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Effects of carbon dioxide and daylength on growth, development and hardiness of Douglas fir (Pseudotsuga… Leadem, Carole Louise Scheuplein 1979

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EFFECTS OF CARBON DIOXIDE AND DAYLENGTH ON GROWTH, DEVELOPMENT AND HARDINESS OF DOUGLAS FIR (Pseudotsuga menziesii) by CAROLE LOUISE (SCHEUPLEIN) LEADEM B.Sc, University of C a l i f o r n i a , Berkeley, C a l i f o r n i a , 1973 A THESIS SUMBITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Faculty of Graduate Studies i n the Department of Botany We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1979 © CAROLE LOUISE LEADEM, 1979 I n 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 t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e 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 a g r e e t h a t t h e L i b r a r y s h a l l m a k e 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 a n d s t u d y . I f u r t h e r a g r e e 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 m a y b e g r a n t e d b y t h e H e a d o f my D e p a r t m e n t o r b y 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 n o t b e 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 . D e p a r t m e n t of BOTANY T h e 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 2 0 7 5 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V 6 T 1W5 D a t e 1 May 1979 ABSTRACT Seedlings and one-year-old trees of Douglas-fir (Pseyidotsuga  menziesii) were grown in chambers with atmospheres maintained at 0.03% to 5.0% CO2 (by volume). Carbon dioxide treatments were given i n conjunction with daylengths of 8 or 16 hours and light intensities -2 -2 which varied from 3.4 mW cn to 7.2 mW cm (400 - 700 nm). The duration of treatment varied from 30 days to 12 weeks. When plants were treated with 0.1% CX^ both seedlings and trees showed enhanced growth, as demonstrated by increases i n dry weight and internodal elongation. C0£ enrichment caused growth enhancement to a degree that other factors became limiting, e.g., irradiance. Carbon dioxide concentrations of 1.0% CC^ and higher generally inhibited growth, as shown by decreases i n internodal elongation, dry weight, and leaf area. Plants grown under high carbon dioxide levels ceased active growth and exhibited increased budset and frost hardiness. High CO2 levels appeared to override photoperiodic control of budset by promoting budset even under warm temperatures and long days. COj-induced frost hardiness appears to require an active metabolism, indicating that the mode of CO2 action i s through increased production of cryoprotectents, such as amino acids and sugars. ' • ' .'• 1 •'•')I.'- ; In some cases, carbon dioxide may substitute in part for the light requirements of photosynthesis when light i s limiting. Thus, an increase in daylength may reduce the level of COj required for a particular effect, e.g., the required C 0 2 levels for inducing frost hardiness are reduced from 1.0% to 0.1% C 0 9 i f long days are i i i provided. Plants which have been grown under normal air (0.03% CO2) have higher photosynthetic rates than enriched plants when a l l plants are measured in normal a i r ; the concentration under which plants are measured appears to have more effect on photosynthetic rates than the CO2 concentration under which the plants are grown. Under long days and high CO2 (1.0% CO2 and higher), plants show reduced diffusion resistance, but the beneficial effects on photo-synthesis due to potential reduction of CO2 diffusion resistance are lost as a result of increased rates of respiration under high CO2. Thus, the enhancement of growth under 0.1% Ct>2j and the inhibition of growth under 1.0% CO2, appear to be mostly related to differences in respiration under the various carbon dioxide treatments. A l l effects of carbon dioxide may not be due to gas exchange characteristics alone, but may result from changes in levels of growth inhibitors, such as abscisic acid. The effects of CO2 on growth and development were examined over a range of CO2 concentrations. Over the entire range CO 2 was found to effect both growth and development and the processes underlying growth and development. TABLE OF CONTENTS Page ABSTRACT i i ACKNOWLEDGEMENTS . . . . . . . . x DEDICATION x i i PREFACE • x i i i CHAPTER I. Effects of carbon dioxide and daylength on growth of Douglas-fir (Pseudotsuga menziesii) INTRODUCTION . 1 MATERIALS AND METHODS 5 RESULTS A. HEIGHT 1. One-year-old trees (a) Effects of 8- and 16-hour daylengths and two carbon dioxide levels . . . . . 10 (b) Effects of 16-hour daylength and three carbon dioxide levels 13 2. Six-month-old seedlings: experimental design (a) Effects of daylength 16 (b) Effects of carbon dioxide 16 (c) Effects of carbon dioxide and daylength . . 19 (d) Effects of temperature . . . . . . 23 3. Summary: Effects of carbon dioxide and daylength on height 23 B. WEIGHT 1. One-year-old trees (a) Effects of 8- and 16-hour daylengths and two carbon dioxide levels 25 (b) Effects of 16-hour daylength and three carbon dioxide levels . . . . . 29 (c) Effects of 8-hour days and four carbon dioxide concentrations . 33 2. Six-month-old seedlings 35 3. Summary: Effects of carbon dioxide and daylength on weight (a) Leaves 41 (b) Stems 42 (c) Roots 42 (d) Biomass Distribution 42 DISCUSSION 44 CHAPTER II. Effects of carbon dioxide and daylength on dormancy and hardiness of Douglas-fir INTRODUCTION . . . . MATERIALS AND METHODS RESULTS V Page 56 60 69 FLUSHING AND BUDSET 1. One-year-old trees (a) Effects of 8- and 16-hour daylengths and two carbon dioxide levels . 67 (b) Effects of 16-hour daylengths and three carbon dioxide levels . . . . . . . . ... . (c) Effects of 8-hour daylength and four carbon dioxide levels 69 2. Six-month-old seedlings (a) General flushing and budset behaviour . . . 72 (b) Maximum budset 75 (c) Earliest date of budset 75 (d) Effects of temperature on plants grown under a i r (0.03% C02) 79 B. COLD HARDINESS 1. Effects of carbon dioxide (a) Preliminary study 79 (b) Plants grown under 8-hour days 82 (c) Plants grown under 16-hour days . 82 2. Effects of post-treatment (a) Plants grown under 8-hour days 86 (b) Plants grown under 16-hour days 89 3. Effects of temperature . 89 4. Effects of C02 and daylength on amino acids • '. • • 92 DISCUSSION 95 CHAPTER III. Effects of carbon dioxide and daylength on gas exchange of Douglas f i r INTRODUCTION 110 MATERIALS AND METHODS 112 RESULTS 116 A. PHOTOSYNTHESIS 1. Plants grown under 8-hour days and four C O 2 levels 2. Plants grown under 16-hour days and four levels B. RESPIRATION CO-C. TRANSPIRATION 1. One-year-old trees 2. Six-month-old seedlings D. TOTAL DIFFUSIVE RESISTANCE DISCUSSION . . . . . . . . . . . . CONCLUSIONS LITERATURE CITED . . APPENDICES TABLE A TABLE B TABLE C TABLE D TABLE E Ranking Codes Spectral Distribution of Sherer Growth Cabinet. Spectral Distribution of Mercury Vapor Lamps. . v i Page ,119 ' 123 123 125 131 133 146 148 Summary of Experiments # -,57 158 Schematic Diagram for Experiment Number 5 . 159 160 161 LIST OF TABLES v i i Table Page 1 Growth of trees grown under 8- and 16-hour photoperiods with either 0.03% or 1.0% CO2 12 2 Growth of trees grown under 16-hour photoperiods with either 0.03%, 0.1%, or 1.0% C0 2 1 5 3 Growth of seedlings. Effect of daylength 17" 4 Growth of seedlings. Effect of carbon dioxide • • • 18 5 Growth of seedlings. Effect of carbon dioxide and daylength . . 20 6 Growth of seedlings. Effect of temperature 24 7 Dry weight of trees grown under 8- and 16-hour photoperiods with either 0.03% or 1.0% C0 2 27 8 Shoot:root of trees grown under 8- and 16-hour photoperiods with either 0.03% or 1.0% C0 2 28 9 Shoot:root of trees grown under 16-hour photoperiods with either 0.03%, 0.1%, or 1.0% C0 2 32 10 Dry weight of trees grown under 16-hour photoperiods with either 0.03%, 0.1%, or 1.0% C0 2 3 4 11 Needle dry weight and area of trees grown under 8-hour photo-periods with 0.03%, 0.1%, 1.0%, or 5.0% C0 2 36 12 Regression of leaf area and leaf weight of trees 37 13 Fresh weight of seedlings grown under 8- and 16-b.our daylengths and 0.03%, 0.1%, 1.0%, or 5.0% C0 2 38 14 Proportional weight distribution of seedlings 40 15 Degree of lateral budset i n trees grown under 8- or 16-hour daylengths with 0.03% or 1.0% C0 2 68 16 Degree of budset in trees grown under 16-hour daylengths with 0.03%, 0.1%, or 1.0% C0 2 70 17 Degree of budset in trees grown under 8-hour daylengths with 0.03%, 0.1%, 1.0%, or 5.0% C0 2 71 18 Date of f i r s t budset in trees grown under 8-hour daylengths with 0.03%, 0.1%, 1.0%, or 5.0% C00 .73 Table Pag 19 Maximum number of buds set in seedlings grown under 8- or 16-hour daylengths with 0.03%, 0.1%, 1.0%, or 5.0% C02 . . . 77 20 Earliest date of budset in seedlings grown under 8- or 16-hour daylengths with 0.03%, 0.1%, 1.0%, or 5.0% C0 2 78 21 Earliest date of budset in seedlings grown under 8-hour daylengths with three temperatures 81 22 Frost hardiness study of trees 83 23 Cold hardiness of seedlings. Effect of carbon dioxide . . . . 84 24 Cold hardiness of seedlings grown under 16-hour days. Effect of carbon dioxide 85 25 Cold hardiness of seedlings grown under 8-hour days with 0.03%, 0.1%, 1.0%, or 5.0% C0 2 > Effect of post-treatment . . 87 26 Cold hardiness of seedlings grown under 16-hour days with 0.03%, 0.1%, 1.0%, or 5.0% C02. Effect of post-treatment . . 90 i 27 Cold hardiness of seedlings grown under 8-hour daylengths with three temperatures. Effect of temperature 93 28 Amino acids in leaf extracts of 1-year-old trees 94 29 Rates of photosynthesis of seedlings grown under 8-hour days . 118 30 Rates of photosynthesis of seedlings grown under 16-hour days . 121 31 Rates of transpiration of seedlings grown under 8-hour days . . 128 32 Rates of transpiration of seedlings grown under 16-hour days . 130 ix LIST OF FIGURES Figure Rage 1 Height increases after treatment with C^-enriched atmospheres (1-year-old Douglas-fir trees) 11 2 Height increases under 16-hr days (1-year-old Douglas-fir trees) • • • • • • ^ 3 Effect of CO2 on height of 6-month-old seedlings 22 4 Shoot fresh weight increases (1-year-old Douglas-fir trees). 30 5 Root fresh weight increases (1-year-old Douglas-fir trees) . 31 6 Degree of budset under 8-hr days (Douglas-fir 6-month-old seedlings) . . 74 7 Degree of budset under 16-hr days (Douglas-fir 6-month-old seedlings) . . . . . 76 8 Degree of budset under 8-hr days and 3 temperatures (Douglas-f i r 6-month-old seedlings) 80 9 Photosynthetic rates of seedlings grown under 8-hr days . . 117 10 Photosynthetic rates of seedlings gronw under 16-hr days . . 120 11 Effect of daylength and CO2 on photosynthesis 122 12 Effect of daylength and on respiration 124 13 Weekly water loss (1-year-old Douglas-fir grown under 8-hr days and 4 C02 levels) 1 2 6 1 2 7 14 Transpiration rates of seedlings grown under 8-hr days . . . 1 2 9 15 Transpiration rates of seedlings grown under 16-hr days . . 16 Effect of daylength and CO2 on total resistance 132 A C K N O W L E D G E M E N T S I would like to thank my main advisor, Tony Glass, for a l l the work he has done on my behalf in the Department. Since I was an off-campus student he took care of many tasks which I would have found very d i f f i c u l t to do without his help. I sincerely appreciate a l l of his efforts to bring this work to completion. I also wish to give my thanks and appreciation to Peter J o l l i f f e who so kindly took over as my advisor after the death of Bruce Tregunna. His guidance during the later part of my research and his many suggestions during the writing of this thesis were invaluable in completing my work. I am very grateful to Pawan Bassi, with whom I shared the laboratory. His good humor and encouragement helped me over many of the hard times. The following people are only a few of those many kind friends and associates who gave me assistance during my research, but my thanks are extended to a l l those whom I did not have room to mention specifically. I would like to thank: Geoff Lister, who advised me on various aspects of gas exchange and who graciously allowed me to examine his unpublished data on western hemlock. the members of my committee: Andy Black, who taught me not to fear mathematical equations; and Ron Foreman, who always found time to give me help and advice. Mel Davies, the Botany Department Saviour of Machines and Equipment, who nursed my ail i n g mechanical monsters countless times. x i Our discussions, both s c i e n t i f i c and philosophical, contributed greatly to my education during my four years at U.B.C. my children, John and Lauryn, for their moral support and for their assistance in the laboratory, even though they could never understand why Mom went to school when she didn't have to. Finally, I would like to thank Tim whose love and moral support kept me going, and whose tangible support—assisting me i n the laboratory, taking photographs, drafting figures, c r i t i c a l l y reading and re-reading this manuscript—helped me finish. For their contributions of Douglas f i r plants used i n this research I would like to thank the B.C. Forest Service and Weyerhaeuser Company, Centralia, Washington. DEDICATION x i i This thesis is dedicated to the memory of E. Bruce Tregunna, scientist, teacher, and friend. Bruce Tregunna was a truly creative, open-minded scientist, and was worthy of emulation. Bruce created a supportive atmosphere in his laboratory and was largely responsible for the co-operative interaction of a l l members of his group. He promoted the exchange of ideas from researchers outside his laboratory as well, and whenever visitors came to campus we would a l l gather in the coffee room for informal discussions. These dis-cussions were a valuable educational experience which served to broaden both my intellectual and s c i e n t i f i c perspectives. As my major advisor, Bruce was at a l l times enthusiastic and encouraging. His support was invaluable to me during the progress of this research. In our personal discussions, Bruce was an attentive listener and receptive to new ideas. He provided guidance when needed but he also allowed me the maximum amount of freedom for research and problem-solving. In addition, Bruce was a compassionate and thoughtful human being for whom I had the greatest admiration. The thought of his death shall always inspire me with a sense of loss. x i i i PREFACE In the laboratory of E. Bruce Tregunna, work on the effects of carbon dioxide enrichment was just beginning when I began my graduate studies. Bruce Tregunna and Aditya Purhoit were screening plants for their response to CO^. and as part of this work, they grew Douglas f i r seedlings under high CO^ levels. After 90 days they examined the plants and found that several plants grown under 1.0% CO,, had formed curious clusters of bract-like leaves which resembled the early developmental stages of female cones. This finding was quite different from the usually observed effects of favourable levels of CO2 enrichment, i.e., the enhancement of dry weight and internodal elongation. It appeared that carbon dioxide which is a relatively simple, molecule basic to plant metabolism could affect plant growth and development in ways other than providing essential nutrition. Intrigued by these pre-liminary results I became interested in further exploring carbon dioxide effects on growth and development of Douglas f i r . Other members of our research team investigated other carbon dioxide effects on physiological events, such as the interaction of C0 2 and phytochrome. I was able to only sporadically reproduce the effects of C0 2 on bud development which originally had caught my interest in this research however, other effects of C0 2 which I observed during the course of my investigations turned out to be equally as interesting. This thesis includes experiments relating to several different effects of carbon dioxide on growth and development of Douglas f i r . The experiments were designed to define the ranges in which C02 enhances growth and the ranges in which C0 2 inhibits growth, xiv as well as explore the interactions between CO^ and other factors influencing growth. The effects of CO^ on budset and on freezing resistance were also studied. An attempt was made to determine the relationships between growth, degree of budset, and freezing resistance. In addition, I investigated the effects of carbon dioxide on photo-synthesis, respiration, and transpriation and made an effort to determine the interrelationships between the different components of gas exchange. 1 CHAPTER ONE EFFECTS OF CARBON DIOXIDE AND DAYLENGTH ON GROWTH OF DOUGLAS FIR (Pseudotsuga menziesii) I N T R O D U C T I O N Investigation of the e f f e c t s of carbon dioxide enrichment on plant growth began about the turn of t h i s century. As early as 1903 there were reports of dry weight increases of as much as 158% when carbon dioxide was added to the plant atmosphere (48). Much uncertainty surrounded this early work because other workers reported negative effects of enrichment, such as reduction i n l e a f area, slow development of internodes, and retardation of flower and f r u i t development. In retrospect, not a l l of these problems may have been due to CO^, but to other factors, such as contaminants i n the gases used f o r enrichment, and excessive humidity i n the treatment chambers. C o n f l i c t i n g reports may have also been due to d i f f i c u l t i e s with accurately measuring the CO^ concentrations around the plant. I t was not then known that s p e c i f i c plants may exhibit a f a i r l y well-defined optimum range for carbon dioxide, and that i n h i b i t o r y e f f e c t s can quickly increase once the optimum i s exceeded. Work i n the f i e l d continued, however, u n t i l about 1930 when i t gradually declined. Interest i n CO^ enrichment was revived when i n 1964 Wittwer and Robb (85) d e t a i l e d the many advantages that h o r t i -c u l t u r i s t s could achieve by using CO^ enrichment i n the greenhouse atmosphere. In cucumber, there were more p i s t i l l a t e flowers which resulted i n a 72% increase i n f r u i t (85) and i n petunias, a c c e l e r a t i o n s i n flower development (38). Lettuce i s an i d e a l crop f o r enrichment, as CO^ promotes increases i n both l e a f s i z e and thickness, plus 2 accelerating growth, making i t possible to grow four crops i n one year rather than just three (83). Benefits from enrichment are realized in many fru i t s , with improvements i n f r u i t color and f r u i t shape, and reduction in the number of scars (37). Outside the horticultural f i e l d , only limited work with carbon dioxide enrichment has been done, but of special interest i s work relating to enhancing the growth of trees. Carbon dioxide has been found to improve rooting of softwood, conifer, and herbaceous cuttings (50). Levels of 1000 ppm CO^  doubled height and growth of white pine seedlings (22), while levels of 900 and 1500 ppm CO^  increased dry weight 30% to 80% in 3-week-old seedlings of white spruce, Norway spruce, jack pine and Scots pine (86). Other reports show positive CO^ effects on the growth of Douglas f i r , western hemlock, noble f i r , white f i r , ponderosa pine, and lodgepole pine (E. B. Tregunna, un-published data, 1974; 71, 72), but the potential benefits of applying carbon dioxide enrichment to economically important trees are far from being realized. To my knowledge there have been few studies on optimum levels for tree growth enrichment, and no exploration of levels at which carbon dioxide becomes inhibitory to growth. The highest reported levels of CO2 used i n tree growth studies have not exceeded 1500 ppm CO^  (71, 72). Daylength also has important implications relating to seasonal and annual cycles of growth, and to the development of perennial species which undergo alternating periods of active growth and dormancy. There has been some work done in the past on the effects of different light 3 intensities in conjuction with several levels of carbon dioxide, but no work has been reported on the effects of different daylengths combined with several carbon dioxide levels. The broad objective of the research presented in this chapter was to determine how carbon dioxide and daylength influenced the growth processes of Douglas f i r . I attempted to answer the following questions: What concentrations of CO^ promoted growth, and what concentrations inhibited growth? Did carbon dioxide affect the biomass distribution within the plant? What were the effects of light on growth, and did irradiance levels or photoperiod affect the expression of the CO^ effect? Did CC^ interact with light or any other factors to influence growth? Finally, could the effects of CO,, persist after enrichment terminated? To accomplish my research objective I conducted a series of experiments. Each experiment was designed to contribute a part to the cumulative knowledge required to answer the questions above. I n i t i a l l y I wished to determine the general ranges of daylength and carbon dioxide which would promote growth of 1-year-old Douglas f i r trees. Two different photoperiods (8 and 16 hours) and two different carbon dioxide levels were used for the f i r s t experiment (0.03% and 1.0% CO,,). The plants were grown for only 30 days, but some general observations on the effects of CO,, and daylength could be made. The observations were used to plan a second experiment in which both the duration and the number of CO,, levels were increased; thus plants were grown for 90 days under three C0 2 levels (0.03%, 0.1% and 1.0% C0 2). The dual objectives were to 4 observe the long-term effects of CO,, on height and weight, and to determine the best CO^  l e v e l for enhanced plant growth. Together the results of experiments 1 and 2 provided enough data to determine the ranges i n which CO,, promoted growth, but only limited data on the point at which (X^ would begin to i n h i b i t growth. Accordingly, i n experiment 3 a higher CO,, l e v e l (5.0% CO,,) was added to the e x i s t i n g three C0 2 treatments. Since l i g h t levels had previously been l i m i t i n g plant response, irradiance was also increased. The cumulative data gathered from the results of the f i r s t three experiments presented a general picture of the response of 1-year-old Douglas f i r trees to CO,, and daylength, however, I had l i t t l e knowledge of how CO^ daylength affected plants younger than one year. There were several reasons for being interested i n the effects of CO^ on young plants. CO,, enrichment during the seedling stage probably would produce the greatest growth benefits since young plants generally respond more readily to environmental changes, and have greater relative growth rates. Smaller plants are easier to handle because of their s i z e , and f i t more e a s i l y into controlled environment chambers. Using younger plant material would also make i t possible to increase sample sizes, consequently reducing t o t a l v a r i a b i l i t y within the sample. F i n a l l y , seedling research has good potential for p r a c t i c a l benefit i n B r i t i s h Columbia where forest nurseries require large numbers of trees each year. Reforestation nursery programs are always seeking new techniques for accelerating seedling growth i n order to produce plantable trees i n the shortest possible time. 5 With the above considerations i n mind, 6-month-old seedlings were grown f o r s i x weeks with e i t h e r 8- or 16-hour photo-periods under four d i f f e r e n t CC^ l e v e l s , then t r a n s f e r r e d to normal a i r for another four weeks (Appendix, Table A, Exp. No. 5). The f i r s t part of the f i n a l experiment provided data on the general e f f e c t s of CC^ and daylength on seedling growth, whereas the second part provided additional data on the persistence of CO^ e f f e c t s a f t e r the plants were grown i n normal a i r . The completion of the experiments described i n t h i s chapter supplied most of the r e s u l t s needed to determine how carbon dioxide and daylength influenced some of the growth processes of Douglas f i r . Most of my i n i t i a l questions were answered although some of the r e s u l t s raised even more questions. Regardless, the cumulative data did enable me at least to formulate a tentative p i c t u r e of CC^ e f f e c t s and i t s interactions with other factors i n f l u e n c i n g growth. MATERIALS AND METHODS Plant M a t e r i a l One-year-old Douglas f i r trees (1-0 stock) were obtained from the B r i t i s h Columbia Forest Service from e i t h e r t h e i r South Surrey or Green Timbers bare root nursery. The coastal provenance trees had been grown from seeds o r i g i n a l l y gathered at an elev a t i o n of approximately 500 m on the east coast of Vancouver Island. Trees were grown i n 15 cm pots f i l l e d with Mica-Peat (a s t e r i l i z e d peat moss and v e r m i c u l i t e mixture) obtained from Langley Peat Limited, Fort Langley, B.C. Seedlings 6 were supplied by Weyerhaeuser Company, Centralia, Washington, and were also coastal provenance Douglas f i r from Twin Harbours, Aberdeen, Washington (elevation 300 m t 60 m) . The plants had been seeded into individual f l e x i b l e p l a s t i c plugs f i l l e d with a standard potting mixture. The seedlings were not transplanted since the plugs were of s u f f i c i e n t volume to allow for reasonable root expansion. Both trees and seedlings were well-watered and f e r t i l i z e d every two weeks with Hi-Sol 20-20-20 (N-P-K) solution. Terminology To simplify the terminology throughout t h i s thesis, CO^ concentrations cf 0.03% CO^ (v/v) w i l l be referred to as either "atmospheric", "normal", or "low" C0 2 l e v e l s ; 0.1% CC>2 w i l l be called "intermediate" C0 2 l e v e l s ; and 1.0% or 5.0% CC>2 w i l l be c a l l e d "high" C0 2 levels. Exact concentrations w i l l be stated i n instances when descriptive labels might otherwise be misleading or ambiguous. Days with l i g h t periods of 8 hours w i l l be called "short days" and those with 16 hours w i l l be called "long days". Experimental Design A general summary of a l l experiments, given i n the Appendix, Table A, can be used for reference when needed. The basic purpose of Experiment 1 was to compare the effects on growth of normal and enriched C0 2 levels under two d i f f e r e n t photoperiods. One-year-old Douglas f i r trees were grown under 8- and 16-hour daylengths with either 0.03% or 1.0% C0 2 i n chambers maintained 7 25 C/20 C. Experimental plants were screened to determine that i n i t i a l l y a l l trees were of uniform size. I n i t i a l height and weight measurements were taken at the beginning of treatment and again after 7, 14, 21, and 30 days of treatment. Height measurements in this experiment and a l l others were taken from the root collar to the tip of the main stem. Dry weights were determined after plant material had been dried at 85 C for 48 hours. In Experiment 2, the number of CO^ levels was expanded to determine the effects of varying concentrations, and to establish the optimum concentration for growth enhancement. Trees were grown under 0.03%, 0.1%, and 1.0% CO^. hut photoperiod (16-hour days) was kept constant. In addition, the duration of the experiment was extended to 90 days to assess long-range growth patterns and responses. Height and weight samples were taken at the beginning of treatment and again after 15, 30, 60, and 90 days. The number of CO levels was again expanded i n Experiment 3, but in this experiment the purpose was to ascertain the point at which would begin to inhibit growth. The focus of the experiment was on growth inhibition, therefore short photoperiods (8 hours) were chosen in conjunction with CO^  concentrations of either 0.03%, 0.1%, 1.0%, or 5.0% After treatment for 21 days, needles from each of the four treatments were sampled and dry weights and areas were determined. Regressions based upon the data were also performed for each of the four treatments. 8 The o v e r a l l objective of the f i n a l experiment was to study the growth of 6-month-old Douglas f i r trees (Appendix, Table A, Exp. No.5). There were two main parts to the experiment, a pre-treatment and a post-treatment (see schematic, Appendix, Table C). The purpose of the f i r s t part was to determine the effects of CO^ and photoperiod on growth, and consisted of pretreating plants for s i x weeks with either 8- or 16-hour days (25 C/20 C) and one of four C0 2 levels (0.03%, 0.1%, 1.0%, or 5.0% C0 2). During the second part of the experiment, a l l plants were transferred to normal a i r and maintained under short warm days (25 C) and long cold nights (5 C) for four weeks. Additional controls were grown continuously under short days and normal a i r , but given thermoperiods of either 25 C/5 C or 5 C/5 C. (The f i r s t number given i s the temperature during the l i g h t period, while the second number i s the temperature during the dark period). The primary purpose of the post-treatment was to establish whether the effects of CC^ pre-treatment persisted beyond the actual CO^  enrichment period, but there was a secondary purpose as w e l l . In nature, short warm days and long cold nights promote the cessation of growth and the onset of dormancy. Many of the plants had already been pre-treated under conditions which enhanced growth, however seedlings were also to be post-treated with conditions which induced dormancy. Thus, the secondary objective of the post-treatment was to ascertain whether pre-treatment or whether post-treatment would exert the greatest influence i n controlling plant growth response. For t h i s experiment height and weight samples were taken after s i x weeks of pre-treatment and after four weeks of post-treatment. 9 Growth Chambers Experiments 1, 2 and 4 (as l i s t e d i n Table A, Appendix) were conducted i n Sherer growth cabinets sealed to maintain ambient CO^ concentrations at the required l e v e l s . Cabinets were illuminated with mixed incandescent and flourescent l i g h t s . Radiant energy at mid-plant l e v e l i n the photosynthetically active region (400-700 mm) was 2 3.4- mW/cm as measured by the spectral radiometer described by Burr and Duncan (89). A sample spectral d i s t r i b u t i o n curve appears i n the Appendix, Table D. Carbon dioxide was supplied from cylinders (Liquid Carbonic or Matheson Supply Company) and a Matheson Rotameter (a precision flowmeter with an adjustable valve) regulated the flow. Gas within the chamber was monitored p e r i o d i c a l l y with a Beckman IR-215 infrared gas analyzer. Experiments 3 and 5 were conducted i n eight s p e c i a l l y constructed Plexiglas chambers (59). A thermistor monitored a i r temperature and a cooling c o i l provided humidity control within each chamber. Condensed water collected at the base of the cooling c o i l and drained to the outside of the chamber through Tygon tubing to another c o l l e c t i n g vessel. A small fan mounted on the Plexiglas w a l l assured that a i r within each chamber was well mixed. A thermostatically cooled c i r c u l a t i n g water bath maintained at a depth of 7 cm reduced infrared radiation from the two 1000 W mercury vapour flourescent lamps (Duroglo GA 217G2) mounted above the eight chambers. Carbon dioxide from cylinders mixed with a i r pumped from outside the laboratory provided the gas supply to the chambers. A i r from each of the eight chambers was 10-automatically sampled i n turn using an aquarium pump inside each chamber; pumped gas was directed to a Beckman Model IR-215 infrared gas analyzer. Solenoid valves were activated whenever the m i l l i v o l t output from the gas analyzer f e l l below the set point, i n j e c t i n g additional CO^  into the chamber, and thus providing a constant CO^ l e v e l . There were occasional problems maintaining CC^ l e v e l s due to failures in equipment however, CC^ concentrations were checked at least daily and usually several times a day and adjustments were made as necessary. Occasional fluctuations of CO^ levels would probably have minimum effect on the o v e r a l l r e s u l t s of long-term experiments lasting a month or more. In any case, CO^ fluctuations would be of importance primarily i n explaining th'e lack of results due to carbon dioxide enrichment, and not for explaining the p o s i t i v e effects of C0„. RESULTS A. HEIGHT 1. One-year-old trees (a) Effects of 8- and 16-hour daylengths and 2 CO,., levels  on height I n i t i a l l y , plants i n a l l four treatments were approximately the same height, but by the th i r d week s i g n i f i c a n t differences between the treatments were apparent (Fig 1, Table I ) . Trees continuously maintained under atmospheric C0 0 and long days (16 hours) were noticeably Figure 1. Mean cumulative elongation for 1-year-old Douglas f i r continuously supplied with C02 at the levels shown, and maintained under either 8- or 16-hour photoperiods (25 C/20 C) with an irradiance of 3.4 mW cm - 2. 7.0 6.0 ~ 5 . 0 E u LU 2 4 - 0 o 6 3 . 0 U l 2.0 1.0 I— b - 02% C02 L D .02% C02 S O 1.0% co2 S O 1.0% C02 L D 3.0 4.0 1.0 2.0 c n - Q t. i - s T I M E (weeks) IL^v!^ f r " 7 8 = I d/yS d 3 t a P ° l n t S S h ° W n l n t h e f i& u r e a r e t h e m e ^ s of 8 plants. Analysis of Variance was used for determining s t a t i s t i c a l differences. Values bearing the same l e t t e r within the same column are not s i g n i f i c a n t l y different at p = 0.05. 12 TABLE I CUMULATIVE GROWTH INCREASE OF CO -ENRICHED DOUGLAS FIR TREES (1-0 Stock) Grown for 30 days under 8- and 16-hour photoperiods (25 C/20 C) with either 0.03% or l.D% C0 2 Irradiance = 3.4 mW cm - 2 TREATMENT MEAN LENGTH OF NEW GROWTH (cm ) Daylength CO cone. 7 Days 14 Days 21 Days 28 Days (h) lO) 8 0.03 1.563 3.75a 5.05a. 6.03ab 8 1.0 2.093 3.98a 5.01a 5.79 a b 16 0.03 2.03a 4.56a 6.04b 6.41b 16 1.0 1.94a 3.86a 5.08a 5.50a The data points shown in the table are the means of 8 plants. Analysis of variance was used for determining s t a t i s t i c a l differences. Values bearing the same letter within the same column are not significantly different from each other at p = 0.05. 13 taller than trees grown under the other three conditions. In contrast, trees grown under high CC^ and long days were the shortest of a l l four treatments. Atmospheres containing high CC^ tended to inhibit stem growth under both short and long days, but the differences were only statistically significant under long days. (b) Effects of 16-hour daylength and 3 C0„ levels on height Significantly different trends between high and low CO,, plants were observable by 30 days (Figure 2, Table II). After that time, extension growth levelled off i n both the low and intermediate groups, but around 60 days they resumed growth. Plants grown under high CO^  grew slower than the other two groups but continued at a fairly constant rate. Trees supplied with intermediate CO^  levels were the tallest at the end of 90 days, although they were relatively late in responding to CQ^ enrichment. High CO 2 levels appeared to inhibit elongation since the shortest trees were those grown under 1.0% CO2. Trees grown under atmospheric conditions were intermediate in height between the other two groups. In comparing Experiments 1 and 2, growth trends for the f i r s t 30 days were similar. In Experiment 1 the greatest differences between trees grown under normal and high CO2 levels occurred at three weeks, whereas in Experiment 2 the greatest differences between normal and high CO2 levels occurred at four weeks. Trees grown in normal air showed exponential and plateau phases in both studies, although the Figure 2. Mean cumulative elongation of 1-year-old Douglas f i r continuously supplied with CO at the levels shown, and maintained under 16-hour photoperiods (25 C/20 C) with an irradiance of 3.4 mW cm - 2. 0 1 5 30 45 60 75 90 TIME (days) The data points shown i n the figure are the means of 5 plants. Analysis of Variance was used for determining s t a t i s t i c a l differences. Values bearing the same l e t t e r within the same column are not s i g n i f i c a n t l y d i f f e r e n t at p = 0.05. 15 TABLE II CUMULATIVE GROWTH INCREASE OF CO -ENRICHED DOUGLAS FIR TREES (1-0 Stock) Grown for 90 days under 16-hour photoperiods (25r.C/20 C) with either 0.03%, 0.1% or 1.0% CO Irradiance = 3.4 mW cm TREATMENT MEAN LENGTH OF NEW GROWTH (cm ) 15 Days 30 Days 60 Days 90 Days 0.03% c o 2 4.38a 6.92b 8.08a 11.78 a b 0.1% c o 2 3.42a 6.04ab 7.14a 14.30b 1.0% c o 2 3.26a 4.62a 6.38a 8.60a The data points shown in the table are the means of 5 plants. Analysis of Variance was used for determining s t a t i s t i c a l differences Values bearing the same letter within the same column are not significantly different from each other at p = 0.05. 16 pattern was more cl e a r l y discernable i n the 90-day study. Trees i n high increased at a lower rate, but grew continuously, and thus did not demonstrate a marked plateau phase. 2. Six-month-old seedlings (a) Effects of daylength on height Plant heights under a l l CO^ levels were averaged, then analyzed according to the daylength under which they had been grown (Table I I I ) . After s i x weeks with warm day and night temperatures (25 C/20 C), new growth of seedlings maintained under short days was not s i g n i f i c a n t l y different from that of seedlings grown under long days. The effects of long days were not apparent u n t i l the plants were transferred to a i r and given short warm days' and long cold nights which i n nature would o r d i n a r i l y promote growth cessation and dormancy onset i n Douglas f i r . In this instance, however, plants previously grown under long days continued to grow even i n non-inducing circumstances. (b) Effects of C0„ on height Individual data points for a l l plants were averaged, then analyzed on the basis of the i r growth regime, regardless of the photoperiod they had received. Data analyzed i n t h i s manner demonstrated that CO^  effects could be seen both during the pre-treatment and post-treatment periods (Table IV). Seedlings given intermediate CO^ levels exhibited the best growth during pre-treatment and post-treatment and 17 TABLE III CUMULATIVE GROWTH INCREASE OF CO^ENRICHED DOUGLAS FIR SEEDLINGS A. EFFECT OF DAYLENGTH Irradiance = 7.2 mW cm TREATMENT MEAN LENGTH OF NEW GROWTH (cm ) Daylength (25/20 C) 8-hr day for 6 weeks and (25/5 C) TOTAL for 4 wks 8-hour 0.98a 0.08a 1.06a 16-hour 0.82a 0.86b 1.68b The data points shown in the table are the means of 48 plants. Analysis of Variance was used for determining s t a t i s t i c a l differences. Values bearing the same letter within the same column are not significantly different from each other at p = 0.01. Mean cumulative elongation of 6-month-old Douglas f i r averaged for a l l C02 levels under each daylength. Plants were pre-treated for six weeks under 8-and 16-hour days (25 C/20 C) and four C02 levels (0.03%, 0.1%, 1.0%, and 5.0% CO2). At the end of pre-treatment plants were transferred to a four week post-treatment of 8-hour days and normal air (0.03% CO2) (25 C/5 C). 18 TABLE IV CUMULATIVE GROWTH INCREASE OF C02~ENRICHED DOUGLAS FIR SEEDLINGS B. EFFECT OF CARBON DIOXIDE -2 Irradiance = 7.2 mW cm TREATMENT MEAN LENGTH OF NEW GROWTH (cm ) Averaged for both daylengths  (25/20 C) 8 - h r d a y s for 6 weeks af <fC) TOTAL for 4 wks. 0.03% C02 0.75a 0.59a 1.34a 0.1% C02 1.29b 0.98b 2.27b 1.0% C02 0.693 0.633 1.32a 5.0% C02 0.65a 0.62a 1.27a The data points shown in the table are the means of 60 plants. Analysis of Variance was used for determining s t a t i s t i c a l differences. Values bearing the same letter within the same column are not significantly different from each other at p = 0.05. Mean cumulative elongation of 6-month-old Douglas f i r averaged for 8— and 16-hour daylengths under each C02 level. Plants were pre-treated for six weeks under 8- and 16-hour days (25 C/20 C) and four C02 levels (0.03%, 0.1%, 1.0%, and 5.0% CO2). At the end of pre-treatment plants were transferred to a four week post-treatment of 8-hour days and normal air (0.03% CO2) (25 C/5 C). 19 were s i g n i f i c a n t l y d i f f e r e n t from plants grown under low and high CO l e v e l s . These r e s u l t s were s i m i l a r to those obtained for 1-year-old Douglas f i r (see Figure 2). (c) Effects of CO,, and daylength on height The combined e f f e c t s of photoperiod and carbon dioxide should be considered, since the separate e f f e c t s of these two parameters have already been examined (Table V). Long days and intermediate CO,, l e v e l s were most e f f e c t i v e i n promoting growth during the pre-treatment period ( i . e . , the i n i t i a l six weeks). However, while the mean height increase for seedlings grown under 16-hour days and 0.1% CO^ was greater than the means of a l l other groups, i t was not s i g n i f i c a n t l y d i f f e r e n t from the means of short-day plants grown under low or intermediate CO,, l e v e l s . At the end of post-treatment (during which seedlings v/ere grown i n normal a i r ) three s t a t i s t i c a l l y d i f f e r e n t groups could be distinguished. The smallest growth increments occurred i n the f i r s t group, consisting of plants pre-treated with short days. No d i f f e r e n c e s i n the short-day group could be a t t r i b u t a b l e to CO., pre-treatment, but under long days responses to carbon dioxide enrichment could be found. Moderate growth occurred i n the second group and was shown by those plants pre-treated with long days and e i t h e r low or high C0 2 l e v e l s . The greatest growth occurred i n the t h i r d group and was exhibited by plants pre-treated with long days and intermediate C0 2 l e v e l s . Plants pre-treated with long days and intermediate C0 2 also demonstrated the greatest t o t a l growth during the 12 week period, and were s i g n i f i c a n t l y d i f f e r e n t from a l l other treatment groups. 20 TABLE V CUMULATIVE GROWTH INCREASE OF CO^ENRICHED DOUGLAS FIR SEEDLINGS C. EFFECT OF CARBON DIOXIDE AND DAYLENGTH _2 Irradiance = 7.2 mW cm TREATMENT MEAN LENGTH OF NEW GROWTH (cm ) Daylength (h) COo cone. (%) (25/20 C) for 6 weeks 8-hr days and (25/5 C) for 4 wks TOTAL 8 0.03 0.99C 0.03a 1.02a 8 0.1 1.22c 0.073 1.29a 8 1.0 0.78 a b 0.14a 0.92a 8 5.0 0.81 a b 0.10a 0.91 a 16 0.03 0.69b. 0.73b 1.42a 16 0.1 1.30C 1.24C 2.54b 16 1.0 0.67 0.73b 1.40a 16 5.0 0.62 0.72b 1.34a The data points shown i n the table are the means of 15 plants. Analysis of Variance was used for determining s t a t i s t i c a l differences. Values bearing the same l e t t e r within the same column are not s i g n i f i c a n t l y different from each other at p = 0.05. Plants were pre-treated for s i x weeks under 8- and 16-hour days (25 C/20 C) and four C02 levels (0.03%, 0.1%, 1.0%, and 5.0% CO2). At the end of pre-treatment plants were transferred to a four week post-treatment of 8-hour days and normal a i r (0.03% C02) (25 C/5 C) . 2 1 General growth responses were the same for short-day and long-day plants when height increases were plotted as a function of CO^ pre-treatment (Figure 3). Both groups showed a base value at low CO^ l e v e l s , an optimum at intermediate CC^ l e v e l s , and a d e c l i n e at high CC>2 l e v e l s . The t o t a l growth of long-day plants was s u b s t a n t i a l l y greater than that of short-day plants, and the magnitude of the peak value was both larger and more c l e a r l y defined. There were no discernable d i f f e r e n c e s between short-day and long-day plants a f t e r s i x weeks f o r 6—month-old Douglas f i r , as had been observed f o r 1-year-old trees. The absence of notable differences between short- and long-day plants may have been due to experimental r e s t r i c t i o n s regarding use of the conditioning chambers. Plants to be used for the long-day portion of the experiments had to be stored under long days i n the greenhouse while short-day plants were being treated i n the experimental chambers. While under greenhouse conditions the plants continued to f l u s h , which probably made them less responsive to further treatment. I t should be noted, however, that when the long-day seedlings were post-treated under conditions which normally do not promote growth (short warm days and cold n i g h t s ) , long-day plants showed greater t o t a l o v e r a l l growth than short-day plants. I t appears, therefore, the e f f e c t s of long photoperiods can p e r s i s t , extending the period of growth enhancement beyond the time of actual treatment. 22 Figure 3. Mean cumulative elongation of 6-ironth-old Douglas f i r continuously supplied with CO2 at the levels shown, and maintained under either 8- or 16-hour photoperiods with an irradiance of 7.2 mW cm-2, then post-treated with 8-h days (25 C/5 C) for 4 weeks. Increase in height after 6 weeks of CO2 pretreatment plus 4 weeks of post-treatment (8-hour days with 25 C/5 C). The data points shown in the figure are the means of 15 plants. Analysis of Variance was used for determining s t a t i s t i c a l differences. Values bearing the same letter of the same case are not significantly different from each other at p = 0.05. 23 (d) Effects of temperature on height Control plants were grown under atmospheric CC^ levels and three different thermoperiods to examine temperature effects upon growth (Table VI) . Plants with warm days and nights grew nearly twice as much as those grown with cooler temperatures. There was no difference noted between plants grown with warm days and cold nights, and those grown with cold days and nights. A l l these groups of plants were maintained under short days; they responded similarly to the short-day plants discussed previously (in Section c); i.e., most growth occurred during the f i r s t six weeks and l i t t l e growth occurred during post-treatment. 3. Summary: Effects of CO,, and daylength on height Carbon dioxide had similar effects on height when the same CO -daylength combinations were used on 1-year-old and 6-month-old plants. In some cases the data were variable, and although plants given low levels of enrichment might have had the highest mean values, the means could not be shown always to be s t a t i s t i c a l l y different from the means of other treatments. Generally however, low levels of enrichment (0.03% and 0.1% CO^ ) promoted internodal elongation, whereas high levels of enrichment (1.0% and 5.0% CO^ ) inhibited elongation. In experiment 1, long-day plants showed the greatest elongation under low CO^  and the least elongation under high C02« Also in experiment 3, plants grown under low enrichment levels (0.1% CO^ ) were the tallest after 90 days, while those under high enrichment levels (1.0% CO,,) were the shortest. The main difference between the results 24 TABLE VI CUMULATIVE GROWTH INCREASE OF CO^ENRICHED DOUGLAS FIR SEEDLINGS D. EFFECT OF TEMPERATURE (0.03% C0 2 only) Irradiance = 7.2 mW cm" PRE- TREATMENT MEAN LENGTH OF NEW GROWTH (cm ) Thermoperiods After 6 weeks pre-treatment After 4 weeks post-treatment TOTAL 25/20 C 0.991 0.03' 1.02 25/5 C 5/5 C 0.52' 0.43' 0.02' 0.06' 0.54' 0.49' The data points shown in the table are the means of 55 plants. Analysis of Variance was used for determining s t a t i s t i c a l differences. Values bearing the same letter within the same column are not significantly different from each other at p =0.01. Plants were pre-treated for six weeks under 8-hour days and four thermoperiods, as shown above. After pre-treatment plants were transferred to a four week post-treatment of 8-hour days (25 C/5 C). A l l treatments were in normal air (0.03% CO2). 25 of experiments 1 and 2 was that i t took much longer for plants i n experiment 2 to show growth benefits from CC^ enrichment. The delayed response may have been due to the low irradiance under which the trees i n experiment 2 were grown (3.4 mW cm-2). I f so, i t appears that l i m i t i n g l i g h t a v a i l a b i l i t y to enriched plants may also l i m i t t h e i r a b i l i t y . t o benefit from CO^  enrichment. Plants i n experiment 2 did eventually respond to CO^ enrichment, but i t took much longer for effects of treatment to become evident. Six-month-old Douglas f i r responded to the effects of daylength and CO^ sim i l a r l y to 1-year-old Douglas f i r . Photoperiod had some effect on growth, since the mean heights of long-day seedlings were greater than the mean heights of short-day seedlings (Figure 3). The most notable increases however, were attributabl'e to levels of CO,, enrichment. Under both short and long days, seedlings given intermediate CO,, levels were the t a l l e s t , while those given normal CO,, le v e l s were the next t a l l e s t . The shortest seedlings were those grown under high C0 2 levels (1.0% and 5.0% C02> . There were no s t a t i s t i c a l differences between the means of plants grown under 1.0% and 5.0% CO,,, which probably indicates that the maximum ef f e c t i v e degree of i n h i b i t i o n l i e s somewhere between these two levels and that greater i n h i b i t i o n of elongation by higher C0 2 concentrations i s u n l i k e l y . B. WEIGHT 1. One-year-old trees (a) Effects of 8- and 16-hour daylengths and two CO,, levels  on weight An increase i n daylength resulted i n greater shoot dry weight 26 under both low and high CO^ levels (Table VII). However, the highest weights were observed under long days and high CO^, and conversely the lowest weights were observed under short days and low CO^. The s i g n i f i -cant differences in shoot weight under long days and high CC^ were primarily a result of increases in leaf weight. Stem weight also increased under long days and high CO^t but i t could not be shown to be significantly different from the other three treatments. The highest mean values for root dry weight were also observed under long days and high CO^ , but there were no significant differences between the four conditions. Additionally, data for trees grown under the above conditions were analyzed using the mathematical relationship known as the shoot: root ratio (Table VIII). The shoot:root ratio has long been used for analyzing whole plant response to various treatments. This ratio reduces two measurements (shoot weight and root weight) to a single measurement which indicates the relative amounts of biomass distributed between the upper and lower portions of the plant. In Table VIII the lowest ratios were those observed for plants maintained under short days and low CC^ levels while the highest ratios were those observed for plants under long days and high CO^ levels. High ratios indicate that greater biomass accumulated in the shoot relative to the root. However, as a comparison of the data in Tables I and VIII shows high shoot:root ratios do not necessarily indicate that increased internodal elongation has also occurred. Although trees which were grown under long days and high CC^ had the highest shoof.root ratios, they had the TABLE VII DRY WEIGHT—MEAN VALUES DOUGLAS FIR TREES (1-0 Stock) Grown for 30 days under designated daylength/CX^ combinations -2 Irradiance = 3.4 mW cm TREATMENT SHOOT Daylength(h) C02conc. TOTAL (g ) Leaf (mg) Stem (mg) ROOT TOTAL (mg) 8 16 -18 16 0.03 0.03 1.0 1.0 0.79c 0.82 0.87c 1.151 515.8C 551.9' 603.4' 850.0 271.3C 268.4' 261.0 301.lc 407.8' 361.3' 399.6' 420.6C The data points shown in the table are the means of 8 plants. Analysis of Variance was used for determining statistical differences. Values bearing the same letter within the same column are not significantly different at p = 0.01. 28 TABLE VIII SHOOT : ROOT RATIO (Calculated on grams dry weight) DOUGLAS FIR TREES (1-0 Stock) Grown for 30 days under designated daylength/C^ combinations Irradiance _2 = 3.4 mW cm Daylength (h) CO„ cone. (%) Shoot/Root at 30 days 8 0.03 1.94 a 8 1.0 2.18 3 16 0.03 2.27 3 16 1.0 2.74 b I n i t i a l shoot/root = 1.36 for a l l treatments The data points shown in the table are the means of 8 plants. Analysis of Variance was used for determining s t a t i s t i c a l differe Values bearing the same letter are not significantly different at p = 0.01. 29 smallest increases in height. Apparently the CC^ assimilated under long days and high CC^ was used primarily to increase shoot biomass (especially leaf dry weight) rather than to increase plant height. (b) Effects of 16-hour daylength and three CO^  levels  on weight During the f i r s t 15 days of treatment plants with enriched atmospheres showed rapid shoot weight increases which were probably associated with the f i r s t flushing period (Figure 4). For both groups of enriched plants a period of rapid growth was followed by a period of rest which lasted until approximately 60 days. Growth resumed after 60 days and continued u n t i l the end of treatment. Plants under normal air demonstrated only half the increases shown by enriched plants during the f i r s t 15 days of treatment. Unenriched plants grew f a i r l y continuously during the period from 15 to 90 days, and thus there was not an evident rest period. Root growth showed the same general response in a l l three CO^ treatments and thus differed from shoot growth which had varied according to the presence or absence of CO^ enrichment (Figure 5). Shoot:root ratios during the f i r s t 15 days did not vary significantly because dry weights of a l l three treatments increased at approximately the same rate (Table IX) . The fact that a l l three groups had shoot:root ratios approaching 2.0 indicated that the greatest proportion of assimilate went into shoot production. By 90 30 Figure 4. Mean cumulative increases i n shoot fresh weight of 1-year-old Douglas f i r continuously supplied with C O 2 at the levels shown, and maintained under 16-hour photoperiods (25 C/20 C) with an irradiance of 3 . 4 mW cm - 2. 15 30 45 60 75 90 TIME (days) The data points shown i n the figure'are the means of 8 plants. Analysi of Variance was used for determining s t a t i s t i c a l differences. Values bearing the same l e t t e r within the same column are not s i g n i f i c a n t l y different at p = 0.05. 31 Figure 5. Mean cumulative increases in root fresh weight of 1-year-old Douglas f i r continuously supplied with CC>2 at the levels shown, and maintained under 16-hour photoperiods (25 C/20 C) with an irradiance of 3.4 mW cm 30 75 90 45 60 T I M E (days) Data points are the means of 8 plants. Analysis of Variance was used to determine s t a t i s t i c a l differences. Values within the same column bearing the same letter are not significantly different from each other at p = 0.05. 32 TABLE IX SHOOT : ROOT RATIO * , (Calculated on grams dry weight) DOUGLAS FIR TREES (1-0 Stock) Grown for 90 days under 16-hour photoperiods -2 Irradiance = 3.4 mW cm TIME (days) TREATMENT 15 30 90 0.03% C02 1.773 2.47a 2.82a 0.1% C02 2.09a 3.19a 4.03b 1.0% C02 1.72a 3.03a 2.59a Initial shoot:root =0.96. The data points shown in the table are the means of 8 plants. Analysis cf Variance was used for determining statistical differences. Values within the same column bearing the same letter are not significantly different at p = 0.05. 33 days the shoot:root r a t i o for 0.1% CO^ plants was 4 times greater than i t s i n i t i a l value; this increase was not matched by 0.03% and 1.0% plants whose r a t i o s at 90 days were only 2h times t h e i r i n i t i a l values. The increases i n shoot:root r a t i o s were due mainly to combined increases i n stem and leaf dry weights rather than to decreases i n root weights (Table X). Both stem and leaf weights of 0.1% CO^  plants were s i g n i f i c a n t l y greater than the other two treatments at 90 days, and the cumulative effects of both increases resulted i n the r e l a t i v e l y large shoot:root r a t i o s observed for 0.1% CO^ plants. This observation should be qualif i e d somewhat since shoot growth patterns were variable and plant response was delayed during the treatment period. However, f i n a l results of this experiment did follow trends observed i n Experiment 1 i n that shoot:root ratios and shoot growth were greater under CO^ enrichment (1.0% CO^ i n experiment 1 and 0.1% CO^ i n experiment 2) than i n the absence of CQ„ enrichment (0.03% CO^ i n experiments 1 and 2). In both experiments 1 and 2 root growth was l i t t l e affected by CO^ l e v e l s . (c) Effects of 8-hour days and 4 CO^  levels on weight In the preceding work I had observed that leaves appeared to be a s i t e often affected by CO^ enrichment (Tables VII and X). Therefore I f e l t that leaves would be a good system i n which to study the effects of (X^ on some growth patterns. An examination of leaf growth could provide some worthwhile information with which to study the effects of C0o on general growth patterns. Accordingly, needles 34 TABLE X DRY WEIGHT—MEAN VALUES DOUGLAS FIR TREES (1-0 Stock) Grown for 90 days under 16-hour photoperiods Irradiance = 3.4 mW cm TREATMENT SHOOT ROOT TOTAL(g ) LEAF(g ) STEM(g .) TOTAL (g. ) 0.03% C0„ 1.92' 1.37£ 0.55' 0.68 0.1% C0r 2.70 1.86 0.84 0.67' 1.0% CO, 1.84' 1.22 0.62' 0.71' The data points shown in the table are - the means of 8 plants. Analysis of variance was used for determining s t a t i s t i c a l differences. Values within the same column bearing the same letter are not significan different at p = 0.05. 35 were randomly sampled from each of the CC^ treatments and measured for area and dry weight (Table XI). As noted before leaf weight was greater under 0.1% C0 2 and the same under 0.03% and 1.0% C0 2 (Tables X and XI) . Leaf areas followed the same trends. Both leaf weight and area were further reduced by 5.0% C0 2 l e v e l s ; they were s i g n i f i c a n t l y less than areas and weights for the three lower C0 2 concentrations. A regression of leaf area on leaf weight produced four d i f f e r e n t first-order equations (Table X I I ) . Higher order regressions were attempted but they did not substantially improve r 2 . Regressions for plants grown under 0.03%, 0.1%, and 5.0% were di f f e r e n t from one another, while the regression for plants grown under 1.0% C0 2 was similar to regressions of most other treatments. Generally speaking, slopes of the four regressions increased with increasing C0 2 concentrations while intercepts of the regressions decreased with increasing C0 2 concentrations. These results indicated that C0 2 could affect leaf growth patterns such that, at a given dry weight and depending on r e l a t i v e changes i n slope and intercept, leaf area would decrease with increasing C0 2. 2. Effects of C0 2 and daylength on weight of 6-month-old seedlings Seedling response to C0 2 and daylength was simi l a r to the response described e a r l i e r for 1-year-old trees (Table X I I I ) . Several general points about the effects of treatment on weight could be made: photoperiod had significant effects on growth; C0 2 enrichment was ef f e c t i v e i n en-hancing growth under long days; and growth of a l l plant organs were affected by photoperiod and C0 o enrichment. 36 TABLE XI NEEDLE DRY WEIGHT AND AREA—MEAN VALUES DOUGLAS FIR TREES (1-0 Stock) Grown for 21 days under 8-hour photoperiods 2 Irradiance = 3.4 mW cm '2 \ TREATMENT AREA (cm2) WEIGHT (mg) 0.03% C0 2 0.31a 4.48a 0.1% C0o 0.44b 6.74b 1.0% C0 o 0.29a 4.26a 5.0% C0o 0.21° 2.91C The data points shown in the table are the means of 10 plants. Analysis of Variance was used for determining s t a t i s t i c a l differences. Values within the same" column bearing the same letter are not significantly different at p = 0.05. 37 TABLE XII REGRESSION OF LEAF AREA AND LEAF WEIGHT DOUGLAS FIR TREES (1-0 Stock) Grown for 21 days under 8-hour photoperiods TREATMENT REGRESSION OF LEAF AREA (y) on , r 2 LEAF WEIGHT (x) 0.03% C02 Y = 0.151 + 0.035 X 3 0.669 0.1% C02 Y = 0.183 + 0.038 X b 0.916 1.0% C0 2 Y = 0.045 + 0.059 X a c 0.946 5.0% C0 2 Y = 0.054 + 0.054 X C 0.889 Leaf area = dependent variable Leaf weight = independent variable Regressions with the same letter are not significantly different at p = 0.05 Least squares regression, Analysis of Variance N = 10 TABLE XIII FRESH WEIGHT—MEAN VALUES DOUGLAS FIR (6-month-old seedlings) Grown for six weeks under designated daylength/CO„ combinations -2 Irradiance = 7.2 mW cm TREATMENT SHOOT(g ) Daylength (h) CO?conc.(%)  LEAF(g ) STEM(mg) BRANCH(mg) ROOT(g ) 8 0.03 1.41a 1.17a 202a 41.4a 0.51a 8 0.1 1.30a 1.09a 175a 36.3a 0.54a 8 •1.0 1.43a 1.20a 176a 48.0a 0.40a 8 5.0 1.56 a b 1.29a 214a 54.3a 0.53a 16 0.03 1.84 b d 1.32ab 454b 59.9a 1.80b 16 0.1 2.23° 1.54 b c 596° 87.6b 2.52b 16 1.0 2.03 c d 1.47 b c 463b 96.8b 1.96b 16 5.0 2.23C 1.63c 400b 103.6b 1.76b The data points shown in the table are the means of 10 plants. Analysis of Variance was used for determining s t a t i s t i c a l differences. Values bearing the same letter with the same column are not significantly different at p = 0.05. 39 Under short days no significant differences between CO^ treatment groups were found, although i t should be noted that the mean shoot weight tended to increase in seedlings grown under 5.0% CO^. Root weight showed l i t t l e response to CO^ enrichment. Under long days significant differences could be found between shoots of enriched and atmospheric plants; the increases i n enriched plants were primarily due to greater leaf and branch weights which were highest under 5.0% CO^  and lowest under 0.03% CO^. Stem weights were significantly higher under 0.1% CO^ than under any other C0 2 concentration. Root weights were not significantly different from one another, but the highest mean weight was associated with plants grown under 0.1% co2. Carbon dioxide and photoperiod influenced seedling weight both quantitatively and qualitatively (Table XIV). F i r s t , total plant weight was greater under long days than i t was under short days. Second, biomass distribution was different under long days than under short days; under long days roots accounted for half of the total plant weight while under short days roots accounted for only one-fourth of the total plant weight. Third, 0.1% C0 2 increased the relative proportions of biomass distributed to the roots. Increases in root biomass were accompanied by corresponding decreases in biomass distributed to leaves, even though leaves accounted for noticeable absolute shoot weight increases (Table XIII). Biomass distribution in stems and branches appeared to be relatively constant and unaffected by either daylength or C0„. TABLE XIV BIOMASS DISTRIBUTION DOUGLAS FIR (6-month-old seedlings) Grown for six weeks under designated daylength/CO- combinations -2 1 Irradiance = 7.2 mW cm RATIO TO TOTAL PLANT WEIGHT TREATMENT Daylength C02 cone, (h) (%) SHOOT 'TOTAL LEAF STEM BRANCH ROOT TOTAL TOTAL WEIGHT (g ) 8 0.03 .73 .61 .10 .02 .27 1.92 8 0.1 .71 •59 .10 .02 .29 1.84 8 1.0 .78 .66 .10 .03 .22 1.83 8 5.0 .75 .62 .10 .03 .25 2.09 16 0.03 .51 .36 .12 .02 .49 3.64 16 0.1 . .47 .32 .13 .02 .53 4.75 16 1.0 .51 .37 .12 .02 .49 3.99 16 5.0 .56 .41 .13 .03 .44 3.99 The ratios shown i n the table are the means of 10 plants. 41 3. Summary: Effects of carbon dioxide and daylength on weight (a) Leaves Carbon dioxide enrichment primarily affected shoot weight of 1-year-old trees, and especially affected the weight of leaves as can be seen when weights measured after 90 days of long days (Table X) are compared with weights measured after 21 days of short days (Table VI) . In both cases leaves were l i g h t e s t when grown under high CO^ enrichment. Note that under short days the effects of enrichment were apparent after only 21 days, whereas under long days i t took much longer for demonstratable weight gains under 0.1% CO^. The fact that short-day trees were maintained under twice the irradiance of long-day trees may account for the greater growth rates of short-day trees. The delayed response of plants grown under low irradiances emphasizes how important l i g h t regime can be when enrichment i s used to enhance growth. Irradiance levels should be maintained at high enough levels to receive the f u l l benefits from enrichment. A comparison between the leaf growth of 1-year-old and 6-month-old trees may also be made to examine the effects of age on plant response. (Tables VII and X I I I ) . When the same daylengths and CO^ levels are compared (short and long days with 0.03% and 1.0% CO^), simi l a r trends can be seen i n responses to CO^ enrichment. For plants of both ages, the lowest leaf weights occurred when the trees were given low CO^  and short days, while the highest leaf weights occurred when plants were given high CO and long days. The results indicate that the response of Douglas f i r to C0 o enrichment and daylength i s consistent with age, at least for the age range used i n this study. 42 (b) Steins No differences i n stem weight could be found when 1-year-old trees were grown under normal or high CO i n both experiments 1 and 2 (Tables VII and X). However, stem weights of 0.1% CO^ plants grown in experiment 2 were s i g n i f i c a n t l y different from weights of normal or high plants. CO^ levels used i n experiment 1 (0.03% and 1.0% CC^) apparently did not stimulate stem growth, and thus i t appeared that CO^ did not affect stem weight u n t i l the intermediate lev e l was used i n experiment 2. Six-month-old seedlings followed the pattern observed for 1-year-old trees (Table X I I I ) . Stem weights of plants grown under 0.1% CO2 and long days were s i g n i f i c a n t l y higher than weights of plants grown under the other CO2 treatments. (c) Roots Root weights of 1-year-old trees were not affected by either daylength or CO2 enrichment (Tables VII and V). However, roots of younger trees grown under long days were s i g n i f i c a n t l y greater than those grown under short days (Table X I I I ) . (d) Biomass D i s t r i b u t i o n Carbon dioxide levels were more important than daylengths i n influencing biomass d i s t r i b u t i o n of 1-year-old Douglas f i r . In experiment 1, trees under long days and high CO2 levels had s i g n i f i c a n t l y more biomass in the shoot than trees under the other three CO2 - daylength combinations (Table V I I I ) . In experiment 2, plants grown under long 43 days and intermediate CO^  levels showed greater shoot production than the other treatments (Table IX). For younger trees photoperiods were more important than CO^ levels in influencing biomass distribution (Table XIV). Under short days about three times more biomass went into shoot production than into root production while under long days shoot and root production were approximately equal. 44 DISCUSSION Growth Enhancement Most investigators have found that levels of 0.1% CO^ are the most favourable for crop plants (84), and on the basis of the results reported i n th i s chapter i t appears that 0.1% CO^ (1000 ppm) is also the best l e v e l for growth enhancement of Douglas f i r . In some instances however, CO^ enrichment greater than 1000 ppm has also enhanced growth. For example, the y i e l d of grain from sorghum grown under 2400 ppm CO2 was approximately twice that under 300 ppm (14) . Also in cucumber under moderate l i g h t i n t e n s i t y no growth i n h i b i t i o n was found at levels of 8000 ppm (84). Douglas f i r responded favorably to levels of 10,000 ppm CO^ as long as l i g h t i n t e n s i t y was low (Table VII, this chapter; E.B. Tregunna, unpublished report). Tomatoes and other vegetable crops have been grown at concentrations as high as 30,000 ppm CO^  with no i l l effects (37). Seedlings did not respond as w e l l to CO^ enrichment as 1-year-old trees. Although enriched seedlings had mean weights greater than those of unenriched seedlings, s i g n i f i c a n t differences could be shown only on the basis of daylength and not on the basis of CO^ l e v e l (Table XIII). However, Tanaka was able to increase shoot dry weights of Douglas f i r seedlings by 37% and root dry weights by 66% by enriching the greenhouse to 1500 ppm C0 2 (72). He also found that age affected seedling response i n a l l species tested (Douglas f i r , western hemlock, noble f i r , white f i r , ponderosa pine, and lodgepole pine); seedlings which were six months old at the end of treatment responded less favourably to C0 o than did four-month-old seedlings. Increased age 45 could account for the lack of seedling response i n my study since trees were approximately eight months old at the end of treatment. Another factor may have been that the seedlings had been maintained under long daylengths i n the greenhouse p r i o r to CO^ treatment and had been growing almost continuously from the time of germination to onset of CO^  enrichment. In addition, dioxide enrichment seems to be most effective when given during a period of rapid growth. Since 1-year-old trees responded favourably when CO^  treatment was given at the beginning of a new cycle of growth, seedlings probably would have shown a greater response i f the CO^ enrichment had been given at an early part of their growth period. However, growth enhancement due to CO,, enrichment was evident i n spite of low o v e r a l l seedling response since seedlings grown under long days and 0.1% C0 2 s t i l l showed a 70% increase i n height and a 30% increase i n t o t a l weight above that for plants grown under long days and normal a i r (Tables IV and VIII) . Growth I n h i b i t i o n Carbon dioxide concentrations higher than 0.1% CO,, generally are inhibitory (37, 48, 59, 85). Under long days and high C0 2 (1.0% C0 2) trees exhibited less growth than those under normal a i r (Table 1). However, seedlings showed approximately the same amount of growth under 0.03%, 1.0%, and 5.0% CO,,, with no apparent harmful effects from the higher C0 2 concentrations (Table IV). Regardless, others have reported negative effects of high CO,, levels on some other plants. Cucumber plants suffered chlorosis and leaf necrosis when grown i n atmospheres enriched with 2000 to 3000 ppm C0 0 (16). The authors suspected that calcium 46 deficiency caused some of the injury yet they were able to minimize the deleterious effects by reducing enrichment to only 1000 ppm CO^ . Nevertheless they could not determine the reason for the shoot damage. Decrease in leaf area and weight may constitute another potential problem with CO,, enrichment. An increase in leaf weight without a corresponding increase in leaf area may cause a decrease in overall growth since photosynthesis may eventually be restricted by the area available for energy capture i f dry matter is not used to expand the photosynthetic surface. In 1-year-old Douglas f i r , leaf. area and dry weight increased under 0.1% CO^j but as CO^  levels became higher, area and dry weight decreased (Table XI). As regressions based upon leaf area and weight data indicate, higher CO^  concentrations decreased leaf areas proportionately more than leaf weights. Thus area:weight ratios were also decreased. High carbon dioxide levels 2 -1 have similarly affected leaf area:weight ratio (dm g ) i n the following vegetable and horticultural crops. Leaf arearweight ratios decreased in beans and tomato when CO^  was increased from 300 to 1000 ppm (84). In Pharbitis n i l ratios also decreased when CO,, was increased from 1000 to 15,000 ppm (59). Similar decreases have been noted for soyabean at 1350 ppm CO,, (15) ; Chrysanthemum morif olium at 1500 ppm C02 (29, 30); tomatoes at 1000 ppm C02 (31); and sugar-beet, barley, kale, and maize at 1000 or 3300 ppm (21). Purhoit and Tregunna suggested that i f net photsynthesis was also increased under higher CO,, levels, higher net assimilation rates may compensate for decreased leaf area (59). Yet Hurd noted that tomatoes with decreased leaf area:weight ratios s t i l l 4 7 showed depressed growth rates after several weeks despite higher net assimilation rates (31). However, i t should be noted that carbon dioxide is not the only factor which may reduce leaf area:weight ratios in Douglas f i r . Brix found that high light intensity also decreased the amount of leaf area relative to the dry matter produced (8). Thus light can in many cases mimic the effects of CO^  on growth and development. This point w i l l be discussed in a later section dealing with CO^  interactions. Growth and Biomass Distribution Reports of carbon dioxide effects on growth and biomass distribution have been contradictory. I found that the 1-year-old Douglas f i r enrichment affect shoots more than roots. Root weights remained a relatively constant proportion of total plant weight so that differences in shoot:root ratio reflected increases in the proportion of assimilate going to the shoot rather than decreases in amounts going to the root (Tables IX and X). For younger trees photoperiods were more important than CC^ levels in influencing growth and biomass distribution (Tables XIII, XIV). However, CO^  did have some effect on biomass distribution. Seedlings under the same daylength grown under intermediate CO^ levels had relatively more biomass in their roots than did seedlings grown under other C00 levels and the same daylength. (Table XIV). 48 Therefore, at least i n this study CO^ enrichment of 1-year-old Douglas f i r appears to primarily affect shoot growth rather than root growth. The shoot absorbs atmospheric CO^ and reduces CO^ to primary photosynthetic products; thus enrichment would be expected to have the greatest immediate effect upon the upper plant portion. However, i n 6-month-old Douglas f i r CO^ enrichment also changed the r e l a t i v e distribution of biomass within the plant, thus i t i s possible that CC^ might affect translocation of organic products as w e l l as af f e c t i n g the production of primary products. Others have found that carbon dioxide increased root weight more than shoot weight (conifers, 71, 72; barley, sugar-beets, kale, 21). Tregunna found that 1.0% CO^ combined with high nitrogen levels substantially promoted both root mass and root elongation of Douglas f i r seedlings (unpublished report). He f e l t , however, that the temperature under which plants were grown was more important than CO^  i n controlling shoot and root growth. Tregunna also noted that root or shoot growth usually would occur at the expense of the other plant part; shoot and root weights did not increase simultaneously. Wittwer showed for beans and tomatoes that CO^ affected roots more than shoots (84). He suggested that the rapid recovery observed after plants had been transplanted might be due to root growth stimulated by carbon dioxide. If Wittwer's observation i s correct, pre-treatment with high CO^  levels could conceivably prove useful i n conditioning plant material preparatory to rooting of cuttings and i n improving drought resistance. 49 In fact CO^enriched atmospheres and carbonated mists have been reported to improve rooting of cuttings (50). Treated cuttings of Chrysanthemum, Weigela, P o t e n t i l l a f r u t i c o s a , Juniperus h o r i z o n t a l i s , and Thuja occidentalis rooted faster and produced more roots per cutting than controls without CO^ treatment. A third view i s that CO^ does not affect biomass relationships and weight d i s t r i b u t i o n remains the same before and after CO^ treatment. -2 -1 For instance when tomatoes were grown under low l i g h t (59 J cm day ) carbon dioxide did not change biomass d i s t r i b u t i o n (31). From the c o n f l i c t i n g results of the reports cited above i t appears that biomass d i s t r i b u t i o n i s not a predictable measure of carbon dioxide effects on plant growth. In addition, other variables must be considered when making comparisons of how CO^ affects growth. These variables include the plant species being compared, the effects of nitrogen and other f e r t i l i z a t i o n treatments, and the part of the growing season during which comparisons are being made. Interactions between carbon dioxide and other factors Plant response as we perceive i t experimentally i s the cumulative result of factors which we have chosen to manipulate, the interactions of those factors, plus other factors over which we may or may not have control. Some of the effects of increasing the carbon dioxide concentrations around the plant are known, but there are probably other factors of which we are not yet aware. Also, as with most other b i o l o g i c a l phenomena, CO effects can be modified by interactions with other 50 environmental factors. Light, temperature, and inorganic n u t r i t i o n figure importantly i n studies of CO,, enrichment since they have been found to interact with the effects of carbon dioxide. Light Light, both intensity and duration, has often been included as an additional variable i n CO^ enrichment studies. An interesting carbon dioxide-light interaction i s the fact that carbon dioxide can in part substitute for the l i g h t requirement when l i g h t i s l i m i t i n g . -2 There i s a range of l i g h t i n t e n s i t y between about 1.0 to 10.0 mW cm in which the rate of photosynthesis can be increased by either an increase i n l i g h t intensity or an increase i n CO^ concentration (23). -2 For example, doubling the l i g h t i n tensity from 2.6 to 5.4 mW cm affected dry weight, leaf area, and net assimilation rates, and was equivalent to a tenfold increase i n CO^ concentration (sugar-beets, barley, kale, and maize, 21). Even when l i g h t i s l i m i t i n g crops can respond to CO^ enrichment; addition of (X^ to greenhouse crops i n mid-winter was found to p a r t i a l l y compensate for reduced photosynthesis due to lack of l i g h t (85). Just as CO^ can at times substitute for lig h t when l i g h t i s l i m i t i n g , so also can l i g h t i n some cases demonstrate effects which mimic the effects of CO,, enrichment; e.g., either increased CO2 or increased l i g h t reduced leaf area weight r a t i o s (see the section on Growth I n h i b i t i o n . ) . Because CO^ enrichment also increases photosynthetic l i g h t saturation levels, CO^  i s most e f f e c t i v e l y u t i l i z e d when i t i s supplied during the mid-day period when l i g h t i n t e n s i t i e s are at their maximum (57). Even i n cloudy weather however, there i s no evidence that daylight 51 is so poor that plants cannot u t i l i z e additional carbon dioxide (84). Effects of light are generally considered to be additive to rather than synergistic to the effects of carbon dioxide (85). The use of only supplemental lighting of 700 ft-c for 16 hours a day stimulated the growth of six conifer species more than when only 0.15% CO^ was used. However i f both treatments were used in combination, the additive effects of CO^  and light could reduce the time required to produce a f a l l crop by two months (72). There was a strong positive interaction between light and CO^  when chrysanthemums were grown under a range of -2 -1 light intensities (125 to 375 J cm 8-hr day ) and carbon dioxide concentrations (0.033% to 0.15% CO^). Plants showed greater f i n a l dry weights and flower dry weights at the highest light intensity and highest CO,, level (30). Other positive interactions of light (both daylength and light intensity) with carbon dioxide could be seen in the results just described for Douglas f i r . Seedlings exhibited greater internodal elongation when both daylength and carbon dioxide levels were increased (Table V). In 1-year-old Douglas f i r dry weights of shoots and leaves were greatest when the longest daylength was combined with the highest CO^ concentration (Table VII). There i s some indication that light intensity may be even more effective than daylength in promoting CO^-enhanced growth. Short-day plants which received fewer hours of light each day but twice the light intensity of long-day plants were able to respond more quickly to treatment (Table X and XI, discussion, p. 41 ) . 52 Temperature Temperature can also play an important part i n CO^ enrichment programs. At l i g h t saturation and low CO,, levels (0.03% CO,,) photo-synthesis i s almost temperature independent, but at elevated CO,, le v e l s photosynthesis may be limited by temperature, or more exactly by the capacity of the biochemical process. The above has been shown for cucumber by Gaastra (23) and for Pinus halepensis by Whiteman and Roller (81). In tomatoes supplemental carbon dioxide had the greatest effect when supplied within the temperature range of 15 to 25 C, but the most suitable temperature within t h i s range depends upon l i g h t i n t e n s i t y (37). Low temperatures are recommended for low l i g h t i n t e n s i t i e s and thereafter temperatures and i n t e n s i t i e s should be increased simultaneously. For Douglas f i r grown i n unenriched a i r (0.03% C0 2) temperature influenced the r e l a t i v e amounts of biomass going to the shoot or the root (70), and thus temperature might influence Douglas f i r growth i n enriched environments as w e l l . Temperature was not included i n my studies of Douglas f i r growth under CO,, enrichment, but i n order to optimize the benefits of CO,, enrichment i n this species i t should be considered i n any future work. Inorganic n u t r i t i o n The acceleration of growth and development induced by CO,, may cause inorganic nutrients to l i m i t the growth of enriched plants (84). Soybeans were enriched with 0.14% CO,, and 21 days l a t e r began to show signs of nitrogen deficiency (15). In Douglas f i r high nitrogen was combined with high C0 2 levels (0.1% and 1.0% C0 2); t h i s experiment resulted i n greatly improved root mass and root elongation (E.B. Tregunna, 53 unpublished data). However, total dry weight did not appear to be significantly affected by the different nitrogen and carbon dioxide combinations; significant differences in total dry weight were due to CC^ levels only. In another study of Douglas f i r grown under normal air, biomass distribution was affected by the amounts of applied f e r t i l i z e r (78). The results of Tregunna indicate that f e r t i l i z a t i o n level may have the same indirect effects on CO^-enriched plants. Implications It is generally accepted that atmospheric CO^ levels are limiting to growth. Bonner, for instance, computed that the theoretical efficiency of light u t i l i z a t i o n could be doubled i f CC^ were not limiting (7). Most plants.will benefit from small amounts of supple-mental CO^  and w i l l respond with increased growth and vigor. As a result, CO2 enrichment of greenhouses i s now a f a i r l y standard practice in both North America and Europe. Carbon dioxide is usually added to the air by burning propane and other gases cr by slowly releasing pure CO^ through small perforations in pipes near the s o i l . There are some problems with these methods: fans must be used to insure uniform mixing; contaminants from leakage or incomplete combustion of burned gases may be highly injurious to plants; and vents closed to retain the supplemental carbon dioxide can result in the detrimental accumulation of heat and humidity in the greenhouse. Other methods for increasing atmospheric CO in the greenhouse have been somewhat successful: 54 carbonized water (obtained by bubbling CO^  gas through sprinkler water) has been shown to give beneficial results (40); organic f e r t i l i z e r placed beneath greenhouse plants can slowly release beneficial amounts of respiratory CO^  close to plant assimilatory organs; and dry ice production of pure can be used both to enrich and to cool the atmosphere near the plants. In spite of these limitations carbon dioxide enrichment currently is being successfully used on flower and vegetable crops, and i t s use is expected to increase i n the future. A notable example i s the waste-heat greenhouse recently built in Saskatoon, Saskatchewan (91). Exhaust gases from a turbine fired with natural gas were used for heating and CO^  enrichment in a polyethylene covered greenhouse. The system resulted in significantly greater yields of marketable tomatoes as well as substantially reduced heating costs. However, the benefits of using CO^ for improving growth and development of conifers and other trees are just beginning to be realized. There are questions which must be answered before CO^  enrichment can reliably and predictably be used to manipulate growth and development of Douglas f i r , but some advantages of have already been discovered. CO^  enrichment has been shown to enhance root growth in several studies even though increased root growth was not demonstrated in my studies (21, 72, 84). The use of CO^  to increase root size and vigor of seedlings before they are planted in f i e l d sites would improve both their recovery from transplanting and their resistance to drought. In sites where irrigation was not possible improved root growth would be especially important. 55 In addition, enrichment could improve seedling performance by production of trees with greater biomass and thicker stems. Sturdier plants would be able to better withstand biological and physical stresses in the f i e l d . Finally, economic advantages could be realized from the increased growth and assimilation rates of enriched plants because time in the nursery could be shortened and stock-handling expenses could be reduced. However, there are several precautions regarding the use of CO^  enrichment. The correct choice of CC^ level i s important in the use of enrichment to induce a desired effect on growth or development. An example can illustrate this point. The data in Figure 3 are f a i r l y typical representations of plant response to carbon dioxide. Under short and long daylengths there are sharply defined optima at intermediate CO^  levels while growth at low and high CC^ levels i s about the same. By selecting a CO^  level which was either too low or too high one could easily conclude that carbon dioxide had no effect on the parameter under consideration. This situation occurred while analyzing stem weight data. At f i r s t i t appeared that CO^ did not affect stem weight; however, a subsequent experiment showed that the CO^ levels used i n i t i a l l y were on either side of the optimum concentration for a demonstrable effect. Finally, one should take the usual precaution when extrapolating controlled environment studies to natural f i e l d conditions. My experiments give indications of the potential of CO^ enrichment, but how enriched seedlings perform in the f i e l d remains to be tested. 56 CHAPTER TWO EFFECTS OF CARBON DIOXIDE AND DAYLENGTH ON DORMANCY AND HARDINESS OF DOUGLAS FIR INTRODUCTION Although growth i n h i b i t i o n by high CO,, levels has been noted previously (31, 37, 48, 59, 84, 85), l i t t l e or no systematic investigatL has been conducted on this topic. Carbon dioxide enrichment studies have focused on growth enhancement of h o r t i c u l t u r a l and vegetable crops since most applied researchers are primarily interested i n enhancing rather than i n h i b i t i n g growth, and very high CO^ levels are d i f f i c u l t to maintain i n the greenhouse or i n open growing areas. Nevertheless, the effects of very high C0 2 levels are s t i l l of theoretical and p r a c t i c a l interest. In 1973 and 1974 several C0 2 enrichment studies were conducted on western hemlock and Douglas f i r . The trees were grown for three months i n controlled environment chambers and were enriched with very high C0 2 levels (E.B. Tregunna, unpublished data). Additional plants were grown under low and intermediate C0 2 levels. Enrichment by intermediate CO,, levels enhanced growth while high C0 2 levels appeared to l i m i t growth. These results were si m i l a r to those reported i n Chapter one. However, high C0 2 levels also increased the number of terminal buds formed by both western hemlock and Douglas f i r . Although growth i n h i b i t i o n had often been observed under high CO,, l e v e l s , CO,,-induced buset was unusual. The above report and my own evidence of reduced growth under high C0 o levels (Chapter One) 57 suggested that CO^  might effect developmental processes other than growth. However, before I proceed further in discussing the influence of CCv, on development, I should elaborate on the usual patterns of growth in Douglas f i r . Obvious seasonal growth of Douglas f i r and most other temperate trees is said to begin when bud scales covering the shoot apex are pushed open (bud burst) as the young shoot emerges and elongates (flushes). The new shoot continues growth u n t i l i t forms another bud and internodal elongation ceases. Within the same growing season this quiescent (temporary) bud may burst and another period of internodal elongation may begin. Douglas f i r often flushes twice during one growing season, and in some instances there may be as many as three flushes during one season. This alternating pattern of flushing and budset occurs on both terminal buds (buds at the apex of the main stem) and lateral buds (buds along branches). Buds formed late i n the f a l l do( not demonstrate this repeated flushing behaviour, and they do not flush even when given favourable growing conditions. These buds are called resting buds because they usually occur when the plant i s i n true winter rest (32, 56). In nature, short autumn daylength is an important environmental signal which acts to induce dormancy and budset in woody species. However, high carbon dioxide levels can also induce budset. Thus there are two factors which may promote the formation of buds. As a test of which of these two factors was the most influencial, experiment 1 was performed. 58 The objective of this experiment was to establish the r e l a t i v e effectiveness of photoperiod and CO^ levels for inducing budset i n Douglas f i r . Thus plants were grown under short and long days with low and high CO^ le v e l s . In addition, the experiment attempted to resolve whether high CO^ levels alone were s u f f i c i e n t for bud induction or i f high CC^ levels were only eff e c t i v e i n conjunction with short days. In the second experiment bud development was observed over a three-month period. The purpose of the experiment was to ascertain (1) whether the buds formed under high CO^ were quiescent (temporary) or resting (overwintering) buds, and (2) whether flushing would resume once buds were formed under elevated CO^ concentrations. In this experiment plants were grown under long days and low, intermediate, and high CO^ levels. Long days were chosen since bud formation had occurred under 16-hour days i n the previous experiment and because flushing would be more l i k e l y to occur under long photoperiods. The resu l t s of experiment 2 would possibly give some indication of the extent of bud formation and the degree of flushing under various CO^ le v e l s . Yet the type of buds being formed under high CO^ levels s t i l l had to be distinguished. In order to make this d i s t i n c t i o n i t would be necessary to establsh that CC>2 was inducing true rest (dormancy which cannot be broken by the usual environmental signals promoting growth (80)) i n addition to inducing budset. Under natural conditions acclimation to winter rest i s a gradual and continuous process. However, for explanatory purposes acclimation and dormancy have been separated into three phases (80) . 59 The f i r s t stage is induced by short days with relatively warm temperatures. Plants are only moderately frost resistant during this period, yet additional resistance of even a few degrees is important as i t may mean the difference between death and survival. Metabolic activity initiated during the f i r s t stage f a c i l i t a t e s the plant's response to the second stage. Low temperatures induce the second stage of acclimation. The hardiness achieved in stage two is sufficient for most plants in cold climates to survive the severities of winter. However, some plants enter a third stage of acclimation which is a greater deepening brought about by even lower temperatures. Once acclimated to this third stage, species are able to withstand extremely low temperatures (-50 C and below) and to recover without damage. In a standard test to assess true dormancy the temperature of a plant is f i r s t lowered to values below 0 C, and then slowly raised. The amount of "hardiness" the plant is said to possess is determined by the minimum temperature which the plant can endure without being k i l l e d or by the amount of damage the plant sustains i f i t is not k i l l e d . Using the standard test i t would be possible to assess the hardiness of treated and untreated plants to determine i f trees which had formed buds under high CO^ were in true rest. A preliminary study was conducted to test the hardiness of CO^-enriched trees. Douglas f i r were pre-treated with either normal a i r or high CO^ levels and then exposed to very low temperatures (-10 C and -20 C) . Plants pre-treated with high CO^ had higher survival rates and exhibited less damage than plants pre-treated with normal air. Thus there were indications that 60 carbon dioxide could induce dormancy as well as budset. However, more testing was required before this observation couldbe substantiated. Consequently, several experiments were performed under different photoperiods and carbon dioxide levels to determine which treatment combinations would be most effective in promoting CO^-induced freezing resistance. The effects of different pre-treatment temperatures were also examined. Finally, the persistence of (X^ -induced freezing resistance was investigated. The degree of freezing resistance remaining after plants were removed from enrichment and the use of special post-treatments were also examined. The results from the experiments reported in this chapter indicated that carbon dioxide affected developmental processes other than those promoting growth. Previously the manipulation of growth and development in conifers had been accomplished by adjusting photoperiod (18) or applying hormones (93). Instead of using photoperiods or hormones for manipulating growth and development, various CO^ con-centrations could be used alone or in combination with other treatments; and thus could potentially prove to be a worthwhile tool in the experimental control of l i f e cycles. MATERIALS AND METHODS Plant Material The experimental material used in this research was Douglas f i r from coastal provenances of British Columbia and Washington. 61 Material which is not ordinarily frost hardy was used for dormancy and hardiness studies since treatments on naturally hardy material could be inconclusive. Inland varieties are adapted to relatively extreme continental climates and consequently have greater frost and drought resistance. Pseudotsuga menziesii of coastal regions has only a limited frost and drought hardiness, a limitation which i s common among plants of maritime climates (58). Additional information on plant material can be found in the Materials and Methods of Chapter one. Experimental Conditions One-year-old trees were grown in experiment 1 to determine which photoperiod and carbon dioxide combinations would be most l i k e l y to induce budset. Plants were maintained for 30 days under 8- and 16-hour daylengths (25 C/20 C), with levels of 0.03% and 1.0% C0 2, and with -2 irradiance of 3.4 mW cm . Observations were, made on flushing and budset. Trees were then scored for bud growth and date of budset using the ranking scales detailed in the Appendix, Table B. In experiment 2 trees were grown for 90 days under 16-hour days (25 C/ 20 C), with three C0 2 concentrations (0.03%, 0.1%, and 1.0%'C02), and -2 with irradiance of 3.4 mW cm . In experiment 3, trees were grown for 30 days under 8-hour photoperiods (25 C/20 C), with four C0 2 levels (0.03%, 0.1%, 1.0%, and 5.0% CO.), and with irradiance of 7.2 mW cm"2. 62 Experiment 4 was a preliminary study to determine whether increased budset induced by high CO^ levels was related to increased frost hardiness. Trees were grown under short, warm days (8-hour days with 20 C/15 C) for three months with either 0.03% or 1.0% C0 2. The trees were then transferred to short, cold days (5 C/5 C) for three weeks. This treatment sequence has already been shown to induce hardiness i n Douglas f i r (1, 12). Plants were tested by lowering the temperature to -10 C or -20 C after several hours the trees were gradually warmed to room temperature (20 C). Subsequent s u r v i v a l and damage was noted. The remainder of the f r o s t hardiness work was performed on six-month-old seedlings i n conjunction with additional work on budset. Experiment 5 was designed to determine which combinations of photoperiod and carbon dioxide would be most effec t i v e i n promoting C0 2-induced freezing resistance. The schematic diagram i n the Appendix, Table C may be useful i n understanding the experimental design. Plants were grown for s i x weeks under 8- and 16-hour days (25 C/20 C) and four C0 2 levels (0.03%, 0.1%, 1.0%, and 5.0% C0 2). At the end of s i x weeks sub-samples from each of the photoperiod and C0 2 treatments were removed and given freezing tests. The remaining plants were transferred to a four week post-treatment i n which they were grown under 8-hour days and normal a i r . Day and night temperatures were 25 C and 5 C respectively. Warm, short days and cool nights were employed i n order to approximate environments which had been shown to increase hardiness i n Douglas f i r . The main purpose of post-treatment was to establish how long CO -induced freezing resistance would persist once 63 the plants were removed from enriched atmospheres. Freezing tests after termination of post-treatment would demonstrate i f there were interactions between pre-treatments and post-treatment, and i f these interactions affected the degree of hardinss. Freezing tests would also indicate whether pre-treatment or post-treatment was the more important i n determining the hardiness response. Controls grown i n normal a i r were run concurrently during the pre-treatment and post-treatment periods. The seedlings were grown under 8-hour days and three d i f f e r e n t thermoperiods: warm days/warm nights (25 C/20 C) ; warm days/cool nights (25 C/5 C); and cool days/cool nights (5 C/5 C) . The objective was to determine how temperature affected hardiness and budset. In addition, the controls would be used to gauge whether carbon dioxide or temperature was more ef f e c t i v e i n increasing freezing resistance. • Seedlings were frozen at two dif f e r e n t temperatures (-6 C and -10 C), f i r s t after s i x weeks of pre-treatment, then after four weeks of post-treatment, and f i n a l l y after s i x weeks of post-treatment. Records were also kept of flushing and budset. Trees were scored using the scales detailed i n the Appendix, Table B. Freezing tests Plants to be frozen were stored i n a cold room at 5 C for at least 12 hours prior to testing. These plants were then placed i n a freezer chest with insulating material surrounding the roots. They were then cooled at a rate of 7 C/hr, l e f t at the minimum temperature 64 for one hour, and rewarmed at a rate of 20 C/hr. This freezing procedure vas identical to the one performed by Timmis and Worrall (73). Temperature within the freezer was lowered using a small heater and a rheostat so that cooling would not progress top quickly. Fans within the freezing compartment ensured uniform temperature distribution. Four thermistors placed among the plants monitored the temperature during the freezing period. After testing, plants were removed from the freezer and placed in a cold room at 5 C for 24 hours. They were then transferred to a growth room with warm, long-day conditions (25 C and 16-hour light periods). After three days, plants were assigned their f i r s t scores which were based on a scale from 1 (no visible damage) to 5 (dead, no recovery) (Appendix, Table B). The plants were scored again at two and six weeks to verify the validity of the assigned scores. The scores were then analyzed by means of a nonparametric analysis of . variance, the Kruskal-Wallis H statistic, which is described i n the next section. Kruskal-Wallis One-way Analysis of Variance for Ranks Most of the.data in this chapter is based upon the scores derived from the ranks which are described in Table B of the Appendix. The data are analyzed by means of the Kruskal-Wallis one-way analysis of variance, one of the most powerful alternatives to the simple analysis of variance for parametric data (36, 69). It is the preferred nonparametric statistic to use when ranking information is available, and since i t uses a l l the order information, i t is a more powerful tool than other nonparametric tests. 65 The basic premise behind the Kruskal-Wallis a n a l y s i s i s that i f a l l the test groups contain random samples from the same general popu-l a t i o n , the average rank i n each group should be about the same s i z e as the average rank i n every other group. Further, the o v e r a l l average rank should be about the same as the average rank f o r each group, and therefore, the expected average rank can be compared with the observed average rank. If the n u l l hypothesis i s to be tested, the s t a t i s t i c "H", which follows the chi-square d i s t r i b u t i o n with k - 1 degrees of freedom, can be computed as follows: k H = 1 2 N ( N + 1 ) R2. 1 - 3 ( N + 1 ) i i n . J = 1 L j where N i s the t o t a l number of cases i n a l l groups Rj i s the sum of ranks i n the j t h group . n^ i s the number of cases i n the j t h group k i s the number of groups In a computation of the Kru s k a l - W a l l i s t e s t each o b s e r v a t i o n i s replaced by a rank; i . e . , a l l scores from a l l the groups are combined and ranked i n a s i n g l e s e r i e s . The smallest score i s repl a c e d by rank 1 ' and the next to smallest by rank 2 u n t i l a l l scores have been ranked. Once a l l scores have been assigned ranks, the sum of the ranks i n each group i s found. The Kruskal-Wallis t e s t determines whether the sums of ranks are l i k e l y to have come from the same population, or whether the sums are so d i f f e r e n t that they more l i k e l y have come from s e p a r a t e populat ions. 66 Ranking Scales Data used to assess bud development, date of budset, and frost damage were derived from ranks described i n the Appendix, Table B. Before raw data could be analyzed by means of the Kruskal-Wallis H s t a t i s t i c , the raw data had to be transformed into a suitable form. An i l l u s t r a t i o n of the procedure follows using the data for date of budset. To determine which treatments induced the e a r l i e s t budset, each seedling was assigned a rank from 2 to 10 based upon the date that budset was f i r s t noted. Rank 2 was given to plants which had set bud by f i v e days, while rank 10 was given to plants which had set but by 45 days. Intermediate ranks were assigned to dates at 5-day i n t e r v a l s between days 5 and 45. A rank of '1' was assigned to plants which did not flush during the 45-day treatment period, while a rank of '11' was assigned to plants which did not set bud during the treatment period. The Kruskal-Wallis H test was performed on the data from a l l plants. F i r s t the plants were ranked according to budset date numbers (numbers 1 to 11) previously assigned; then a composite rank (Rank Sum) for a l l plants i n each group was calculated; and f i n a l l y the group rank sums were analyzed to determine i f there were differences between the groups. These are the results which appear i n Table XX. In the table, treatments have been reordered according to the rank they received i n the Kruskal-Wallis H test. The treatment with the lowest rank appears f i r s t , the treatment with the next lowest rank appears second, etc. A low rank indicates that a greater proportion of the plants i n that group set bud at an early date, while a high rank indicates that a greater proportion of the plants i n the group set bud at a l a t e r date. 67 Data for bud development were handled i n a s i m i l a r manner except that low scores for bud development indicated a greater degree of budset than that for high scores. For f r o s t damage low scores indicated less damage and higher survival than that for high scores. Amino acid analysis Ethanol extracts of needles were made according to the procedure of Salminen and Koivistoinen (67) and were then dried i n a f l a s h evaporator. Five ml of pH 2.2 buffer was f i r s t added to the dried residue, the solution was then centrifuged for 2 minutes at 600 g i n a c l i n i c a l centrifuge, and the sample was f i n a l l y loaded d i r e c t l y onto the column of a Beckman Model 120 C amino acid analyzer. RESULTS A. FLUSHING AND BUDSET 1. One-year-old trees (a) Effects of 8- and 16-hour daylengths and 2 CO^ levels Trees given high under both daylengths flushed e a r l i e r than trees given low CO^  (87% of high CO^ plants had flushed by day 5, whereas only 62% of low CO2 plants had flushed by day 5 - but these differences were not shown to be s t a t i s t i c a l l y s i g n i f i c a n t ) . Terminal buds did not set within the 30-day period, but measurement of l a t e r a l budset showed a d e f i n i t e pattern (Table XV); l a t e r a l budset has been observed to precede terminal budset (Roger Timmis, personal communication). When plants were ranked according to the number of buds set, those 68 TABLE XV DEGREE OF LATERAL BUDSET IN DOUGLAS FIR TREES (1-0 Stock) Grown for 30 days under 8- or 16-hour daylengths with either 0.03% C0 2 or 1.0% C0 2 -2 Irradiance = 3.4 mW cm TREATMENT RANK ^ NUMBER OF BUDS Daylength (h) C0 2 cone. (%) SUM SET (N = 80) 16 1.0 11326 43 8 0.03 12468 30 8 1.0 14984 27 16 0.03 16167 9 According to Kruskal-Wallis H test (Nonparametric Analysis of Variance) Kruskal-Wallis test s t a t i s t i c = 32.51 p (assuming chi-square distn. with df, 3) = 0.0000 Rank sums shown in the table are based upon ranks of 20 plants and are in order by increasing rank sum. 69 grown under long days and high C00 showed the. greatest degree of budset (lowest rank number). Plants grown under long days and low CC^ showed the smallest degree of budset (highest rank number). Plants grown under short days with both low and high CC^  levels were intermediate in their degree of budset. (b) Effects of 16-hour daylength and three CO levels Plants in a l l treatments began to flush soon after the beginning of experiment (Table XVI). By 40 days plants in a l l treatments began to reset buds although significant differences were not noted until after 50 days. At 56 days plants grown under low and intermediate levels had approximately the same number of buds. Plants grown under high CO^  levels had twice as many buds as plants grown with the lower CO^  levels. After 56 days plants which had set bud under low levels flushed for the second time; 1.0% CO2 plants did not flush, but remained in budset during the remainder of the measurement period. (c) Effects of 8-hour daylength and four CO^  levels At the start of the experiment a l l plants had buds, but soon afterward plants under a l l four (X^ treatments flushed (Table XVII). However, plants grown under 5.0% broke bud, elongated to about 1 cm by 7 days, and did not elongate further throughout the measurement period. CO2 primarily affected budset. The greatest degree of budset was noted under low C09, and the degree of budset successively decreased 70 TABLE XVI DEGREE OF BUDSET IN DOUGLAS FIR TREES (1-0 Stock) Grown under 16-hour daylengths with 0.03%, 0.1%, or 1.0% C0 2 Irradiance = 3.4 mW cm -2 TREATMENT 0.03% CO, 0.1% C0r NUMBER OF BUDS SET 56 Days 67 Days 73 Days 80 Days 1.0% CO, 16 16 16 16 Goodman's & Kruskal's Gamma 0.56 1.00 1.00 Probability of Gamma 0.007 0.001 0.005 1.00 0.001 When two individuals are. chosen at random from the population, Goodman's and Kruskal's gamma tests how much more probable i t i s to get l i k e than unlike orders i n the two c l a s s i f i c a t i o n s . The data points shown i n the table are the means of 20 plants. 71 TABLE XV.II DEGREE OF BUDSET IN DOUGLAS FIR TREES (1-0 Stock) Grown under 8-hour daylengths with 0.03%, 0.1%, 1.0%, or 5.0% C0 2 "Irradiance = 3.4 mW cm NUMBER OF BUDS SET TREATMENT 0 Days 7 Days 21 Days 28 Days 0.03% C0 2 16 0 14 16 0.1% co 2 16 0 9 16 1.0% co 2 16 0 7 15 5.0% C0 2 16 * 0 0 0 Goodman's & Kruskal's Gamma - - 0.81 1.00 Probability of Gamma - - 0.000 0.000 shoots burst bud, but did not elongate beyond 1 cm. When two individuals are chosen at random from the population, Goodman's and Kruskal's gamma tests how much more probable i t is to get like than unlike orders in the two classifications. The data points shown in the table are the means of 16 plants. 72 as CC^ concentrations increased. Under long days however, budset was the greatest under the highest CO^ concentration (1.0% CO^); the results of CO^  enrichment on budset under short days were thus opposite to the results obtained under long days. I t i s suspected that these results may be anomolous due to the possible contamination of the gas supply since plants grown under 5.0% CO^ were arrested during the f i r s t week of flushing and were not able to proceed with development. Carbon dioxide also influenced the rate at which buds were set (Table XVIII). More plants set buds at e a r l i e r dates when treated with low levels of (X^ (0.03% c ^ ^ ) . Budset tended to occur l a t e r as concentration increased i n the atmosphere around the plant, and with 5.0% CO2 l e v e l s , no buds were set within the measurement period. 2. Six-month-old seedlings (a) General flushing and budset behaviour Under short days, there were no s i g n i f i c a n t differences i n the rates of budset among seedlings treated with d i f f e r e n t levels of CO2. However, there were trends which associated budset with CO2 levels (Figure 6). Generally the highest C0 2 levels (1.0% and 5.0% C02) promoted buset while levels of 0.1% CO2 i n h i b i t e d budset. For example, this i n h i b i t i o n was evident at 35 days when most other treatments had achieved close to their maximum degree of budset. Plants given 0.03% CO2 were intermediate i n their budset response. None of the plants under short days flushed again once buds had been set. 73 TABLE XVIII DATE OF FIRST TERMINAL BUDSET DOUGLAS FIR TREES (1-0 Stock) Grown under 8-hour daylengths with 0.03%, 0.1%, 1.0%, or 5.0% C0 2 IRRADIANCE = 3.4 mW cm"2 NUMBER OF BUDS SET TREATMENT Date on which trees f i r s t set bud Day 21 Day 28 No Budset 0.03% C0 2 14 2 0 0.1% c o 2 9 7 0 1.0% c o 2 8 8 0 5.0% C0 2 0 0 16 Goodman's & Kruskal's Gamma =0.85 Probability of Gamma = 0.0000 When two individuals are chosen at random from the population, Goodman's and Kruskal's gamma tests how much more probable i t i s to get like than unlike orders in the two classifications. The data points shown in the table are the means of 16 plants. Figure 6. Mean cumulative budset of 6-month-old Douglas f i r continuously supplied with C02 at the levels shown, and maintained tinder 8-hour photoperiods (25 C/20 C) _ a l t i h I r r a d i a n c e o f 7.2 mW C U T 2 - — , , 75 Under long days budset generally occurred later (Figure 7). Budset peaked sharply at 35 days then quickly declined as plants burst bud and resumed elongation growth. The overall effect of CO^  was similar to that observed under short days; plants treated with 5.0% CO^  had the greatest degree of budset followed in decreasing order by 1.0% C02, 0.03% C02, and 0.1% C0 2. (b) Maximum budset of six-month-old seedlings The degree of budset of six-month-old seedlings was primarily influenced by the daylength under which they had been grown (Table XIX); more buds were set under short days than under long days. Although C n 2 concentration had no discernable effect on degree of budset in the short-day group, i t did appear to influence the degree of budset within the long-day group. Long-day plants treated with 0.1% C0 2 had about half the number of buds set as the other three C0 2 treatments. It could be expected that 0.1% C0 2 would be the concentration under which there would be the least budset since earlier experiments had shown that 0.1% C0 2 level was most effective in promoting growth (Figures 2, 3, 4; Tables II, IV, V, X, XI). (c) Earliest date of budset of six-month-old seedlings A l l plants grown under short days set buds earlier than those grown under long days (Table XX). Within daylength groups, the timing of budset was influenced by the following C0 2 levels given i n descending order of effectiveness: 5.0%, 1.0%, 0.03%, and 0.1% C02. This order of C0 2 treatment was the same for both short and long days. Figure 7. Mean cumulative budset of 6-month-old Douglas f i r continuously supplied with C0 2 at the levels shown, and maintained under 16-hour photoperiods (25 C/20 C) with an irradiance of 7.2 mW cm - 2. 77 TABLE XIX MAXIMUM NUMBER OF BUDS SET IN DOUGLAS FIR SEEDLINGS Grown under 8- or 16-hour daylengths with 0.03%, 0.1%, 1.0%, or 5.0% C0 2 and 25 C/20 C NUMBER OF BUDS SET CARBON DIOXIDE CONCENTRATION 8-hour daylengths 16-hour daylengths 0.03% C0 2 50 29 0.1% c o 2 50 16 1.0% c o 2 52 29 5.0% C0 2 48 32 The data points shown in the table are the means of 54 plants. 78 TABLE ,XX EARLIEST DATE OF BUDSET V DOUGLAS FIR SEEDLINGS Grown under 8- or 16-hour daylengths with 0.03%, 0.1%, 1.0%, or 5.0% CO _2 Irradiance 7.2 mW cm TREATMENT ^ S U M * Daylength (h) C02 Cone. (%) 8 5.0 7150 8 1.0 8094 8 0.03 10129 8 0.1 10546 16 5.0 11100 16 1.0 12728 16 . 0.03 14033 16 0.1 14631 * According to Kruskal-Wallis H test (Nonparametric Analysis of Variance) Kruskal-Wallis test s t a t i s t i c = 60.55 p (assuming chi-square d i s t n . with df, 7) = 0.0000 Rank sums shown i n the table are based upon ranks of 54 plants and are i n order by increasing rank sums. 79 (d) Effects of temperature on six-month-old seedlings grown under normal air (0.03% COJ 2~ General flushing and budset behaviour for a l l temperature treatments were similar to those observed for other plants given short-day treatments as noted i n Section 2(a) (Figure 8). Almost total budset was achieved by 35 to AO days and no further flushing occurred. Maximum budset (35 buds set out of 54 plants) was obtained under the 25 C day/5 C night combination which approximates the situation in nature in the f a l l and which has been reported to be conducive to budset in Douglas f i r (42). Second highest budset (29 buds set) was obtained under the 25 C day/20 C night combination; the lowest budset (20 buds set) was obtained under th 5 C day/5 C night combination. The condition closest to the natural condition for induction of budset (25 C day/5 C night) also yielded the earliest budset (Table XXI). Plants grown under 25 C day/20 C night were the next earliest to set bud. Plants grown under 5 C day/5 C night set buds last. The production of factors which promote dormancy and budset in Douglas f i r requires short, warm days (41). The temperatures of the 5 C day/5 C night treatment undoubtedly limited the a b i l i t y of the plants so treated to produce the elements necessary for dormancy induction. B. COLD HARDINESS 1. Effects of carbon dioxide (a) Preliminary study A l l plants grown under normal a i r (0.03% C09) eventually Figure 8. Mean cumulative budset of 6-month-old Douglas f i r grown in air under 8-) photoperiods with day/night temperatures as shown. •hour 81 TABLE XXI EARLIEST DATE OF BUDSET DOUGLAS FIR SEEDLINGS Grown under 8-hour daylengths with three temperatures for six weeks TREATMENT RANK SUM* 25 C/5 C 3186 25 C/20 C 4594 5 C/5 C 4781 According to Kruskal-Wallis H test (Nonparametric Analysis of Variance) Kruskal-Wallis H test statistic =13.42 p (assuming X distn. with df,2) = 0.0012 Rank sums shown in the table are based upon ranks of 54 plants and are in order by increasing rank sum. 82 died, whether tested at -10 C or -20 C (Table XXII). However, a l l plants grown under 1.0% CO^ and given freezing temperatures as low as -10 C survived with only a s l i g h t amount of damage to fo l i a g e . Plants grown under 1.0% CO^ and exposed to -20 C showed a 50% s u r v i v a l rate. (b) Plants grown under 8-hour days Six-month-old seedlings tested at -5 C after s i x weeks of CO^  showed the least damage (lowest rank) i f grown under 1.0% C0 2 and the greatest damage (highest rank) i f grown under 5.0% C0 2 (Table XXIII). Plants grown under 0.03% and 0.1% C0 2 had approximately the same rank but 0.03% C0 2 plants performed s l i g h t l y better. Plants tested at -10 C were not s i g n i f i c a n t l y d i f f e r e n t , but th e i r ranks followed a s i m i l a r order to the ranks of plants tested at -6 C. Plants grown under 0.03% and 1.0% CO^  exhibited less damage from freezing than plants grown under 5.0% and 0.1% CO . (c) Plants grown under 16-hour days Six-month-old seedlings grown under long days and 0.1% C0 2 showed the least amount of cold injury when tested both at -6 C and -10 C, but only the scores for the -10 C test were s i g n i f i c a n t l y different. When the results for seedlings under both short and long days and after being tested to -6 C were compared (Tables XXIII and XXIV), short-day seedlings had lower scores than plants grown under long-days; furthermore, the lowest scores for a l l groups tested at -6 C 83 TABLE XXII FROST HARDINESS STUDY DOUGLAS FIR TREES (1-0 Stock) FREEZING TEST CARBON DIOXIDE TREATMENT Minimum Duration of temperature min. temp. -20 C -10 C 8 hrs. 1 hr. 0.03% CO, 1.0% CO, A l l plants died 50% death in in 7 days 75 days A l l plants died No death at in 60 days 135 days Slight damage observed after 60 days Trees were grown for 3 months under 8-hour days with 20 C/15 C; then 8-hour days with 5 C/5 C for 3 weeks prior to freeze testing. The data points shown in the table are the means of 4 plants. 84 TABLE XXIII COLD HARDINESS OF CO^ENRICHED DOUGLAS FIR SEEDLINGS A. EFFECT OF CARBON DIOXIDE Grown for 6 weeks with 8-hour days (25 C/20 C) TREATMENT FREEZE TESTING to -6 C TREATMENT FREEZE TESTING to -10 C 1.0% c o 2 0.03% C02 0.1% c o 2 5.0% CO„ 154 188 196 282 0.03% CO, 1.0% c o 2 5.0% c o 2 0.1% c o „ 180 191 203 247 Kruskal-Wallis H 8.27 * probability 0.041 Kruskal-Wallis H 2.91 * probability 0.41 2 Assuming X distn. with df = 3 Rank sums shown in the table are derived from the Kruskal-Wallis H test (Nonparametric Analysis of Variance) and are in order by increasing rank sum. The data are based upon ranks of 10 plants. 85 TABLE XXIV COLD HARDINESS OF C02-ENRICHED DOUGLAS FIR SEEDLINGS A. EFFECT OF CARBON DIOXIDE Grown for 6 weeks with 16-hour days (25 C/20 C) TREATMENT RANK SUMS (Frozen to -6 C) TREATMENT RANK SUMS (Frozen to -10 C) 0.1% CO, 0.03% CO, 1.0% CO, 5.0% CO, 196 204 208 213 0.1% CO, 0.03% CO, 1.0% CO, 5.0% CO, 55 255 255 255 Kruskal-Wallis H 0.13 * probability 0.99 Kruskal-Wallis H 39.00 probability 0.000 * 2 Assuming X distn. with df = 3 Rank sums shown in the table are derived from the Kruskal-Wallis H test (Nonparametric Analysis of Variance) and are in order by increasing rank sum. The data are based upon ranks of 10 plants. 86 were for those seedling grown under 1.0% CX^ and short days. When seedlings were tested to -10 C however, the lowest scores were obtained by seedlings grown under 0.1% C0^ and long days. 2. Effects of post-treatment (a) Plants grown under 8-hour days After six weeks of pre-treatment plants grown under short days showed significant differences between CO^ treatments when a l l plants were tested at -6 C (Table XXV). Seedlings pre-treated with 1.0% CO^ exhibited the least damage (lowest sums of ranks (of a l l groups tested). However, by four weeks of post-treatment there were no differences between the four CO^  pre-treatments. Plants which had been grown under 1.0% and 0.03% CO,, s t i l l had the lowest ranks, but they sustained greater damage than had been shown at the end of pre-treatment. By six weeks some general effects on hardiness due to post-treatment could be seen: (1) there was less damage in a l l CO^  pre-treatment groups and (2) the effects of CO^  pre-treatment was no longer readily discernable. Plants tested at -10 C after six weeks of pre-treatment showed no significant differences in damage and survival between the four CO^ treatments. Post-treatment had no apparent effect on hardiness of plants tested at -10 C since there were no significant differences between CO^  groups after four weeks of post-treatment, nor was there a substantial reduction in damage scores. TABLE XXV COLD HARDINESS OF CO^ENRICHED DOUGLAS FIR SEEDLINGS B. EFFECT OF POST-TREATMENT 2 FREEZE TESTING TO -6 C 0 WEEKS 3 A WEEKS 3 6 WEEKS 3 c o 2 Pretreatment RANK SUM 4 CO Pretreatment RANK SUM 4 CO Pretreatment RANK SUM 4 1.0% c o 2 154 0.03% C02 193 0.03% C02 109 0.03% C02 188 1.0% c o 2 194 1.0% c o 2 120 0.1% c o 2 196 0.1% c o 2 205 5.0% C02 147 5.0% C02 282 5.0% C02 228 0.1% c o 2 186 Kruskal-Wallis H 8.27 Kruskal-Wallis H 0.78 Kruskal-Wallis H 7.13 probability ^  0.041 probability ^  0.854 probability 0.068 2 PRETREATMENT: 6 weeks with 8-hour days (25 C/20 C) and four levels of CO 3 POSTTREATMENT: 6 weeks with 8-hour days (25 C/5 C) and 0.03% CO- only 2 ^ Length of posttreatment 2 5 Rank assigned according to Kruskal-Wallis H Test (Nonparametric Analysis of Variance) Assuming chi-squared distribution with df = 3 TABLE XXV (Continued) COLD HARDINESS OF CO^ENRICHED DOUGLAS FIR SEEDLINGS B. EFFECT OF POST-TREATMENT 2 FREEZE TESTING TO -10 C 0 WEEKS 3 4 WEEKS 3 c o 2 Pretreatment RANK SUM 4 c o 2 Pretreatment RANK SUM 4 0.03% C02 180 1.0% c o 2 173 1.0% c o 2 191 0.1% c o 2 191 5.0% C02 203 0.03% C02 225 0.1% c o 2 247 5.0% C02 231 Kruskal-Wallis H 2.91 Kruskal-Wallis H 1.99 probability 0.41 probability 0.57 PRETREATMENT: 6 weeks with 8-hour days (25 C/20 C) and four levels of C02 FOSTTREATMENT: 6 weeks with 8-hour days (25 C/5 C) and 0.03% C02 only Length of posttreatment co Rank assigned according to Kruskal-Wallis H Test(Nonparametric Analysis of Variance) 0 0 Assuming chi-squared distribution with df = 3 89 (b) Plants grown under 16-hour days Plants previously grown under long days and tested at -6 C after six weeks of CO^  treatment showed no s t a t i s t i c a l differences between CO^  treatment groups (Table XXVI). After four weeks of post-treatment with short, warm days and cool nights, there were s t i l l no statistically significant differences among the groups. After six weeks' treatment, significant differences due to post-treatment could be found in that plants pre-treated with 1.0% CO^ sustained the least damage of a l l plants tested. When plants were tested at -10 C at six weeks of pre-treatment, 1.0% seedlings did markedly better than the other three treatment groups; they showed very l i t t l e damage, whereas plants in the other three groups were highly damaged. After four weeks of post-treatment, plants grown under 0.1% CO2 demonstrated greatest hardiness while other CO^ treatment groups showed only slight reductions in their damage scores. By six weeks of post-treatment, the four CO2 groups tested at -10 C were not s t a t i s t i c a l l y different. Most of the effects of CO^ treatment had been obscured in the period between four, and six weeks. Plants which had previously been given enrichment however, had the least amount of damage; seedlings grown under normal air had noticeably higher scores than those which had received treatment. 3. Effects of temperature After six weeks with no post-treatment, those plants which TABLE XXVI COLD HARDINESS OF CO -ENRICHED DOUGLAS FIR SEEDLINGS B. EFFECT OF POST-TREATMENT 2 FREEZE TESTING TO -6 C 0 WEEKS 3 4 WEEKS 3 6 WEEKS 3 c o 2 Pretreatment RANK SUM 4 CO Pretreatment RANK SUM 4 CO Pretreatment RANK SUM 4 0.1% c o 2 196 0.03% C02 140 1.0% c o 2 122 0.03% C02 204 5.0% C02 211 0.03% C02 181 1.0% c o 2 208 1.0% c o 2 230 5.0% C02 248 5.0% C02 213 0.1% c o 2 240 0.1% c o „ 270 Kruskal-Wallis H 0.13 Kruskal-Wallis H 5.09 Kruskal-Wallis H 11.58 probability 0.99 probability 0.17 probability 0.009 1 2 3 4 5 PRETREATMENT: 6 weeks with 16-hour days (25 C/20 C) and four levels of CO POSTTREATMENT: 6 weeks with 8-hour days (25 C/ 5 C) and 0.03% C02 only Length of posttreatment Rank assigned according to Kruskal-Wallis H Test (Nonparametric Analysis of Variance) Assuming chi-squared distribution with df = 3 N = 10 o TABLE XXVI (Continued) COLD HARDINESS OF CO^ENRICHED DOUGLAS FIR SEEDLINGS B. EFFECT OF POST-TREATMENT 2 FREEZE TESTING TO -10 C 0 WEEKS 4 WEEKS 6 WEEKS co 2 Pretreatment RANK SUM CO Pretreatment RANK SUM co 2 Pretreatment RANK SUM 0.1% co 2 55 0.1% co 2 107 1.0% co 2 172 0.03% C0 2 255 0.03% C0 2 211 5.0% C0 2 189 1.0% co 2 255 5.0% C0 2 245 0.1% co 2 194 5.0% C0 2 255 1.0% co 2 257 0.03% C0 2 266 Kruskal-Wallis H 39.00 Kruskal-Wallis H 12.12 Kruskal-Wallis H 5.34 probability ^ 0.0000 probability ~* 0.007 probability 0.148 PRETREATMENT: 6 weeks with 16-hour days (25 C/20 C) and four levels of C0o POSTTREATMENT: 6 weeks with 8-hour days (25 C/5 C) and 0.03% C0 2 only N = 10 £ Length of posttreatment Rank assigned according to Kruskal-Wallis H Test (Nonparametric Analysis of Variance) Assuming chi-squared distribution with df = 3 92 had been grown under short, warm days and cool nights as well as those plants which had been grown under cool days and nights showed the least cold damage (Table XXVII). Plants grown under warm days and nights showed the most damage. After two weeks of post-treatment, effects of the previous temperature were s t i l l evident. Plants which had previously been given 8-hour days of 25 C /5 C showed considerably less damage than the other two temperature combinations. Plants previously given 8-hour days with 5 C/5 C had. intermediate scores, and plants previously given 8-hour days with 25 C/20 C had the highest scores (highest damage) . By four weeks in the new thermoperiods however, there were no significant differences among the three groups and the order of ranks had changed. The main conclusions from the above results are that: (1) warm short days and cool nights (25 C /5 C) are most effective in inducing hardiness; and (2) the effects of a previous themoperiod are persistent for two to four weeks after a change in temperature regime. 4. Effects of CO,, and daylength on amino acids Under short and long days total amino acids increased i n leaves grown with high CO,, levels (1.0% CO^ ) as compared to those found i n leaves grown with normal air. The changes were due to increases both i n acidic and neutral fractions and in basic fractions. Under long days greater amounts of the following amino acids were found: glutamine, proline, isoleucine, leucine, phenylalanine, histidine and arginine. Under short days proline and arginine showed the greatest increases. Very high CO,, levels (5.0% CO,,) affected the amino acid contents of leaf extracts by greatly decreasing the total amounts present. 93 TABLE XXVII COLD HARDINESS OF 6-month-old DOUGLAS FIR SEEDLINGS C. EFFECT OF TEMPERATURE PRETREATMENT KRUSKAL-WALLIS RANKS AFTER FREEZE TESTING TO -6 C 0 WEEKS 2 WEEKS 2 4 WEEKS 25 C/5 C 5 C/5 C 25 C/20 C 135 135 195 88 177 201 157 130 119 Kruskal-Wallis H 3 probability 8.92 0.012 10.21 0.006 1.95 0.38 PRETREATMENT: Grown for 6 weeks with 8-hour days and 3 thermoperiods with 0.03% C02 Length of posttreatment. POSTTREATMENT: 6 weeks with 8-hour days, (25 C/5 C), and 0.03% C02 Assuming chi-squared distribution with df = 2 Rank sums shown in the table derived from the Kruskal-Wallis H test (Nonparametric Analysis of Variance) and are in order by increasing rank sum. The data are based upon ranks of 10 plants. 94 TABLE XXVIII AMINO ACIDS IN LEAF EXTRACTS OF 1-YEAR-OLD DOUGLAS FIR Data expressed i n ng/mg 8-HOUR DAYS 16-HOUR DAYS 0.03% C0 2 1.0% C0 2 0.03% C0 2 1.0% C0 2 5.0% C0 2 asn 36.73 9.98 73.00 98.55 48.17 thr - - - - -ser - - - 110.44 -gin 148.07 162.74 194.53 282.66 106.65 pro 125.93 358.04 34.68 56.89 57.28 giy 11.43 6.84 3.14 15.84 3.12 ala 26.93 24.94 35.97 36.61 11.15 v a l 0.99 12.16 7.08 10.73 4.80 met 3.88 - 4.16 -i l e 3.71 3.98 4.87 9.96 2.91 leu 5.07 3.71 3.90 6.33 2.59 tyr 6.47 2.77 10.92 19.07 3.47 phe 8.01 1.50 9.25 15.56 5.54 TOTAL ACIDICS a NEUTRALS 377.22 586.66 377.34 666.80 245.68 lys 52.99 156.29 384.27 203.24 109.17 his - 13.67 73.96 -arg 328.50 960.39 505.33 895.44 257.34 TOTAL BASICS 381.09 1116.68 903.27 1172.64 366.51 IaDS(n^/mg) 7 5 8 ' 3 1 1703.34 1280.61 1839.44 612.19 95 DISCUSSION Budset The effects of carbon dioxide enrichment on budset complement the effects of carbon dioxide on growth discussed i n Chapter One. Growth was shown to be enhanced by 0.1% CO^ and depressed under high CO2 (1.0% and 5.0% CO^), but the C0? effects on budset were the inverse of i t s effects on growth. In .most cases high concentrations of CO^ (1.0% and 5.0% CO^ ) promoted budset whereas low concentrations (0.03% and 0.1% CO^) inhibited budset. Carbon dioxide hastened budset of 6-month-old seedlings grown under short and long days i n the following descending order: 5.0%, 1%, 0.03%, and 0.1% C02 (Table XX). Daylength did not appear to alter the rate at which buds were set, since CO^ affected budset i n the same manner under both short and long days. These results reveal two points about the effects of carbon dioxide on cessation of growth and development of buds. F i r s t , by promoting development of buds under long days, carbon dioxide appears to override the photoperiodic control which o r d i n a r i l y induces the f i r s t stage of dormancy i n most woody species (56, 80). However, although photoperiod i s generally recognized as the key agent c o n t r o l l i n g growth cessation and development of dormancy, there have been many reports that photoperiodic control may be bypassed by many other factors, some of which are temperature, l i g h t i n t e n s i t y , nutrients, and water supply (56). I t now seems that carbon dioxide also may be added to the l i s t of agents bypassing photoperiodic control. Second, the effects of carbon dioxide on budset appear to be dependent on photosynthesis. Development of buds 96 and induction of rest are generally considered to be active metabolic processes. Biochemical events which occur in the autumn as plants prepare for winter rest result in changes which involve a l l major groups of plant compounds—nucleic acids, proteins, carbohydrates, and li p i d s (1, 52, 54). Environmental factors which limit metabolic activity also tend to retard induction of dormancy so that plants which are severely depleted in photosynthetic reserves cannot acclimate (80). On the other hand, environmental factors which enhance metabolic activity tend to promote cold hardening. McGuire and F l i n t (47) found for four different conifers that hardening was increased by light; they concluded that light operated via photosynthesis. Van den Driessche (76, 77) observed that Douglas f i r seedlings hardened more under short photoperiods i f light intensity was high, whereas at low light intensity hardening increased with increasing daylength. Further support for the role of metabolism during early dormancy comes from research on the effect of low temperatures during this period. In spruce, low temperature during short-day treatments tended to delay bud formation. However, when the temperature was raised, buds were formed more rapidly (27). Dormancy was postponed in Douglas f i r under 9-hour days when seedlings were grown under low temperatures (42). I observed the same delay with 6-month-old seedlings (Section B (3) of this chapter). The fact that the C0^ effect on budset of 1-year-old trees was clearly demonstrated under long light periods but not under short light periods, suggested that light was a limiting factor and that low light intensity could be compensated for by the use of longer daylengths (Table XV). C09 enrichment also promoted budset of 6-month-old seedlings 97 under short and long days when light intensity was increased. These observations indicate that CO^  may be acting via photosynthesis to produce effects on budset, and that this is an active process. In experiment 2, budset under 1.0% CO,, was not only quanti-tatively but qualitatively different from budset under 0.03% and 0.1% CO^ (Table XVI). Buds formed under low C0 2 (0.03% and 0.1% C02) appeared to / be quiescent (temporary) buds while those formed under 1.0% C0 2 appeared to be resting (dormant) buds. The number of buds set under 1.0% C0 2 at 56 days was twice that set under the lower C0 2 concentrations. In addition, trees with 1.0% C0 2 which had set bud did not break bud during the period from 56 to 90 days, whereas trees with lower C0 2 levels did break bud and flush. Six-month-old seedlings also showed qualitative differences in budset, but these differences were primarily due to photo-period rather than to CO,, treatments (Figures 6 and 7). Under short days, a l l plants demonstrated maximum budset at approximately 35 days and did not flush during the remainder of the treatment period. Under long days, plants also exhibited budset by 35 days, but plants with a l l C0 2 treatments began flushing within the next five days. In the examples cited above, C0 2 inhibited bud break in the f i r s t case while short photoperiods inhibited bud break in the second case. These cases may represent another instance of the carbon dioxide-light interaction discussed at the end of Chapter One; that is,.C0 2 can sometimes sustitute for the light requirement of photosynthesis when light i s limited (23). If the separate inhibition of bud break by high CO,, and by short photoperiods is the result of a C0,,-light interaction, then the above cases may be further support for the assumption that photosynthesis is mediating the effects of C0 9 on bud development. 98 The effects of temperature on bud formation agree with results previously reported i n the l i t e r a t u r e for Douglas f i r (Figure 8 and Table XXI). Under short days, warm days and cool nights promoted budset as noted elsewhere (Lavender et a l (4))); low day and night temperatures inhibited budset, as also reported by Lavender and Overton (42). Some comparisons can be made between the results mentioned above and those obtained when seedlings which had been treated with various CO2 levels were, maintained under conditions which did not favor budset (Figures 7 and 8). Neither photoperiod nor temperature favored bud formation since seedlings were grown under long days with warm day and night temperatures (25 C/20 C) . Yet at 35 days, seedlings grown under 5.0% CO2 had set 32 buds . This number almost matched the maximum of 35 buds obtained under the 'conditions most favorable for budset i n Douglas f i r (short warm days with cool nights) . Experimental control of l i f e cycle The effectiveness of carbon dioxide, both i n promoting growth and i n promoting cessation of growth, as determined by the concentrations of CO2 used, gives some indication of the potential of (X^ as a to o l i n the experimental control of l i f e cycles. The manipulations of growth and development i n conifers have been primarily accomplished by (1) adjusting daylengths and thermoperiods, or (2) by applying hormones to various plant parts, or (3) by using a combination of the f i r s t two methods. Control of the vegetative cycle of Picea abies by regulation of daylength and thermoperiod i s an example of the f i r s t approach. Dormling et a l (18) were able to control time of budset, size of buds formed, 99 duration of budset, and time, duration, and amount of growth during the subsequent flushing period. The objective of the authors was to a r t i f i c i a l l y age young seedlings to bring on the adult flowering stage sooner. Increasing the number of growth cycles per year would effectively shorten the length of the juvenile period and hasten the onset of flowering which in Picea.abies does not ordinarily occur u n t i l 20 to 30 years. Exogenously applied hormones were used by Ross and Pharis (65) to promote flowering in coastal Douglas f i r . They applied gabberellic acid to sexually mature, non-flowering four-year-old grafts (on two-year-old rootstock) and significantly promoted male and female cone production. Carbon dioxide enrichment by i t s e l f or i n combination with other treatments may provide another method for controlling growth and development. Intermediate CO^  levels (0.1% CO^)'!!! conjunction with long days would allow young seedlings to substantially increase their height and weight during the growing season. When plants were ready to be l i f t e d and stored during the winter months, levels of 1.0% CO,, and short days could be used to cease growth and induce budset. CO^  enrichment could be used to promote root growth prior to spring planting in order to help prevent transplant damage and assist in the establishment of young seedlings. There is even the possibility that carbon dioxide enrichment could be used to promote differentiation of buds into male and female st r o b i l i . Buds resembling the buds of female s t r o b i l i were produced on one-year-old Douglas-fir trees grown under 1.0% CO2 for one month (E.B. Tregunna, unpublished data). This observation was not replicated however, and is only mentioned here to indicate what future work in this area may include. The use of C0„ enrichment in combination with hormones, 100 non-destructive g i r d l i n g , and other methods could perhaps promote cone production i n grafts as young as two years. The a b i l i t y to produce viable seed i n a conifer i n a period as short as two to three years would have tremendous importance i n tree breeding and improvement programs. Frost Hardiness Before commencing, i t should be noted that there are c e r t a i n q u a l i f i c a t i o n s with respect to the data upon which t h i s d i s c u s s i o n on hardiness i s based. Scores for f r e e z i n g tests are based upon a ranking scale and therefore are o r d i n a l i n nature. There are two p o s s i b l e sources of error i n analyzing o r d i n a l data: (1) the error introduced when one t r i e s to f i t injury, which i s a c t u a l l y a continuum, into a r b i t r a r i l y d i s c r e t e ranks; and (2) judgemental errors which occur when one attempts to c o r r e c t l y c l a s s i f y plants according to predetermined ranks or c l a s s e s . It i s possible that the inherent systematic er r o r of having one i n d i v i d u a l c l a s s i f y a l l plants could skew the data and lead to erroneous conclusions. To obviate some of these d i f f i c u l t i e s I used a sample s i z e of ten plants for each freezing t e s t and waited u n t i l s i x weeks before I assigned a f i n a l rank to each plant to ensure that any f r e e z i n g i n j u r y would be apparent. In addition, non-parametric s t a t i s t i c a l tests which I used for data analysis make few assumptions so estimates of s i g n i f i c a n t differences are f a i r l y conservative. 101 Effects of daylength and carbon dioxide enrichment (Refer to Tables XXIII and XXIV). At the less severe test of -6 C, photoperiod was an important factor in hardening plants. For most CO,, treatments, seedlings grown under short days showed less overall damage than those under long days. However, the most hardy of a l l plants tested were 1.0% CO,, seedlings which had been grown under short days; thus the treatment which combined the most effective photoperiodic and C02 conditions for increasing hardiness resulted i n the lowest damage scores. When a l l plants grown under short days were tested at -6 C, seedlings which had received 1.0% CO^  showed the least injury. For plants grown under long days and tested at -6 C, 0.1% CO,, seedlings exhibited the least injury even though there were not s t a t i s t i c a l differences between the. four C02 treatments. As discussed previously (Chapter One) carbon dioxide again appeared to be interacting with li g h t . In this instance, CO,, seemed to be a partial substitute for the reduced daylength which was experienced by short day plants. When plants were grown under long days intermediate CO,, levels were evidently sufficient to provide protection from frost injury, but when plants were grown under short days CO,, had to be increased in order to give the same relative protection. For plants frozen at the more severe temperature (-10 C), the greatest hardiness was shown by intermediate C0 2 seedlings grown under long days. In Chapter One growth was also found to be enhanced by intermediate C02 levels and long days. The increase i n hardiness under the same conditions was probably facilitated by the reserves acquired during a pre-treatment which promoted growth. The cessation of growth i s not a pre-requisite for hardiness induction according to results reported for Norway spruce (13). The fact that Douglas f i r plants maintained under 102 conditions favoring growth demonstrated greater resistance to frost injury than those plants given less favourable conditions agrees with the results for Norway spruce. Effects of post-treatment Post-treatments (short days with 25 C/5 C) did appear to be somewhat beneficial to seedlings which had previously been given CO^ enrichment under short days (Table XXV). The addition of cool night temperatures for six weeks resulted in lower injury scores for a l l groups of plants. Carbon dioxide treatment effects were replaced or overriden by effects of post-treatment so that by six weeks of post-treatment the effects of previous CO^ treatment were no longer discernable. When plants were frozen to -6 C and -10 C, damage scores were quite variable after six weeks' post-treatment (Table XXVI). Half of the CO2 treatments showed decreased damage as a result of post-treatment, and half of the treatments showed increased damage. Post-treatment appeared to be most beneficial to plants pre-treated with 1.0% CO^, and most detrimental to plants pre-treated with 0.1% CO^. Intermediate C 0 2 plants had the least damage at the end of pre-treatment, but at the end of post-treatment had more damage than any other treatment after freezing to -6 C. After freezing to -10 C, 0.1% CO2 plants sustained close to the maximum damage reported for the four groups. 103 Results i n Table XXVII d e t a i l temperature e f f e c t s on hardiness; the data confirm r e s u l t s reported elsewhere for Douglas f i r which found that short days with cool nights were most e f f e c t i v e i n inducing f r e e z i n g resistance and that cool days with cool nights delayed or i n h i b i t e d the induction of hardiness (42). Generally plants maintained i n conditions which.favor growth and n u t r i t i o n e x h ibit greater r e s i s t a n c e to winter injury (56). Thus post-treatment was most e f f e c t i v e f o r those plants which had been pre-treated with warm days and nights. A f t e r four weeks of post-treatment, they showed the l e a s t i n j u r y and the greatest improvement i n scores. The increased responsiveness to post-treatment shown by plants previously maintained under warm days and nights i s l i k e l y due to growth temperatures which favored metabolism and which allowed f o r an accumulation of reserves p r i o r to hardiness-inducing conditions. Conversely, plants pre-treated with warm days/cold nights (25 C/5 C) cr cool days/cool nights (5 C/5 C) did not show improved scores as a r e s u l t of post-treatment. In summary, (1) post-treatments of warm short days and cool nights, conditions normally found to be e f f e c t i v e i n inducing dormancy and hardiness of Douglas f i r , did not improve f r e e z i n g r e s i s t a n c e of a l l the Douglas f i r plants grown under the various CO^, daylength, and temperature pre-treatments; and (2) the e f f e c t s of CO2 and daylength pre-treatments were us u a l l y obscured by the e f f e c t s of post-treatment within four to six weeks. 104 Speculations on the mode of action of carbon dioxide on hardiness In 1964 Heber and Santarius (24) proposed the following hypothesis to account for frost injury in the c e l l : as water i s removed by crystallization during freezing, hydrogen bonds between water and proteins are broken which then result i n alterations to sensitive lipopro-teins. Hydroxy1-containing compounds such as sugars act as protective substances through their a b i l i t y to retain or substitute water via hydrogen bonding to protein structures sensitive to dehydration. During freezing, hydrogen bonds are formed between hydroxyls of the sugar and the functional water of the membrane system. These stabilizing bonds are not broken because sugars do not crystallize out as water does during freezing. Upon thawing, water again becomes available and hydrogen bonds between proteins and water are re-established. On the basis of this theory, efficient frost-hardening of the plant c e l l requires (1) production of enough soluble sugars or similar substances to afford protection and (2) a suitable distribution of these substances in the protoplasm or near sensitive sites. In dormant tissue the accumulation of sugars and other protective substances in the protoplasm can occur because metabolism remains low. Heber et a l . (25) later included certain amino acids in the l i s t of cryoprotectants, and then classified them into three groups based upon their relative effectiveness. Proline, threonine, y-aminobutyric acid, arginine succinate, and lysine HCl were the amino acids noted for their a b i l i t y to protect thylakoids from inactivation of photophosphorylation due to alterations of the membrane during freezing. 105 Keeping the aforementioned work i n mind, i t now seems appropriate to speculate how carbon dioxide might be acting to increase freezing resistance. I have already indicated that carbon dioxide appears to be affecting hardiness v i a photosynthesis. I t i s possible that photosynthesis i s affording protection from f r o s t injury by increasing the amounts of cryoprotectants i n the tissue. As suggested by Heber et^ a l . (24), the protective substances could be elevated amounts of sugars or amino acids which had been formed under carbon dioxide enrichment; but i t seems most l i k e l y that sugars and amino acids are acting together to increase freezing resistance. There are two i n d i r e c t l i n e s of evidence which indicate that amino acids and sugars do increase i n carbon dioxide enriched plants. The f i r s t l i n e of evidence concerns amino acid analyses made of ethanol extracts of leaves of one-year-old Douglas f i r (Table XXVIII). Although freezing tests were not performed on the plants used i n the analyses, tests were performed on other plants of the same age i n a preliminary study (Table XXII) and on 6-month-old seedlings (Tables XXIII and XXIV). Results of a l l tests suggest that levels around 1.0% increased hardiness of plants grown under both short and long days. Assuming that hardiness i s increased by high CO^ l e v e l s , one would s t i l l have to establish a relationship between amino acid levels and (X>2 enrichment le v e l s . Therefore, although t o t a l amino acids increased under high CO,, levels i n both short and long days, the amino acids of primary interest would be those which had been previously found to be 106 associated with increased cold hardiness. Extracts from leaves grown under long days and high contained increased amounts of glutamine, isoleucine, leucine, phenylalanine, histidine, arginine, and proline. For leaves grown under short days and high CO^, there were notable increases in proline and arginine. Increases in cold hardiness have been related to increases in a l l of the amino acids l i s t e d above (glutamine, phenylalanine, leucine, and isoleucine i n red osier dogwood, 43; arginine and histidine in winter weat, 55; glutamine and proline in perennial ryegrass, 19). Proline has been specifically mentioned by Heber et a l . (25) as a cryoprotectant. In addition, proline has been frequently mentioned as an amino acid which accumulates in large quantities in plants grown under short days and which also increases in plants during bud development. Furthermore, proline acts as an inhibitor to growth and respiration (Sergeev and Sergeeva, 1963; Sergeev, 1964a, and 1964b, as cited by Alden and Hermann (1)). The role of proline is s t i l l uncertain, but the fact that i t so often fluctuates with changes in environmental parameters does indicate that i t probably performs an important function for the plant. The second piece of evidence concerns light micrographs which were taken of 1-year-old trees. The micrographs showed large accumlations of starch in needles of plants which had been grown under long days. The amount of starch increased as levels were increased from 0.03% CO2 to 1.0% CO2. Starch at 5.0% CO2 was approximately equal to that at 1.0% CO2. Accumulations of starch and sugar as a result of CO2 enrichment have also been reported in tomato (46) and in beans, sugar-beets, and barley (51). Carbohydrates are very effective cryoprotectants (25, 87; 107 spruce and pine, 2), therefore i t may be the accumulation of starch under high CO levels that protects CO -enriched plants against freezing injury. Short-day plants did not show large starch accumulations regardless of enrichment level, but since the photographed plants were not freeze-tested i t is not known whether they were hardy or not. Conversely, freeze-tested plants were not examined microscopically so i t is d i f f i c u l t to determine i f lack of starch accumulation i s a consistent feature for short-day plants. If this i s true, some factor other than sugar may be responsible for freezing resistance in short-day plants. Comparison of various plant responses to CO,., enrichment Cessation of growth, development of buds, increased resistance to freezing injury, and winter rest a l l take place approximately at the same time. Since a l l these developments are closely associated in time the phenonmena have been assumed to be causally related, but evidence to the contrary shows that this assumption i s incorrect. In Acer negundo and Viburnum for instance, frost hardiness was independent of bud dormancy; substantial hardiness levels were attained without dormancy development as a prerequisite(32). In Douglas f i r , budset was not necessarily correlated with freezing resistance (10). The situation i s further complicated by the fact that all parts of the plant do not harden at the same time (56). Nevertheless, a comparison of internodal elongation, bud development, and freezing injury of 6-month-old seedlings given different daylengths, CO^ levels, and thermoperiods serves to consolidate and summarize some of the data presented thus far. The use of the same plants throughout these measurements was an advantage because i t 108 increased the validity of the comparisons and eliminated some of the variation due to the use of different material. Under short days, there was no relationship between internodal elongation, budset, and freezing injury (Tables V, XIX, and XXIII). For example, a comparison of freezing injury and internodal elongation showed that the highest and lowest freezing scores at —6 C belonged to plants grown under 5.0% and 1.0% CO^  respectively, but there were no differences between the two treatments in their amount of growth. The number of buds set under a l l carbon dioxide levels was approximately the same, regardless of treatment and thus no correlation between budset and freezing resistance could be made. Under long days, there were trends associating elongation growth, number of buds set, and freezing scores '(Tables V, XIX, and XXIV); however, the trends were in a direction opposite to one which would correlate hardiness with cessation of growth. The greatest degree of freezing resistance was observed i n plants with the least budset and the most internodal elongation; i.e., plants grown under long days and 0.1% C02-There was no correlation between budset and freezing resistance of seedlings given different temperature treatments (Figure 8 and Table XXVII). The same injury score was received by plants grown under 25 C/ 5 C and 5 C/5 C, yet plants given 25 C/5 C had the greatest number of buds set (36 buds). There was some indication of a correlation between freezing resistance and internodal elongation however, since plants given 25 C/5 C and 5 C/5 C sustained the least amount of freezing injury, 109 and also had the least growth (Tables VI and XXVII); plants given warm temperatures (25 C/20 C) sustained greatest injury and had most growth. The comparisons just made further i l l u s t r a t e that events included under the general heading of phenomena associated with dormancy cannot be used alone to predict freezing resistance. What the comparisons do indicate however, i s that an active metabolism is necessary to establish carbon dioxide-induced resistance to freezing injury. 110 CHAPTER THREE EFFECTS OF CARBON DIOXIDE AND DAYLENGTH ON GAS EXCHANGE OF DOUGLAS FIR INTRODUCTION Evidence from Chapter One regarding growth and Chapter Two regarding bud development and hardiness indicated that photosynthesis might be the primary mode of action for the effects of high CO^. However, i t became apparent while I was investigating the effects of CO2 on growth and development of Douglas f i r that there was limited gas exchange information upon which to base conclusions regarding some of my results. Most studies dealing with the effects of CO^ on gas exchange have been conducted on plants which had been grown in normal air but only exposed for short periods to high CO^  levels. However, some investigators who were primarily interested in the growth aspects of CO^  enrichment determined assimilation rates after crops had been grown in C02~enriched atmospheres (6, 9, 21). Although most researchers reported that photosynthetic rates were enhanced by CO2 enrichment, there are a few reports that elevated CO^ levels did not enhance photosynthesis. Photosynthetic rates of cucumbers grown at increased CO2 levels were lower than rates of cucumbers grown at normal CO2 levels (90). Photo-synthetic rates determined by G.R. Lister (unpublished data) for western hemlock after prolonged exposure to CO2 also indicated that high enrichment levels could depress assimilation rates. The fact that Lister used higher than usual CO2 levels (1.0% CO2) and longer period of enrichment probably accounts for the differences between his results and those of I l l others who reported enhanced photosynthesis under high CO^  levels. Nevertheless, there was limited information available on the effects of high CO^ levels on the major components of gas exchange, especially respiration and transpiration. The major objective of the research reported in this chapter was to determine how CO^ enrichment and daylength affected the gas exchange of Douglas f i r seedlings. To achieve this objective seedlings were grown under several photoperiods and CC^ levels. After treatment for six weeks the plants were placed in a cuvette and rates of photosynthesis, transpiration, and respiration were determined. Once these gas exchange rates were known, diffusion resistances could be calculated. The concept of total resistance is useful for visualizing several different processes which are occurring at the same time, namely, photosynthesis and transpiration, both of which are affected by changes in resistance to gas flow. In addition, changes in total resistance could possibly explain some of the observed CO^  effects on photosynthesis and transpiration. Finally, the inter-relationships of the different components of gas exchange under CO,, enrichment were examined. Knowledge of the manner in which carbon dioxide enrichment affected primary components of gas exchange could also lead to a better understanding of CO^ effects on plant growth and development. 112 MATERIALS AND METHODS Plant Material and Experimental Conditions Douglas f i r seedlings were obtained from sources described in the Materials and Methods of Chapter One. Plants were grown under 8- or 16-hour photoperiods (25 C/20 C) with an irradiance of 7.2 mW -2 . . . cm and four carbon dioxide levels (0.03%, 0.1%, 1.0% and 5.0% C0 2). After six weeks plants were removed from C0 2 treatment, placed, individua in a cuvette, and measured to determine rates of photosynthesis, respiration, and transpiration. The experimental design i s schematically presented i n Table C of the Appendix, and other experimental information i s given i n Table A of the Appendix. i Gas Exchange Measurements The major components of the closed gas exchange system consisted of Plexiglas cuvette, a thermocouple with a microvoltmeter, an e l e c t r i c hygrometer, a gas-drying tube containing s i l i c a g e l , an injection port for adding C0 2, an infrared gas analyzer, an a i r pump, and an adjustable flowmeter. Volume of the system was 1.14 l i t e r including the cuvette volume of 0.79 l i t e r . Relative humidity varied from 60-75%, and the flow rate of the gas i n the system was maintained at 4 1/min with a pump and Matheson rotometer. Light was provided by three 300 W General E l e c t r i c Cool-Beam lamps and was f i l t e r e d through a 10 cm-deep waterbath placed between the l i g h t s and the cuvette to reduce infrared radiation from the lamps. Radiant energy was measured by means of a YSI-Kettering Model 65 radiometer. Irradiance was varied 113 by using neutral density f i l t e r s placed between the lamps and the cuvette. Leaf temperature was measured by in s e r t i n g the welded bead of a copper-constantan thermocouple j u s t under the surface of the underside of a needle. The reference junction was i n an icewater bath (0 C) and the voltage d i f f e r e n t i a l between the two junctions was read with a Keithly Instruments microvoltmeter. Transpiration was determined by a d i f f e r e n t i a l system which measured the absolute water content of the a i r entering and ex i t i n g the cuvette with an E.G. & G. International, Inc. Model 880 dew-point hygrometer. Carbon dioxide concentration was measured with a Beckman Model IR-215 infrared gas analyzer. Apparent photosynthesis and transpiration of seedlings was measured i n normal a i r at four different irradiances (15, 26, 44, and 77 mW cm ). Rates were measured f i r s t at the highest irradiance and l a s t at the lowest irradiance. After a l l the l i g h t measurements were completed the cuvette was covered with a dark cloth and the dark respiratory r i s e of CO2 was recorded. Total Diffusive Resistance Diffusive resistance to gas exchange of water vapor and CO^ was calculated using the e l e c t r i c a l analog model of r e s i s t o r s i n series. The t o t a l resistance to gases entering and leaving the stomates i s the sum of a l l the resistances i n the path r = r *F r r t o t a l boundary layer stomata mesophyll Total resistance can be calculated using the following relationship 114 r t o t a l Pvapor, leaf Pvapor, air (2) Transpiration rate where pvapor, leaf water vapor density of absolute humidity (g H^ O cm~3) inside the leaf vapor, air water vapor density (g H2O cm--3) outside the laminar boundary layer of the leaf -2 -1 If tranpsiration rate i s given in units of g cm s, then v t o t a i has units of s cm \ Leaf area on a projected or planar area i s used i n this thesis and was determined using a photocell planimeter as described in the following section. Since stomata of Douglas f i r only occur on the lower surface of the needle, leaf area was not doubled in any calculations. Of the three components of total resistance to water vapor diffusion, boundary l a y e r w a s considered to be very small compared to r t o t a l o r t o rstomata a n d 'mesophyll' S m a 1 1 o b J e c t s s u c h a s needles have correspondingly small boundary layers as long as a i r speed exceeds 1 1/min. A theoretical calculation using a Reynolds number for a small cylinder the size of a needle under approximately the same experimental conditions as those used to determine transpiration rates showed that the boundary layer resistance would be less than 1 sec cm ^  (88). Mesophyll resistance may or may not be small compared to r but r , ,, i s J J r stomata' mesophyll generally of much more importance to CO diffusion than i t i s to water vapor diffusion. Therefore, the total resistance to H^ O vapor diffusion is primarily an indication of how C0„ affects r . Data obtained in 2 stomata the cuvette from 6-month-old trees grown under 8- and 16-hour days were used to calculate total diffusive resistance to R^ O vapor from equation (2). P vapor leaf 'i'S u s u a H y assumed to be equal to the saturation water vapor density at leaf temperature. 115 Leaf Area Determinations A photocell planimeter (Type AAM-5, Hayaski Denko Company, Tokyo) was used to determine the planar area of needles. Needles were removed from each seedling and measured at least six times. The mean value was used to obtain the best possible determination of leaf area. Drew and Running (20) found that of two methods for the determination of leaf area of conifers, optical planimetry was more precise, but the glass bead method was more accurate. When they used a needle cross-sectional correction factor (determined to be 1.16 for Douglas f i r ) the accuracy of the optical method was improved to a level comparable with the glass bead method. The glass bead method was found to be impi -actical because of the time required to make a single measurement. In addition, the method could not be used for branches with more than 20 needles due to the difficulty of covering needles uniformly with a monolayer of beads. Optical planimetry has also been used successfully by Ludlow and Jarvis (45) for Sitka spruce. Water Loss Measurements One-year-old trees were grown for 30 days under 8-hour days and four C02 levels (0.03%, 0.1%, 1.0% and 5.0% CO^. Weekly records were kept of total water loss from each plant chamber. Two methods of calculating water loss were used: (1) after being condensed on cooling coils,water inside the gas-tight plant chamber was collected into flasks which were weighed daily to determine total water transpired per week per chamber. (2) after pots were watered to f i e l d capacity each week and weighed, the exposed s o i l surface was covered and sealed with t i n f o i l , 116 and the pot replaced inside the chamber. After one week, the pot was removed and weighed again to determine weekly water loss. While data points for the two methods were always within 10%, the second method was probably the more accurate of the two since there was less chance for evaporative water loss; therefore only results from method (2) w i l l be presented. RESULTS A. APPARENT PHOTOSYNTHESIS 1. Plants grown under 8-hour days and four CO,, levels Plants grown under short days with normal air had significantly higher photosynthetic rates under a l l irradiances than plants grown under 1.0% or 5.0% C0 2 (Figure 9 and Table XXIX). Seedlings grown under 1.0% and 5.0% C0 2 were not significantly different from one another and showed similar photosynthetic responses to increasing irradiance. Plants grown under low and intermediate CO^ levels reached approximately 70% of their maximum photosynthetic rates at the lowest _2 irradiance (15 mW cm ) which was closest to that under which they had been grown. Plants grown under 1.0% and 5.0% C0 2 reached about 40% of their maximum photosynthetic rates at the lowest irradiance. None of plants in the four CO^  treatments reached light saturation at the highest irradiance, although seedlings grown under high CO^ (1.0% and 5.0% C00) appeared to approach a light saturation level. 117 Figure 9. Apparent photosynthesis of 6-month-old Douglas f i r continuously supplied with CO2 at the. levels shown, and maintained under 8-hour photoperiods (25 C/20 C) for six weeks with an irradiance of 7.2 mW cm-2. 10.0 20.0 30.0 40.0 50.0 60.0 70.0 LIGHT INTENSITY (mW cm"2) Data points are the means of three plants. Analysis of Variance was used to determine s t a t i s t i c a l differences. Values within the same column bearing the same letter are not significantly different from each other at p = 0.05. TABLE XXIX RATES OF PHOTOSYNTHESIS Douglas F i r Seedlings Grown for six weeks under 8-hour days and designated C0_ levels PHOTOSYNTHESIS (ug CO, cm"2 min"1) TREATMENT -RATES @ EACH LIGHT INTENSITY (N = 3) RATES FOR ALL LIGHT , c -T -2 -2 T T - 2 7 . T T -2 INTENS. COMBINED (N = 12) 15 mw cm 26 mw cm 44 mw cm 71 mW cm ' 0.03% C0 2 • 639a .741a .802a .948a .783a 0.1% co 2 .479 a b .559 a b .549 a b .663 a b .563b 1.0% co 2 .112b .214b .223b .290b .210C 5.0% C0 2 • 156b .219b .288b .331b .248° Analysis of Variance was used for determining s t a t i s t i c a l differences. Values within the same column bearing the same letter are not significantly different from each other at p = 0. 05. 119 2. Plants grown under 16-hour days and four CO levels 2 Under long days, plants grown under normal a i r also had significantly higher photosynthetic rates than those grown under high C02 (1.0% and 5.0% C02> (Figure 10 and Table XXX). Photosynthetic rates of seedlings grown under 1.0% and 5.0% C0 2 were significantly different from one another only at the lowest irradiance. Photosynthetic rates of plants grown under 0.1% C0 2 were generally higher than, but not significantly different from plants grown under 1.0% or 5.0% C02> This was probably due to the high v a r i a b i l i t y of individual measurements. Plants grown under 0.03%, 0.1%, and 1.0% C0 2 reached 65% to 85% of their maximum photosynthetic rates at the lowest irradiance -2 (15 mW cm ). Plants grown under 5.0% C0 2 showed no apparent photo-synthesis at the lowest irradiance and demonstrated only a very low -2 positive photosynthetic rate at the next higher irradiance (26 mW Cm ). Under long days, a l l four C0 2 groups reached light saturation -2 -2 levels between 26 mW cm and 44 mW cm . However, for short-day plants light saturation levels were not reached for any of the four C0 2 groups _2 even at the highest irradiance (71 mW cm ). A direct comparison of the effects of daylength and C0 2 on apparent photosynthesis was made by plotting apparent photosynthesis as a function of the C0 2 concentration under which the seedlings were grown (Figure 11). For each C0 2 concentration, photosynthetic rates under a l l light intensities were averaged into one measurement for that particular C0 2 level. The daylength under which seedlings are grown does influence photosynthetic rates of plants which are both grown and measured under normal a i r . The results show that plants grown under long days showed significantly higher photosynthetic rates than those 120 Figure 10. Apparent photosynthesis of 6-month-old Douglas f i r continuously supplied with C02 at the levels shown, and maintained under 16-hour photoperiods (25 C/20 C) for s i x weeks with an irradiance of 7.2 mW cm - 2. 1.6 I— ~ 0.8 I— V. \ o.i% co LU 3= Data points are the means of- three plants. Analysis of Variance was used to determine s t a t i s t i c a l differences. Values within the same column 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 from each other at p = 0.05. TABLE XXX RATES OF PHOTOSYNTHESIS DOUGLAS FIR SEEDLINGS Grown for six weeks under 16-hour days and designated C0? levels PHOTOSYNTHESIS (u.g C0 2 cm min ) TREATMENT RATES @ EACH LIGHT INTENSITY (N = 3) RATES FOR ALL LIGHT 15 mW cm"2 26 mW cm"2 44 mW cm"2 71 mW cm"2 INTENS. COMBINED (N = 12) 0.03% C0 2 1.168a 1.443a 1.367a 1.284a 1.316a 0.1% c o 2 .421a .606b • 641b ,657 a b .581b 1.0% c o 2 .372a •i436b .365b .361b .384 b c 5.0% C0 2 . o o o b .082b .422b .268b :i93 c Analysis of Variance was used to determine s t a t i s t i c a l differences. Values within the same column bearing the same letter are not significantly different from each other at p = 0.05. 122 Figure 11. Effect of daylength and C02 on apparent photosynthesis. Douglas f i r were grown for 6 weeks under 8- and 16-hour photoperiods with C02 at the levels shown. Irradiance 7.2, _n mw cm z 14.0 12.0 10.0 C B o U l ac t-z: >-in o o a: O -< £ Ou < 0.03% CO. 0.2% C0„ 2.0% CO. S.0% C0„ C0 2 CONC (log scale) Data points are the means of three plants. Analysis of Variance was used to determine s t a t i s t i c a l differences. Values within the same column 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 from each other at p = 0.05. 123 grown under short days. However, for plants grown under CO^ enrichment the results are more d i f f i c u l t to interpret. There are significant differences between daylength groups for plants grown under high CC^, but no differences between daylength groups for plants grown under intermediate CC^. At very high CO,, levels (5.0% CO^) there are no significant differences i n photosynthetic rates among daylength groups. The overall effect of increased CO^  under both daylengths was to reduce apparent photosynthetic rates when the rates were measured under normal a i r . Perhaps other differences due to CO^  treatment could have been" shown i f the plants had been measured under the same CO^ l e v e l as they had been grown, instead of being measured under normal a i r . B. RESPIRATION For plants grown under short days, there were no s i g n i f i c a n t differences i n r e s p i r a t i o n between the four CO^ levels under which the plants were grown (Figure 12). For long-day plants there was a d i f f e r e n t response. Respiration rates of plants grown under normal a i r were s i m i l a r under both short and long days. When the was increased to 0.1% CO^  however, respiration of long-day seedlings was s i g n i f i c a n t l y lower than that of short-day seedlings. When CO^ levels were raised to 5.0% CO2 the respiration of seedlings grown under long days was s i g n i f i c a n t l y higher than that of seedlings grown under 5.0% CO^ and short days. C. TRANSPIRATION 1. One-year-old trees The CO,, concentration under which trees were grown c l e a r l y 124 Figure 12. Effect of daylength and CO2 on respiration. Douglas f i r were grown under 8- and 16-hour photoperiods with CO2 at the levels shown for six weeks with an irradiance of 7.2 mW cm-2. 0.03% CO 0% CO. 5.0% C0r '2 "2 C0 2 CONC (log scale) Data points are the means of eight plants. Analysis of Variance was used to determine s t a t i s t i c a l differences. Values within the same column bearing the same letter are not significantly different from each other at p = 0.05. 125 affected the amount of water lost by transpiration (Figure 13). As the CO^  concentration in the atmosphere increased, the amount of transpired water decreased. Less water was transpired with each successive week during the course of the experiment, but the relative amounts of water lost weekly by plants remained essentially the same among the different treatments. As has been observed in other conifers, a reduction in trans-piration during the last weeks of the experiment probably occurred because the young shoots completed flushing and entered a less active growth period (12). Plants grown under 5.0% C0^ were always s t a t i s t i c a l l y distinguishable from plants grown under 0.03% C0 2 > but transpiration rates of plants grown under intermediate CO,, levels (0.1% and 1.0% CO,,) generally overlapped values shown by plants grown under either 0.03% or 5.0% C02-2. Six-month-old seedlings Under short days, plants grown under 0.03% C0 2 transpired most and those grown under 5.0% CO,, transpired least (Figure 14 and Table XXXI). Transpiration rates of plants grown under 0.1% and 1.0% CO,, were intermediate to those observed for plants grown under 0.03% and 5.0% CO,,. Transpiration rates obtained for six-month-old seedlings agree with water loss rates observed for one-year-old trees since in both cases the greatest amount of transpiration occurred with 0.03% C0 2 plants and the least amount occurred with 5.0% C0 2 plants. Under long days, plants grown under 5.0% C0 2 showed the greatest amount of transpiration (Figure 15 and Table XXXII). Plants grown under the other three C0o concentrations showed lower rates of 126 Figure 13. Weekly water loss. One-year-old Douglas f i r were grown for four weeks under 8-hour photoperiods with C O 2 at the levels shown. Irradiance was 7.2 mW cm-2. 290 270 250 230 b> 210 190 5t 170 150 130 0.03% CO. 0.1% CO. 1.0% CO. 5.0% C0„ T_ cn 2.0 4.0 3.0 TIME (weeks) Data points are the means of eight plants. Analysis of Variance was used to determine s t a t i s t i c a l differences. Values within the same column are not significantly different at p = 0.05. 127 Figure 14. Transpiration fates of 6-month-old Douglas f i r continuously supplied with CO2 at the levels shown, and maintained under 8-hour photoperiods (25 C/20 C) for six weeks with an irradiance of 7.2 mW cm-2. 0.9 -0.8 0.7 h -0.6 h ~ 0.5 E o o CM ^0.4 20.3 DL. 0.2 0.1 r ~ 10.0 20.0 3 0 . 0 4 0 . 0 5 0 . 0 6 0 . 0 7 0 . 0 LIGHT INTENSITY (mW cm-') Data points are the means of three plants. Analysis of Variance was used for determining s t a t i s t i c a l differences. Values bearing the same letter within the same column are not significantly different at p = 0.05. TABLE XXXI RATES OF TRANSPIRATION DOUGLAS FIR SEEDLINGS Grown for s i x weeks under 8-hour days and designated C0_ levels TRANSPIRATION ( ug P^ O cm"2 s- - 1 ) TREATMENT RATES @ EACH LIGHT INTENSITY (N = 3) RATES FOR ALL LIGHT -2 -2 -2 -2 15 mW cm 26 mW cm 44 mW cm 71 mW cm INTENS. COMBINED (N = 12) 0.03% C0 2 .344a .422a .672a .910a ,587a 0.1% c o 2 .302 a .386a .563 a b .577 a b •457 a b 1.0% co 2 .I88 a .367a .426 a b .609 a b • 397 b 5.0% CO • 288 a • 351 a .297b .395b .333 b Analysis of Variance was. used to determine s t a t i s t i c a l differences. Values within the same column bearing the same l e t t e r are not s i g n i f i c a n t l y different from each other at p = 0.05. 1 2 9 Figure 15. Transpiration rates of 6-month-old Douglas f i r continuously supplied with CO2 at the .levels shown, and maintained under 16-hour photoperiods (25 C/20 C) , for six weeks with an irradianr-p of 7.? mW rm-2. 0 . 8 0 . 7 0 . 6 0 . 5 0 . 4 3. O 2 0 . 3 0 . 2 o.ifr-1 30.0 40.0 50.0 60.0 70.0 10.0 20.0 LIGHT INTENSITY (mW cm"2) Data points are the means of three plants. Analysis of Variance was used for determining s t a t i s t i c a l differences. Values bearing the same letter within the same column are not significantly different at p = 0.05. TABLE XXXII RATES OF TRANSPIRATION ^ DOUGLAS FIR SEEDLINGS Grown for six weeks under 16-hour days and designated CO2 levels TREATMENT TRANSPIRATION (ug HoO cm 2 sec" 1) RATES @ EACH LIGHT INTENSITY (N = 3) -2 -2 -2 15 mW cm 26 mW cm 44 mW cm 71 mW cm -2 RATES FOR ALL LIGHT INTENS. COMBINED (N = 12) 0.03% C0 2 .268a •422 a b .460a ,534a .421a 0.1% c o 2 .207a .226a .3l9 a .511a • 316 a 1.0% c o 2 • 242a .368 a b .4l5 a • 4l6 a .360a 5.0% C0 2 .491b • 660b .789a • 769a .677b Analysis of Variance used to determine s t a t i s t i c a l differences. Values within the same column bearing the same letter are not significantly different from each other at p = 0.05. 131 transpiration and were not s t a t i s t i c a l l y d i f f e r e n t from one another D. TOTAL DIFFUSIVE RESISTANCE Resistance has been plotted as a function of the natural log of the C02 concentration i n Figure 16. For each CO,, concentration resistances under a l l irradiances were averaged into one meaBurement for that CO,, l e v e l . Carbon dioxide did not appear to have any offeet on t o t a l resistance except under the highest CO^  concentration (5 0% CO ) The ef f e c t s of CO,, appear at 1.0% CO,,, but there are no s t a t i s t i c a l differences between plants grown under short days and long day« u n t i l the C0 2 concentrations reach 5.0% CO,,. At 5.0% C02, plants grown under long days show decreased resistance to gas flow. Rates of photosynthesis were not af f e c t e d by changes i n resistance since the biggest changes in photosynthetic rates occurred in those carbon dioxide l e v e l s where t o t a l r e s i s t a n c e was not s i g n i f i c a n t l y d i f f e r e n t under both short and long days, i.e., 0.03% to 1.0% C0 2 (Tables XXIX and XXX). 132 Figure 16. Effect of daylength and C02 on t o t a l resistance. Douglas f i r were grown for 6 weeks under 8- and 16--hour photoperiods with CO2 at the levels shown. Irradiance was 7.2 mW cm-2. 50 40 30 W u < H w H CO w Pi 20 10 8-hr day ®a day 0.03% C0Z 0.1% C02 1.0% C02 5.0% C02 CO2 CONC (log scale) Data points are the means of 12 plants. Analysis of Variance was used to determine s t a t i s t i c a l differences. Values within the same column are not s i g n i f i c a n t l y d ifferent at p = 0.01. 133 D I S C U S S I O N Plant Variability One of the limitations in interpreting data in this investi-gation of gas exchange was the greatly variable response of seedlings to similar treatments. This v a r i a b i l i t y was mentioned as a problem with Douglas f i r by Kreuger and Ferrell (39) who found that differences between seedlings of the same provenance could be greater than differences between two varieties. Coastal varieties of Douglas f i r are more variable than interior varieties; De-Vescovei and Sziklai (17) found that coastal varieties had greater nuclear volume and DNA content than interior varieties-the implication was that more genetic p o s s i b i l i t i e s exist for coastal types. Coastal climates, although not as severe as interior climates, are more locally variable in their microclimates so greater genetic potential would clearly have adaptive significance. The usual method of minimizing the effects of the inherent variability of Douglas f i r i s to increase sample size. As sample size increases, the mean of the sample population (x) approaches the true mean of the population ( u ) , and the standard deviation about the mean decreases. In the growth and dormancy studies (Chapters One and Two) samples sizes were ten or more, but larger sample sizes in gas exchange measurements were not practical due to the length of time required to obtain a complete set of measurements for each seedling; therefore, gas exchange measurements were based upon samples of three seedlings for each treatment. The number of replications was also restricted by the large number of different growth conditions under which the plants were 134 raised (four CO^  concentrations X two daylengths). A possible solution to the above problem would have been a restriction of the number of conditions upon which determinations were made; for instance, a test of only short-day plants or a measurement of only the minimum and maximum CO^  concentrations in each daylength could have been conducted. However, a l l combinations of CO^  and daylength were of interest because the effects of carbon dioxide varied at each concentration. Accordingly, accepting the inherent v a r i a b i l i t y associated with small sample sizes was considered to be an adequate compromise. Another solution would have been to combine several seedlings into a single cuvette and this would have reduced both inherent v a r i a b i l i t y and measurement time. The only major modification to the gas exchange system would have been the use of a cuvette large enough to accommodate the additional seedlings. Increased system volume could have been compensated for by greater gas exchange rates from the increase in seedling material. However, the advantage derived from the increased system volume would be partially lost due to the necessity of also increasing the flow rate within the system. An increase in flow rate would decrease the magnitude of the potential CC^ difference across the cuvette and consequently reduce the ease i n resolving determinations of gas exchange. Thus, the potential advantage of reduced v a r i a b i l i t y which would be gained by the use of additional seedlings would be partially offset by the increased d i f f i c u l t y in determining gas exchange rates. 135 The inherent v a r i a b i l i t y of this material made i t d i f f i c u l t at times to make clear d i s t i n c t i o n s among the various treatment groups. With larger sample sizes more d e f i n i t i v e statements possibly could have been made regarding the effects of carbon dioxide and daylength. Apparent Photosynthesis When measured i n normal a i r , seedlings grown under long days and 0.03% CC^ had the highest photosynthetic rates (Figures 9, 10, and -2 -1 11). The maximum rate for seedlings of 8.4 mg CO^ cm hr , was only -2 -1 s l i g h t l y lower than the maximum rate of 12.0 mg CO^ dm hr reported by Erix (8) for 100-day-old Douglas f i r seedlings. Younger conifer seedlings tend to have higher photosynthetic and respiratory rates than older seedlings (Rutter, 1957, as c i t e d by 8). The rate reported by Brix i s thus i n f a i r agreement with the rate which I obtained for 6-month-v old seedlings. Plants grown under enriched atmospheres and transferred to atmospheric CO^  conditions usually demonstrate photosynthetic rates similar to those shown by plants grown continuously i n normal a i r (6, 21). The photosynthetic rates of recently transferred plants are dependent upon the new CO^ concentration and not upon the o r i g i n a l concentration. Gas exchange of Douglas f i r seedlings i n t h i s study was measured only under atmospheric CO^  and not under the CO^ concentrations i n which they had grown. The fact that seedlings were not measured and grown under the same levels p a r t i a l l y accounts for photosynthetic rates of enriched seedlings being lower than rates of seedlings grown in 0.03% C0„. 136 Most reports indicate that increasing CO^ i n the a i r surrounding the plant increases assimilation rates. For sugar-beet, barley, and kale, apparent photosynthetic rates i n 0.1% and 0.33% CO^ were about 20% and 30% respectively greater than rates i n 0.03% CO,, (21). The effects of CO^  on flowering of P h a r b i t i s , Xanthium, and Silene were considered by Purhoit and Tregunna (59) to be due to promotion of photosynthesis as a result of CO^  enrichment. Three-week-old soybeans grown i n a i r and measured at different CO,, concentrations had photosynthetic rates at low l i g h t i n t e n s i t i e s that were CO^ saturated at 0.04% C0 o, but at high l i g h t i n t e n s i t i e s photosynthesis increased eight-fold and was not CO^ saturated at 0.16% C0 2 (9). Bishop and Whittingham (6) grew tomatoes i n normal a i r and i n a i r enriched with 0.1% CO,,; both groups increased t h e i r photosynthetic rates when measured under 0.1% CO,,. Increased l i g h t intensity also increased photosynthetic rates of both groups whether measured under normal or enriched a i r . Interactions between l i g h t and CO,, were evident since the highest assimilation rates were found when 4 plants were measured under the highest l i g h t i n t e n s i t y (7.5 x 10 ergs -2 -1 cm sec ) and the highest CO,, concentration (0.1% CO,,). There i s some evidence that plants grown under high CO^ levels (1.0% or more) for long periods eventually decrease t h e i r a s s i m i l a t i o n rates under a l l C0 2 l e v e l s . L i s t e r (unpublished data) grew western hemlock seedlings for three months under 0.03%, 0.1% and 1.0% C0 2. Phosynthetic rates were determined at the end of one month, two months, and three months. At the end of the f i r s t month, photosynthetic rates tended to f a l l into groups based upon the C0 o l e v e l under which the seedlings 137 were measured rather than the l e v e l under which they were grown, (e.g., plants grown under 0.03%, 0.1% and 1.0% CO^, but measured for photo-synthesis under 0.03%, formed one group; those measured under 0.1% CO^ formed a second group, etc.). Within each group, the highest rates were exhibited by those plants which had been grown and measured at the same CO^  concentration. At the end of two months, plants grown under 0.03% CO^  had the highest rates regardless of the CO^ l e v e l under which they were measured. Western hemlock seedlings grown under l'.;0% C0 0 had the lowest rates i n each of the three measurement groups. At the end of three months, plants grown under 0.1% CO^ had the highest photosynthetic rates i n each of the measurement groups, while 1.0% CO^ seedlings s t i l l had the lowest rates. Results were sim i l a r when photosynthetic data for western hemlock (measured under 0.03% C^) were compared with my data for Douglas f i r . After long periods of growth under high CO^ concentrations (1.0% CO^  or more), Douglas f i r seedlings exhibited a s s i m i l a t i o n rates that were lower than rates of plants grown under low concentrations (0.03% and 0.1% CO^). I t i s not known why prolonged periods of high CO^ levels should cause declines i n CO^ assimilation, but a possible explanation i s that CO^ acts by decreasing stomatal aperture. Transpiration measure-ments (Figures 13, 14, 16) for plants grown under 8-hour days confirm that C0„ could be increasing r (assuming that r ^ i s greater 2 b stomata stomata & than r i n transpiration); however, measurements for plants mesophyll grown under 16-hour days (Figures 15, 16) do not confirm t h i s explanation mainly because 5.0% CO resulted i n very high transpiration and therefore 138 very low r .. Other possible explanations for the CO.-induced S t OHlcLtlcL 2. decline in CO^  assimilation could be that CO^  reduces stomatal reactivity, either by affecting the efficiency of carboxylation reactions, or by creating a buildup of wax in stomatal antechambers as has been observed in aging needles of Sitka spruce (35) . Frydrych (90) suggested that an adaptation of the leaf tissue to increased CO^  levels was responsible for photosynthetic declines but did not speculate on what the adaptations to increased CO^  concentrations might be. Daylength had two main effects on photosynthetic rates of plants grown under various levels of CO^: (1) photosynthetic rates were generally higher for long-day plants than for short-day plants; (2) plants grown under short days and low CO^ concentrations (0.03% and 0.1% CO ) did not reach light saturation at the highest irradiance -2 (71 mW cm ). Under long days a l l (X^ treatment;groups reached light saturation at approximately half that irradiance. This could be attributed to the fact that under long-day plants were photosynthesizing at almost their maximum rates, while short-day plants were light limited due to reduced daylength. Consequently, short-day plants were able to respond to extra light offered i n the form of increased irradiance. Respiration Under short days, respiratory rates remained essentially the same regardless of the CO^ concentration under which the plants were grown. However, this was not the case for long-day plants, which in comparison to plants grown under 0.03% C0_ showed decreased respiration 139 under 5.0% C^. Differences in respiration under various CO^ levels could account for the growth depression noted in plants grown under 5.0% CO^ , as well as the growth enhancement noted for plants grown under 0.1% CO2 (Chapter One). Respiration rates have been shown to have significant effects on growth. For instance, in two varieties of corn, Heichel found that the more rapid dry matter accumulation of the faster growing variety was correlated with i t s lower rate of respiration (26). The higher respiration rates observed i n plants grown under 5.0% C0 2 could be due to the accumulation of carbohydrates. High respiratory rates which have been found in conifers grown in cold environments (Douglas f i r , 70; Pinus radiata, 64; Sitka spruce, 75) have been postulated to be related to the high levels of soluble sugars found in cold-adapted plants. Low temperatures may limit metabolism and translocation of photosynthetic products from sites of synthesis and thus allow sugar to be accumulated and then burned inef f i c i e n t l y . Increased respiration may also be a stress reaction to above-normal levels of carbon dioxide. Miller (48) noted that low, non-injurious concentrations of CO2 could bring about retardation of respiration, but that very high levels of CO2 could increase respiration of plant organs which normally had low respiration rates. It is not known which one of these p o s s i b i l i t i e s represents the actual reason for changes in respiratory rates due to increased levels of CO2, but the influence of respiration would be important in determining whether there would be a net gain or loss in apparent photo-synthesis. Ultimately major changes in respiration would result in a significant change in growth. 140 Transpiration Carbon dioxide affected transpiration rates of both trees and seedlings. For 1-year-old trees, transpired water decreased as CO2 levels in the plant atmosphere increased. Transpiration is mainly controlled through changes in stomatal aperture (3). Increased CO2 levels have been reported to result in increased stomatal closure in a number of species (spruce, pine, 12; several monocots and dicots, 53; pepper plants, 34; various crop plants, 23). Thus the reduced water loss observed under enrichment is probably due to reductions in stomatal aperture under high CO2 levels. Furthermore, i t appears that when plants have been grown with elevated CO^ levels for a relatively long time (X^ effects on stomatal aperture can persist even after removal of the plants from the enriched atmosphere. When transpiration rates of seedlings were measured in normal air one or two days after termination of CO2 enrichment, the effects of pre-treatment were s t i l l evident. For short-day seedlings CO^ effects on transpiration were the same as those observed for 1-year-old trees; transpiration rates were high under low CX^ levels (Figure 14). On the other hand, for long-day plants the effects of CO2 on transpiration were the reverse; transpiration rates were high under high CO2 levels (5.0% CO2). These findings seem paradoxical since one assumes that long-day plants grown under high CO2 would behave similarly to short-day plants under high CO2 by exhibiting stomatal closure when treated with high CO2 levels. It is possible that CO2 only affects stomatal closure within a particular range of concentrations. Very high CO levels may exceed the upper threshold in which carbon dioxide may influence stomatal aperture. 141 Total Resistance Generally high carbon dioxide levels (5.0% CO,,) affected t o t a l C0 2 d i f f u s i o n resitance by an increase i n resistance under short days, and a decrease i n t o t a l resistance under long days (Figure 16). Under short days, the effects of carbon dioxide v/ere l i k e l y due to stomatal closure under high CO^ concentrations. This i s reflected i n the low transpiration and photosynthetic rates of short-day plants grown under 5.0% CO^  (Figures 9 and.14). The greatest apparent photosynthetic rates at high l i g h t i n t e n s i t i e s shown by plants grown under normal a i r are due to the combined effects of high transpiration (low stomatal resistance) and re l a t i v e l y low respiration rates for short-day plants under a l l CO2 l e v e l s . Under long days, high carbon dioxide levels resulted i n decreased apparent photosynthesis and increased dark respiration (Figure 11 and 12) even though high transpiration rates indicated that stomatal resistance was r e l a t i v e l y low (Figure 16) . Increased photorespiration could be responsible for the declines i n apparent photosynthesis since photosynthetic rates declined i n 0.03% and 5.0% CO2 plants when l i g h t i n t e n s i t i e s were greater than 26 -2 mW cm (Figure 10). Non-photosynthetic effects of CO2 Some of the observed effects of carbon dioxide appear to be non-photosynthetic since they occur outside the range of enhanced photo-synthesis (Figure 11). High carbon dioxide levels (1.0% and 5.0% CO2) depressed photosynthetic rates of seedlings below those rates shown by seedlings grown under low or intermediate C0 2 levels (0.03% and 0.1% CO2) , yet as shown e a r l i e r carbon dioxide affected growth and development 142 significantly. Many of the high CO^  effects were similar to effects associated with abscisic acid (ABA). Abscisic acid has recently been implicated i n control of stomatal opening and closing through sensitization of stomata to CO,, (60) . In the interaction between ABA and CO both must be present for CO^-induced stomatal closure to occur. For Xanthium strumarium L. and some other species, ABA must be present before stomata are sensitive to CO^ , and stomata only respond to ABA i f CO^  Is available. ABA accumulates rapidly in wilting leaves; Raschke (60) postulates that abscisic acid i s synthesized in the mesophyll and subsequently transported to the guard cells where i t can prevent accumulation of K+. Thus ABA causes stomatal closure and restoration of the water balance within the guard c e l l s . High carbon dioxide levels under short days appear t o ^ f f e c t stomatal closure and to reduce the transpiration loss in Douglas f i r seedlings and trees (Figure 13, 14). In Douglas f i r seedlings, the effects of high CO^ levels on transpiration persisted even after the seedlings were removed from the high CO^ environment and were measured in normal a i r . CO^-induced increases in abscisic acid levels may explain why stomata remain closed after plants have been removed from the enriched atmosphere. Abscisic acid and stomatal closure have also been related to low temperature effects i n various plants. Wilson (82) found that abscisic acid sprayed on leaves of Phaseolus vulgaris was e-fective in closing stomata and preventing c h i l l i n g injury. Christersson (12) also speculated on the action of ABA and i t s importance in stomatal closure in relation to the induction of frost hardiness of spruce and pine. Irving and Lanphear (33) demonstrated that by applying ABA to Acer negundo they could induce hardiness 143 under long days. When they analyzed l e a f extracts of untreated trees, they discovered large amounts of ABA produced under short days but only small amounts of ABA produced under long days. Stomata i n Xanthium strumarium L . , which are not o r d i n a r i l y s e n s i t i v e to closure by high l e v e l s of CC^, became s e n s i t i v e to CO,, a f t e r c h i l l i n g pre-treatment. C h i l l i n g increased a b s c i s i c a c i d l e v e l s i n Xanthium leaves which i n turn increased stomatal s e n s i t i v i t y to CC^. On the other hand, high C0 2 l e v e l s (1.0% C02> were e f f e c t i v e i n preventing i n j u r y when seedlings grown under short days were freeze-tested at -6 C. (Tables XXIII and XXIV). The p a r a l l e l e f f e c t s of high C0 2 l e v e l s and ABA i n p r o t e c t i n g plants from f r e e z i n g injury implies that there may be some degree of a b s c i s i c a c i d involvement i n COr,-induced hardiness. The balance between ABA and growth promotors has been shown by many to be associated with growth i n h i b i t i o n and dormancy i n woody species (Alnus and Betula, 28; Douglas f i r , 11, 41). High concentrations of carbon dioxide were also associated with increased dormancy i n Douglas f i r seedlings. Under short and long days, buds set e a r l i e r when seedlings were grown under 1.0% and 5.0% C0 2 (table XX). Further, high carbon dioxide was able to overcome e f f e c t s of both temperature and photoperiod by promoting a high degree of budset under non-inducing conditions, perhaps by increasing endogenous l e v e l s of ABA or reducing l e v e l s of growth promotors. ABA has also been shown to simulate the e f f e c t s of short days and low temperatures by reducing g i b b e r e l l i c a c i d content and modifying the morphology of two v a r i e t i e s of a l f a l f a grown under non-inducing 144 conditions (79). Even under inducing conditions, one a l f a l f a variety was not able to develop the customary rossette growth and low temperature resistance until ABA had been added to the nutrient solution (63). High levels of carbon dioxide were also able to produce changes in the morphology of Douglas f i r ; internodal elongation was inhibited in trees given 1.0% C0 2 (Table II) and seedlings given 1.0% and 5.0% C0 2 (Figure 3). Comparisons between the action of abscisic acid and high carbon dioxide levels are intriguing, but are at this point only speculative. The similar effects of CC>2 and ABA may be only coincidental responses to stress. However carbon dioxide and plant hormone inter-actions are not necessarily limited to those between carbon dioxide and abscisic acid. There are other possible ways that C0 2 and plant hormone interactions could affect plant growth and development. For instance, the action of C0 2 in overriding the photoperiodic control of budset may not be related to the effects of C0 2 on photosynthesis, but instead may be due to the interaction of carbon dioxide with phytochrome. Phytochrome has already been shown to be involved in the frost hardiness response of Douglas f i r (73), and although a C02-phytochrome relationship has yet to be demonstrated for Douglas f i r , a C0 2 requirement for phytochrome action in the flowering of the short-day plant Xanthium has been demonstrated (4, 5). The exact role of C0 2 i n the phytochrome response was not known, but the C0 2 requirement was shown to be non-photosynthetic. Just on the basis of these few comparisons i t is apparent that we do not yet know enough about C0 2 and i t s interactions with endogenous plant factors; however, there are good indications that further 145 investigation of the effects of high CO^ levels on various growth and developmental processes may aid our understanding of aspects of hormonal action and regulation. CONCLUSIONS 1 . Levels of 0 . 1 % C O 2 are best for enhancement of growth processes, such as dry weight and internodal elongation, and inhibitory to non-growth processes, such as budset. 2 . Concentrations of 1 . 0 % C O 2 and higher generally inhibit growth processes such as internodal elongation, dry weight, and leaf area, but are effective in promoting budset. 3. C O 2 enrichment enhances growth to a degree that other factors may become limiting, e.g., light intensity. 4 . Higher carbon dioxide levels ( 1 . 0 % and 5.0% C O 2 ) may override photoperiodic control of budset by promoting budset under warm temperatures and long days. 5. In some cases, carbon dioxide may substitute in part for the light requirement of photosynthesis when light i s limiting. Thus, an increase in daylength may reduce the level of C O 2 required for a particular effect, e.g., the required C O 2 levels for inducing frost hardiness are reduced from 1 . 0 % to 0 . 1 % C O 2 i f long days are provided. 6. An active metabolism appears to be necessary to establish CC^-induced frost hardiness. The mode of CO 2 action is l i k e l y through increased production of cryoprotectents, such as amino acids and sugars. 7. Plants which have been grown under normal a i r ( 0.03% CC^) have higher photosynthetic rates than enriched plants when a l l plants are measured in normal a i r ; the C 0 2 concentration under which plants are measured appears to have more effect on photosynthetic rates than the C O 2 concentration under which the plants are grown. 147 8. Plants grown under long days and high CO^ have lower CO diffusion resistance (as determined from transpiration rates). However, the potential benefits to net photosynthesis from reduced CC^ diffusion resistance are not realized. This i s due to increased respiration under high CO^, and possibly also due to high CO^ mesophyll resistance which was not included in the total transpiration resistance. 9. 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TABLE A SUMMARY OF EXPERIMENTS EXPERIMENT DAYLENGTH NUMBER CARBON DIOXIDE CONCENTRATION LIGHT INTENSITY DURATION OF TREATMENT 8 hours and 16 hours 0.03% and 1.0% C0„ 3.4 mW cm -2 30 Days AGE OF PRIMARY PURPOSE PLANTS OF STUDY 1 year Growth & Development 16 hours 0.03%, 0.1%, & 1.0% CO-3.4 mW cm -2 90 Days 1 year Growth & Development 8 hours 0.03%, 0.1%, 1.0% & 5.0% C0 2 7.2 mW cm 30 Days 1 year Frost Hardiness 8 hours & 16 hours 0.03%, 1.0%, & 5.0% CO, 6.0 mW cm 12 Weeks 1 year Frost Hardiness, Amino Acid Analysis 8 hours & 16 hours 0.03%, 0.1%, 1.0%, & 5% C0 2 7.2 mW cm 12 Weeks 6 months Frost Hardiness, Growth, Development of Seedlings 157 TABLE B RANKING CODES * Bud growth 1 Dormant pyramid-shaped bud 2 Bud swollen larger than base diameter, or with leaves just visible in the open end of the bud 3 Leaves protruding from the bud, but s t i l l parallel to the stem axis 4 Leaves extending horizontally away from the stem * Categories of bud growth are a modification of the classification system used by Tregunna and Crown (74). Date of budset 1 Did not flush within the 45-day period 2 Set bud at Day 5 3 Set bud at Day 10 4 Set bud at Day 15 5 Set bud at Day 20 6 Set bud at Day 25 7 Set bud at Day 30 8 Set bud at Day 35 9 Set bud at Day 40 10 Set bud at Day 45 11 Did not set bud within the 45-day period Frost damage 1 No damage 2 Very slight damage (less than 10% of the needles) 3 Moderate damage (approximately 20-30% of the needles) 4 Severe damage (approximately 50% of the needles) but plant w i l l survive 5 A l l needles brown and damaged, tree dead, no recovery 158 TABLE C SCHEMATIC DIAGRAM FOR EXF. NUMBER 5 Douglas-fir six-month-old seedlings CARBON DIOXIDE PRETREATMENT A = Freeze-tested to - 6°C. B •» Freeze-tested to - 10°C. 8 HOURS LIGHT (25 C.) 16 HOURS DARK (20°C.) 0.03%, 0.1%, 1.0%, or 5.0% CO, TRANSFER THERMOPERIOD POST-TREATMENT 8 HOURS LIGHT (25 C.) + 16 HOURS DARK (5°C.) 0.03% C0 2 ONLY 0 WEEKS 2 WEEKS 4 WEEKS 6 WEEKS FOR 6 WEEKS UP TO 6 WEEKS A B A B A B A B * Sequence was repeated using 16 hours l i g h t (25°C.) and 8 hours dark (20°C.) APPENDIX D -- SPECTRAL DISTRIBUTION OF SHERER GROWTH CABINET Experiments 1, 2, and 4 (See Table A) 3.4 MILL!WATTS/CM" (401 - 698) TOTAL ENERGY 1 -| 1 1 1 h——I 1 1 1 i 1 1 ]• 1 401 420 439 459 479 499 519 540 558 579 601 618 639 660 680 698 WAVELENGTH (nm) -3.0 -4.0 \ 1 1 1 j 1 1 1 1 ) 1 1 1 1 1 h -A01 420 439 459 479 499 519 540 558 579 601 618 639 660 680 698 WAVELENGTH (ran) 

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