increased CO^  concentration); (2) those associated with adaptation of CO^  exchange systems to growth at supra-normal levels of CO^  (e.g. the changes in photosynthetic and photorespiratory metabolism demonstrated in Part I I ) ; (3) those which show a requirement for levels of CO^  approximately equal to atmospheric concentrations (ca. 0.01 to 0.03%)'(e.g. the stimulation of coleoptile elongation (40) , stomatal closure (122), and light-break mediated flowering inhibition in Xanthium (13)); (4) those which result in modification of growth and development when the CO^  concentration is raised to approximately 1% (e.g. the effects on flowering in Pharbitis, Siline, and Xanthium (Part I I I , 51, 234)). Within each category there may be responses involving different species and different aspects of physiology. For example, in addition to the very high C02 concentration effects on flowering in Pharbitis, S'iiene, and Xanthium, 1% C02 has been shown to influence budset and winter hardiness in Douglas Fir seedlings (247). The mechanisms involved in the effects of classes (3) and (4) are uncertain at present, and the possibility that some of these responses are mediated through similar physiological reactions cannot be discounted. As discussed in the preceding sections, the interaction of C02 and ethylene may underly the effects of both classes (2) and (3). Furthermore, dark C02 fixation has previously been implicated only in low concentration responses (class 2), although i t has also been observed 234 that changes in protein and organic acid synthesis occur at much higher CG^  concentrations (260). Such qualitative and quantitative differences between the products of dark CO^ fixation at low (0.03%) and high (1-5%) concentrations may relate to the effects on flowering and vegetative development in Pharbitis (class 3). Undoubtedly some overlapping also occurs between class (1) and several of the other categories. It is generally considered, for example, that the relationship between stomatal opening and atmospheric CO^  concentration (class 3) depends partly upon the net assimilation response of the guard c e l l chloroplasts (235; class 1). The fact that this i s not the only factor involved is demonstrated by the requirement for C O 2 to induce closure in stomata in complete darkness (123). As indicated in previous discussions, the interaction of CO^  and ethylene is another possible mechanism underlying the effects of both classes 2 and 3. The categorization of C O 2 effects creates arbitrary distinctions which may prove to be inaccurate as further studies are carried out. This approach does, however, underline the varied role of C O 2 in mediating the known physiological responses of plants. In view of this, i t is interesting to consider how the effects demonstrated in this research relate to current practices in crop production, and whether CX^-control has potential as a tool in modifying plant growth. Reduction of photorespiration and the inhibition of photosynthesis in crop plants would probably result in significant increases in productivity (62, 192). The results of the present studies have shown that both these processes may be reduced by atmospheric CO —enrichment to 0.1%. The practical significance of these findings 235 rests f i r s t l y on the effectiveness of this method of controlling photorespiration relative to other approaches, and, secondly, on the f e a s i b i l i t y of C02_enrichment for crop plants. To date, attempts to reduce photorespiration have concentrated on chemical treatments (307, 309), and screening species of crop plants for mutant individuals which show low photorespiratory activity (200, 293). Although some success has been reported for the control of glycolate synthesis by application of glycidate (309), recent results have raised serious doubts about the effectiveness of this chemical in reducing photorespiration (61). Screening programs have so far met with l i t t l e success (53, 62, 209) despite the testing of numerous crop species and selections. In view of these findings, i t seems that (^—enrichment may be, at present, the most effective means of reducing photorespiration. This method not only results in increases in instantaneous rates of photosynthesis, but also persistent changes in the photosynthetic system of enriched plants, which improve the efficiency of CO^  fixation. There is l i t t l e doubt of the fe a s i b i l i t y of CO^-enrichment for greenhouse crops, but the question of whether these procedures are applicable in the open f i e l d is more contentious. Many major food crops respond to CO- enrichment by increased dry matter production and yield (66, 164, 208, 296, 302, 135) and, as discussed above, i t seems li k e l y that they may also exhibit the same physiological modifications shown by tomato, in the present experiments. The main 236 problem clearly l i e s in the rapid atmospheric dilution of supplementary when the gas is applied in a f i e l d situation (208). Some tests have shown, however, that the procedure may be feasible in the vicin i t y of large natural CT^ sources (296). At present, i t seems that the practical constraints on the use of CO^-enrichment in the f i e l d may be too great for i t s adoption on a large scale. These constraints are not encountered in crop growing enclosures, but the problems associated with CO^-enrichment in these situations nevertheless require close attention. The results presented in Part II suggest that, in vegetable crops, careful control of a 0.1% C O 2 level is advisable. The lack of information on CO^ concentrations in commercial greenhouses is regrettable, especially since few growers are fortunate enough to have a feedback control system integrated with enrichment equipment. Knowledge of ambient CT^ levels may be of importance especially where concentrations can achieve naturally high levels, as in cucumber houses using traditional straw/soil mixtures in the cropping beds. Conductimetric analysis systems similar to that described in Part I of this thesis, may f i l l present needs for C O 2 monitoring and control equipment in greenhouses. It is doubtful from the results of experiments concerned with the effects of CX^  on flowering in Pharbitis, whether adequate control of blooming in flower crops can be achieved by this method. It may be that other plants w i l l show complete suppression of 237 flowering in inductive photoperiods at 1 - 5% CC^  (as already demonstrated with Xanthium. at a CC^  concentration of 10%; 51). The practical and economic f e a s i b i l i t y of this technique would, in that case, rest on the exposure time necessary for effective flowering control. The potential uses and control of CO^  in agriculture have been considered in the light of present day crop production techniques and limitations on productivity. As a f i n a l consideration on the implication of this research i t is interesting to note some recent predictions which have stated that the ambient atmospheric CO^ content could increase from the present 0.03%, to 0.06% by the year 2020 (300). 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Interactions of carbon dioxide concentration, l i g h t intensity and temperature on plant resistances to water vapour and carbon dioxide d i f f u s i o n . New Phytol. 66:463-473. 292. WHITEMAN, P.C. and D. ROLLER. 1968. Estimation of mesophyll resistance to d i f f u s i o n of carbon dioxide and water vapour. In: Eckardt, F.E. (editor). Functioning of T e r r e s t r i a l Ecosystems at the Primary Production Level. Unesco, Paris, pp. 415-419. 293. WIDHOLM, J.M. and W.L. OGREN. 1969. Photorespiratory-induced senescence of plants under conditons of low carbon dioxide. Proc. Nat. Acad. U.S.A. 63:668-675. 294. WISHNICR, M. and M.D. LANE. 1971. Ribulose diphosphate carboxylase from spinach leaves. In: San Pietro, A. (editor). Methods i n Enzymology. Vol. XXIII Photosynthesis. Part A. Academic Press, New York. pp. 570-577. 295. WITTWER, S.H. 1967. Carbon dioxide and i t s role i n plant growth. Proc. XVII Int. Hort. Cong. 111:311-322. 296. WITTWER, S.H. 1974. 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APPENDIX 1 The following l i s t of experiments performed i n the course of this thesis research, i s presented as an aid to reference i n the text. Experiment Number Experiment T i t l e 1 Measurement of stomatal resistances i n leaves of a greenhouse cucumber crop; concurrent measurement of greenhouse CO^ concentration. 2 Growth and y i e l d analysis of tomato.crops, grown i n two greenhouses without C02~ enrichment (preliminary to Experiment 3). 3 Leaf development and y i e l d analysis of greenhouse tomato crops grown with and without atmospheric C0^~enrichment. 4 Gas exchange measurements on greenhouse tomato crops grown with and without atmospheric CO^-enrichment. 5 Growth analysis of tomato plants grown at 3 CO^  concentrations i n chambers. 6 CO^ exchange measurements on leaf 3 of tomato plants grown at 3 concentrations i n Chambers, at 2 stages of development. 262 Experiment Number Experiment T i t l e 7 Replacement Experiment: . CO^ exchange measurements on tomato plants grown at 3 CC>2 concentrations i n chambers, 14 days after replacement i n a normal (0.03%) C O 2 atmosphere. 8 Assays of RuBP-case and GaO a c t i v i t i e s i n leaves of tomato plants grown at 3 C O 2 concentrations i n chambers, at 2 stages of development. 9 Analysis of flowering and vegetative development i n Pharbitis nil plants grown i n LD and SD at 4 C O 2 concentrations. 10 Gas exchange measurements on Pharbitis nil plants grown under 0.03% or 1.0% C O 2 i n LD and SD. 263 APPENDIX 2 The behavioral equation for net photosynthesis of in d i v i d u a l leaves"*" used i n the analysis of data i n Experiment 4, may be summarized as follows: F I M / E I + ^p " V 1 / E c + ( C I " r ) -2 -1 Where: F = Flux density of CO^  (g m s ) I = absorbed quantum f l u x density (400 - 700 nm) -2 -1 (yEinstein m s ) - i n this study incident radiant f l u x density was taken to equal t h i s parameter. IQ = the compensation point for incident photosynthetically -2 -1 active .: radiation (yEinstein m s ) C\ = CO^ concentration i n the i n t e r c e l l u l a r spaces within the -3 leaf (g m ) '.- calculated by the methods of Moss and Rawlins^ -3 r = (X>2 compensation point (g m ) F^ M = maximum possible CO^ fl u x from any one leaf (obtained at saturating levels of atmospheric concentration and T w ~ 2 I ) (g m s ) E j = e f f i c i e n c y of u t i l i z a t i o n of radiant energy obtained at CO 2 saturation, when C places no l i m i t a t i o n on the photosynthetic process (g yEinstein "*") 264 = effic i e n c y of CC^ u t i l i z a t i o n obtained at saturating 3 -1 levels of I (m g ) In this study, the terms I p , F and T: were measured, 1^ was obtained by interpolation between values of dark respiration, and -2 -1 net photosynthesis at an irradiance of 50 uE m s , and C., F T W , l IM E^ . and E^ , were calculated. At each l e v e l of irradiance for in d i v i d u a l leaves, l i n e a r regressions were calculated between reciprocal values of ( C - T) and F. From these plots, values of CO^ f l u x at an extrapolated value of saturating C. (designated F ), and E were X X L * obtained from the intercept ^  and (slope ^ x intercept), respectively. A mean value for E^ , was obtained for each leaf age i n each growth regime (except i n the case of apical leaves grown i n normal a i r where lack of a consistent lin e a r relationship between F and (C - T) "*" precluded further analysis of the data). Other regressions were calculated between values of ( Ip - IQ) and F^ . ^  to give F ^ and E^ -1 -1 from the intercept and slope . Again, average values were obtained from a l l available regression data for each treatment. For a more complete discussion and val i d a t i o n of the model, see reference"*". Van Bavel, C. H. M. 1975. A behavioral equation for leaf carbon dioxide assimilation and a test of i t s v a l i d i t y . Photosynthetica 9: 165-176. 2 Moss, D. N. and S. L. Rawlins. 1963. Concentration of carbon dioxide inside leaves. Nature 197: 1320-1321. APPENDIX 3 As a preliminary to the enzyme a c t i v i t y assays described i n Materials and Methods, Part I I , Section 2 of this thesis, pH optima were determined using the assay procedures of Wishnick and 1 2 Lane (RuBP-case) and Baker and Tolbert .(GaO). Optimum substrate concentration was also determined for RuBP-case, but not for GaO since, i n th i s case, the amount of sodium glycolate added i n the extraction medium was s u f f i c i e n t to saturate a c t i v i t y . The t r i a l s were conducted on leaf material of a uniform age from plants grown i n the greenhouse under the pretreatment conditions mentioned i n the text (Materials and Methods, Part I I , Section 2). 1. RuBP-case The following reaction mixture was used x: 1.0 M Tris (Cl") Buffer (100 ymoles) 0.005 M D-ribulose-l,55blphosphate 14 0.5 M sodium bicarbonate,(( C) (25 ymoles) 0.5 M magnesium chloride (5 ymoles) 0.006 M EDTA (3 ymoles) + enzyme extract i n a t o t a l volume of 0.5 ml Wishnick, M. and M. D. Lane, 1972. Ribulose diphosphate carboxylase from spinach leaves. In: San Pie t r o , A. (editor). Methods i n enzymology. Vol. XXIII Photosynthesis. Part A. Academic Press, New York. pp. 570-577. 2 Baker, A. L.„and N. E. Tolbert, 1966. Glycolate oxidase (Ferredoxin containing form). In: Wood, W. A. (editor). Methods i n enzymology Vol. IX.Carbohydrate metabolism. Academic Press, New York, pp. 338-342. Figure A3-1 depicts the relationship between RuBP concentration and enzyme a c t i v i t y at a temperature of 30°C and pH 7.8. Saturating substrate concentration was found to be approximately 0.4 mM (equivalent to 0.2 ymoles per 0.5 ml of reaction mixture. The relationship between enzyme a c t i v i t y and pH at a temperature of 30°C and saturating substrate concentration i s shown i n Figure A.3-2. Optimum pH was approximately '7.8 which corresponds closely with that determined by Wishnick and Lane'*'. In Experiment 8 Tris (Cl~) buffer, pH 7.8 and 0.25 ymoles RuBP were used for a l l RuBP-case determinations. GaO 2 The following reaction mixture was used : 0.04 M Sodium glycolate (20 ymoles) 0.1 M cysteine-HCl (10 ymoles) 0.1 M phenylhydrazine-HCl (10 ymoles) 0.1 M potassium phosphate buffer (200 ymoles) + enzyme extract i n a t o t a l volume of 2.9 ml The relationship between a c t i v i t y and pH for this enzyme i s shown i n Figure A3-3. pH optimum was estimated to l i e between 8.0 and 8.5. In Experiment 8 potassium phosphate buffer, pH 8.3 was used for a l l GaO determinations. FIGURE A3-1: RuBP-case a c t i v i t y i n r e l a t i o n to substrate concentration at pH 7.8, 30°C. 267 267a FIGURE A3-2: RuBP-case a c t i v i t y i n r e l a t i o n to pH at saturating substrate concentration, 30°C. 268a FIGURE A3-3: GaO a c t i v i t y i n r e l a t i o n to pH at saturating substrate concentration, 27°C. A C T I V I T Y 1 1 (jjmoles glyoxylate formed g fw~' min ) o o o o o o o o O —-V* _ i 1 I I I i i i i i ' 

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