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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 . S c , U n i v e r s i t y 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 t h e s i s as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA June, 1979 ©  CAROLE LOUISE LEADEM, 1979  In  presenting  this  an a d v a n c e d  degree  the  shall  I  Library  further  for  agree  thesis  in  at  University  the  make  that  his  of  this  written  may  representatives. thesis  for  be  It  financial  of  1 May  1979  of  Columbia,  British  by  the  understood  gain  BOTANY  The U n i v e r s i t y o f B r i t i s h 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  of  extensive  granted  is  fulfilment  available for  permission.  Department  Date  freely  permission for  s c h o l a r l y purposes  by  it  partial  Columbia  shall  Head  be  requirements  reference copying  that  not  the  of  agree  and  of my  I  this  or  allowed  without  that  study. thesis  Department  copying  for  or  publication my  ABSTRACT Seedlings and one-year-old trees of Douglas-fir (Pseyidotsuga menziesii) were grown i n 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 l i g h t intensities -2  which varied from 3.4 mW cn  to 7.2 mW cm  -2  (400 - 700 nm). The  duration of treatment varied from 30 days to 1 2 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.  C 0 £ enrichment caused growth  enhancement to a degree that other factors became l i m i t i n g , 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 i n part for the l i g h t requirements of photosynthesis when light i s l i m i t i n g . Thus, an increase i n daylength may reduce the l e v e l of COj required for a particular effect, e.g., the required C 0 levels for inducing 2  frost hardiness are reduced from 1 . 0 % to 0 . 1 % C 0 i f long days are 9  ;  iii provided. Plants which have been grown under normal a i r (0.03% CO2) have higher photosynthetic rates than enriched plants when a l l plants are measured i n 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 photosynthesis 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 i n h i b i t i o n of  growth under 1.0% CO2, appear to be mostly related to differences i n 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 i n levels of growth i n h i b i t o r s , 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  . . . . . . . .  DEDICATION  x  xii  PREFACE  •  xiii  CHAPTER I. Effects of carbon dioxide and daylength on growth of Douglas-fir (Pseudotsuga menziesii) INTRODUCTION  .  MATERIALS AND METHODS  1 5  RESULTS A.  HEIGHT 1. One-year-old trees (a) Effects of 8- and 16-hour daylengths and two carbon dioxide l e v e l s . . . . . 10 (b) Effects of 16-hour daylength and three carbon dioxide l e v e l s 13 2.  3.  Six-month-old seedlings: experimental design (a) E f f e c t s of daylength (b) E f f e c t s of carbon dioxide (c) Effects of carbon dioxide and daylength . . (d) Effects of temperature . . . . . .  16 16 19 23  Summary: E f f e c t s 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 l e v e l s 25 (b) E f f e c t s of 16-hour daylength and three carbon dioxide l e v e l s . . . . . 29 (c) Effects of 8-hour days and four carbon dioxide concentrations . 33 2.  Six-month-old seedlings  35  3.  Summary: E f f e c t s of carbon dioxide and daylength on weight (a) Leaves 41 (b) Stems 42 (c) Roots 42 (d) Biomass D i s t r i b u t i o n 42  DISCUSSION  44  V  Page  CHAPTER I I . Effects of carbon dioxide and daylength on dormancy and hardiness of Douglas-fir INTRODUCTION  . . . .  56  MATERIALS AND METHODS  60  RESULTS FLUSHING AND BUDSET 1. One-year-old trees (a) E f f e c t s of 8- and 16-hour daylengths and two carbon dioxide l e v e l s . (b) Effects of 16-hour daylengths and three carbon dioxide l e v e l s . . . . . . . . ... . (c) Effects of 8-hour daylength and four carbon dioxide l e v e l s 2.  Six-month-old seedlings (a) General flushing and budset behaviour . . . (b) Maximum budset (c) E a r l i e s t date of budset (d) E f f e c t s of temperature on plants grown under a i r (0.03% C0 ) 2  B.  COLD HARDINESS 1. Effects of carbon dioxide (a) Preliminary study (b) Plants grown under 8-hour days (c) Plants grown under 16-hour days 2. Effects of post-treatment (a) Plants grown under 8-hour days (b) Plants grown under 16-hour days 3. Effects of temperature 4. Effects of C02 and daylength on amino acids  DISCUSSION  .  67  69 69 72 75 75 79  79 82 82 86 89 . 89  • '. • • 92 95  CHAPTER I I I . 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  vi 2. B.  RESPIRATION  C.  TRANSPIRATION 1. One-year-old trees 2.  D.  Page  Plants grown under 16-hour days and four COlevels  ,119 ' 123 123  Six-month-old seedlings  125  TOTAL DIFFUSIVE RESISTANCE  DISCUSSION  131  . . . . . . . . . . . .  133  CONCLUSIONS  146  LITERATURE CITED  . .  148  APPENDICES TABLE A  Summary of Experiments  TABLE B  Ranking Codes  TABLE C  Schematic Diagram f o r Experiment Number 5  TABLE D  Spectral D i s t r i b u t i o n of Sherer Growth Cabinet.  160  TABLE E  Spectral D i s t r i b u t i o n of Mercury Vapor Lamps. .  161  #  -,57 158 . 159  vii LIST OF TABLES  Table 1  Page  Growth of trees grown under 8- and 16-hour photoperiods with either 0.03% or 1.0% CO 2  2  12  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. E f f e c t of daylength  4  Growth of seedlings. E f f e c t of carbon dioxide  5  Growth of seedlings. E f f e c t of carbon dioxide and daylength . . 20  6  Growth of seedlings. E f f e c t of temperature  7  Dry weight of with either Shoot:root of with either  8 9  trees 0.03% trees 0.03%  17" • • • 18  24  grown under 8- and 16-hour photoperiods or 1.0% C0 grown under 8- and 16-hour photoperiods or 1.0% C0 2  27  2  28  Shoot:root of trees grown under 16-hour photoperiods with either 0.03%, 0.1%, or 1.0% C0  32  Dry weight of trees grown under 16-hour photoperiods with either 0.03%, 0.1%, or 1.0% C0  34  2  10  2  11  Needle dry weight and area of trees grown under 8-hour photoperiods with 0.03%, 0.1%,  1.0%,  or 5.0% C0  2  12  Regression of l e a f area and leaf weight of trees  13  Fresh weight of seedlings grown under 8- and 16-b.our daylengths and 0.03%, 0.1%,  1.0%,  or 5.0% C0  Proportional weight d i s t r i b u t i o n of seedlings  15  Degree of l a t e r a l budset i n trees grown under 8- or 16-hour daylengths with 0.03% or 1.0% C0 Degree of budset i n trees grown under 16-hour daylengths with 0.03%, 0.1%, or 1.0% C0 Degree of budset i n trees grown under 8-hour daylengths with 0.03%, 0.1%, 1.0%, or 5.0% C0 2  2  17  2  18  37  38  2  14  16  36  Date of f i r s t budset i n trees grown under 8-hour daylengths with 0.03%, 0.1%, 1.0%, or 5.0% C0 0  40  68 70 71  .73  Table 19  Pag  Maximum number of buds set i n seedlings grown under 8- or 16-hour daylengths with 0.03%, 0.1%, 1.0%, or 5.0% C0 . . .  77  Earliest date of budset i n seedlings grown under 8- or 16-hour daylengths with 0.03%, 0.1%, 1.0%, or 5.0% C0  78  Earliest date of budset i n 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  24  Cold hardiness of seedlings grown under 16-hour days. Effect of carbon dioxide  85  Cold hardiness of seedlings grown under 8-hour days with 0.03%, 0.1%, 1.0%, or 5.0% C0 Effect of post-treatment . .  87  2  20  2  21  25  ....  84  2>  26  Cold hardiness of seedlings grown under 16-hour days with 0.03%, 0.1%, 1.0%, or 5.0% C0 . Effect of post-treatment . . i Cold hardiness of seedlings grown under 8-hour daylengths with three temperatures. Effect of temperature  93  28  Amino acids i n leaf extracts of 1-year-old trees  94  29  Rates of photosynthesis of seedlings grown under 8-hour days  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  2  27  90  . 118  . 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 (Douglasf i r 6-month-old seedlings)  9  80  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  124  13  Weekly water loss (1-year-old Douglas-fir grown under 8-hr days and 4 C0 levels)  on respiration  1 2 6  2  14  Transpiration rates of seedlings grown under 8-hr days  ...  15 16  Transpiration rates of seedlings grown under 16-hr days Effect of daylength and CO2 on total resistance  . .  1  27  1  29  132  ACKNOWLEDGEMENTS  I would l i k e to thank my main advisor, Tony Glass, f o r a l l the work he has done on my behalf i n 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 l a t e r part of my research  and his many suggestions during the writing of t h i s thesis were invaluable i n 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 specifically.  to a l l those whom I did not have room to mention I would l i k e to thank:  Geoff L i s t e r , who advised me on various aspects of gas exchange and who graciously allowed me to examine h i s 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 a i l i n g mechanical monsters countless times.  xi  Our discussions, both s c i e n t i f i c and p h i l o s o p h i c a l , contributed greatly to my education during my four years at U.B.C. my children, John and Lauryn, f o r their moral support and for their assistance i n the laboratory, even though they could never understand why Mom went to school when she didn't have to. F i n a l l y , I would l i k e to thank Tim whose love and moral support kept me going, and whose tangible s u p p o r t — a s s i s t i n g me i n the laboratory, taking photographs, d r a f t i n g f i g u r e s , c r i t i c a l l y reading and re-reading this manuscript—helped me f i n i s h . For their contributions of Douglas f i r plants used i n this research I would l i k e to thank the B.C. Forest Service and Weyerhaeuser Company, Centralia, Washington.  xii DEDICATION  This thesis i s dedicated to the memory of E. Bruce s c i e n t i s t , teacher, and friend.  Tregunna,  Bruce Tregunna was a t r u l y c r e a t i v e ,  open-minded s c i e n t i s t , and was worthy of emulation. created a supportive atmosphere i n his laboratory  Bruce  and was l a r g e l y  responsible for the co-operative i n t e r a c t i o n of a l l members of h i s group.  He promoted the exchange of ideas from researchers  outside  his laboratory as w e l l , and whenever v i s i t o r s came to campus a l l gather i n the coffee room for informal discussions.  we would  These d i s -  cussions were a valuable educational experience which served to broaden both my i n t e l l e c t u a l 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 t h i s research. listener  In our personal discussions, Bruce was an attentive and receptive to new ideas.  He provided guidance when  needed but he also allowed me the maximum amount of freedom f o r research and problem-solving.  In addition, Bruce was a compassionate  and thoughtful human being f o r whom I had the greatest  admiration.  The thought of his death s h a l l always i n s p i r e me with a sense of l o s s .  xiii PREFACE  In the laboratory of E. Bruce Tregunna, work on the e f f e c t s 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^ l e v e l s . After 90 days they examined the plants and found that several plants grown under 1.0% CO,, had formed curious clusters of b r a c t - l i k e leaves which resembled the early developmental stages of female cones.  This finding was quite d i f f e r e n t from the  usually observed e f f e c t s of favourable l e v e l s of CO2 enrichment, i . e . , the enhancement of dry weight and internodal elongation.  I t appeared  that carbon dioxide which i s a r e l a t i v e l y simple, molecule basic to plant metabolism  could a f f e c t plant growth and development i n ways  other than providing e s s e n t i a l n u t r i t i o n . liminary results  Intrigued by these pre-  I became interested i n further exploring carbon  dioxide effects on growth and development of Douglas f i r .  Other  members of our research team investigated other carbon dioxide e f f e c t s on physiological events, such as the i n t e r a c t i o n of C0 I was able to only sporadically  2  and  reproduce the e f f e c t s of C0  phytochrome. 2  on  bud development which o r i g i n a l l y had caught my i n t e r e s t i n this research  however, other e f f e c t s of C0  2  which I observed during the  course of my investigations turned out to be  equally as i n t e r e s t i n g .  This thesis includes experiments r e l a t i n g to several different  effects of carbon dioxide on growth and development of  Douglas f i r . The experiments were designed to define the ranges i n which C0  2  enhances growth and the ranges i n which C0  2  i n h i b i t s growth,  as well as explore the interactions between CO^ and other factors influencing growth.  xiv  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 In addition, I investigated  resistance.  the e f f e c t s of carbon dioxide on photo-  synthesis, respiration, and transpriation and made an e f f o r t to determine the i n t e r r e l a t i o n s h i p s between the d i f f e r e n t components of gas exchange.  1  CHAPTER  ONE  EFFECTS OF CARBON DIOXIDE AND GROWTH OF DOUGLAS FIR  DAYLENGTH ON  (Pseudotsuga  menziesii)  INTRODUCTION  I n v e s t i g a t i o n of the e f f e c t s of carbon d i o x i d e enrichment on p l a n t growth began about the t u r n o f t h i s c e n t u r y .  As e a r l y as  1903  there were r e p o r t s of d r y weight i n c r e a s e s of as much as 158% when carbon d i o x i d e was  added  to the p l a n t atmosphere  (48).  Much u n c e r t a i n t y  surrounded t h i s e a r l y work because o t h e r workers r e p o r t e d e f f e c t s of enrichment, such as r e d u c t i o n i n l e a f a r e a , of i n t e r n o d e s ,  and r e t a r d a t i o n o f f l o w e r  and f r u i t  r e t r o s p e c t , not a l l of these problems may other  negative  slow  development  development.  In  have been due to CO^,  but to  f a c t o r s , such as contaminants i n t h e gases used f o r enrichment,  and excessive h u m i d i t y i n the t r e a t m e n t chambers. may have a l s o been due to d i f f i c u l t i e s CO^ c o n c e n t r a t i o n s p l a n t s may  with  around the p l a n t . I t was  exhibit a f a i r l y well-defined  Conflicting  reports  a c c u r a t e l y measuring not then known t h a t  the specific  optimum range f o r carbon d i o x i d e ,  and that i n h i b i t o r y e f f e c t s can q u i c k l y i n c r e a s e once the optimum i s exceeded.  Work i n the f i e l d  continued,  I n t e r e s t i n CO^  however, u n t i l  about 1930 when  it  gradually declined.  in  1964 Wittwer and Robb (85) d e t a i l e d the many advantages  c u l t u r i s t s could a c h i e v e atmosphere.  by u s i n g CO^  enrichment was  enrichment i n the  In cucumber, t h e r e were more p i s t i l l a t e  r e s u l t e d i n a 72% i n c r e a s e i n f r u i t i n flower development as CO^ promotes  (38).  Lettuce  r e v i v e d when horti-  greenhouse  flowers  (85) and i n p e t u n i a s , i s an i d e a l crop  that  which  accelerations  f o r enrichment,  i n c r e a s e s i n b o t h l e a f s i z e and t h i c k n e s s ,  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  i n many f r u i t s , with improvements i n f r u i t color and f r u i t shape, and reduction i n the number of scars (37). Outside the h o r t i c u l t u r a l 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, unpublished 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 i n the past on the effects of different l i g h t  3  intensities i n conjuction with several l e v e l s of carbon dioxide, but no work has been reported on the e f f e c t s of d i f f e r e n t daylengths combined with several carbon dioxide l e v e l s . The broad objective of the research presented i n 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 a f f e c t the biomass d i s t r i b u t i o n  within the plant?  What were the e f f e c t s of l i g h t on growth, and did  irradiance levels or photoperiod a f f e c t the expression of the CO^  effect?  Did CC^ interact with l i g h t or any other factors to influence growth? F i n a l l y , could the effects of CO,, p e r s i s t a f t e r enrichment To accomplish my research objective I conducted experiments.  terminated? a series of  Each experiment was designed to contribute a part to the  cumulative knowledge required to answer the questions above.  Initially  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. different photoperiods  Two  (8 and 16 hours) and two d i f f e r e n t carbon dioxide  levels were used for the f i r s t experiment (0.03% and 1.0%  CO,,).  The  plants were grown f o r 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 i n which both the duration and the number of CO,, levels were increased; thus plants were grown f o r 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 e f f e c t s of CO,, on height and weight, and  to  determine the best CO^ l e v e l f o r enhanced plant growth. Together the r e s u l t s of experiments 1 and 2 provided enough data to determine the ranges i n which CO,,  promoted growth, but only l i m i t e d 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 l e v e l s had p r e v i o u s l y been l i m i t i n g  plant response, i r r a d i a n c e was also increased. The cumulative data gathered from the r e s u l t s of the f i r s t three experiments presented a general p i c t u r e of the response of 1year-old Douglas f i r trees to CO,, and daylength, however, I had  little  knowledge of how CO^ daylength a f f e c t e d plants younger than one  year.  There were several reasons f o r being i n t e r e s t e d i n the e f f e c t s of on young plants.  CO^  CO,, enrichment during the seedling stage probably  would produce the greatest growth b e n e f i t s since young plants generally respond more r e a d i l y to environmental changes, and have greater r e l a t i v e growth r a t e s .  Smaller plants are easier to handle because of  t h e i r s i z e , and f i t more e a s i l y i n t o c o n t r o l l e d environment chambers. Using younger plant m a t e r i a l would a l s o make i t p o s s i b l e to increase sample s i z e s , consequently reducing t o t a l v a r i a b i l i t y w i t h i n the sample. F i n a l l y , seedling research has good p o t e n t i a l f o r p r a c t i c a l b e n e f i t i n B r i t i s h Columbia where f o r e s t nurseries require large numbers of trees each year.  Reforestation nursery programs are always seeking  new  techniques for a c c e l e r a t i n g seedling growth i n order to produce plantable trees i n the shortest possible time.  5  With  t h e above c o n s i d e r a t i o n s i n mind, 6-month-old  s e e d l i n g s were grown f o r s i x weeks w i t h e i t h e r 8- o r 16-hour  photo-  p e r i o d s under f o u r 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 t o normal air  f o r another f o u r weeks (Appendix,  f i r s t p a r t of the f i n a l experiment of  T a b l e A, Exp. No. 5 ) . The  p r o v i d e d data on t h e g e n e r a l e f f e c t s  CC^ and daylength on s e e d l i n g growth, whereas t h e second  part provided  a d d i t i o n a l data on the p e r s i s t e n c e o f CO^ e f f e c t s a f t e r t h e p l a n t s were grown i n normal a i r . The completion o f the experiments  described i n this  s u p p l i e d most of the r e s u l t s needed to determine  chapter  how carbon d i o x i d e  and daylength i n f l u e n c e d some of t h e growth p r o c e s s e s o f Douglas f i r . Most o f my i n i t i a l q u e s t i o n s were answered r a i s e d even more q u e s t i o n s .  a l t h o u g h some o f t h e r e s u l t s  R e g a r d l e s s , the c u m u l a t i v e data d i d  enable me a t l e a s t t o f o r m u l a t e a t e n t a t i v e p i c t u r e o f CC^ e f f e c t s and its  i n t e r a c t i o n s w i t h o t h e r f a c t o r s i n f l u e n c i n g growth.  MATERIALS AND METHODS Plant One-year-old  Douglas f i r t r e e s (1-0 s t o c k ) were o b t a i n e d  from the B r i t i s h Columbia or  Material  F o r e s t S e r v i c e from e i t h e r  Green Timbers bare r o o t n u r s e r y .  t h e i r South  Surrey  The c o a s t a l provenance t r e e s had  been grown from seeds o r i g i n a l l y gathered a t an e l e v a t i o n o f a p p r o x i m a t e l y 500 m on t h e east c o a s t o f Vancouver I s l a n d . pots f i l l e d with Mica-Peat  T r e e s were grown i n 15 cm  (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) o b t a i n e d from L a n g l e y Peat L i m i t e d , F o r t L a n g l e y , B.C. S e e d l i n g s  6  were supplied by Weyerhaeuser Company, C e n t r a l i a , Washington, and were also coastal provenance Douglas f i r from Twin Harbours, Aberdeen, Washington (elevation 300 m t 60 m) .  The p l a n t s had been seeded i n t o  i n d i v i d u a l 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 p o t t i n g mixture.  The seedlings were not transplanted since the plugs were of  s u f f i c i e n t volume to allow f o r reasonable root expansion.  Both trees  and seedlings were well-watered and f e r t i l i z e d every two weeks w i t h Hi-Sol 20-20-20 (N-P-K) s o l u t i o n .  Terminology To s i m p l i f y the terminology throughout t h i s t h e s i s , concentrations c f 0.03% "atmospheric",  2  levels.  (v/v) w i l l be r e f e r r e d to as e i t h e r  "normal", or "low" C0  "intermediate" C0 C0  CO^  2  CO^  2  l e v e l s ; 0.1% CC> w i l l be c a l l e d 2  l e v e l s ; and 1.0% or 5.0%  CC> w i l l be c a l l e d "high" 2  Exact concentrations w i l l be stated i n instances when  descriptive l a b e l s might otherwise be misleading or ambiguous. with l i g h t periods of 8 hours w i l l be c a l l e d "short days" and  Days those  with 16 hours w i l l be c a l l e d "long days".  Experimental Design A general summary of a l l experiments,  given i n the Appendix,  Table A, can be used f o r reference when needed. The basic purpose of Experiment 1 was to compare the e f f e c t s on growth of normal and enriched C0 photoperiods. 16-hour  2  l e v e l s under two d i f f e r e n t  One-year-old Douglas f i r trees were grown under 8-  daylengths w i t h e i t h e r 0.03% or 1.0% C0  2  and  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 i n this experiment and a l l others were taken from the root collar to the t i p 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 i n 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 i n 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 o b j e c t i v e 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 e f f e c t s of  CO^  and photoperiod on growth, and consisted of p r e t r e a t i n g p l a n t s f o r s i x weeks with e i t h e r 8- or 16-hour days (25 C/20 l e v e l s (0.03%, 0.1%,  1.0%,  or 5.0% C0 ). 2  C) and one of four  C0  2  During the second part of the  experiment, a l l plants were t r a n s f e r r e d to normal a i r and  maintained  under short warm days (25 C) and long cold nights (5 C) f o r four weeks. Additional controls were grown continuously under short days and normal a i r , but given thermoperiods of e i t h e r 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 p e r i o d , while the second number i s the temperature during the dark p e r i o d ) . The primary purpose of the post-treatment  was  to e s t a b l i s h whether  the e f f e c t s of CC^ pre-treatment p e r s i s t e d beyond the a c t u a l enrichment period, but there was a secondary purpose as w e l l .  CO^ In  nature, short warm days and long cold nights promote the cessation of growth and the onset of dormancy.  Many of the p l a n t s had already been  pre-treated under conditions which enhanced growth, however seedlings were also to be post-treated w i t h conditions which induced dormancy. Thus, the secondary o b j e c t i v e of the post-treatment whether pre-treatment or whether post-treatment influence i n c o n t r o l l i n g plant growth response.  was  to a s c e r t a i n  would exert the greatest For t h i s experiment  height and weight samples were taken a f t e r s i x weeks of pre-treatment and a f t e r 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 concentrations at the required l e v e l s .  CO^  Cabinets were i l l u m i n a t e d w i t h  mixed incandescent and flourescent l i g h t s .  Radiant energy at mid-  plant l e v e l i n the p h o t o s y n t h e t i c a l l y a c t i v e region (400-700 mm) 2  was  3.4- mW/cm as measured by the s p e c t r a l radiometer described by Burr and Duncan (89).  A sample s p e c t r a l 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 c y l i n d e r s ( L i q u i d  Carbonic  or Matheson Supply Company) and a Matheson Rotameter (a p r e c i s i o n flowmeter w i t h an adjustable valve) regulated the flow.  Gas w i t h i n  the chamber was monitored p e r i o d i c a l l y w i t h a Beckman IR-215 i n f r a r e d gas analyzer. Experiments 3 and 5 were conducted i n eight s p e c i a l l y constructed P l e x i g l a s chambers (59).  A thermistor monitored a i r temperature and a  cooling c o i l provided humidity c o n t r o l w i t h i n each chamber.  Condensed  water c o l l e c t e d at the base of the c o o l i n g 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 v e s s e l .  A  small fan mounted on the P l e x i g l a s w a l l assured that a i r w i t h i n each chamber was w e l l mixed.  A t h e r m o s t a t i c a l l y cooled c i r c u l a t i n g water  bath maintained at a depth of 7 cm reduced i n f r a r e d r a d i a t i o n from the two 1000 W mercury vapour flourescent lamps (Duroglo GA 217G2) mounted above the eight chambers.  Carbon d i o x i d e from c y l i n d e r s  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 i n s i d e each chamber; pumped gas was d i r e c t e d to a Beckman Model IR-215 i n f r a r e d gas analyzer.  Solenoid valves were a c t i v a t e d whenever the m i l l i v o l t  output from the gas analyzer f e l l below the set p o i n t , i n j e c t i n g a d d i t i o n a l CO^ i n t o the chamber, and thus providing a constant CO^ level.  There were occasional problems maintaining CC^ l e v e l s due  to f a i l u r e s i n equipment however, CC^ concentrations were checked at l e a s t d a i l y and u s u a l l y several times a day and adjustments were made as necessary.  Occasional f l u c t u a t i o n s of CO^ l e v e l s would  probably have minimum e f f e c t on the o v e r a l l r e s u l t s of long-term experiments l a s t i n g a month or more.  In any case, CO^ f l u c t u a t i o n s  would be of importance p r i m a r i l y i n explaining th'e l a c k of r e s u l t s due to carbon dioxide enrichment, and not f o r 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,., l e v e l s 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 t h i r d week  s i g n i f i c a n t d i f f e r e n c e s between  the treatments were apparent ( F i g 1, Table I ) . maintained under atmospheric C0  0  Trees continuously  and long days (16 hours) were n o t i c e a b l y  Figure 1. Mean cumulative elongation f o r 1-year-old Douglas f i r continuously supplied with C02 a t the l e v e l s shown, and maintained under e i t h e r 8- or 16-hour photoperiods (25 C/20 C) with an i r r a d i a n c e of 3.4 mW cm . -2  7.0  b  6.0  - 02%  C02  LD  .02%  C02  SO  1.0%  co2  SO  1.0%  C02  LD  ~5.0 E u LU  24-0 o  63.0 Ul  2.0  1.0  — I  1.0  IL^v!^ cn  -  Q t.  i  2.0  - s  3.0  T I M E  4.0  (weeks)  fr" I / ° ° & ^ s of 8 plants. Analysis of Variance was used f o r determining s t a t i s t i c a l d i f f e r e n c e s . Values bearing the same l e t t e r w i t h i n 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. 7 8  =  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  12  TABLE I CUMULATIVE GROWTH INCREASE OF CO -ENRICHED DOUGLAS FIR  TREES  (1-0 Stock)  Grown f o r 30 days under 8- and 16-hour photoperiods (25 C/20 C) with either 0.03% or l.D% C 0 Irradiance = 3.4 mW c m -2  2  TREATMENT Daylength (h)  MEAN LENGTH OF NEW GROWTH (cm ) CO  cone.  lO)  7 Days  14 Days  21 Days  28 Days  3.75  a  5.05 . a  6.03  ab  a  5.01  a  5.79  ab  8  0.03  1.56  8  1.0  2.09  16  0.03  2.03  16  1.0  1.94  3  3  3.98  a  4.56  a  6.04  a  3.86  a  5.08  b  a  6.41  b  5.50  a  The data points shown i n the table are the means of 8 plants. Analysis of variance was used f o r 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 from each other at p = 0.05.  13  t a l l e r 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 i n h i b i t stem  growth under both short and long days, but the differences were only s t a t i s t i c a l l y s i g n i f i c a n t under long days.  (b)  Effects of 16-hour daylength and 3 C0„ l e v e l s on height S i g n i f i c a n t l y d i f f e r e n t trends between high and low CO,,  plants were observable by 30 days (Figure 2, Table I I ) .  After that  time, extension growth l e v e l l e d o f f 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 f a i r l y constant rate.  Trees supplied with intermediate CO^ l e v e l s  were the t a l l e s t at the end of 90 days, although they were r e l a t i v e l y late i n responding to CQ^ enrichment.  High CO 2 l e v e l s appeared to  i n h i b i t elongation since the shortest trees were those grown under 1.0% CO2.  Trees grown under atmospheric conditions were intermediate  i n height between the other two groups. In comparing Experiments 1 and 2, growth trends f o r the f i r s t 30 days were similar.  In Experiment 1 the greatest differences between  trees grown under normal and high CO2 l e v e l s occurred at three weeks, whereas i n Experiment 2 the greatest differences between normal and high CO2 levels occurred at four weeks.  Trees grown i n normal a i r  showed exponential and plateau phases i n both studies, although the  Figure 2. Mean cumulative elongation of 1-year-old Douglas f i r continuously supplied w i t h CO at the l e v e l s shown, and maintained under 16-hour photoperiods (25 C/20 C) w i t h an i r r a d i a n c e of 3.4 mW cm . -2  0  1 5  30  45  TIME  60  75  90  (days)  The data points shown i n the f i g u r e are the means of 5 p l a n t s . A n a l y s i s of Variance was used f o r determining s t a t i s t i c a l d i f f e r e n c e s . Values bearing the same l e t t e r w i t h i n 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 a t 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  6.92  b  8.08  a  11.78  a  6.04  7.14  a  14.30  a  4.62  6.38  a  8.60  0.03%  co  2  4.38  0.1%  co  2  3.42  1.0%  co  2  3.26  a  ab  a  ab  b  a  The data points shown i n the table are the means of 5 plants. Analysis of Variance was used f o r 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.  16  pattern was more c l e a r l y discernable i n the 90-day study. high  Trees i n  increased at a lower r a t e , but grew continuously, and thus d i d  not demonstrate a marked plateau phase.  2. Six-month-old  seedlings  (a) E f f e c t s of daylength on height Plant heights under a l l CO^ l e v e l s were averaged, then analyzed according to the daylength under which they had been grown (Table I I I ) . (25 C/20  A f t e r s i x weeks w i t h warm day and night temperatures  C), new growth of seedlings maintained under short days 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 that of seedlings grown under long days.  The effects of long days were not apparent u n t i l the p l a n t s  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 c e s s a t i o n and dormancy onset i n Douglas f i r .  In t h i s instance, however, p l a n t s  previously grown under long days continued to grow even i n noninducing circumstances.  (b) E f f e c t s of C0„ on height Individual data points f o r a l l plants were averaged, then analyzed on the basis of t h e i r photoperiod they had received.  growth regime, regardless of the Data analyzed i n t h i s manner demonstrated  that CO^ e f f e c t s could be seen both during the pre-treatment treatment periods (Table IV).  and post-  Seedlings given intermediate CO^  exhibited the best growth during pre-treatment  and post-treatment  levels and  17  TABLE  III  CUMULATIVE GROWTH INCREASE OF CO^ENRICHED DOUGLAS FIR SEEDLINGS  A.  EFFECT OF DAYLENGTH Irradiance = 7.2 mW cm  TREATMENT Daylength  MEAN LENGTH OF NEW GROWTH (cm ) (25/20 C) for 6 weeks  8-hour  0.98  16-hour  0.82  8-hr day and (25/5 C) for 4 wks  a  0.08  a  0.86  TOTAL  a  1.06  b  1.68  a  b  The data points shown i n 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 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 from each other at p = 0.01. Mean cumulative elongation of 6-month-old Douglas f i r averaged f o r a l l C0 levels under each daylength. Plants were pre-treated f o r 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% CO2) (25 C/5 C). 2  18  TABLE IV CUMULATIVE GROWTH INCREASE OF C0 ~ENRICHED 2  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) for 6 weeks  0.03% C0  2  0.75  a  8 - h r a  d a y s  f <fC) for 4 wks.  TOTAL  0.59  a  1.34  b  2.27  b  3  1.32  a  a  1.27  a  0.1% C0  2  1.29  0.98  1.0% C0  0.69  3  2  0.63  5.0% C0  0.65  a  2  0.62  b  a  The data points shown i n 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 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. Mean cumulative elongation of 6-month-old Douglas f i r averaged for 8— and 16-hour daylengths under each C02 l e v e l . 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 a i r (0.03% CO2) (25 C/5 C).  19  CO  were s i g n i f i c a n t l y d i f f e r e n t from p l a n t s grown under low and h i g h levels.  These r e s u l t s were s i m i l a r  to those o b t a i n e d  for  1-year-old  Douglas f i r (see F i g u r e 2 ) .  (c) E f f e c t s of CO,, and The  daylength  on h e i g h t  combined e f f e c t s of p h o t o p e r i o d  should be c o n s i d e r e d , s i n c e the s e p a r a t e parameters have a l r e a d y been examined Long days and  weeks).  carbon d i o x i d e  e f f e c t s of these  two  (Table V ) .  i n t e r m e d i a t e CO,,  l e v e l s were most  i n promoting growth d u r i n g the pre-treatment six  and  period  effective  (i.e.,  the  initial  However, w h i l e t h e mean h e i g h t i n c r e a s e f o r s e e d l i n g s grown  under 16-hour days and groups, i t was  0.1%  CO^  was  g r e a t e r than  the means of a l l o t h e r  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 s h o r t -  day p l a n t s grown under low o r i n t e r m e d i a t e CO,, At the end of p o s t - t r e a t m e n t  levels.  ( d u r i n g which s e e d l i n g s v/ere  grown i n normal a i r ) t h r e e s t a t i s t i c a l l y d i f f e r e n t groups c o u l d distinguished.  The  s m a l l e s t growth increments  group, c o n s i s t i n g of p l a n t s p r e - t r e a t e d w i t h in  the short-day  occurred  s h o r t days.  group c o u l d be a t t r i b u t a b l e to CO.,  i n the  be first  No d i f f e r e n c e s  pre-treatment,  but under long days responses to carbon d i o x i d e enrichment c o u l d found.  Moderate growth o c c u r r e d  i n the second group and was  those p l a n t s p r e - t r e a t e d w i t h l o n g days and levels.  The g r e a t e s t growth o c c u r r e d  levels.  shown by  e i t h e r low or h i g h  i n the t h i r d group and  e x h i b i t e d by p l a n t s p r e - t r e a t e d w i t h l o n g days and P l a n t s p r e - t r e a t e d w i t h l o n g days and  be  C0 was  intermediate  intermediate C0  2  C0  2  also  demonstrated the g r e a t e s t t o t a l growth d u r i n g the 12 week p e r i o d , 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 o t h e r treatment  2  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 Daylength (h)  MEAN LENGTH OF NEW GROWTH (cm ) 8-hr days and (25/5 C) f o r 4 wks  COo cone. (25/20 C) (%) f o r 6 weeks  TOTAL  8  0.03  0.99  C  0.03  a  1.02  a  8  0.1  1.22  c  0.07  3  1.29  a  8  1.0  0.78  ab  0.14  a  0.92  8  5.0  0.81  ab  0.10  a  0.91  16  0.03  0.69 . b  0.73  b  1.42  16  0.1  1.30  C  1.24  C  2.54  b  16  1.0  0.67  0.73  b  1.40  a  16  5.0  0.62  0.72  b  1.34  a  a  a  a  The data points shown i n the table are the means of 15 p l a n t s . Analysis of Variance was used f o r determining s t a t i s t i c a l d i f f e r e n c e s . Values bearing the same l e t t e r w i t h i n 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 from each other a t p = 0.05. Plants were pre-treated f o r s i x weeks under 8- and 16-hour days (25 C/20 C) and four C0 l e v e l s (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% C0 ) (25 C/5 C) . 2  2  21 General growth responses were the same f o r s h o r t - d a y day p l a n t s when h e i g h t  i n c r e a s e s were p l o t t e d as a f u n c t i o n of  pre-treatment ( F i g u r e 3).  Both groups showed a base v a l u e  l e v e l s , an optimum at i n t e r m e d i a t e  CC>2  levels.  greater  The  l e v e l s , and  p l a n t s , and  both l a r g e r and more c l e a r l y There were no  and  CC^  CO^  a t low  a decline at  CO^  high  the magnitude of the peak  defined.  d i s c e r n a b l e d i f f e r e n c e s between  long-day p l a n t s a f t e r s i x weeks  had been observed f o r 1 - y e a r - o l d d i f f e r e n c e s between s h o r t - and experimental  f o r 6—month-old Douglas f i r ,  trees.  The  absence of  long-day p l a n t s may  r e s t r i c t i o n s regarding  short-day  use  as  notable  have been due  to  of the c o n d i t i o n i n g chambers.  P l a n t s to be used f o r the long-day p o r t i o n of the experiments had be stored under l o n g days i n the greenhouse w h i l e being  t r e a t e d i n the e x p e r i m e n t a l  c o n d i t i o n s the p l a n t s continued l e s s responsive  chambers.  short-day  to f l u s h , which probably  to f u r t h e r treatment.  do not promote growth  long-day p l a n t s showed g r e a t e r plants.  p l a n t s were  made them  I t s h o u l d be n o t e d , however, conditions  ( s h o r t warm days and  t o t a l o v e r a l l growth than  cold nights), short-day  I t appears, t h e r e f o r e , the e f f e c t s of long p h o t o p e r i o d s  p e r s i s t , extending  the p e r i o d of growth enhancement beyond the  of a c t u a l treatment.  to  While under greenhouse  that when the long-day s e e d l i n g s were p o s t - t r e a t e d under which normally  long-  t o t a l growth of long-day p l a n t s was s u b s t a n t i a l l y  than that of short-day  v a l u e was  and  can  time  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 i n 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 i n 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 l e t t e r of the same case are not s i g n i f i c a n t l y 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 s i m i l a r l y to the shortday 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 posttreatment.  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-monthold 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 i n  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 only) 2  Irradiance = 7.2 mW cm"  PRE- TREATMENT Thermoperiods  MEAN LENGTH OF NEW GROWTH (cm ) After 6 weeks pre-treatment  After 4 weeks post-treatment  TOTAL  25/20 C  0.99  0.03'  1.02  25/5 C  0.52'  0.02'  0.54'  5/5 C  0.43'  0.06'  0.49'  1  The data points shown i n the table are the means of 55 plants. Analysis of Variance was used f o r 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 a t p =0.01. Plants were pre-treated f o r s i x 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 i n normal a i r (0.03% CO2).  25  of experiments 1 and 2 was that i t took much longer f o r p l a n t s i n experiment 2 to show growth b e n e f i t s from CC^ enrichment.  The delayed  response may have been due to the low i r r a d i a n c e 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 p l a n t s may a l s o 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 d i d  eventually respond to CO^ enrichment, but i t took much longer f o r effects of treatment to become evident. Six-month-old Douglas f i r responded to the e f f e c t s of daylength and CO^ s i m 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 l e v e l s of CO,, enrichment.  Under both short and long days, seedlings given intermediate  CO,, l e v e l s were the t a l l e s t , while those given normal CO,, l e v e l s were the next t a l l e s t . C0  2  The shortest seedlings were those grown under high  l e v e l s (1.0% and 5.0% C0 > . There were no s t a t i s t i c a l d i f f e r e n c e s 2  between the means of plants grown under 1.0% and 5.0% CO,,, which probably indicates that the maximum e f 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 l e v e l s and that greater i n h i b i t i o n of elongation by higher C0 concentrations i s u n l i k e l y . 2  B. WEIGHT 1. One-year-old trees (a) Effects of 8- and 16-hour daylengths and two CO,, l e v e l s on weight An increase i n daylength r e s u l t e d i n greater shoot dry weight  26  under both low and high CO^ l e v e l s (Table V I I ) .  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 i n shoot weight under long days and high CC^ were primarily a result of increases i n leaf weight.  Stem weight also  increased under long days and high CO^t but i t could not be shown to be s i g n i f i c a n t l y d i f f e r e n t from the other three treatments.  The  highest mean values f o r root dry weight were also observed under long days and high CO^, but there were no s i g n i f i c a n t differences between the four conditions. Additionally, data f o r trees grown under the above conditions were analyzed using the mathematical relationship known as the shoot: root r a t i o (Table VIII).  The shoot:root r a t i o has long been used f o r  analyzing whole plant response to various treatments.  This r a t i o reduces  two measurements (shoot weight and root weight) to a single measurement which indicates the r e l a t i v e amounts of biomass d i s t r i b u t e d between the upper and lower portions of the plant.  In Table VIII the lowest  ratios were those observed f o r plants maintained  under short days  and low CC^ levels while the highest r a t i o s were those observed f o r plants under long days and high CO^ l e v e l s .  High r a t i o s i n d i c a t e  that greater biomass accumulated i n the shoot r e l a t i v e to the root. However, as a comparison of the data i n 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 r a t i o s , 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 SHOOT  TREATMENT Daylength(h) C02conc.  TOTAL (g )  ROOT Leaf (mg)  Stem (mg)  TOTAL (mg)  8  0.03  0.79  515.8  271.3  407.8'  16  0.03  0.82  551.9'  268.4'  361.3'  -18  1.0  0.87  603.4'  261.0  399.6'  16  1.0  1.15  850.0  301.l  420.6  c  c  1  C  C  c  The data points shown i n 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 at p = 0.01.  C  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.  8  0.03  1.94  a  8  1.0  2.18  3  16  0.03  2.27  3  16  1.0  2.74  b  (%)  Shoot/Root at 30 days  I n i t i a l shoot/root = 1.36 for a l l treatments The data points shown i n 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 l e t t e r are not s i g n i f i c a n t l y different at p = 0.01.  29  smallest increases i n height. long days and high CC^ was  Apparently  the CC^ assimilated under  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 u n t i l approximately 60 days.  Growth resumed a f t e r  60 days and continued u n t i l the end of treatment.  Plants under normal  air demonstrated only h a l f 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 i n 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 s i g n i f i c a n t l y because dry weights of a l l three treatments increased at approximately the same rate (Table IX) . groups had shoot:root  ratios approaching 2.0  The f a c t that a l l three indicated that the  greatest proportion of assimilate went into shoot production.  By  90  30  Figure 4. Mean cumulative increases i n shoot f r e s h weight of 1-year-old Douglas f i r continuously supplied w i t h C O 2 a t the l e v e l s shown, and maintained under 16-hour photoperiods (25 C/20 C) w i t h an i r r a d i a n c e of 3 . 4 mW cm . -2  15  30  45  TIME  60 (days)  75  90  The data points shown i n the f i g u r e ' a r e the means of 8 p l a n t s . A n a l y s i of Variance was used f o r determining s t a t i s t i c a l d i f f e r e n c e s . Values bearing the same l e t t e r w i t h i n 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.  31  Figure 5. Mean cumulative increases i n root fresh weight of 1-year-old Douglas f i r continuously supplied with CC>2 at the l e v e l s shown, and maintained under 16-hour photoperiods (25 C/20 C) with an irradiance of 3.4 mW cm  30  45 T I M E  60  75  90  (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 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.  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  0.03% C0  30  1.77  3  2  90  2.47  2.82  a  a  0.1% C0  a  2  2.09  3.19  4.03  1.0% C0  a  2  1.72  3.03  2.59  a  a  b  a  I n i t i a l shoot:root =0.96. The data points shown i n the table are the means of 8 plants. Analysis cf 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.  33  days the shoot:root r a t i o f o r 0.1% CO^ plants was 4 times greater than i t s i n i t i a l value; t h i s 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 l e a f dry weights rather than to decreases i n root weights (Table X ) .  Both stem and l e a f 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 a t 90 days, and the cumulative e f f e c t s of both increases r e s u l t e d i n the r e l a t i v e l y large shoot:root r a t i o s observed f o r 0.1% CO^ p l a n t s .  This observation  should be q u a l i f i e d somewhat s i n c e shoot growth patterns were v a r i a b l e and plant response was delayed during the treatment period.  However, f i n a l  r e s u l t s of t h i s experiment d i d follow trends observed i n Experiment 1 i n that shoot:root r a t i o s 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).  I n both  experiments 1 and 2 root growth was l i t t l e a f f e c t e d by CO^ l e v e l s .  (c) E f f e c t s of 8-hour days and 4 CO^ l e v e l s 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 V I I and X).  Therefore  I f e l t that leaves would be a good system i n which to study the e f f e c t s of (X^ on some growth patterns.  An examination of l e a f  growth could provide some worthwhile information w i t h which to study the e f f e c t s of C0 on general growth patterns. o  A c c o r d i n g l y , 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  SHOOT  ROOT  TREATMENT TOTAL(g )  LEAF(g )  STEM(g .)  TOTAL (g. )  0.03% C0„  1.92'  1.37  0.55'  0.68  0.1% C0  2.70  1.86  0.84  0.67'  1.84'  1.22  0.62'  0.71'  r  1.0% CO,  £  The data points shown i n 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 f o r area and dry weight (Table X I ) . As noted before l e a f weight was greater under 0.1% C0 and the same under 0.03% and 1.0% C0 2  Leaf areas followed the same trends.  2  (Tables X and X I ) .  Both l e a f weight and area were  further reduced by 5.0% C0 l e v e l s ; they were s i g n i f i c a n t l y l e s s than 2  areas and weights f o r the three lower C 0  concentrations.  2  A regression of l e a f area on l e a f weight produced four d i f f e r e n t f i r s t - o r d e r equations (Table X I I ) .  Higher order regressions were attempted  but they did not s u b s t a n t i a l l y improve r .  Regressions f o r p l a n t s grown  2  under 0.03%, 0.1%, and 5.0% were d i f f e r e n t from one another, w h i l e the regression for plants grown under 1.0% C 0 was s i m i l a r to regressions 2  of most other treatments.  Generally speaking, slopes of the four  regressions increased w i t h i n c r e a s i n g C0 concentrations w h i l e i n t e r c e p t s 2  of the regressions decreased with i n c r e a s i n g C 0 concentrations. 2  These  r e s u l t s indicated that C 0 could a f f e c t l e a f growth patterns such t h a t , at 2  a given dry weight and depending on r e l a t i v e changes i n slope and i n t e r c e p t , leaf area would decrease with i n c r e a s i n g C0 . 2  2. E f f e c t s of C0 and daylength on weight of 6-month-old seedlings 2  Seedling response to C0 and daylength was s i m i l a r to the response 2  described e a r l i e r f o r 1-year-old trees (Table X I I I ) .  Several general  points about the e f f e c t s of treatment on weight could be made: photoperiod had s i g n i f i c a n t e f f e c t s on growth; C 0 enrichment was e f f e c t i v e i n en2  hancing growth under long days; and growth of a l l plant organs were affected by photoperiod and C0 enrichment. o  36  TABLE XI NEEDLE  DRY WEIGHT AND AREA—MEAN VALUES  DOUGLAS FIR TREES (1-0 Stock) Grown f o r 21 days under 8-hour photoperiods 2 Irradiance = 3.4 mW cm  TREATMENT  0.03%  C0  2  AREA (cm ) 2  0.31  a  0.1% C0 '2  0.44  1.0% C0  o  0.29  5.0% C0  o  0.21°  o  \  b  a  WEIGHT (mg)  4.48  a  6.74  b  4.26  a  2.91  C  The data points shown i n the table are the means of 10 plants. Analysis of Variance was used f o r determining 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 at p = 0.05.  37  TABLE XII REGRESSION OF LEAF AREA AND LEAF WEIGHT DOUGLAS FIR TREES (1-0 Stock) Grown f o r 21 days under 8-hour photoperiods  TREATMENT  0.03%  C0  2  REGRESSION OF LEAF AREA (y) on , LEAF WEIGHT (x)  r  2  Y = 0.151 + 0.035 X  3  0.669 0.916  0.1% C0  Y = 0.183 + 0.038 X  b  2  1.0% C0  Y = 0.045 + 0.059 X  a  2  5.0% C0  Y = 0.054 + 0.054 X  C  2  c  0.946 0.889  Leaf area = dependent variable Leaf weight = independent v a r i a b l e Regressions with 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 a t p = 0.05 Least squares regression, Analysis of Variance  N = 10  TABLE XIII FRESH WEIGHT—MEAN VALUES DOUGLAS FIR (6-month-old seedlings) Grown f o r 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)  8  0.03  1.41  8  0.1  1.30  8  •1.0  1.43  8  5.0  1.56  ab  1.29  16  0.03  1.84  bd  1.32  ab  454  16  0.1  2.23°  1.54  bc  596°  87.6  16  1.0  2.03  1.47  bc  463  b  96.8  16  5.0  2.23  400  b  103.6  a  1.17  a  1.09  a  1.20  C  cd  1.63  ROOT(g )  a  202  a  41.4  a  0.51  a  175  a  36.3  a  0.54  a  a  176  a  48.0  a  0.40  a  a  214  a  54.3  a  0.53  a  b  59.9  a  1.80  b  b  2.52  b  b  1.96  b  1.76  c  a  b  b  The data points shown i n the table are the means of 10 plants. Analysis of Variance was used f o r determining s t a t i s t i c a l differences. Values bearing the same l e t t e r with 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.  39  Under short days no s i g n i f i c a n t differences between CO^ treatment groups were found, although i t should be noted that the mean shoot weight tended to increase i n seedlings grown under 5.0% CO^. Root weight showed l i t t l e response to CO^ enrichment. Under long days s i g n i f i c a n t 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  s i g n i f i c a n t l y higher under 0.1% CO^ than under any other C0  2  concentration.  Root weights were not s i g n i f i c a n t l y d i f f e r e n t from one another, but the highest mean weight was associated with plants grown under 0.1%  co . 2  Carbon dioxide and photoperiod quantitatively  influenced seedling weight both  and q u a l i t a t i v e l y (Table XIV).  F i r s t , t o t a l plant  weight was greater under long days than i t was under short days. Second, biomass d i s t r i b u t i o n was d i f f e r e n t under long days than under short days; under long days roots accounted f o r half of the t o t a l plant weight while under short days roots accounted f o r only one-fourth of the t o t a l plant weight.  Third, 0.1% C0  proportions of biomass distributed  2  increased the r e l a t i v e  to the roots.  biomass were accompanied by corresponding  Increases i n root  decreases i n biomass d i s t r i b u t e d  to leaves, even though leaves accounted for noticeable absolute shoot weight increases (Table X I I I ) .  Biomass d i s t r i b u t i o n i n stems and branches  appeared to be r e l a t i v e l y constant and unaffected by either or C0„.  daylength  TABLE XIV BIOMASS DISTRIBUTION DOUGLAS FIR (6-month-old seedlings) Grown f o r s i x weeks under designated daylength/CO- combinations -2 Irradiance = 7.2 mW cm 1  RATIO TO TOTAL PLANT WEIGHT TREATMENT SHOOT Daylength C02 cone, 'TOTAL (h) (%)  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 r a t i o s shown i n the table are the means of 10 plants.  41  3. Summary: E f f e c t s of carbon dioxide and daylength on weight (a) Leaves Carbon dioxide enrichment p r i m a r i l y affected shoot weight of 1-year-old trees, and e s p e c i a l l y a f f e c t e d the weight of leaves as can be seen when weights measured a f t e r 90 days of long days (Table X) are compared with weights measured a f t e r 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 e f f e c t s of enrichment were apparent a f t e r only 21 days, whereas under long days i t took much longer f o r demonstratable weight gains under 0.1% CO^.  The f a c t that short-day trees were maintained  under twice the i r r a d i a n c e of long-day trees may account f o r 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 l e v e l s should be  maintained a t high enough l e v e l s to r e c e i v e the f u l l b e n e f i t s from enrichment. A comparison between the l e a f growth of 1-year-old and 6-monthold trees may also be made to examine the e f f e c t s of age on p l a n t response. (Tables V I I and X I I I ) .  When the same daylengths and CO^ l e v e l s  are compared (short and long days with 0.03% and 1.0% CO^), s i m i l a r trends can be seen i n responses to CO^ enrichment.  For p l a n t s of both  ages, the lowest leaf weights occurred when the trees were given low CO^ and short days, while the highest l e a f weights occurred when plants were given high CO  and long days.  The r e s u l t s i n d i c a t e that the  response of Douglas f i r to C0 enrichment and daylength i s consistent o  with age, at l e a s t f o r the age range used i n t h i s 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 2 (Tables V I I and X ) .  i n both experiments 1 and  However, stem weights of 0.1% CO^ plants grown  i n experiment 2 were s i g n i f i c a n t l y d i f f e r e n t or high  plants.  from weights of normal  CO^ l e v e l s used i n experiment 1 (0.03% and  1.0% CC^) apparently d i d not stimulate stem growth, and thus i t appeared that CO^ d i d not a f f e c t stem weight u n t i l the intermediate l e v e l was used i n experiment 2. Six-month-old seedlings followed the p a t t e r n observed f o r 1-year-old trees (Table X I I I ) .  Stem weights of p l a n t s 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 a f f e c t e d by e i t h e r daylength or CO2 enrichment (Tables V I I 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 l e v e l s 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 l e v e l s had s i g n i f i c a n t l y more biomass i n the shoot than trees under the other three CO2 - daylength combinations (Table V I I I ) .  In experiment 2, p l a n t s grown under long  43  days and intermediate CO^ l e v e l s showed greater shoot production than the other treatments (Table IX).  For younger trees photoperiods were  more important than CO^ l e v e l s i n influencing biomass d i s t r i b u t i o n (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 i n v e s t i g a t o r s have found that l e v e l s of 0.1% CO^ are the most favourable f o r crop plants (84), and on the b a s i s of the r e s u l t s reported i n t h i s chapter i t appears that 0.1% CO^ (1000 ppm) i s also the best l e v e l f o r 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  i n 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 l e v e l s 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 V I I , this chapter; E.B. Tregunna, unpublished r e p o r t ) . Tomatoes and other vegetable crops have been grown at concentrations as high as 30,000 ppm CO^ with no i l l e f f e c t s (37). Seedlings did not respond as w e l l to CO^ enrichment as 1-yearold trees.  Although enriched seedlings had mean weights greater than  those of unenriched seedlings, s i g n i f i c a n t d i f f e r e n c e s could be shown only on the basis of daylength and not on the basis of CO^ l e v e l (Table X I I I ) .  However, Tanaka was able to increase shoot dry weights  of Douglas f i r seedlings by 37% and root dry weights by 66% by e n r i c h i n g the greenhouse to 1500 ppm C0  2  (72).  He a l s o found that age a f f e c t e d  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 p i n e ) ; seedlings which were s i x months old at the end of treatment responded l e s s favourably to C0 than d i d four-month-old seedlings. o  Increased age  45  could account f o r the lack of seedling response i n my study since trees were approximately eight months o l d 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 a d d i t i o n , dioxide enrichment seems to be most  e f f e c t i v e when given during a period of r a p i d 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 t h e i r growth period.  However, growth enhancement due to  CO,, enrichment was evident i n s p i t e 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 f o r plants grown under long days and normal a i r (Tables IV and V I I I ) .  Growth I n h i b i t i o n Carbon dioxide concentrations higher than 0.1% CO,, g e n e r a l l y are i n h i b i t o r y (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 e f f e c t s from the higher C0 concentrations (Table IV). 2  Regardless, others have reported negative  effects of high CO,, l e v e l s on some other p l a n t s .  Cucumber p l a n t s  suffered c h l o r o s i s and l e a f 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 i n leaf area and weight may constitute another potential problem with CO,, enrichment.  An increase i n leaf weight  without a corresponding increase i n leaf area may cause a decrease i n overall growth since photosynthesis may eventually be restricted by the area available for energy capture i f dry matter i s 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 C0  2  (29, 30); tomatoes at 1000 ppm C0  2  (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  47  showed depressed growth rates after several weeks despite higher net assimilation rates (31). However, i t should be noted that carbon dioxide i s not the only factor which may  reduce leaf area:weight r a t i o s i n Douglas f i r .  found that high l i g h t i n t e n s i t y also decreased relative to the dry matter produced (8).  Brix  the amount of leaf area  Thus l i g h t can i n many cases  mimic the effects of CO^ on growth and development.  This point w i l l  be discussed i n a l a t e r section dealing with CO^ i n t e r a c t i o n s .  Growth and Biomass D i s t r i b u t i o n Reports of carbon dioxide e f f e c t s on growth and biomass distribution have been contradictory.  I found that the  Douglas f i r enrichment a f f e c t shoots more than roots.  1-year-old Root weights  remained a r e l a t i v e l y constant proportion of t o t a l plant weight so that differences i n shoot:root r a t i o r e f l e c t e d increases i n the proportion of assimilate going to the shoot rather than decreases i n amounts going to the root (Tables IX and X). For younger trees photoperiods  were more important  than CC^  levels i n influencing growth and biomass d i s t r i b u t i o n (Tables XIII, XIV).  However, CO^ did have some effect on biomass d i s t r i b u t i o n .  Seedlings under the same daylength grown under intermediate CO^  levels  had r e l a t i v e l y more biomass i n their roots than did seedlings grown under other C0  0  levels and the same daylength.  (Table XIV).  48  Therefore, at l e a s t i n t h i s study CO^ enrichment of 1-yearold Douglas f i r appears to p r i m a r i l y a f f e c t 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 e f f e c t upon the upper plant p o r t i o n .  However, i n  6-month-old Douglas f i r CO^ enrichment a l s o changed the r e l a t i v e d i s t r i b u t i o n of biomass w i t h i n the p l a n t , thus i t i s p o s s i b l e that CC^ might a f f e c t t r a n s l o c a t i o n of organic products as w e l l as a f 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 ( c o n i f e r s , 71, 72; b a r l e y , sugar-beets, k a l e , 21).  Tregunna found that 1.0% CO^ combined with high nitrogen l e v e l s  s u b s t a n t i a l l y promoted both root mass and root elongation of Douglas f i r seedlings (unpublished r e p o r t ) .  He f e l t , however, that the  temperature under which plants were grown was more important than CO^ i n c o n t r o l l i n g shoot and root growth.  Tregunna a l s o noted that  root or shoot growth u s u a l l y would occur at the expense of the other plant part; shoot and root weights d i d not increase simultaneously. Wittwer showed f o r beans and tomatoes that CO^ a f f e c t e d roots more than shoots (84). He suggested that the rapid recovery observed a f t e r p l a n t s had been transplanted might be due to root growth stimulated by carbon dioxide.  I f Wittwer's observation i s c o r r e c t , pre-treatment w i t h high  CO^ l e v e l s could conceivably prove u s e f u l i n conditioning plant m a t e r i a l preparatory to rooting of cuttings and i n improving drought r e s i s t a n c e .  49  In fact CO^enriched atmospheres and carbonated mists have been reported to improve rooting of cuttings (50). Treated c u t t i n g s 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 f a s t e r and produced more roots per c u t t i n g than controls without CO^ treatment. A t h i r d view i s that CO^ does not a f f e c t biomass r e l a t i o n s h i p s and weight d i s t r i b u t i o n remains the same before and a f t e r CO^ treatment. -2 For instance when tomatoes were grown under low l i g h t (59 J cm  -1 day  )  carbon dioxide d i d 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 r e s u l t s of the reports c i t e d above i t appears that biomass d i s t r i b u t i o n i s not a p r e d i c t a b l e measure of carbon dioxide e f f e c t s on plant growth.  In a d d i t i o n , other v a r i a b l e s  must be considered when making comparisons of how CO^ a f f e c t s growth. These variables include the plant species being compared, the e f f e c t s 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 f a c t o r s Plant response as we perceive i t experimentally i s the cumulative r e s u l t of factors which we have chosen to manipulate, the i n t e r a c t i o n s of those factors, plus other f a c t o r s over which we may or may not have control.  Some of the e f f e c t s of increasing the carbon dioxide  concentrations around the plant are known, but there are probably other factors of which we are not yet aware. phenomena, CO  A l s o , as with most other b i o l o g i c a l  e f f e c t s can be modified by i n t e r a c t i o n s with other  50  environmental f a c t o r s .  L i g h t , 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 i n t e r a c t with the e f f e c t s of carbon d i o x i d e .  Light L i g h t , both i n t e n s i t y and duration, has o f t e n been included as an a d d i t i o n a l v a r i a b l e i n CO^ enrichment s t u d i e s .  An i n t e r e s t i n g  carbon d i o x i d e - l i g h t i n t e r a c t i o n i s the f a c t that carbon d i o x i d e can i n part s u b s t i t u t e f o r 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 t o 10.0 mW cm i n which the rate of photosynthesis  can be increased by e i t h e r an  increase i n l i g h t i n t e n s i t y or an increase i n CO^ concentration (23). -2 For example, doubling the l i g h t i n t e n s i t y from 2.6 to 5.4 mW cm affected dry weight, l e a f area, and net a s s i m i l a t i o n rates, and was equivalent to a tenfold increase i n CO^ concentration barley, k a l e , and maize, 21).  (sugar-beets,  Even when l i g h t i s l i m i t i n g crops can  respond to CO^ enrichment; a d d i t i o n of (X^ to greenhouse crops i n mid-winter was found to p a r t i a l l y compensate f o r reduced due to lack of l i g h t (85).  photosynthesis  Just as CO^ can a t times s u b s t i t u t e f o r  l i g h t when l i g h t i s l i m i t i n g , so a l s o can l i g h t i n some cases demonstrate effects which mimic the e f f e c t s of CO,, enrichment; e.g., e i t h e r increased CO2 or increased l i g h t reduced l e a f 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 a l s o increases photosynthetic  light  saturation l e v e l s , 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 t h e i r maximum (57).  Even i n cloudy weather however, there i s no evidence that d a y l i g h t  51  i s so poor that plants cannot u t i l i z e a d d i t i o n a l 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 l i g h t i n g of 700 f t - c f o r 16 hours a day stimulated the growth of s i x conifer species more than when only 0.15% CO^ was used. However i f both treatments were used i n combination, the a d d i t i v e effects of CO^ and l i g h t could reduce the time required to produce a f a l l crop by two months (72). There was a strong p o s i t i v e i n t e r a c t i o n between l i g h t and CO^ when chrysanthemums were grown under a range of -2 light i n t e n s i t i e s (125 to 375 J cm concentrations  -1 8-hr day  (0.033% to 0.15% CO^).  ) and carbon dioxide  Plants showed greater f i n a l  dry weights and flower dry weights at the highest l i g h t i n t e n s i t y and highest CO,, l e v e l (30).  Other p o s i t i v e interactions of l i g h t  (both  daylength and l i g h t i n t e n s i t y ) with carbon dioxide could be seen i n 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 l i g h t i n t e n s i t y may be even more e f f e c t i v e than daylength i n promoting CO^-enhanced growth. Short-day plants which received fewer hours of l i g h t each day but twice the l i g h t i n t e n s i t y of long-day plants were able to respond more quickly to (Table X and XI, discussion, p.  41 ) .  treatment  52  Temperature Temperature can also play an important part i n CO^ enrichment programs.  At l i g h t s a t u r a t i o n and low CO,, l e v e l s  (0.03% CO,,) photo-  synthesis i s almost temperature independent, but at elevated CO,, l e v e l s photosynthesis may be l i m i t e d by temperature, or more e x a c t l y by the capacity of the biochemical process.  The above has been shown f o r  cucumber by Gaastra (23) and f o r Pinus halepensis by Whiteman and R o l l e r (81). In tomatoes  supplemental carbon dioxide had the greatest  effect when supplied w i t h i n the temperature range of 15 to 25 C, but the most s u i t a b l e temperature w i t h i n 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 f o r 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 ) temperature influenced 2  the r e l a t i v e amounts of biomass going to the shoot or the root (70), and thus temperature might i n f l u e n c e 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 t h i s species i t should be considered i n any future work.  Inorganic n u t r i t i o n The a c c e l e r a t i o n of growth and development induced by CO,, may cause inorganic n u t r i e n t s to l i m i t the growth of enriched p l a n t s (84).  Soybeans were enriched w i t h 0.14% CO,, and 21 days l a t e r began to  show signs of nitrogen d e f i c i e n c y (15). I n Douglas f i r h i g h n i t r o g e n was combined with high C0  2  l e v e l s (0.1% and 1.0% C 0 ) ; t h i s experiment 2  resulted i n greatly improved root mass and root elongation (E.B. Tregunna,  53  unpublished data).  However, t o t a l dry weight d i d not appear to be  s i g n i f i c a n t l y affected by the d i f f e r e n t nitrogen and carbon dioxide combinations; s i g n i f i c a n t differences i n t o t a l dry weight were due to CC^ levels only. In another study of Douglas f i r grown under normal a i r , biomass d i s t r i b u t i o n 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 i n d i r e c t effects on CO^-enriched plants.  Implications It i s generally accepted that atmospheric CO^ l e v e l s are l i m i t i n g to growth.  Bonner, f o r instance, computed that the t h e o r e t i c a l  efficiency of l i g h t u t i l i z a t i o n could be doubled i f CC^ were not l i m i t i n g (7). Most p l a n t s . w i l l benefit from small amounts of supplemental 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 i n both North America and Europe.  Carbon dioxide i s usually  added to the a i r by burning propane and other gases cr by slowly releasing pure CO^ through small perforations i n 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 r e s u l t i n the detrimental accumulation of heat and humidity i n the greenhouse. increasing atmospheric CO  Other methods for  i n 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 i s being successfully used on flower and vegetable crops, and i t s use i s expected to increase i n the future.  A notable example  i s the waste-heat greenhouse recently b u i l t i n Saskatoon, Saskatchewan (91).  Exhaust gases from a turbine fired with natural gas were used  for heating and CO^ enrichment i n a polyethylene covered greenhouse. The system resulted i n 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 r e l i a b l y 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 i n several studies even though increased root growth was not demonstrated i n my studies (21, 72, 84). The use of CO^ to increase root size and vigor of seedlings before they are planted i n f i e l d sites would improve both their recovery from transplanting and their resistance to drought.  In sites where i r r i g a t i o n  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 b i o l o g i c a l and p h y s i c a l stresses i n the f i e l d .  F i n a l l y , economic advantages could be r e a l i z e d  from the increased growth and a s s i m i l a t i o n rates of enriched plants because time i n the nursery could be shortened  and  stock-handling  expenses could be reduced. However, there are several precautions regarding the use of enrichment.  The correct choice of CC^ l e v e l i s important  i n the use of  enrichment to induce a desired e f f e c t on growth or development. example can i l l u s t r a t e this point.  CO^  The data i n Figure 3 are  An  fairly  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^ l e v e l s i s about the same.  By s e l e c t i n g a CO^ l e v e l which was  e i t h e r too low or  too high one could e a s i l y conclude that carbon dioxide had no e f f e c t on the parameter under consideration. analyzing stem weight data.  This s i t u a t i o n occurred  while  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 e f f e c t .  F i n a l l y , 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 p o t e n t i a l of  enrichment, but how to be tested.  CO^  enriched seedlings perform i n the f i e l d remains  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,, l e v e l s has been noted previously (31, 37, 48, 59, 84, 85), l i t t l e or no systematic  investigatL  has been conducted on t h i s t o p i c . 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 p r i m a r i l y i n t e r e s t e d i n enhancing rather than i n h i b i t i n g growth, and very high CO^ l e v e l s are d i f f i c u l t to maintain i n the greenhouse or i n open growing areas. Nevertheless, the e f f e c t s of very high C 0 l e v e l s are s t i l l of 2  t h e o r e t i c a l and p r a c t i c a l i n t e r e s t .  In 1973 and 1974 s e v e r a l C 0  2  enrichment studies were conducted on western hemlock and Douglas f i r . The trees were grown f o r three months i n c o n t r o l l e d environment chambers and were enriched w i t h very high C 0 l e v e l s (E.B. Tregunna, unpublished 2  data).  A d d i t i o n a l plants were grown under low and intermediate C 0  levels.  2  Enrichment by intermediate CO,, l e v e l s enhanced growth while  high C 0 levels appeared to l i m i t growth. These r e s u l t s were s i m i l a r 2  to those reported i n Chapter one.  However, high C 0 l e v e l s a l s o 2  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 o f t e n 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 C 0 l e v e l s (Chapter One) o  57  suggested that CO^ might effect developmental growth.  processes other than  However, before I proceed further i n discussing the influence  of CCv, on development, I should elaborate on the usual patterns of growth i n Douglas f i r . Obvious seasonal growth of Douglas f i r and most other temperate trees i s 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 quiescent  this  (temporary) bud may burst and another period of internodal  elongation may begin. Douglas f i r often flushes twice during one growing season, and i n 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 l a t e r a l buds (buds along branches).  Buds formed l a t e i n the f a l l do  (  not demonstrate this repeated flushing behaviour, and they do not f l u s h even when given favourable growing conditions. These buds are c a l l e d resting buds because they usually occur when the plant i s i n true winter rest (32, 56). In nature, short autumn daylength i s an important  environmental  signal which acts to induce dormancy and budset i n woody species. However, high carbon dioxide levels can also induce budset. factors which may promote the formation of buds.  Thus there are two As a test of which of  these two factors was the most i n f l u e n c i a l , experiment  1 was performed.  58  The objective of t h i s experiment was to e s t a b l i s h the r e l a t i v e effectiveness of photoperiod and CO^ l e v e l s f o r inducing budset i n Douglas f i r .  Thus p l a n t s were grown under short and long days with  low and high CO^ l e v e l s .  In a d d i t i o n , the experiment attempted to  resolve whether high CO^ l e v e l s alone were s u f f i c i e n t f o r bud i n d u c t i o n or i f high CC^ l e v e l s were only e f f e c t i v e i n conjunction w i t h short days. In the second experiment bud development was observed over a three-month period.  The purpose of the experiment was to a s c e r t a i n  (1) whether the buds formed under high CO^ were quiescent (temporary) or r e s t i n g (overwintering) buds, and (2) whether f l u s h i n g would resume once buds were formed under elevated CO^ concentrations. In t h i s experiment plants were grown under long days and low, intermediate, and high CO^ levels.  Long days were chosen s i n c e bud formation had occurred under  16-hour days i n the previous experiment and because f l u s h i n g would be more l i k e l y to occur under long photoperiods.  The r e s u l t s of experiment  2 would possibly give some i n d i c a t i o n of the extent of bud formation and the degree of f l u s h i n g under various CO^ l e v e l s .  Yet the type  of buds being formed under high CO^ l e v e l s s t i l l had to be d i s t i n g u i s h e d . In order to make t h i s d i s t i n c t i o n i t would be necessary to establsh that CC>2 was inducing true r e s t (dormancy which cannot be broken by the usual environmental s i g n a l s promoting growth (80)) i n a d d i t i o n to inducing budset. Under n a t u r a l conditions a c c l i m a t i o n to winter r e s t i s a gradual and continuous process.  However, f o r explanatory purposes  acclimation and dormancy have been separated i n t o three phases (80) .  59  The f i r s t stage i s induced by short days with r e l a t i v e l y warm temperatures.  Plants are only moderately  f r o s t r e s i s t a n t during this  period, yet additional resistance of even a few degrees i s important as i t may mean the difference between death and s u r v i v a l .  Metabolic  a c t i v i t y i n i t i a t e d 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. stage of acclimation.  Low  temperatures  induce the second  The hardiness achieved i n stage two i s s u f f i c i e n t  for most plants i n cold climates to survive the s e v e r i t i e s of winter. However, some plants enter a t h i r d stage of acclimation which i s a greater deepening brought about by even lower temperatures. acclimated to this t h i r d stage, species are able to withstand low temperatures  (-50  Once extremely  C and below) and to recover without damage.  In a standard test to assess true dormancy the temperature of a plant i s f i r s t lowered to values below 0 C, and then slowly raised. The amount of "hardiness" the plant i s said to possess i s 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 i s 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 i n true rest. study was conducted  A preliminary  to test the hardiness of CO^-enriched  f i r were pre-treated with either normal a i r or high CO^ exposed to very low temperatures  (-10  C and -20 C) .  trees.  Douglas  l e v e l s and  then  Plants pre-treated  with high CO^ had higher s u r v i v a l rates and exhibited less damage than plants pre-treated with normal a i r . 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 d i f f e r e n t photoperiods and carbon dioxide l e v e l s to determine which treatment combinations would be most e f f e c t i v e i n promoting CO^-induced freezing resistance.  The effects of d i f f e r e n t pre-treatment temperatures were  also examined. F i n a l l y , the persistence of (X^ induced freezing resistance -  was investigated. The degree of freezing resistance remaining a f t e r plants were removed from enrichment and the use of s p e c i a l posttreatments were also examined. The results from the experiments reported i n this chapter indicated that carbon dioxide affected developmental processes other than those promoting growth. Previously the manipulation of growth and development i n 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^ concentrations could be used alone or i n combination with other treatments; and thus  could p o t e n t i a l l y prove to be a worthwhile tool i n the  experimental control of l i f e cycles.  MATERIALS AND METHODS  Plant Material The experimental material used i n this research was Douglas f i r from coastal provenances of B r i t i s h Columbia and Washington.  61  Material which i s not o r d i n a r i l y f r o s t hardy was used for dormancy and hardiness studies since treatments on naturally hardy material could be inconclusive.  Inland v a r i e t i e s are adapted to r e l a t i v e l y  extreme continental climates and consequently have greater f r o s t and drought resistance.  Pseudotsuga menziesii of coastal regions has only  a limited frost and drought hardiness, a l i m i t a t i o n which i s common among plants of maritime climates (58). Additional information on plant material can be found i n the Materials and Methods of Chapter one.  Experimental Conditions One-year-old  trees were grown i n 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 l e v e l s of 0.03% and 1.0% C0 , and with 2  -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 i n the Appendix, Table B. In experiment 2 trees were grown for 90 days under 16-hour days (25 C/ 20 C), with three C0 concentrations (0.03%, 0.1%, and 1.0%'C0 ), and -2 2  with irradiance of 3.4 mW cm  2  .  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^ l e v e l s was r e l a t e d to increased f r o s t hardiness.  Trees were grown under short, warm days (8-hour  days with 20 C/15 C) f o r three months w i t h e i t h e r 0.03% or 1.0%  C0 . 2  The trees were then transferred to short, cold days (5 C/5 C) f o r 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 a f t e r 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 w i t h a d d i t i o n a l work on budset. Experiment 5 was designed to determine which combinations of photoperiod and carbon dioxide would be most e f f e c t i v e i n promoting C0 -induced freezing r e s i s t a n c e . 2  The schematic diagram i n the Appendix,  Table C may be u s e f u l 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  l e v e l s (0.03%, 0.1%, 1.0%, and 5.0% C 0 ) . 2  At the end of s i x  weeks sub-samples from each of the photoperiod and C0 removed and given freezing t e s t s .  2  treatments were  The remaining plants were t r a n s f e r r e d  to a four week post-treatment i n which they were grown under 8-hour days and normal a i r . respectively.  Day and night temperatures were 25 C and 5 C 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  e s t a b l i s h how long CO -induced f r e e z i n g r e s i s t a n c e would p e r s i s t once  63  the plants were removed from enriched atmospheres.  Freezing t e s t s  a f t e r termination of post-treatment would demonstrate i f there were interactions between pre-treatments  and post-treatment, and i f these  i n t e r a c t i o n s a f f e c t e d the degree of hardinss. indicate whether pre-treatment  Freezing t e s t s would also  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 o b j e c t i v e was to determine how temperature a f f e c t e d  hardiness and budset.  In a d d i t i o n , the c o n t r o l s would be used to  gauge whether carbon d i o x i d e or temperature was more e f f e c t i v e i n increasing f r e e z i n g r e s i s t a n c e . • Seedlings were frozen at two d i f f e r e n t temperatures (-6 C and -10 C), f i r s t a f t e r s i x weeks of pre-treatment, then a f t e r four weeks of post-treatment, and f i n a l l y a f t e r s i x weeks of post-treatment. Records were a l s o kept of f l u s h i n g and budset. Trees were scored using the scales d e t a i l e d i n the Appendix, Table B.  Freezing t e s t s Plants to be frozen were stored i n a cold room a t 5 C f o r at least 12 hours p r i o r to t e s t i n g .  These p l a n t s were then placed i n  a freezer chest with i n s u l a t i n g m a t e r i a l surrounding the roots.  They  were then cooled at a r a t e 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 i n 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 v a l i d i t y of the assigned scores.  The  scores were then analyzed by means of a nonparametric analysis of . variance, the Kruskal-Wallis H s t a t i s t i c , which i s described i n the next section.  Kruskal-Wallis One-way Analysis of Variance for Ranks Most of the.data in this chapter i s 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 i s the preferred nonparametric s t a t i s t i c to  use when ranking information is available, and since i t uses a l l the order information, i t i s a more powerful tool than other nonparametric tests.  65  The b a s i c premise b e h i n d t h e K r u s k a l - W a l l i s a n a l y s i s i s t h a t i f a l l the t e s t groups c o n t a i n random samples  from t h e same g e n e r a l popu-  l a t i o n , the average rank i n each group s h o u l d be about t h e same s i z e as the  average rank i n e v e r y o t h e r group.  F u r t h e r , the o v e r a l l average  rank  should be about t h e same as t h e average rank f o r each group, and t h e r e f o r e , the  expected average rank can be compared w i t h t h e o b s e r v e d a v e r a g e rank.  I f the n u l l h y p o t h e s i s i s t o be t e s t e d , t h e s t a t i s t i c the  c h i - s q u a r e d i s t r i b u t i o n w i t h k - 1 degrees  "H", w h i c h  o f freedom,  follows  c a n be computed  as f o l l o w s :  k H =  R. 2  1 2  - 3  1  N ( N + 1 )  i  J = 1  i  n  L  ( N + 1 )  .  j  where N  i s t h e t o t a l number o f c a s e s i n a l l groups  Rj  i s t h e sum o f ranks i n the j t h group  . n^ k  i s t h e number o f c a s e s i n t h e j t h group i s the number o f groups  I n a computation o f t h e K r u 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 r e p l a c e d by a rank; i . e . , a l l s c o r e s from a l l t h e groups a r e combined a n d ranked i n a s i n g l e  series.  The s m a l l e s t s c o r e i s r e p l a c e d b y r a n k 1 '  and t h e n e x t t o s m a l l e s t by r a n k 2 u n t i l a l l s c o r e s h a v e b e e n r a n k e d . Once all  s c o r e s have been a s s i g n e d r a n k s , t h e sum o f t h e r a n k s i n e a c h  group i s found. The K r u s k a l - W a l l i s  t e s t d e t e r m i n e s whether  ranks are l i k e l y  the same p o p u l a t i o n , o r w h e t h e r t h e  to have come from  sums a r e so d i f f e r e n t populat ions.  that  t h e sums o f  they more l i k e l y have come from s e p a r a t e  66  Ranking Scales Data used to assess bud development, date of budset, and f r o s t damage were derived from ranks described i n the Appendix, Table B. Before raw data could be analyzed by means of the K r u s k a l - W a l l i s H s t a t i s t i c , the raw data had to be transformed i n t o a s u i t a b l e form. An i l l u s t r a t i o n of the procedure follows using the data f o r 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 a t 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 d i d not f l u s h during the 45-day treatment p e r i o d , while a rank of '11' was assigned to plants which d i d not set bud during the treatment p e r i o d . The Kruskal-Wallis H t e s t was performed on the data from a l l p l a n t s . 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) f o r a l l plants i n each group was c a l c u l a t e d ; and f i n a l l y the group rank sums were analyzed t o determine i f there were d i f f e r e n c e s between the groups. These are the r e s u l t s which appear i n Table XX.  In the t a b l e , treatments  have been reordered according to the rank they received i n the K r u s k a l 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 s e t bud at an early date, while a high rank i n d i c a t e s that a greater proportion of the plants i n the group set bud at a l a t e r date.  67  Data f o r bud development were handled i n a s i m i l a r manner except that low scores f o r bud development i n d i c a t e d a greater degree of budset than that f o r high scores.  For f r o s t damage low  scores  indicated less damage and higher s u r v i v a l than that f o r high  scores.  Amino a c i d a n a l y s i s Ethanol extracts of needles were made according to the procedure of Salminen and K o i v i s t o i n e n (67) and were then dried i n a f l a s h evaporator.  Five ml of pH 2.2 b u f f e r was f i r s t added to the d r i e d  residue, the s o l u t i o n was  then centrifuged f o r 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 a c i d analyzer.  RESULTS A. FLUSHING AND BUDSET 1. One-year-old trees (a) Effects of 8- and 16-hour daylengths and 2 CO^ l e v e l s Trees given high than trees given low CO^  under both daylengths flushed e a r l i e r  (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 d i d not set w i t h i n 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 f o r 30 days under 8- or 16-hour daylengths with either 0.03% C0 or 1.0% C0 2  2  -2 Irradiance = 3.4 mW cm  TREATMENT Daylength (h) C0 cone. (%) 2  16  RANK ^ SUM  NUMBER OF BUDS SET (N = 80)  1.0  11326  43  8  0.03  12468  30  8  1.0  14984  27  0.03  16167  9  16  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 d i s t n . with df, 3) = 0.0000 Rank sums shown i n the table are based upon ranks of 20 plants and are i n order by increasing rank sum.  69  grown under long days and high C0 showed the. greatest degree of budset 0  (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 i n a l l treatments began to flush soon after the beginning of experiment (Table XVI). By 40 days plants i n a l l treatments began to reset buds although significant differences were not noted u n t i l 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 i n budset during the remainder of the measurement period. (c) Effects of 8-hour daylength and four CO^ levels At soon afterward  the start of the experiment a l l plants had buds, but 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 C0 , and the degree of budset successively decreased 9  70  TABLE XVI DEGREE OF BUDSET IN DOUGLAS FIR TREES (1-0 Stock) Grown under 16-hour daylengths w i t h 0.03%, 0.1%, or 1.0% C0  2  -2  Irradiance = 3.4 mW cm  NUMBER OF BUDS SET TREATMENT 56 Days  67 Days  73 Days  80 Days  0.03% CO, 0.1% C0  r  1.0% CO, Goodman's & Kruskal's Gamma P r o b a b i l i t y of Gamma  16  0.56 0.007  16  1.00 0.001  16  1.00 0.005  16  1.00 0.001  When two i n d i v i d u a l s are. chosen at random from the p o p u l a t i o n , 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 p l a n t s .  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  0.03% C0 0.1% c o  2  1.0% c o 5.0% C0  2  2  2  Goodman's & Kruskal's Gamma Probability of Gamma  7 Days  21 Days  28 Days  16  0  14  16  16  0  9  16  16  0  7  15  16  -  * 0  0  0  -  0.81 0.000  1.00 0.000  shoots burst bud, but d i d 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 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 16 plants.  72  as CC^ concentrations increased.  Under long days however, budset was  the greatest under the highest CO^ concentration (1.0% CO^); the r e s u l t s of CO^ enrichment on budset under short days were thus opposite to the r e s u l t s obtained under long days.  I t i s suspected  that these  r e s u l t s may be anomolous due to the p o s s i b l e contamination  of the gas  supply since plants grown under 5.0% CO^ were arrested during the f i r s t week of f l u s h i n g and were not able to proceed w i t h development. Carbon dioxide also influenced the rate at which buds were s e t (Table XVIII).  More plants set buds at e a r l i e r dates when treated  with low l e v e l s of (X^ (0.03% ^ ^ ) . c  Budset tended to occur l a t e r as  concentration increased i n the atmosphere around the p l a n t , and with 5.0% CO2 l e v e l s , no buds were set w i t h i n the measurement p e r i o d .  2. Six-month-old seedlings (a) General f l u s h i n g and budset behaviour Under short days, there were no s i g n i f i c a n t d i f f e r e n c e s i n the rates of budset among seedlings treated with d i f f e r e n t l e v e l s of CO2. However, there were trends which associated budset w i t h CO2 l e v e l s (Figure 6).  Generally the highest C0 l e v e l s (1.0% and 5.0% C0 ) 2  2  promoted buset while l e v e l s of 0.1% CO2 i n h i b i t e d budset.  For example,  t h i s i n h i b i t i o n was evident at 35 days when most other treatments had achieved close to t h e i r maximum degree of budset. CO2 were intermediate i n t h e i r budset response. short days flushed again once buds had been set.  P l a n t s given 0.03% None of the p l a n t s under  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  0.03% C0 0.1%  c o  2  1.0%  c o  2  5.0% C0  2  2  Day 28  No Budset  14  2  0  9  7  0  8  8  0  0  0  16  Goodman's & Kruskal's Gamma =0.85 P r o b a b i l i t y 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 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 16 plants.  Figure 6.  Mean cumulative budset of 6-month-old Douglas f i r continuously supplied with C02 at the l e v e l s shown, and maintained tinder 8-hour photoperiods (25 C/20 C) _altih  Irradiance  o  f 7. 2  mW C U T 2 -  — ,  ,  75  Under long days budset generally occurred l a t e r (Figure 7). Budset peaked sharply at 35 days then quickly declined as plants burst bud and resumed elongation growth.  The o v e r a l l e f f e c t of CO^  was  similar to that observed under short days; plants treated with CO^ had the greatest degree of budset followed i n decreasing by 1.0%  C0 , 2  0.03%  C0 , 2  and 0.1%  5.0%  order  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 e f f e c t 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%  about half the number of buds set as the other three C0 It could be expected that 0.1%  C0  2  2  C0  2  had  treatments.  would be the concentration under  which there would be the least budset since e a r l i e r experiments had shown that 0.1% C0  2  l e v e l was most e f f e c t i v e i n promoting growth (Figures  2, 3, 4; Tables I I , IV, V, X, XI).  (c) E a r l i e s t date of budset of six-month-old  seedlings  A l l plants grown under short days set buds e a r l i e r than those grown under long days (Table XX). budset was  Within daylength groups, the timing of  influenced by the following C0  of effectiveness: 5.0%, treatment was  1.0%,  2  l e v e l s given i n descending order  0.03%, and 0.1%  C0 . 2  the same for both short and long days.  This order of  C0  2  Figure 7.  Mean cumulative budset of 6-month-old Douglas f i r continuously supplied with C0 at the l e v e l s shown, and maintained under 16-hour photoperiods (25 C/20 C) with an irradiance of 7.2 mW cm . 2  -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  0.03% C0  2  8-hour daylengths  16-hour daylengths  50  29  0.1%  c o  2  50  16  1.0%  c o  2  52  29  48  32  5.0% C0  2  The data points shown i n the table are the means of 54 plants.  78  TABLE ,XX EARLIEST DATE OF BUDSET DOUGLAS FIR SEEDLINGS  V  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 Daylength (h) C02 Cone. (%)  ^  S U M  8  5.0  7150  8  1.0  8094  8  0.03  10129  8  0.1  10546  16  5.0  11100  16  1.0  12728  0.03  14033  0.1  14631  16 16  .  *  * According to K r u s k a l - W a l l i s H t e s t (Nonparametric A n a l y s i s of Variance) Kruskal-Wallis t e s t s t a t i s t i c = 60.55 p (assuming chi-square d i s t n . w i t h 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 a i r (0.03% COJ  2~  General flushing and budset behaviour for a l l temperature treatments were similar to those observed for other plants given shortday treatments as noted i n Section 2(a) (Figure 8). Almost t o t a l 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 i n the f a l l and which has been reported to be conducive to budset i n 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 l a s t .  The production  of factors which promote dormancy and budset i n 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% C0 ) eventually 9  Figure 8.  Mean cumulative budset of 6-month-old Douglas f i r grown i n a i r under 8-)•hour photoperiods with day/night temperatures as shown.  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 f r e e z i n g temperatures as low as -10 C survived with only a s l i g h t amount of damage to f o 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 r a t e .  (b) Plants grown under 8-hour days Six-month-old seedlings tested at -5 C a f t e r s i x weeks of CO^ showed the l e a s t damage (lowest rank) i f grown under 1.0% C0 and 2  the greatest damage (highest rank) i f grown under 5.0% C 0  2  (Table X X I I I ) .  Plants grown under 0.03% and 0.1% C 0 had approximately the same rank 2  but 0.03% C0 plants performed s l i g h t l y b e t t e r . 2  Plants tested a t -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 t h e i r ranks followed a s i m i l a r order to the ranks of plants tested a t -6 C.  Plants grown under 0.03%  and 1.0% CO^ exhibited l e s s damage from f r e e z i n g 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 l e a s t amount of cold i n j u r y when tested both at -6 C and -10 C, but only the scores f o r the -10 C t e s t were s i g n i f i c a n t l y different. When the r e s u l t s f o r seedlings under both short and long days and a f t e r being tested to -6 C were compared (Tables XXIII and XXIV), short-day seedlings had lower scores than plants grown under longdays; furthermore, the lowest scores f o r a l l groups tested a t -6 C  83  TABLE XXII FROST HARDINESS STUDY DOUGLAS FIR TREES (1-0 Stock)  FREEZING TEST  CARBON DIOXIDE TREATMENT  Minimum temperature  Duration of min. temp.  0.03% CO,  -20 C  8 hrs.  A l l plants died i n 7 days  50% death i n 75 days  -10 C  1 hr.  A l l plants died  No death at  i n 60 days  1.0% CO,  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 i n 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 f o r 6 weeks with 8-hour days (25 C/20 C)  TREATMENT  1.0%  c o  2  0.03% C0 0.1%  c o  2  2  5.0% CO„ Kruskal-Wallis H * probability  FREEZE TESTING to -6 C  TREATMENT  154  0.03% CO,  180  188  1.0%  c o  2  191  196  5.0%  c o  2  203  282  0.1%  co„  247  8.27 0.041  Kruskal-Wallis H * probability  FREEZE TESTING to -10 C  2.91 0.41  2 Assuming X d i s t n . with df = 3 Rank sums shown i n the table are derived from the KruskalWallis H test (Nonparametric Analysis of Variance) and are i n order by increasing rank sum. The data are based upon ranks of 10 plants.  85  TABLE XXIV  COLD HARDINESS OF C0 -ENRICHED DOUGLAS FIR SEEDLINGS 2  A.  EFFECT OF CARBON DIOXIDE  Grown for 6 weeks with 16-hour days (25 C/20 C)  TREATMENT  RANK SUMS (Frozen to -6 C)  RANK SUMS (Frozen to -10 C)  0.1% CO,  196  0.1% CO,  0.03% CO,  204  0.03%  1.0% CO,  208  1.0% CO,  255  5.0% CO,  213  5.0% CO,  255  Kruskal-Wallis H probability  CO,  55 255  0.13  Kruskal-Wallis H  39.00  0.99  probability  0.000  *  *  TREATMENT  2 Assuming X d i s t n . with df = 3 Rank sums shown i n the table are derived from the Kruskal-Wallis H test (Nonparametric Analysis of Variance) and are i n 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 s i x weeks of pre-treatment plants grown under short days showed s i g n i f i c a n t 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 l e a s t 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 e f f e c t s on hardiness due  to post-treatment could be seen: (1) there was less damage i n a l l CO^ pre-treatment groups and (2) the e f f e c t s of CO^ pre-treatment was no longer readily discernable. Plants tested at -10 C a f t e r s i x weeks of pre-treatment showed no s i g n i f i c a n t differences i n damage and s u r v i v a l between the four CO^ treatments.  Post-treatment had no apparent effect on hardiness of  plants tested at -10 C since there were no s i g n i f i c a n t differences between CO^ groups after four weeks of post-treatment, nor was there a substantial reduction i n 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 co  2  Pretreatment 1.0%  c o  2  0.03% C0 0.1%  c o  5.0% C0  2  2  2  A WEEKS  3  RANK SUM  4  CO Pretreatment  154  0.03% C0  188  1.0%  c o  196  0.1%  c o  282  5.0% C0  6 WEEKS  3  RANK SUM  CO Pretreatment  4  193  0.03% C0  2  194  1.0%  2  205  5.0% C0  228  0.1%  2  2  c o  c o  2  2  2  2  3  RANK SUM  4  109 120 147 186  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 POSTTREATMENT: 6 weeks with 8-hour days (25 C/5 C) and 0.03% CO- only Length of posttreatment Rank assigned according to Kruskal-Wallis H Test (Nonparametric Analysis of Variance) Assuming chi-squared distribution with df = 3 2  3  ^ 5  2  TABLE XXV (Continued) COLD HARDINESS OF CO^ENRICHED DOUGLAS FIR SEEDLINGS B. EFFECT OF POST-TREATMENT  2  FREEZE TESTING TO -10 C  0 WEEKS c o  2  Pretreatment  3  4 WEEKS  RANK SUM  4  c o  2  Pretreatment  3 RANK SUM  180  1.0%  c o  2  191  0.1%  c o  2  203  0.03% C0  247  5.0% C0  Kruskal-Wallis H  2.91  Kruskal-Wallis H  1.99  probability  0.41  probability  0.57  0.03% C0 1.0%  c o  5.0% C0 0.1%  c o  2  2  2  2  4  173 191 2  2  225 231  PRETREATMENT: 6 weeks with 8-hour days (25 C/20 C) and four levels of C0 FOSTTREATMENT: 6 weeks with 8-hour days (25 C/5 C) and 0.03% C0 only Length of posttreatment Rank assigned according to Kruskal-Wallis H Test(Nonparametric Analysis of Variance) Assuming chi-squared distribution with df = 3 2  2  co 0 0  89  (b) Plants grown under 16-hour days Plants previously grown under long days and tested at -6 C after s i x weeks of CO^ treatment showed no s t a t i s t i c a l differences between CO^ treatment groups (Table XXVI).  A f t e r four weeks of post-  treatment with short, warm days and cool nights, there were s t i l l no s t a t i s t i c a l l y s i g n i f i c a n t differences among the groups.  After s i x  weeks' treatment, s i g n i f i c a n t differences due to post-treatment could be found i n 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 s i x weeks of pretreatment, 1.0%  seedlings d i d 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 s l i g h t in their damage scores.  reductions  By s i x 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 d i f f e r e n t .  Most  of the effects of CO^ treatment had been obscured i n 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 a i r had noticeably higher scores than those which had received treatment.  3. Effects of temperature After s i x 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 co  2  Pretreatment 0.1%  c o  2  0.03% C0 1.0%  c o  5.0% C0  2  2  2  3  4 WEEKS  RANK SUM  4  CO Pretreatment  196  0.03% C0  204  5.0% C0  208  1.0%  c o  2  213  0.1%  c o  2  2  2  3  6 WEEKS  RANK SUM  CO Pretreatment  4  140  1.0%  211  0.03% C0  230  5.0% C0  240  0.1%  c o  3  RANK SUM  4  122  2  2  181  2  248  co„  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% C0 only Length of posttreatment Rank assigned according to Kruskal-Wallis H Test (Nonparametric Analysis of Variance) Assuming chi-squared distribution with df = 3 2  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 co Pretreatment 2  0.1% c o  RANK SUM  55  2  0.03% C0  4 WEEKS  2  CO Pretreatment 0.1% c o  2  255  0.03% C0  2  6 WEEKS co Pretreatment  RANK SUM  2  RANK SUM  107  1.0% c o  2  172  211  5.0% C0  2  189  2  194  1.0% c o  2  255  5.0% C0  2  245  0.1% c o  5.0% C0  2  255  1.0% c o  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  p r o b a b i l i t y ~*  0.007  probability  0.148  PRETREATMENT: 6 weeks with 16-hour days (25 C/20 C) and four levels of C0 POSTTREATMENT: 6 weeks with 8-hour days (25 C/5 C) and 0.03% C0 only Length of posttreatment Rank assigned according to Kruskal-Wallis H Test (Nonparametric Analysis of Variance) Assuming chi-squared d i s t r i b u t i o n with df = 3 o  2  N = 10  £  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 i n 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 i n inducing hardiness; and (2) the effects of a previous themoperiod are persistent for two to four weeks after a change i n 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 a i r .  The changes were due to increases both i n acidic  and neutral fractions and i n basic fractions.  Under long days greater  amounts of the following amino acids were found: glutamine, proline, isoleucine, leucine, phenylalanine, h i s t i d i n e 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 t o t a l amounts present.  93  TABLE XXVII COLD HARDINESS OF 6-month-old  DOUGLAS FIR SEEDLINGS  C. EFFECT OF TEMPERATURE  KRUSKAL-WALLIS RANKS AFTER FREEZE TESTING TO -6 C PRETREATMENT 0 WEEKS  2 WEEKS  2  4 WEEKS  25 C/5 C  135  88  157  5 C/5 C  135  177  130  25 C/20 C  195  201  119  8.92  10.21  1.95  0.012  0.006  0.38  Kruskal-Wallis H 3 probability  PRETREATMENT: Grown for 6 weeks with 8-hour days and 3 thermoperiods with 0.03% C0 2  Length of posttreatment. POSTTREATMENT: (25 C/5 C), and 0.03% C0  6 weeks with 8-hour days,  2  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 i n 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 0.03% C 0 asn  36.73  -  thr ser  2  1.0% C 0 9.98  -  16-HOUR DAYS 2  0.03% C 0  2  1.0% C 0  2  5.0% C 0  73.00  98.55  48.17  -  -  -  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  val  0.99  12.16  7.08  10.73  4.80  met  3.88  -  4.16  -  ile  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  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  TOTAL ACIDICS a NEUTRALS  -  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  2  95  DISCUSSION Budset The e f f e c t s of carbon dioxide enrichment on budset complement the effects of carbon dioxide on growth discussed i n Chapter One. was shown to be enhanced by 0.1% CO^ and depressed under high (1.0% and 5.0% CO^), but the C0  ?  Growth CO2  e f f e c t s on budset were the inverse of i t s  e f f e c t s 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^) i n h i b i t e d budset.  Carbon dioxide hastened budset of 6-month-old  seedlings  grown under short and long days i n the f o l l o w i n g descending order: 5.0%,  1%, 0.03%, and 0.1% C0  2  (Table XX).  Daylength d i d not appear to  a l t e r the rate at which buds were s e t , since CO^ a f f e c t e d budset i n the same manner under both short and long days. These r e s u l t s r e v e a l two points about the e f f e c t s 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 d i o x i d e appears to override the photoperiodic c o n t r o l 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 c o n t r o l may be bypassed by many other f a c t o r s , some of which are temperature, l i g h t i n t e n s i t y , n u t r i e n t s , and water supply (56). I t now seems that carbon d i o x i d e a l s o may be added to the l i s t of agents bypassing photoperiodic c o n t r o l . Second, the e f f e c t s of carbon d i o x i d e 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 i n the autumn as plants prepare  for winter rest result i n changes which involve a l l major groups of plant compounds—nucleic acids, proteins, carbohydrates, and l i p i d s (1, 52, 54). Environmental factors which l i m i t metabolic a c t i v i t y also tend to retard induction of dormancy so that plants which are severely depleted i n photosynthetic reserves cannot acclimate (80). On the other hand, environmental factors which enhance metabolic a c t i v i t y tend to promote cold hardening.  McGuire and F l i n t (47) found for four different conifers  that hardening was increased by l i g h t ; they concluded that l i g h t operated via photosynthesis.  Van den Driessche (76, 77) observed that  Douglas f i r seedlings hardened more under short photoperiods i f l i g h t intensity was high, whereas at low l i g h t 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 i n 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).  C0  9  enrichment also promoted budset of 6-month-old seedlings  97  under short and long days when l i g h t i n t e n s i t y was increased.  These  observations indicate that CO^ may be acting v i a photosynthesis to produce effects on budset, and that this i s an a c t i v e process. In experiment 2, budset under 1.0% CO,, was not only quantitatively but q u a l i t a t i v e l y d i f f e r e n t from budset under 0.03% and 0.1% (Table XVI).  Buds formed under low C0  2  (0.03% and 0.1% C0 ) appeared to 2  be quiescent (temporary) buds while those formed under 1.0% C0 to be resting (dormant) buds. C0  2  The number of buds set under  at 56 days was twice that set under the lower C0  In addition, trees with 1.0% C0  2  2  2  /  appeared  1.0%  concentrations.  which had set bud did not break bud  during the period from 56 to 90 days, whereas trees with lower C0 did break bud and flush.  CO^  2  levels  Six-month-old seedlings also showed q u a l i t a t i v e  differences i n budset, but these differences were primarily due to photoperiod 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 treatments began flushing within the next f i v e days. cited above, C0  2  C0  2  In the examples  i n h i b i t e d bud break i n the f i r s t case while short  photoperiods inhibited bud break i n the second case.  These cases may  represent another instance of the carbon d i o x i d e - l i g h t i n t e r a c t i o n discussed at the end of Chapter One; that i s , . C 0  2  can sometimes s u s t i t u t e  for the l i g h t requirement of photosynthesis when l i g h t i s l i m i t e d (23). If the separate i n h i b i t i o n of bud break by high CO,, and by short photoperiods i s the r e s u l t of a C0,,-light i n t e r a c t i o n , then the above cases may be further support for the assumption mediating the effects of C0  9  on bud  development.  that photosynthesis i s  98  The e f f e c t s of temperature on bud formation agree w i t h r e s u l t s previously reported i n the l i t e r a t u r e f o r 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 i n h i b i t e d budset, as a l s o reported by Lavender and Overton (42). Some comparisons can be made between the r e s u l t s mentioned above and those obtained when seedlings which had been treated with various CO2 levels were, maintained not favor budset (Figures 7 and 8).  under conditions which d i d Neither photoperiod nor  temperature favored bud formation since seedlings were grown under long days with warm day and night temperatures (25 C/20 C) . seedlings grown under 5.0% CO2 had set 32 buds .  Yet at 35 days,  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 w i t h cool nights) .  Experimental c o n t r o l of l i f e c y c l e The effectiveness of carbon d i o x i d e , both i n promoting growth and i n promoting cessation of growth, as determined by the concentrations of CO2 used, gives some i n d i c a t i o n of the p o t e n t i a l of (X^ as a t o o l i n the experimental c o n t r o l of l i f e c y c l e s .  The manipulations of growth  and development i n c o n i f e r s have been p r i m a r i l y accomplished by (1) adjusting daylengths and thermoperiods,  or (2) by applying hormones to  various plant parts, or (3) by using a combination methods.  of the f i r s t two  Control of the vegetative c y c l e of Picea abies by r e g u l a t i o n of  daylength and thermoperiod i s an example of the f i r s t approach.  Dormling  et a l (18) were able to c o n t r o l time of budset, s i z e of buds formed,  99  duration of budset, and time, duration, and amount of growth during subsequent flushing period.  The objective of the authors was  the  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 e f f e c t i v e l y  shorten  the length of the juvenile period and hasten the onset of flowering which in Picea.abies does not o r d i n a r i l y 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 . mature, non-flowering  They applied g a b b e r e l l i c acid to sexually  four-year-old grafts (on two-year-old  rootstock)  and s i g n i f i c a n t l y promoted male and female cone production. Carbon dioxide enrichment by i t s e l f other treatments may development.  or i n combination with  provide another method for c o n t r o l l i n g growth and  Intermediate CO^  l e v e l s (0.1% CO^)'!!! conjunction with long  days would allow young seedlings to s u b s t a n t i a l l y increase their height and weight during the growing season.  When plants were ready to be  and stored during the winter months, levels of 1.0% could be used to cease growth and induce budset.  lifted  CO,, and short days  CO^  enrichment could  be used to promote root growth p r i o r to spring planting i n order to help prevent transplant damage and a s s i s t i n the establishment seedlings.  of young  There i s even the p o s s i b i l i t y that carbon dioxide enrichment  could be used to promote d i f f e r e n t i a t i o n of buds into male and female strobili.  Buds resembling  one-year-old  the buds of female s t r o b i l i were produced on  Douglas-fir trees grown under 1.0%  (E.B. Tregunna, unpublished  data).  CO2  for one month  This observation was  not r e p l i c a t e d  however, and i s only mentioned here to indicate what future work i n t h i s area may  include. The use of C0„ enrichment i n combination with hormones,  100  n o n - d e s t r u c t i v e g i r d l i n g , and cone p r o d u c t i o n  o t h e r methods c o u l d  i n g r a f t s as young as  two  perhaps promote  years.  produce v i a b l e seed i n a c o n i f e r i n a p e r i o d  as  The  ability  short  as  two  years would have tremendous importance i n t r e e b r e e d i n g and  to to  three  improvement  programs.  Frost  Hardiness  Before commencing, i t s h o u l d be noted t h a t q u a l i f i c a t i o n s with respect h a r d i n e s s i s based. s c a l e and  therefore  are o r d i n a l i n nature. o r d i n a l d a t a : (1)  (2) judgemental  c l a s s i f y plants possible  the  There a r e  two  possible  error introduced  e r r o r s which o c c u r when one  inherent  c l a s s i f y a l l plants  could  s y s t e m a t i c e r r o r of h a v i n g one skew the d a t a and  To o b v i a t e some of these d i f f i c u l t i e s f o r each f r e e z i n g t e s t and f i n a l rank to each p l a n t apparent.  lead  when one  d i f f e r e n c e s are f a i r l y  conclusions.  a  be  t e s t s which I used  assumptions so e s t i m a t e s of s i g n i f i c a n t  conservative.  plants  I assigned  f r e e z i n g i n j u r y would  In a d d i t i o n , n o n - p a r a m e t r i c s t a t i s t i c a l  f o r data a n a l y s i s make few  It i s  individual  to erroneous  s i x weeks b e f o r e  to ensure t h a t any  tries  discrete  I used a sample s i z e of t e n  waited u n t i l  sources  attempts to c o r r e c t l y  a c c o r d i n g to predetermined ranks or c l a s s e s .  that the  on  t e s t s are based upon a r a n k i n g  to f i t i n j u r y , which i s a c t u a l l y a continuum, i n t o a r b i t r a r i l y ranks; and  certain  to the d a t a upon which t h i s d i s c u s s i o n  Scores f o r f r e e z i n g  of e r r o r i n a n a l y z i n g  t h e r e are  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 i n 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 C0  conditions for increasing hardiness resulted i n the lowest damage  2  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 C0 treatments. As discussed previously (Chapter One) carbon dioxide 2  again appeared to be interacting with l i g h t . In this instance, CO,, seemed to be a p a r t i a l substitute for the reduced daylength which was experienced by short day plants. When plants were grown under long days intermediate CO,, levels were evidently s u f f i c i e n t to provide protection from f r o s t injury, but when plants were grown under short days CO,, had to be increased i n 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 seedlings grown under 2  long days. C0  2  In Chapter One growth was also found to be enhanced by intermediate  levels and long days.  The increase i n hardiness under the same  conditions was probably f a c i l i t a t e d by the reserves acquired during a pretreatment 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 f r o s t 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) d i d appear to be somewhat b e n e f i c i a l to seedlings which had previously been given CO^ enrichment under short days (Table XXV).  The addition of cool night  temperatures f o r s i x weeks resulted i n lower i n j u r y scores f o r a l l groups of plants.  Carbon dioxide treatment e f f e c t s were replaced or  overriden by e f f e c t s of post-treatment so that by six weeks of posttreatment the effects of previous CO^ treatment were no longer discernable. When plants were frozen to - 6 C and - 1 0 C, damage scores were quite variable after s i x weeks' post-treatment (Table XXVI).  Half of  the CO2 treatments showed decreased damage as a r e s u l t of post-treatment, and half of the treatments showed increased damage.  Post-treatment  appeared to be most b e n e f i c i a l to plants pre-treated with 1 . 0 % CO^, and most detrimental to plants pre-treated with 0.1% CO^. C0  2  Intermediate  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 a f t e r freezing to -6 C.  After freezing to - 1 0 C, 0.1% CO2 plants sustained  close to the maximum damage reported for the four groups.  103  R e s u l t s i n T a b l e XXVII d e t a i l  temperature  the data c o n f i r m r e s u l t s r e p o r t e d elsewhere  e f f e c t s on h a r d i n e s s ;  f o r Douglas f i r which  found  that s h o r t days w i t h c o o l n i g h t s were most e f f e c t i v e i n i n d u c i n g f r e e z i n g r e s i s t a n c e and that c o o l days with c o o l n i g h t s d e l a y e d or i n h i b i t e d i n d u c t i o n of h a r d i n e s s which.favor injury  (42).  Generally p l a n t s maintained  i n conditions  growth and n u t r i t i o n e x h i b i t g r e a t e r r e s i s t a n c e to w i n t e r  (56).  Thus p o s t - t r e a t m e n t was  most e f f e c t i v e f o r those p l a n t s  which had been p r e - t r e a t e d w i t h warm days and n i g h t s . post-treatment, they showed the l e a s t i n j u r y and i n scores.  the  The  A f t e r f o u r weeks of  the g r e a t e s t improvement  i n c r e a s e d r e s p o n s i v e n e s s to p o s t - t r e a t m e n t  shown by  p l a n t s p r e v i o u s l y m a i n t a i n e d under warm days and n i g h t s i s l i k e l y due growth temperatures which f a v o r e d metabolism accumulation of r e s e r v e s p r i o r  and which a l l o w e d f o r an  to h a r d i n e s s - i n d u c i n g c o n d i t i o n s .  Conversely, p l a n t s p r e - t r e a t e d w i t h warm d a y s / c o l d n i g h t s (25 C/5 c o o l d a y s / c o o l n i g h t s (5 C/5  C) d i d not show improved  C) c r  s c o r e s as a r e s u l t  o f post-treatment. In summary, (1) p o s t - t r e a t m e n t s o f warm s h o r t days and  cool  n i g h t s , c o n d i t i o n s n o r m a l l y found to be e f f e c t i v e i n i n d u c i n g dormancy and hardiness of Douglas f i r , all  d i d n o t improve f r e e z i n g r e s i s t a n c e of  the Douglas f i r p l a n t s grown under the v a r i o u s CO^,  temperature  p r e - t r e a t m e n t s ; and  (2) the e f f e c t s o f CO2  pre-treatments were u s u a l l y obscured w i t h i n f o u r to s i x weeks.  to  by the e f f e c t s o f  daylength, and  and  daylength  post-treatment  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 i n 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 lipoproteins. Hydroxy1-containing compounds such as sugars act as protective substances through their a b i l i t y to retain or substitute water v i a 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 s t a b i l i z i n g bonds are not broken because sugars do not c r y s t a l l i z e 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 i n the protoplasm or near sensitive s i t e s .  In  dormant tissue the accumulation of sugars and other protective substances i n the protoplasm can occur because metabolism remains low. Heber et a l . (25) later included certain amino acids i n the l i s t of cryoprotectants, and then classified them into three groups based upon their r e l a t i v e 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 a c t i n g to increase f r e e z i n g resistance. I have already i n d i c a t e d that carbon d i o x i d e appears to be a f f e c t i n g hardiness v i a photosynthesis.  I t i s p o s s i b l e that  photosynthesis i s a f f o r d i n g p r o t e c t i o n from f r o s t i n j u r y by i n c r e a s i n g the amounts of cryoprotectants i n the t i s s u e .  As suggested by Heber  et^ a l . (24), the p r o t e c t i v e substances could be elevated amounts of sugars or amino acids which had been formed under carbon d i o x i d e enrichment; but i t seems most l i k e l y that sugars and amino acids are a c t i n g together to increase freezing r e s i s t a n c e .  There are two i n d i r e c t l i n e s  of evidence which i n d i c a t e that amino acids and sugars  do increase  i n carbon dioxide enriched p l a n t s . The f i r s t l i n e of evidence concerns amino a c i d analyses made of ethanol extracts of leaves of one-year-old Douglas f i r (Table XXVIII). Although freezing t e s t s were not performed on the plants used i n the analyses, tests were performed on other plants of the same age i n a p r e l i m i n a r y study (Table XXII) and on 6-month-old seedlings (Tables X X I I I and XXIV). Results of a l l t e s t s suggest that l e v e l s 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 e s t a b l i s h a r e l a t i o n s h i p between amino a c i d l e v e l s and (X>2 enrichment l e v e l s .  Therefore, although t o t a l amino acids increased  under high CO,, l e v e l s i n both short and long days, the amino acids of primary i n t e r e s t would be those which had been p r e v i o u s l y found to be  106  associated with increased cold hardiness. grown under long days and high  Extracts from leaves  contained  isoleucine, leucine, phenylalanine,  increased amounts of glutamine,  h i s t i d i n e , arginine, and p r o l i n e .  For leaves grown under short days and high CO^, increases i n proline and arginine.  there were notable  Increases i n cold hardiness  have been  related to increases i n a l l of the amino acids l i s t e d above (glutamine, phenylalanine,  leucine, and isoleucine i n red o s i e r dogwood, 43;  arginine  and h i s t i d i n e i n winter weat, 55; glutamine and p r o l i n e i n perennial ryegrass, 19). Proline has been s p e c i f i c a l l y mentioned by Heber et a l . (25) as a cryoprotectant.  In addition, p r o l i n e has been frequently mentioned  as an amino acid which accumulates i n large quantities i n plants grown under short days and which also increases i n plants during bud development. Furthermore, proline acts as an i n h i b i t o r to growth and r e s p i r a t i o n (Sergeev and Sergeeva, 1963; Alden and Hermann (1)).  Sergeev, 1964a, and 1964b, as cited by  The r o l e of proline i s s t i l l uncertain, but  the fact that i t so often fluctuates with changes i n environmental parameters does indicate that i t probably performs an important function for the plant. The second piece of evidence concerns l i g h t micrographs which were taken of 1-year-old of  trees.  starch i n needles of plants which had been grown under long days.  The amount of starch increased as CO2 to 1.0% 1.0%  The micrographs showed large accumlations  CO2.  CO2.  Starch at 5.0%  l e v e l s were increased from CO2 was  0.03%  approximately equal to that at  Accumulations of starch and sugar as a r e s u l t of CO2 enrichment  have also been reported i n tomato (46) and i n beans, sugar-beets, and barley (51).  Carbohydrates are very e f f e c t i v e cryoprotectants  (25,  87;  107  spruce and pine, 2), therefore i t may be the accumulation of starch under high CO  l e v e l s that protects CO -enriched plants against  freezing injury.  Short-day plants did not show large starch accumulations  regardless of enrichment l e v e l , but since the photographed plants were not freeze-tested i t i s not known whether they were hardy or not. Conversely, freeze-tested plants were not examined microscopically so i t i s 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.  I f this i s true, some factor other than  sugar may be responsible f o r freezing resistance i n 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 c l o s e l y associated i n time the  phenonmena have been assumed to be causally r e l a t e d , but evidence to the contrary shows that this assumption i s i n c o r r e c t .  In Acer negundo and  Viburnum for instance, f r o s t hardiness was independent of bud dormancy; substantial hardiness  l e v e l s were attained without dormancy development  as a prerequisite(32).  In Douglas f i r , budset was not n e c e s s a r i l y  correlated with freezing resistance (10). complicated  The s i t u a t i o n i s further  by the fact that a l l parts of the plant do not harden at  the same time (56).  Nevertheless,  a comparison of internodal elongation,  bud development, and freezing i n j u r y of 6-month-old seedlings  given  different daylengths, CO^ l e v e l s , and thermoperiods serves to consolidate and summarize some of the data presented thus f a r .  The use of the same  plants throughout these measurements was an advantage because i t  108  increased the v a l i d i t y 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 i n 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 i n 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%  C0 2  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 j u s t 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 i s necessary to establish carbon dioxide-induced resistance to freezing i n j u r y .  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 i n normal a i r but only exposed for short periods to high CO^ levels.  However, some  investigators who were primarily interested i n the growth aspects of CO^ enrichment determined assimilation rates after crops had been grown i n 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 L i s t e r 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  Ill  others who  reported enhanced photosynthesis under high CO^ Nevertheless, there was  l i m i t e d information  levels.  a v a i l a b l e on  the effects of high CO^ l e v e l s on the major components of gas exchange, especially respiration and transpiration. research  reported  i n this chapter was  The major objective of  to determine how  CO^  the  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^ l e v e l s . After treatment for s i x weeks the plants were placed in a cuvette and rates of photosynthesis, transpiration, and r e s p i r a t i o n were determined.  Once these gas exchange rates were known, d i f f u s i o n  resistances could be calculated. The concept of t o t a l resistance i s useful for v i s u a l i z i n g several d i f f e r e n t processes which are at the same time, namely, photosynthesis and  occurring  transpiration, both of  which are affected by changes i n resistance to gas flow. In addition, changes i n t o t a l resistance could possibly explain some of the observed CO^ effects on photosynthesis and  transpiration.  F i n a l l y , the i n t e r -  relationships of the d i f f e r e n t components of gas exchange under enrichment were examined.  CO,,  Knowledge of the manner i n which carbon  dioxide enrichment affected primary components of gas exchange could also lead to a better understanding of CO^ development.  e f f e c t s on plant growth and  112  MATERIALS AND METHODS  Plant M a t e r i a l and Experimental  Conditions  Douglas f i r seedlings were obtained from sources i n the Materials and Methods of Chapter One. 8- or 16-hour photoperiods -2 . . . cm  (25 C/20  described  Plants were grown under  C) w i t h an i r r a d i a n c e of 7.2 mW  and four carbon dioxide l e v e l s (0.03%, 0.1%,  1.0% and 5.0%  C0 ). 2  After s i x weeks plants were removed from C 0 treatment, placed, i n d i v i d u a 2  i n a cuvette, and measured to determine rates of photosynthesis, r e s p i r a t i o n , and t r a n s p i r a t i o n . 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 P l e x i g l a s cuvette, a thermocouple w i t h 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 i n j e c t i o n port f o r adding C0 , 2  and an adjustable flowmeter.  an i n f r a r e d gas analyzer, an a i r pump, Volume of the system was 1.14  including the cuvette volume of 0.79  liter.  liter  R e l a t i v e humidity v a r i e d  from 60-75%, and the flow rate of the gas i n the system was at 4 1/min with a pump and Matheson rotometer.  maintained  L i g h t 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 r a d i a t i o n from the lamps. Radiant energy was measured by means of a YSI-Kettering Model 65 radiometer.  Irradiance was v a r i e d  113  by using neutral density f i l t e r s placed between the lamps and cuvette.  the  Leaf temperature was measured by i n s e r t i n g the welded bead  of a copper-constantan of a needle.  thermocouple j u s t under the surface of the  underside  The reference j u n c t i o n 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 j u n c t i o n s was read w i t h a K e i t h l y Instruments microvoltmeter.  T r a n s p i r a t i o n 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 e x i t i n g the cuvette with an E.G. Model 880 dew-point hygrometer.  & G. I n t e r n a t i o n a l , Inc.  Carbon d i o x i d e concentration was  measured with a Beckman Model IR-215 i n f r a r e d gas analyzer.  Apparent  photosynthesis and t r a n s p i r a t i o n of seedlings was measured i n normal a i r at four d i f f e r e n t i r r a d i a n c e s (15, 26, 44, and 77 mW cm  ). Rates were  measured f i r s t at the highest i r r a d i a n c e and l a s t at the lowest i r r a d i a n c e . After a l l the l i g h t measurements were completed the cuvette was covered w i t h a dark c l o t h and the dark r e s p i r a t o r y r i s e of CO2 was  recorded.  T o t a l D i f f u s i v e Resistance D i f f u s i v e r e s i s t a n c e 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 s e r i e s . The t o t a l resistance to gases entering and l e a v i n g the stomates i s the sum of a l l the resistances i n the path  r  =  total  r  boundary l a y e r  *F  r  stomata  r  mesophyll  Total resistance can be c a l c u l a t e d using the f o l l o w i n g r e l a t i o n s h i p  114  r  total  p  vapor, leaf  water vapor density of absolute humidity (g H^O cm~3) inside the leaf  vapor, a i r  water vapor density (g H2O cm - ) outside the laminar boundary layer of the leaf  P  vapor, leaf vapor, a i r Transpiration rate  (2)  P  where  -  -2 If tranpsiration rate i s given i n units of g cm units of s cm \  3  -1 s,  then  v t o t a  i  has  Leaf area on a projected or planar area i s used i n this  thesis and was determined using a photocell planimeter as described i n the following section.  Since stomata of Douglas f i r only occur on the  lower surface of the needle, leaf area was not doubled i n any calculations. Of the three components of t o t a l resistance to water vapor diffusion, boundary l y a  r  total  o r  t o  r  stomata  a n d  e r w a s  considered to be very small compared to  '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 stomata' mesophyll J  J  r  generally of much more importance to CO diffusion than i t i s to water vapor diffusion.  Therefore, the t o t a l resistance to H^O vapor diffusion  i s primarily an indication of how C0„ affects r . Data obtained i n 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 '' H y assumed to be equal to the saturation water vapor density at leaf temperature. i Su s u a  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 s i x 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 d i f f i c u l t y 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 C0 levels (0.03%, 0.1%, 1.0% and 5.0% CO^. 2  Weekly records  were kept of t o t a l 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 l o s s ; therefore only r e s u l t s from method (2) w i l l be presented.  RESULTS A. APPARENT PHOTOSYNTHESIS 1. Plants grown under 8-hour days and four CO,, l e v e l s Plants grown under short days with normal a i r had s i g n i f i c a n t l y higher photosynthetic rates under a l l irradiances than plants grown under 1.0% or 5.0% C0  (Figure 9 and Table XXIX).  2  Seedlings grown under  1.0% and 5.0% C0 were not s i g n i f i c a n t l y d i f f e r e n t from one another and 2  showed similar photosynthetic  responses to increasing irradiance.  Plants grown under low and intermediate CO^ l e v e l s reached approximately 70% of t h e i r maximum photosynthetic  rates at the lowest  _2 irradiance (15 mW cm been grown.  ) which was closest to that under which they had  Plants grown under 1.0% and 5.0% C0  of their maximum photosynthetic  2  reached about 40%  rates at the lowest irradiance.  None  of plants i n the four CO^ treatments reached l i g h t saturation at the highest irradiance, although seedlings grown under high CO^ (1.0% and 5.0% C0 ) appeared to approach a l i g h t saturation l e v e l . 0  117  Figure 9.  Apparent photosynthesis of 6-month-old Douglas f i r continuously supplied with CO2 at the. l e v e l s shown, and maintained under 8-hour photoperiods (25 C/20 C) f o r s i x weeks with an irradiance of 7.2 mW cm . -2  10.0  20.0  30.0  LIGHT  INTENSITY  40.0 (mW  50.0  60.0  70.0  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 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  XXIX  RATES OF PHOTOSYNTHESIS Douglas F i r Seedlings Grown f o r s i x weeks under 8-hour days and designated C0_ levels  PHOTOSYNTHESIS (ug CO, cm" min" ) RATES @ EACH LIGHT INTENSITY (N = 3) RATES FOR ALL LIGHT ,c -2 -2 -2 . -2 INTENS. COMBINED (N = 12) 15 mw cm 26 mw cm 44 mw cm 71 mW cm ' 2  TREATMENT  T  0.03% C0  2  TT  • 639  a  .741  ab  .559  0.1% c o  2  .479  1.0% c o  2  .112  5.0% C0  2  • 156  a  .214  b  b  b  .219  b  ab  7  .802  a  .549 .223  ab  1  TT  .948  a  .783  .663  .563  ab  a  b  b  .290  b  .210  b  .331  b  .248°  .288  C  Analysis of Variance was used f o r determining 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.  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 s i g n i f i c a n t l y higher photosynthetic C0  2  rates than those grown under high  (1.0% and 5.0% C0 > (Figure 10 and Table XXX). 2  Photosynthetic  rates of seedlings grown under 1.0% and 5.0% C0 were s i g n i f i c a n t l y 2  different from one another only at the lowest irradiance.  Photosynthetic  rates of plants grown under 0.1% C0 were generally higher than, but not 2  s i g n i f i c a n t l y different from plants grown under 1.0% or 5.0% C0 > 2  This  was probably due to the high v a r i a b i l i t y of i n d i v i d u a l measurements. Plants grown under 0.03%, 0.1%, and 1.0% C0 reached 65% to 2  85% of their maximum photosynthetic  rates at the lowest irradiance  -2 (15 mW cm  ). Plants grown under 5.0% C0 showed no apparent photo2  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 groups reached l i g h t saturation 2  -2 -2 levels between 26 mW cm and 44 mW cm . However, f o r short-day plants l i g h t saturation l e v e l s were not reached f o r any of the four C0 groups _2 2  even at the highest irradiance (71 mW cm ). A direct comparison of the effects of daylength and C0 on 2  apparent photosynthesis  was made by p l o t t i n g apparent photosynthesis as  a function of the C0 concentration under which the seedlings were grown 2  (Figure 11). For each C0 concentration, photosynthetic 2  rates under  a l l light i n t e n s i t i e s were averaged into one measurement f o r that particular C0 l e v e l . 2  The daylength under which seedlings are grown  does influence photosynthetic measured under normal a i r .  rates of plants which are both grown and  The results show that plants grown under  long days showed s i g n i f i c a n t l y higher photosynthetic  rates than those  120  Figure 10. Apparent photosynthesis of 6-month-old Douglas f i r continuously supplied with C02 a t the l e v e l s shown, and maintained under 16-hour photoperiods (25 C/20 C) f o r s i x weeks with an i r r a d i a n c e of 7.2 mW c m . -2  1.6 I —  ~ 0.8 I— V. LU 3=  \  o.i%  co  Data points are the means of- three p l a n t s . A n a l y s i s of Variance was used to determine s t a t i s t i c a l d i f f e r e n c e s . Values w i t h i n 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 f o r s i x weeks under 16-hour days and designated C0  PHOTOSYNTHESIS TREATMENT 15 mW cm"  2  0.03% C0  2  1.168  a  0.1% c o 2  .421  1.0% c o 2  .372  5.0% C0  .ooob  2  a  a  (u.g C0 cm  min )  RATES @ EACH LIGHT INTENSITY  (N = 3)  26 mW cm"  2  1.443  a  2  44 mW cm"  2  1.367  71 mW cm"  2  1.284  a  ,657  • 641  b  .365  b  .361  .422  b  .268  •i436  .082  b  b  b  b  levels  RATES FOR ALL LIGHT INTENS. COMBINED (N = 12)  1.316  a  b  .606  ?  ab  .581  a  b  .384 :i93  bc  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 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.  122  Figure 11. E f f e c t of daylength and C02 on apparent photosynthesis. Douglas f i r were grown f o r 6 weeks under 8- and 16-hour photoperiods with C02 a t the l e v e l s shown. I r r a d i a n c e 7.2, _n mw cm z  14.0  12.0  10.0 C  B o  Ul  a c tz: > in o o a: O-  <£ Ou  <  0.03% CO.  0.2% C0„  2.0% CO.  C0  2  S.0%  C0„  CONC ( l o g scale)  Data points are the means of three p l a n t s . A n a l y s i s of Variance was used to determine s t a t i s t i c a l d i f f e r e n c e s . Values w i t h i n 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, f o r plants grown under CO^  enrichment the r e s u l t s are more d i f f i c u l t to i n t e r p r e t .  There are  s i g n i f i c a n t differences between daylength groups f o r plants grown under high CC^, but no d i f f e r e n c e s between daylength groups f o r plants grown under intermediate CC^. At very high CO,, l e v e l s (5.0% CO^) there are no s i g n i f i c a n t differences i n photosynthetic  rates among daylength groups.  The o v e r a l l e f f e c t of increased CO^ under both daylengths was t o reduce apparent photosynthetic rates when the rates were measured under normal a i r . Perhaps other d i f f e r e n c e s 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^ l e v e l s 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 t o 0.1%  CO^ however, r e s p i r a t i o n 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^ l e v e l s were r a i s e d to 5.0%  CO2 the r e s p i r a t i o n 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 r e s p i r a t i o n . Douglas f i r were grown under 8- and 16-hour photoperiods with CO2 at the levels shown f o r s i x weeks with an irradiance of 7.2 mW cm 2. -  0.03% CO  0% CO.  '2  "2  C0  2  5.0%  C0  r  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 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.  125  affected the amount of water l o s t by t r a n s p i r a t i o n (Figure 13). As the CO^ concentration i n 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 r e l a t i v e amounts of water lost weekly by plants remained e s s e n t i a l l y the same among the d i f f e r e n t treatments.  As has been observed i n other conifers, a reduction i n trans-  piration during the l a s t weeks of the experiment probably  occurred  because the young shoots completed f l u s h i n g 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% C 0  2>  but t r a n s p i r a t i o n  rates of plants grown under intermediate CO,, l e v e l s (0.1% and 1.0% CO,,) generally overlapped  values shown by plants grown under either 0.03% or  5.0% C0 2  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 l e a s t (Figure 14 and Table XXXI).  Transpiration rates of plants grown under 0.1% and 1.0% CO,,  were intermediate to those observed f o r plants grown under 0.03% and 5.0% CO,,.  Transpiration rates obtained  f o r six-month-old seedlings agree  with water loss rates observed for one-year-old  trees since i n both  cases the greatest amount of t r a n s p i r a t i o n occurred with 0.03% C0 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 t r a n s p i r a t i o n (Figure 15 and Table XXXII). grown under the other three C0  o  2  Plants  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  0.03%  CO.  0.1%  CO.  1.0%  CO.  5.0%  C0„  290  270  250  230  b> 210  190 5t  170  150  130  T_  cn 4.0  3.0  2.0  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 Eo  o  CM  ^0.4  20.3 DL.  0.2  0.1  r~  10.0  20.0  LIGHT  30.0  40.0  INTENSITY (mW  50.0  60.0  70.0  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 s i g n i f i c a n t l y different at p = 0.05.  TABLE XXXI RATES OF TRANSPIRATION DOUGLAS FIR SEEDLINGS Grown f o r s i x weeks under 8-hour  days and designated C0_ l e v e l s  TRANSPIRATION ( g P^O cm" s2  - 1  u  TREATMENT -2 15 mW cm  0.03% C0  2  RATES @ EACH LIGHT INTENSITY (N = 3) -2 -2 -2 26 mW cm 44 mW cm 71 mW cm  RATES FOR ALL LIGHT INTENS. COMBINED (N = 12)  .344  a  .422  a  .672  a  .386  a  .563  ab  .577  ab  •457  ab  a  .367  a  .426  ab  .609  ab  • 397  b  0.1% c o  2  .302  1.0% c o  2  .I88  5.0% CO  )  • 288  a  • 351  a  .297  a  b  .910  .395  a  b  ,587  .333  a  b  A n a l y s i s of Variance was. used to determine s t a t i s t i c a l d i f f e r e n c e s . Values w i t h i n 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.  129  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) , f o r s i x weeks with an irradianr-p of 7.? W rm-2. m  0.8  0.7  0.6  0.5  0.4 3. O  2  0.3  0.2  o.ifr-  1 10.0  20.0 LIGHT  30.0 INTENSITY  40.0 (mW  50.0 cm" )  60.0  70.0  2  Data points are the means of three plants. Analysis of Variance was used f o r 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.  TABLE XXXII RATES OF TRANSPIRATION  ^  DOUGLAS FIR SEEDLINGS Grown for s i x weeks under 16-hour days and designated CO2 levels  TRANSPIRATION  TREATMENT  0.03% C0 0.1% c o  2  1.0% c o  2  5.0% C0  2  2  (ug HoO cm  2  sec" ) 1  RATES @ EACH LIGHT INTENSITY (N = 3) -2 -2 -2 71 mW cm-2 15 mW cm 26 mW cm 44 mW cm .268  a  •422  .207  a  .226  • 242 .491  b  a  ab  .460  a  ,534  RATES FOR ALL LIGHT INTENS. COMBINED (N = 12)  a  .421  a  • 316  a  .3l9  a  .511  .368  .4l5  a  • 4l6  a  .360  • 769  a  .677  ab  • 660  b  .789  a  a  a  a  b  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 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.  131  t r a n s p i r a t i o n 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 p l o t t e d as a f u n c t i o n of the natural l o g of the C0  c o n c e n t r a t i o n i n Figure 16.  2  For each CO,,  concentration  r e s i s t a n c e s under a l l i r r a d i a n c e s were averaged i n t o one meaBurement f o r that CO,, l e v e l .  Carbon d i o x i d e d i d not appear to have any  on t o t a l r e s i s t a n c e except under the h i g h e s t CO^ The e f f e c t s of CO,, appear at 1.0%  CO,,,  offeet  concentration  b u t t h e r e a r e no  (5 0% CO  statistical  d i f f e r e n c e s between p l a n t s grown under s h o r t days and l o n g day« the C0  2  c o n c e n t r a t i o n s r e a c h 5.0%  CO,,.  At 5.0%  long days show d e c r e a s e d r e s i s t a n c e to gas Rates of p h o t o s y n t h e s i s  C0 , 2  until  p l a n t s grown under  flow.  were not a f f e c t e d by changes i  r e s i s t a n c e s i n c e the b i g g e s t changes i n p h o t o s y n t h e t i c  n  rates occurred  i n those carbon d i o x i d e l e v e l s where t o t a l r e s i s t a n c e was n o t s i g n i f i c a n t l y d i f f e r e n t under b o t h s h o r t and l o n g days, i . e . ,  to 1.0% C0  2  (Tables XXIX and XXX).  0.03%  )  132  Figure 16. E f f e c t of daylength and C02 on t o t a l r e s i s t a n c e . Douglas f i r were grown f o r 6 weeks under 8- and 16--hour photoperiods with CO2 at the l e v e l s shown. Irradiance was 7.2 mW cm 2. -  50  40  8-hr day  30  W u  <  H w  20  H CO  w  day  ®a  Pi  10  0.03%  C0  Z  0.1%  C0  2  1.0%  C0  2  5.0%  C0  2  CO2 CONC ( l o g scale) Data points are the means of 12 p l a n t s . Analysis of Variance was used to determine s t a t i s t i c a l d i f f e r e n c e s . Values w i t h i n 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 a t p = 0.01.  133  DISCUSSION  Plant V a r i a b i l i t y  One of the l i m i t a t i o n s i n i n t e r p r e t i n g data i n this i n v e s t i gation of gas exchange was to similar treatments.  the greatly v a r i a b l e response of seedlings  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 F e r r e l l (39) who  found that differences  between seedlings of the same provenance could be greater than differences between two v a r i e t i e s .  Coastal v a r i e t i e s of Douglas f i r are more v a r i a b l e  than i n t e r i o r v a r i e t i e s ; De-Vescovei and S z i k l a i (17) found that coastal v a r i e t i e s had greater nuclear volume and DNA varieties-the implication was coastal types.  content than i n t e r i o r  that more genetic p o s s i b i l i t i e s e x i s t for  Coastal climates, although not as severe as i n t e r i o r  climates, are more l o c a l l y v a r i a b l e i n their microclimates  so greater  genetic potential would c l e a r l y have adaptive s i g n i f i c a n c e . The usual method of minimizing  the e f f e c t s of the inherent  v a r i a b i l i t y of Douglas f i r i s to increase sample s i z e .  As sample s i z e  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 i n gas exchange measurements were not p r a c t i c a l 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 r e p l i c a t i o n s was  also r e s t r i c t e d by  the  large number of d i f f e r e n t growth conditions under which the plants were  134  raised (four CO^ concentrations X two daylengths). A possible s o l u t i o n to the above problem would have been a r e s t r i c t i o n 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 i n each daylength have been conducted. However, a l l combinations of CO^ and  could  daylength  were of interest because the e f f e c t s 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 f o r by greater gas exchange rates from the increase i n seedling material.  However, the advantage derived from the increased  system volume would be p a r t i a l l y l o s t due to the necessity of also increasing the flow rate within the system.  An increase i n flow rate  would decrease the magnitude of the p o t e n t i a l CC^ d i f f e r e n c e across the cuvette and consequently of gas exchange.  reduce the ease i n resolving determinations  Thus, the p o t e n t i a l advantage of reduced v a r i a b i l i t y  which would be gained by the use of a d d i t i o n a l seedlings would be p a r t i a l l y offset by the increased d i f f i c u l t y i n determining exchange rates.  gas  135  The inherent v a r i a b i l i t y of t h i s m a t e r i a l made i t d i f f i c u l t at times to make c l e a r d i s t i n c t i o n s among the various treatment  groups.  With larger sample s i z e s more d e f i n i t i v e statements p o s s i b l y could have been made regarding the e f f e c t s of carbon d i o x i d e 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, -2 11).  The maximum r a t e f o r seedlings of 8.4 mg CO^ cm  -1 hr  , was -1  -2 s l i g h t l y lower than the maximum rate of 12.0 mg CO^ dm by E r i x (8) f o r 100-day-old Douglas f i r seedlings.  and  hr  only  reported  Younger c o n i f e r  seedlings tend to have higher photosynthetic and r e s p i r a t o r y r a t e s than older seedlings (Rutter, 1957, as c i t e d by 8).  The r a t e reported by  B r i x i s thus i n f a i r agreement w i t h the r a t e which I obtained f o r 6-monthv  old seedlings.  Plants grown under enriched atmospheres and t r a n s f e r r e d to atmospheric CO^ conditions u s u a l l y demonstrate photosynthetic rates s i m i l a r to those shown by plants grown continuously i n normal a i r (6, 21).  The photosynthetic rates of r e c e n t l y t r a n s f e r r e d p l a n t s 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  measured only under atmospheric CO^ and not under the CO^ i n which they had grown. grown under the same  was  concentrations  The f a c t that seedlings were not measured and l e v e l s p a r t i a l l y accounts f o r photosynthetic  rates of enriched seedlings being lower than rates of seedlings grown i n 0.03%  C0„.  136  Most reports i n d i c a t e that i n c r e a s i n g CO^ i n the a i r surrounding the plant increases a s s i m i l a t i o n r a t e s .  For sugar-beet, b a r l e y , and  kale, apparent photosynthetic rates i n 0.1% and 0.33% and 30% r e s p e c t i v e l y greater than rates i n 0.03%  CO^ were about 20%  CO,, (21).  The e f f e c t s  of CO^ on flowering of P h a r b i t i s , Xanthium, and S i l e n e were considered by Purhoit and Tregunna (59) to be due to promotion of as a r e s u l t of CO^ enrichment.  photosynthesis  Three-week-old soybeans grown i n a i r and  measured at d i f f e r e n t 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% l i g h t i n t e n s i t i e s photosynthesis saturated at 0.16%  C0  2  (9).  C0 , but at high o  increased e i g h t - f o l d and was not  CO^  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  light  i n t e n s i t y also increased photosynthetic r a t e s of both groups whether measured under normal or enriched a i r . I n t e r a c t i o n s between l i g h t  and  CO,, were evident since the highest a s s i m i l a t i o n r a t e s 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 -2 cm  ergs  -1 sec  ) and the highest CO,, concentration (0.1% CO,,). There i s some evidence that p l a n t s grown under high CO^ l e v e l s  (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  levels.  L i s t e r (unpublished data) grew western  hemlock seedlings f o r 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 r a t e s tended to f a l l i n t o 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 f o r photosynthesis under 0.03%, formed one group; those measured under 0.1% CO^ formed a second group, e t c . ) .  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, p l a n t s 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  had the lowest rates i n each of the three measurement groups.  0  At  the end of three months, p l a n t s 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 s i m i l a r when photosynthetic data f o r western hemlock (measured under 0.03% C^) were compared with my data f o r Douglas fir.  After long periods of growth under high CO^ concentrations (1.0%  CO^ or more), Douglas f i r seedlings e x h i b i t e d a s s i m i l a t i o n rates that were lower than rates of plants grown under low and 0.1% CO^).  concentrations (0.03%  I t i s not known why prolonged periods of high CO^ l e v e l s  should cause declines i n CO^ a s s i m i l a t i o n , but a p o s s i b l e explanation i s that CO^ acts by decreasing stomatal aperture.  T r a n s p i r a t i o n measure-  ments (Figures 13, 14, 16) f o r plants grown under 8-hour days confirm that C0„ could be i n c r e a s i n g r 2 stomata b  (assuming that r ^ stomata  i s greater &  than r i n t r a n s p i r a t i o n ) ; however, measurements f o r p l a n t s mesophyll grown under 16-hour days (Figures 15, 16) do not confirm t h i s explanation mainly because 5.0% CO  r e s u l t e d i n very high t r a n s p i r a t i o n and therefore  138  very low r  .. Other possible explanations  f o r the CO.-induced  S t OHlcLtlcL  2.  decline i n CO^ a s s i m i l a t i o n could be that CO^ reduces  stomatal  r e a c t i v i t y , either by a f f e c t i n g the e f f i c i e n c y of carboxylation reactions, or by creating a buildup of wax i n stomatal  antechambers  as has been observed i n aging needles of Sitka spruce (35) .  Frydrych  (90) suggested that an adaptation of the leaf t i s s u e to increased CO^ levels was responsible f o r photosynthetic on what the adaptations  declines but d i d not speculate  to increased CO^ concentrations might be.  Daylength had two main e f f e c t s on photosynthetic  rates of  plants grown under various levels of CO^: (1) photosynthetic  rates  were generally higher f o r long-day plants than f o r short-day  plants;  (2) plants grown under short days and low CO^ concentrations  (0.03% and  0.1% CO ) did not reach l i g h t saturation at the highest irradiance  -2 (71 mW cm  ).  Under long days a l l (X^ treatment groups reached ;  light saturation at approximately h a l f 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 l i g h t limited due to reduced daylength.  Consequently, short-day  plants were able to  respond to extra l i g h t offered i n the form of increased irradiance. Respiration Under short days, respiratory rates remained e s s e n t i a l l y the same regardless of the CO^ concentration under which the plants were grown.  However, this was not the case f o r long-day plants, which i n  comparison to plants grown under 0.03% C0_ showed decreased r e s p i r a t i o n  139  under 5.0% C^.  Differences i n r e s p i r a t i o n under various CO^  levels  could account for the growth depression noted i n plants grown under  5.0%  CO^,  0.1%  CO2  as well as the growth enhancement noted f o r plants grown under (Chapter One).  Respiration rates have been shown to have s i g n i f i c a n t  effects on growth.  For instance, i n two v a r i e t i e s of corn, Heichel  found that the more rapid dry matter accumulation of the f a s t e r growing variety was  correlated with i t s lower rate of r e s p i r a t i o n (26).  The higher r e s p i r a t i o n 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 i n conifers grown i n cold environments (Douglas f i r , 70; Pinus radiata, 64; Sitka spruce,  75) have  been postulated to be related to the high l e v e l s of soluble sugars found i n cold-adapted  plants.  Low  temperatures may  l i m i t metabolism and  translocation of photosynthetic products from s i t e s of synthesis and thus allow sugar to be accumulated and then burned i n e f f i c i e n t l y . Increased  respiration may  of carbon dioxide.  also be a stress reaction to above-normal l e v e l s  M i l l e r (48) noted that low, non-injurious  concentrations  of CO2 could bring about retardation of r e s p i r a t i o n , but that very high levels of CO2 could increase r e s p i r a t i o n of plant organs which normally had low respiration rates.  It i s 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 i n respiratory rates due to increased levels of CO2, i n determining synthesis.  but the influence of r e s p i r a t i o n would be important  whether there would be a net gain or loss i n apparent photo-  Ultimately major changes i n r e s p i r a t i o n would r e s u l t i n a  significant change i n growth.  140  Transpiration Carbon dioxide affected t r a n s p i r a t i o n rates of both trees and seedlings.  For 1-year-old  trees, transpired water decreased as CO2  in the plant atmosphere increased.  levels  Transpiration i s mainly controlled  through changes i n stomatal aperture  (3).  Increased  CO2  l e v e l s have been  reported to result i n increased stomatal closure i n a number of species (spruce, pine, 12; several monocots and d i c o t s , 53; pepper plants, various crop plants, 23).  34;  Thus the reduced water loss observed under  enrichment i s probably due to reductions i n stomatal aperture under high CO2  levels.  Furthermore, i t appears that when plants have been grown with  elevated CO^ levels for a r e l a t i v e l y long time (X^ e f f e c t s on stomatal aperture can p e r s i s t even a f t e r removal of the plants from the atmosphere.  enriched  When transpiration rates of seedlings were measured i n normal  a i r one or two days a f t e r termination of CO2 pre-treatment were s t i l l  enrichment, the e f f e c t s of  evident.  For short-day seedlings CO^ e f f e c t s on t r a n s p i r a t i o n were the same as those observed f o r 1-year-old high under low CX^ l e v e l s (Figure 14). plants the effects of CO2  trees; t r a n s p i r a t i o n rates were On the other hand, for long-day  on transpiration were the reverse; t r a n s p i r a t i o n  rates were high under high CO2  l e v e l s (5.0% CO2).  These findings seem  paradoxical since one assumes that long-day plants grown under high  CO2  would behave s i m i l a r l y to short-day plants under high CO2 by e x h i b i t i n g stomatal closure when treated with high CO2  levels.  I t i s possible that  CO2 only affects stomatal closure within a p a r t i c u l a r range of Very high CO may  l e v e l s may  influence stomatal  concentrations.  exceed the upper threshold i n which carbon dioxide aperture.  141  Total Resistance Generally high carbon dioxide l e v e l s (5.0% CO,,) a f f e c t e d t o t a l C0 d i f f u s i o n r e s i t a n c e by an increase i n r e s i s t a n c e under short 2  days, and a decrease i n t o t a l r e s i s t a n c e under long days (Figure 16). Under short days, the e f f e c t s of carbon dioxide v/ere l i k e l y due to stomatal closure under high CO^ concentrations.  This i s r e f l e c t e d i n the low  t r a n s p i r a t i o n 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 e f f e c t s of high t r a n s p i r a t i o n (low stomatal r e s i s t a n c e ) and r e l a t i v e l y low r e s p i r a t i o n rates f o r short-day p l a n t s under a l l CO2 l e v e l s . Under long days, high carbon dioxide l e v e l s r e s u l t e d i n decreased apparent photosynthesis and increased dark r e s p i r a t i o n (Figure 11 and 12) even though high t r a n s p i r a t i o n rates i n d i c a t e d that stomatal r e s i s t a n c e was r e l a t i v e l y low (Figure 16) . Increased p h o t o r e s p i r a t i o n could be r e s p o n s i b l e f o r 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 e f f e c t s of CO2 Some of the observed e f f e c t s of carbon dioxide appear to be non-photosynthetic  since they occur outside the range of enhanced photo-  synthesis (Figure 11). High carbon d i o x i d e l e v e l s (1.0% and 5.0% CO2) depressed photosynthetic rates of seedlings below those rates shown by seedlings grown under low or intermediate C0  2  l e v e l s (0.03% and 0.1% CO2) ,  yet as shown e a r l i e r carbon dioxide a f f e c t e d 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 i n wilting leaves; Raschke (60) postulates that abscisic acid i s synthesized i n 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 i n 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 i n 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 i n 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 i n stomatal closure i n 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 a n a l y z e d  d i s c o v e r e d l a r g e amounts of ABA amounts of ABA L.,  l e a f e x t r a c t s of u n t r e a t e d  t r e e s , they  produced under s h o r t days but o n l y  produced under l o n g days.  small  Stomata i n Xanthium strumarium  which are not o r d i n a r i l y s e n s i t i v e to c l o s u r e by h i g h l e v e l s  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 p r e - t r e a t m e n t .  of  Chilling  i n c r e a s e d a b s c i s i c a c i d l e v e l s i n Xanthium l e a v e s which i n t u r n i n c r e a s e d stomatal s e n s i t i v i t y  to CC^.  On  the o t h e r hand, h i g h C 0  levels  2  (1.0% C0 > 2  were e f f e c t i v e i n p r e v e n t i n g i n j u r y when s e e d l i n g s grown under s h o r t days were f r e e z e - t e s t e d at -6 C. e f f e c t s of h i g h C 0  2  ( T a b l e s X X I I I and XXIV).  l e v e l s and ABA  i n j u r y i m p l i e s t h a t t h e r e may  The  parallel  i n p r o t e c t i n g p l a n t s from f r e e z i n g  be some degree of a b s c i s i c a c i d  involvement  i n COr,-induced h a r d i n e s s . The balance between ABA  and  growth promotors has been shown  by many to be a s s o c i a t e d w i t h growth i n h i b i t i o n and dormancy i n woody s p e c i e s (Alnus and B e t u l a , 28; Douglas f i r ,  11,  41).  High  concentrations  of carbon d i o x i d e were a l s o a s s o c i a t e d w i t h i n c r e a s e d dormancy i n Douglas f i r s e e d l i n g s .  Under s h o r t and  when s e e d l i n g s were grown under 1.0% high carbon d i o x i d e was  and  l o n g days, buds s e t 5.0%  C0  2  earlier  ( t a b l e XX).  a b l e to overcome e f f e c t s of both  photoperiod by promoting a h i g h degree of budset under  Further,  temperature  and  non-inducing  c o n d i t i o n s , perhaps by i n c r e a s i n g endogenous l e v e l s o f ABA  or  reducing l e v e l s of growth promotors. ABA and  low  has a l s o been shown to s i m u l a t e the e f f e c t s of s h o r t days  temperatures by r e d u c i n g g i b b e r e l l i c a c i d content  the morphology of two v a r i e t i e s of a l f a l f a grown under  and  modifying  non-inducing  144  conditions (79).  Even under inducing conditions, one a l f a l f a v a r i e t y  was not able to develop the customary rossette growth and low resistance u n t i l ABA  temperature  had been added to the nutrient s o l u t i o n (63).  High  levels of carbon dioxide were also able to produce changes i n the morphology of Douglas f i r ; internodal elongation was given 1.0%  C0  (Table II) and seedlings given 1.0%  2  i n h i b i t e d i n trees  and 5.0%  C0  Comparisons between the action of a b s c i s i c acid and  (Figure 3).  2  high  carbon dioxide l e v e l s are i n t r i g u i n g , but are at t h i s point only speculative.  The s i m i l a r e f f e c t s of CC> and ABA may  be only coincidental  2  responses to stress.  However carbon dioxide and plant hormone i n t e r -  actions are not necessarily l i m i t e d to those between carbon dioxide abscisic acid.  There are other possible ways that C0  2  interactions could a f f e c t plant growth and development. the action of C0  2  i n overriding the photoperiodic  may  not be related to the e f f e c t s of C0  may  be due to the i n t e r a c t i o n  2  and  and plant hormone For  instance,  control of budset  on photosynthesis, but  instead  of carbon dioxide with phytochrome.  Phytochrome has already been shown to be involved i n the f r o s t hardiness response of Douglas f i r (73), and although a C0 -phytochrome 2  relationship has yet to be demonstrated f o r Douglas f i r , a C0  2  requirement  for phytochrome action i n the flowering of the short-day plant Xanthium has been demonstrated (4, 5).  The exact r o l e of C0  response was not known, but the C0 photosynthetic.  2  requirement was  2  i n the phytochrome shown to be  non-  Just on the basis of these few comparisons i t i s  apparent that we do not yet know enough about C0  2  and i t s i n t e r a c t i o n s with  endogenous plant factors; however, there are good indications that further  145  i n v e s t i g a t i o n of the e f f e c t s of high CO^ l e v e l s on various growth and developmental processes may a i d our understanding of aspects of hormonal action and r e g u l a t i o n .  CONCLUSIONS 1.  Levels of 0 . 1 % C O 2 are best f o r enhancement of growth processes,  such as dry weight and internodal elongation, and i n h i b i t o r y to  non-  growth processes, such as budset. 2.  Concentrations  processes  of 1 . 0 % C O 2 and higher generally i n h i b i t growth  such as internodal elongation, dry weight, and leaf area,  but are effective i n promoting budset. 3.  C O 2 enrichment enhances growth to a degree that other factors may  become l i m i t i n g , e.g., 4.  light intensity.  Higher carbon dioxide l e v e l s ( 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  requirement of photosynthesis increase i n daylength may particular effect, e.g.,  substitute i n part f o r the l i g h t  when l i g h t i s l i m i t i n g .  reduce the l e v e l of C O 2 required for a the required C O 2 l e v e l s f o r inducing f r o s t  hardiness are reduced from 1 . 0 % to 0 . 1 % 6.  Thus, an  C O 2 i f long days are  An active metabolism appears to be necessary to e s t a b l i s h  induced frost hardiness.  provided. CC^-  The mode of CO 2 action i s 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 . 0 3 % CC^)  higher photosynthetic  have  rates than enriched plants when a l l plants are  measured i n normal a i r ; the C 0  2  concentration under which plants are  measured appears to have more e f f e c t 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  resistance (as determined from t r a n s p i r a t i o n r a t e s ) .  diffusion  However, the  potential benefits to net photosynthesis from reduced CC^ d i f f u s i o n resistance are not r e a l i z e d . This i s due to increased r e s p i r a t i o n under high CO^, and possibly also due to high CO^ mesophyll resistance which was not included i n the t o t a l transpiration resistance. 9.  Enhancement of growth under 0.1%  and i n h i b i t i o n of growth under  1.0% (X>2 appear to be strongly related to differences i n r e s p i r a t i o n under the various carbon dioxide treatments. 10.  A l l effects of carbon dioxide may not be due to increased photo-  synthesis, but may r e s u l t from changes i n l e v e l s of growth i n h i b i t o r s , such as abscisic acid.  148  LITERATURE CITED 1.  Alden, J . and R.K. Hermann. 1971. Aspects of the cold-hardiness mechanism i n p l a n t s . Bot. Rev. 37: 37-142.  2.  Aronsson, A., Ingestad, T., and Lars-Gfa'ran Lb*8f. 1976. Carbohydrate metabolism and f r o s t hardiness i n pine and spruce seedlings grown at d i f f e r e n t photoperiods and thermoperiods. P h y s i o l . Plant. 36 (2): 127-132.  3.  Babalola, 0., Boersma, L. , and C T . Youngberg. 1968. Photosynthesis and t r a n s p i r a t i o n of Monterey pine seedlings as a f u n c t i o n of s o i l water suction and s o i l temperature. 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Assn. 49: 732-763.  TABLE A SUMMARY OF EXPERIMENTS  EXPERIMENT NUMBER  CARBON DIOXIDE CONCENTRATION  LIGHT INTENSITY  8 hours and 16 hours  0.03% C0„  3 . 4 mW cm  16 hours  0.03%, 0.1%, & 1.0% CO-  3.4 mW cm  8 hours  0.03%, 0.1%, 1.0% & 5.0% C 0  7.2 mW cm  and 1.0%  -2  -2  PRIMARY PURPOSE OF STUDY  30 Days  1 year  Growth & Development  90 Days  1 year  Growth & Development  30 Days  1 year  Frost Hardiness  12 Weeks  1 year  Frost Hardiness, Amino A c i d A n a l y s i s  12 Weeks  6 months  Frost Hardiness, Growth, Development of Seedlings  2  8 hours & 16 hours  0.03%, 1.0%, & 5.0% CO,  6.0 mW cm  8 hours & 16 hours  0.03%, 0.1%, 1.0%, & 5% C 0  7.2 mW cm 2  AGE OF PLANTS  DURATION OF TREATMENT  DAYLENGTH  157  TABLE B RANKING CODES *  Bud growth 1 Dormant pyramid-shaped bud 2 Bud swollen larger than base diameter, or with leaves just v i s i b l e i n the open end of the bud 3 Leaves protruding from the bud, but s t i l l p a r a l l e l to the stem axis 4 Leaves extending horizontally away from the stem * Categories of bud growth are a modification of the c l a s s i f i c a t i o n system used by Tregunna and Crown (74). Date of budset 1 2 3 4 5 6 7 8 9 10 11  Did Set Set Set Set Set Set Set Set Set Did  not bud bud bud bud bud bud bud bud bud not  flush within the 45-day period at Day 5 at Day 10 at Day 15 at Day 20 at Day 25 at Day 30 at Day 35 at Day 40 at Day 45 set bud within the 45-day period  Frost damage 1 2 3 4  No damage Very slight damage (less than 10% of the needles) Moderate damage (approximately 20-30% of the needles) 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 D o u g l a s - f i r six-month-old s e e d l i n g s  CARBON DIOXIDE PRETREATMENT  8 HOURS LIGHT (25 C.) 16 HOURS DARK (20°C.)  FOR 6 WEEKS  0.03%, 0.1%, 1.0%, o r 5.0% CO,  TRANSFER  THERMOPERIOD POST-TREATMENT  8 HOURS LIGHT (25 C.) + 16 HOURS DARK (5°C.)  UP TO 6 WEEKS  0.03% C 0 ONLY 2  0 WEEKS  A B  A = Freeze-tested to - 6°C.  2 WEEKS  B •» Freeze-tested to - 10°C.  4 WEEKS  6 WEEKS  *  Sequence was repeated using 16 hours l i g h t  A B A B A B  (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  ]•  401 420 439 459 479 499 519 540 558 579 601 618 639 660 680 WAVELENGTH  (nm)  1  698  -3.0  -4.0 \ 1 1 1 j 1 1 1 1 ) A01 420 439 459 479 499 519 540 558 579 WAVELENGTH  1 1 1 601 618 639  (ran)  1 1 h660 680 698  

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