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Cold acclimation and freezing in Douglas-fir seedlings Timmis, Roger 1973

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o l n z s 8 COLD ACCLIMATION AND FREEZING IN DOUGLAS-FIR SEEDLINGS by ROGER TIMMIS B.Sc. (For.) U n i v e r s i t y of Aberdeen, 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Faculty of Forestry We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA In presenting this thesis in part ia l fulfilment of the requirements for an advanced degree at the University of Br i t i sh Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of Br i t i sh Columbia Vancouver 8, Canada Date I ABSTRACT Five major i n v e s t i g a t i o n s were conducted on the cold hardiness of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings: (1) to define a hardiness measurement technique; (2) to determine environ-mental c o n t r o l and i n d u c i b i l i t y of hardiness at d i f f e r e n t parts of the f i r s t seasonal growth cycle; and (3) to examine the independence of acclimation, loss of hardiness and f l u s h i n g i n c l i m a t i c a l l y " s p l i t " p lants. G e n e t i c a l l y i d e n t i c a l material from hardy and nonhardy branches of the s p l i t plants was then used i n conductometric and c a l o r i -metric studies of (4) the fr e e z i n g process, and (5) the energy of t i s s u e water, to see i f adaptive changes i n these could account f o r the induced hardiness d i f f e r e n c e s . The measurement technique involved f r e e z i n g samples of excised needles under c o n t r o l l e d conditions to various temperatures, i n j u r y was estimated as the degree of browning a f t e r 7 days, and hardiness defined as the temperature causing 50% i n j u r y . Injury to excised needles was correlated with i n j u r y to attached needles, which was i n turn broadly r e l a t e d to ultimate s u r v i v a l of whole plants frozen at the same temperature. Random err o r and pos s i b l e bias i n hardiness estimates both increased i n hardier populations. Ten percent hardiness d i f f e r e n c e s (e.g., two degrees i n twenty) between populations could be detected with f i f t e e n - p l a n t samples; changes of 1°C could be followed during the course of treatment of an i n d i v i d u a l seedling. II C o n t r o lled environment studies i n the f i r s t growth cycle showed that germinants (1 week) were unable to a t t a i n any f r e e z i n g tolerance under 8-hr days at 2°C even a f t e r 9 weeks, but were k i l l e d whenever ic e formed. However, seedlings older than 3 weeks (1 to 2 cm of e p i -cotyl) could develop true hardiness under the inf l u e n c e of e i t h e r short days (less e f f e c t i v e ) or low temperatures (above 0°C), independently of l i g n i f i c a t i o n , bud s e t t i n g or entry i n t o r e s t . A b i l i t y to acclimate increased gradually with age, and was i n v e r s e l y r e l a t e d to growth and maturation, apparently because the l a t t e r processes had higher tem-perature optima. Photoperiod a f f e c t e d growth and bud formation only above about 15°C, but influenced hardiness at 1°C with a longer inductive daylength at low than at high l i g h t i n t e n s i t i e s (12 and 8 hours r e s p e c t i v e l y ) . I nterruption of the long inductive dark period with 15 min of red l i g h t (650 nm) caused a decrease i n hardiness and bud set, and an increase i n growth. Far-red i n t e r r u p t i o n s (750 nm) alone had no e f f e c t , but enhanced the red l i g h t e f f e c t when applied immediately afterwards. Night f r o s t s (-7°C) caused s i g n i f i c a n t dehydration, and r a p i d l y increased hardiness, only i f both the warm short-day, and c h i l l i n g treatments had been given i n sequence f i r s t , and the d a i l y supply of l i g h t continued. These r e s u l t s are i n general agreement with the hypothesis that cold acclimation takes place i n three p h y s i o l o g i c a l l y d i s t i n c t stages under natural conditions. I l l Studies on 3-year-old seedlings were c a r r i e d out by exposing each branch of a forked plant to a d i f f e r e n t temperature ( 2 ° or 2 0 °C) but s i m i l a r l i g h t conditions f o r periods of one t o f i v e months. The c h i l l -i n g stimulus f o r breaking r e s t and inducing hardiness was confined t o the c h i l l e d branch, but the warm branch apparently transmitted a f a c t o r which prevented f u l l hardening i n the c h i l l e d one. A f a c t o r moving i n the same d i r e c t i o n a l s o promoted f l u s h i n g i n branches c h i l l e d only at night from December t o June (and r e c e i v i n g greenhouse temperatures and n a t u r a l photoperiods by day). This was not replaceable by a s i n g l e i n j e c t i o n of g i b b e r e l l i c a c i d . Factors from the expanding shoot caused loss of short-day-induced hardiness i n previous year's f o l i a g e and stimulated cambial d i v i s i o n . C h i l l i n g at night prevented the dehar-dening but d i d not prevent cambial a c t i v i t y . The dehardening f a c t o r was t r a n s l o c a t e d to an opposite branch whereas movement of cambium stimulator was s t r i c t l y b a s i p e t a l . These r e s u l t s suggest that promoter-i n h i b i t o r l e v e l s c o n t r o l l i n g dormancy are independently regulated, and that a two-stage dehardening process might p r o t e c t against premature l o s s of hardiness i n nature. The progress of f r e e z i n g i n needles of the hardy/nonhardy branch p a i r s was recorded simultaneously by d i f f e r e n t i a l thermal a n a l y s i s and the conductance of low voltage d i r e c t e l e c t r i c current. The r e s u l t s of both methods exhibited the same major patterns. Freezing i n immature leaves was nonequilibrium and i n t r a c e l l u l a r . Freezing i n needles c o l d -acclimated under short days was an e q u i l i b r i u m process preceded by a short non-equilibrium f r e e z i n g of the free i n t e r c e l l u l a r water f r a c t i o n . This pattern d i d not change i n leaves more deeply cold-acclimated by low temperatures. Thawing i n mature needles was characterized by a greater proportion of i c e (than during freezing) at a l l temperatures, with i n d i c a t i o n s that not a l l the o r i g i n a l c e l l water was reabsorbed. Freezing records are int e r p r e t e d as showing that the c e l l membrane became more permeable to ions a f t e r i n j u r i o u s slow f r e e z i n g but retained i t s e s s e n t i a l i n t e g r i t y , whereas rapid f r e e z i n g caused immediate membrane damage. No features of the f r e e z i n g or thawing curves of f i r s t or sub-sequent freeze-thaw cycles were u s e f u l as p r e d i c t o r s of i n j u r y to needles by slow f r e e z i n g . Energy of water i n hardy/nonhardy needle p a i r s was compared by two methods. Heats of vapourization (A H y) of weighed increments of water, removed from excised needles under vacuum, were estimated from the c a l i b r a t e d vapourization endotherms recorded on a d i f f e r e n t i a l thermal analyser. In the second method, needle water contents were measured g r a v i m e t r i c a l l y a f t e r e q u i l i b r a t i o n with l i t h i u m c h l o r i d e solutions of known d e s i c c a t i n g energy. I t was found that A Hv, a proposed measure of water binding near surfaces, increased as the proportion of water remaining i n the l e a f decreased. For each increment removed, A H v was s i g n i f i c a n t l y higher i n hardy needles, notwithstanding various possible sources of error. Hardy needles a l s o retained more water non-osmotically (than nonhardy needles) a f t e r e q u i l i b r a t i o n with L i C l i s o p e i s t i c with t h e i r f r o s t - k i l l i n g temperature. The date suggest V that avoidance of dehydration, principally by non-osmotic lowering of cell water potential, can account for almost half of the 25 centigrade degree difference in hardiness between branches. VI TABLE OF CONTENTS Page L i s t of Tables VIII L i s t of Figures _ IX Acknowledgements XII Introduction 1 Chapter 1 - AN EXCISED-NEEDLE FREEZING TEST OF COLD 2 HARDINESS IN DOUGLAS-FIR Abstract 3 Introduction 4 Materials and Methods 5 Results and Discussion 9 Conclusions 13 References 15 Chapter 2 - ENVIRONMENTAL CONTROL OF COLD ACCLIMATION 18 IN DOUGLAS-FIR DURING GERMINATION, ACTIVE GROWTH AND REST Abstract 19 Introduction 21 General Methods 22 Experiments and Results 24 Discussion 37 References 43 VII Chapter 3 - TRANSLOCATION OF DEHARDENING AND BUD-BREAK 47 PROMOTERS IN CLIMATICALLY SPLIT DOUGLAS-DIR Abstract 48 Introduction - 50 General Methods 50 Experiments and Results 52 Discussion 56 References 61 Chapter 4 - ELECTRICAL AND THERMAL RECORDS OF FREEZING IN 64 DOUGLAS-FIR NEEDLES Abstract 65 Introduction 67 Materials and Methods 69 Results 73 Discussion 77 References 82 Chapter 5 - THE ROLE OF BOUND WATER IN COLD HARDINESS OF 87 DOUGLAS-FIR NEEDLES Abstract 88 Introduction 89 Materials and Methods 91 Results 98 Discussion References 105 VIII LIST OF TABLES Page Chapter 3 I E f f e c t of root and stem temperature on the f o l i a r ' 52 hardiness of warm and c h i l l e d branches Chapter 4 I R e l a t i v e increase i n conductance (M^) of weak e l e c t r i c 75 current through needles a f t e r slow f r e e z i n g II C h a r a c t e r i s t i c s of e l e c t r i c a l conductance records of 76 needles during repeated freeze-thaw cycles i n r e l a t i o n to hardiness and i n j u r y I I I Percent frozen water at completion of detectable 77 exotherm i n hardy and nonhardy needles cooled i n a d i f f e r e n t i a l scanning calorimeter Chapter 5 I Heats of vapourization of water from hardy and nonhardy ^9 needles II Water re t e n t i o n by needles at e q u i l i b r i u m with l i t h i u m iOO chl o r i d e at 0°C. IX LIST OF FIGURES Page 10 10 Chapter 1 1. S e n s i t i v i t y comparison of e l e c t r o l y t i c and v i s u a l measures of free z i n g i n j u r y to excised needles 2. C o r r e l a t i o n between mean e l e c t r o l y t i c and v i s u a l values f o r f r e e z i n g i n j u r y , and ultimate s u r v i v a l of whole plants 3. C o r r e l a t i o n between i n d i v i d u a l e l e c t r o l y t i c and v i s u a l i n j u r y values f o r whole plants 4. C o r r e l a t i o n between fr e e z i n g i n j u r y to excised and H attached needles 5. Age v a r i a t i o n of v i s i b l e f r e e z i n g i n j u r y w i t h i n a plant 12 6. V a r i a t i o n i n v i s i b l e f r e e z i n g i n j u r y between plants and L ^ at d i f f e r e n t hardiness l e v e l s 7. V a r i a t i o n i n cold-acclimation rates of i n d i v i d u a l 13 seedlings 8. C l a s s i f i c a t i o n of v i s i b l e i n j u r y to excised needles and 13 to f o l i a g e of whole plants Chapter 2 1. Freezing i n j u r y to seedlings a f t e r 8 weeks' acc l i m a t i o n 25 under low l i g h t i n t e n s i t y (Experiment 1) 2. Relationship between development and hardiness i n 25 coastal and i n t e r i o r provenances (Expt. 1) 29 30 3. Freezing chamber f o r measuring hardiness, and rate 26 of i c e formation i n very young seedlings (Expt. 2) 4. Cold•acclimation and f r e e z i n g pattern of 1-week 2 7 old seedlings (Expt. 2) 5. Schematic r e l a t i o n s h i p of seedling development and 27 hardiness to timing, duration and s e v e r i t y of cold treatments (Expts. 1 and 2) 6. E f f e c t of l i g h t i n t e n s i t y and photoperiod on 29 hardiness (Expt. 3) 7. Freezing i n j u r y to f o l i a g e a f t e r treatment a t 1°C under a range of photoperiods (Expt. 3) 8. Apparatus f o r n i g h t - i n t e r r u p t i o n treatments during cold hardening (Expt. 4) 9. S p e c t r a l d i s t r i b u t i o n of energy from red and f a r red 30 sources (Expt. 4) 10. E f f e c t of night i n t e r r u p t i o n s on hardiness of two 3^ provenances (Expt. 4) 11. E f f e c t of treatment sequence on hardiness (Expt. 5) 6^ Chapter 3 1. Experimental treatments and t h e i r e f f e c t s on 2^ f l u s h i n g 2. Hardiness of f o l i a g e on c l i m a t i c a l l y s p l i t plants 2^ 3. Hardiness and f l u s h i n g of an i n t e r i o r provenance 54 under natural photoperiods i n a greenhouse 4. Stem cross sections of a c l i m a t i c a l l y s p l i t seedling bearing a f l u s h i n g and a dormant branch Chapter 4 1. Apparatus for obtaining thermal and e l e c t r o -phoretic f r e e z i n g records of needles 2. T y p i c a l records of the r e l a t i v e m o b i l i t y of water during f r e e z i n g of mature and immature needles 3. Relative m o b i l i t y of water, as a function of temperature, i n hardy and nonhardy needles of a s i n g l e s p l i t plant 4. R e l a t i v e m o b i l i t y of water and temperature e l e v a t i o n / depression i n mature needles during three cycles of f r e e z i n g and thawing 5. Freezing i n j u r y to hardy needles as a function of cooling and warming rates Chapter 5 1. Apparatus f o r vacuum d i f f e r e n t i a l scanning calorimatry 2. T y p i c a l vapourization endotherms f o r water evaporated under reduced pressure at 30°C 3. C a l i b r a t i o n curve f o r isothermal vacuum vapourization of water i n the d i f f e r e n t i a l scanning calorimeter 4. Heats of vapourization f o r water removed under vacuum from excised needles 5. Water contents of needles at e q u i l i b r i u m with l i t h i u m chloride solutions XII ACKNOWLE DGEMENTS I gratefully acknowledge the help of the following people and institutions. Mr. J. M. Kinghorn of the Pacific Forest Research Centre, Canadian Forestry Service, Victoria, B.C., initia t e d the research as part of a project for culture techniques in the production of container seedlings, and provided technical help i n i t s early stages. Mr. J. Arnott of this laboratory provided plants for several experiments and Mr. J. C Wiens, also of this institution, drafted seven of the figures. The Canada Department of Agriculture, Vancouver, allowed the use of their cold room. In the University's Faculty of Forestry Dr. P. G . Haddock assisted i n finding space and equipment i n i t i a l l y ; Dr. A. Kozak helped with s t a t i s t i c a l analyses; Drs. J. W. Wilson and B. J. van der Kamp made their growth and cold storage f a c i l i t i e s avail-able, and Dr. J. Worrall provided general supervision and support throughout. The Department of Plant Science made a controlled environ-ment chamber available for use i n " s p l i t plant" experiments, and Dr. F. Eady, formerly of this department, applied gibberellic acid treatments to some s p l i t plants. Dr. L . M. Lavkulich of the Soil Science Department provided a d i f f e r e n t i a l thermal analyser, and Dr. T. A. Black kindly reviewed Chapter 5 i n detail. Mr. E. L. Watson of the Agricultural Engineering Department also loaned electronic recording equipment and cold room space. The British Columbia Forest Service supplied most of the seed and some seedlings. 1 INTRODUCTION The a b i l i t y of plants to withstand f r e e z i n g has been the subject of research f o r over a century, comprising an estimated''' 6,000 2 published papers. Several good reviews are a v a i l a b l e and no general review i s attempted here. The present work was begun i n 1968 to extend the knowledge to small (containerized) c o n i f e r seedlings being used i n r e f o r e s t a t i o n programmes. The research i s presented i n f i v e more or less self-contained sections according to the approximate progression: measurement of hardiness —» environmental c o n t r o l — * aspects of i n t e r n a l c o n t r o l —» f r e e z i n g patterns and c h a r a c t e r i s t i c s of t i s s u e water. Each sec t i o n contains i t s own s p e c i f i c l i t e r a t u r e review, d i s c u s s i o n and abstract. The p r i n c i p l e f i n d i n g s and conclusions of the study as a whole are summarized i n the preceeding abstract. Alden, J . and Hermann, R.K. (1971) Aspects of the cold hardiness mechanism i n plants, Bot. Rev. 37(37-142). L e v i t t , J . (1956) The hardiness of plants. Academic Press, New Y o r k . (1972) Responses of plants to environmental s t r e s s . Academic Press, New York. Mazur, P. (1969) Freezing i n j u r y i n plants. Ann. Rev. Plant P h y s i o l . 20(419-48). Meryman, H J. (1966) Review of b i o l o g i c a l f r e e z i n g . In: H. J. Meryman, ed., Cryobiology. Academic Press, New York (48-58). Olien, CR. (1967) Freezing stresses and s u r v i v a l . Ann. Rev. Plant P h y s i o l . 18(387-408). Parker, J. (1963) Cold r e s i s t a n c e i n woody plants. Bot. Rev. (123-201) . Weiser, C.J. (1970) Cold r e s i s t a n c e and i n j u r y i n woody plants. Science 169(1269-78). 2 CHAPTER L AN EXCISED-NEEDLE FREEZING TEST OF COLD HARDINESS IN DOUGLAS-FIR 3 ABSTRACT A f r e e z i n g t e s t i s described which i s s u i t a b l e f o r measuring hardiness changes i n small c o n i f e r seedlings during p h y s i o l o g i c a l studies. The method involves f r e e z i n g samples of excised needles, under c o n t r o l l e d conditions, to various tempera-tures. Injury i s estimated as the degree of browning a f t e r 7 days. Hardiness i s defined as the temperature causing 50% i n j u r y . Injury to excised needles was c o r r e l a t e d with i n j u r y to attached needles, which was i n turn broadly r e l a t e d to ultimate s u r v i v a l of whole plants frozen at the same temperature. Dexter's e l e c t r o l y t i c method provided a les s s e n s i t i v e measure of i n j u r y , but both v i s u a l and e l e c t r o l y t i c estimates were s i g n i f i c a n t l y c o r r e l a t e d . Random e r r o r and p o s s i b l e bias i n hardiness e s t i -mates both increased i n ha r d i e r populations. Ten percent hardiness d i f f e r e n c e s (e.g., two degrees i n twenty) between populations could be detected with f i f t e e n - p l a n t samples; changes of P C could be followed during the course of treatment of an i n d i v i d u a l seedling. INTRODUCTION During research i n t o the physiology of cold a c c l i m a t i o n i n c o n i f e r seedlings i t was found d e s i r a b l e to have a standard measure of hardiness. I d e a l l y , t h i s needed to be (1) non-de s t r u c t i v e , so that changes i n a s i n g l e plant could be followed; (2) expressible as a " k i l l i n g temperature" so that the information could be r e l a t e d d i r e c t l y to f i e l d conditions and compared with the work of other i n v e s t i g a t o r s using other species; (3) repro-ducible and reasonably quick. These requirements are somewhat c o n f l i c t i n g . To obtain measurements of l e t h a l temperatures by f r e e z i n g whole plants i s d e s t r u c t i v e ; i t requires excessive r e p l i c a t i o n and cumbersome apparatus; and i t does not simulate natural conditions i f roots are frozen as w e l l . Completely non-destructive methods (5, 6 , 1 2 , 1 6 , 2 1 , 23) are e i t h e r too inaccurate, even f o r f i e l d use, or e l s e make questionable assumptions about the process being measured, so that f r e e z i n g t e s t s are u l t i m a t e l y necessary. Excised parts have therefore been used (8, 9, 15, 17), but these undergo chemical changes (7) that might a f f e c t develop-ment of i n j u r y , and may e x h i b i t an a t y p i c a l f r e e z i n g pattern (3, 10, 13). Furthermore, the excised t i s s u e or organ may not be c r i t i c a l to s u r v i v a l of the whole plant (1, 14, 18). The following i s a b r i e f d e s c r i p t i o n of the p a r t l y d e s t r u c t i v e method adopted using small excised-needle samples, the v a r i a t i o n i n f r e e z i n g i n j u r y (assessed by a l t e r n a t i v e c r i t e r i a ) thus obtained, and the p r e c i s i o n with which s u r v i v a l of attached f o l i a g e and whole plants i s predicted. MATER LA LS AND METHODS Plants Pseudotsuga menziesii (Mirb.) Franco seedlings less than one year o l d and from one of two southern Vancouver Island provenances provided most of the data. The two provenances were Mt. Prevost, 500 m ( l a t . 48°50 ' x long. 123°40 ' ) and Mt. Benson, (49°09' x 1 2 4 ° 0 4 ' ) . Data given below r e f e r to the Mt. Prevost provenance unless otherwise stated. Plants were r a i s e d i n 2 x 11 cm pots under favourable conditions i n a greenhouse or under p a r t i a l shade outdoors, with regular watering and n u t r i e n t a p p l i c a t i o n . They were subjected to various c o l d - a c c l i m a t i o n treatments, s p e c i f i e d below, to produce a range of hardiness. Freezing of Whole Plants The aim of a l l t e s t s was to measure low temperature hardiness, not tolerance of abnormally rapid f r e e z i n g or thawing rates (2), which has a d i f f e r e n t p h y s i o l o g i c a l basis and i s of l i m i t e d p r a c t i c a l concern. Whole plants were frozen i n an insu-lated box with i n t e r n a l fan i n a -25°C cold room at 4°C/hr. Subsequently a maximum safe cooling rate of 7°C/hr was estab-l i s h e d , and temperature was p o s i t i v e l y c o n t r o l l e d by small fan-heaters i n s i d e a commercial freezer, the heaters being linked to a temperature programmer. Plants i n the i n s u l a t e d box warmed at 10°C/hr, and the others at 20°C/hr a f t e r attainment of the minimum temperature (injury was shown to be independent of warming rates l e s s than 40°C/hr). Temperatures monitored by thermocouples placed i n various parts of the f o l i a g e d i f f e r e d by a maximum of 1.5°C i n the box, and 1.0°C i n the f r e e z e r . Plant p o s i t i o n i n g was randomized to avoid b i a s . Pots were soaked with water f o r sev e r a l hours before f r e e z i n g to equalize sap tension, turgor, s o i l f r e e z i n g , atmospheric humidity and therefore to a c e r t a i n extent, supercooling. Excised-Needle Freezing Test Samples consisted of 7 to 10 needles removed from equ a l l y spaced p o s i t i o n s down the stem so as to transect age v a r i a t i o n . For each plant, three or more such samples were taken and each placed base-down i n a labeled, clean 4.5 ml v i a l containing a few drops of d i s t i l l e d water. Samples at t h i s stage could be conveniently stored overnight at 1°C without s i g n i f i c a n t e f f e c t . The number of samples per plant depended on abundance of needles and foreknowledge of the approximate k i l l i n g temperature; care-f u l removal of a large proportion of the needles a f f e c t e d neither a c c l i m a t i o n of the remainder nor subsequent bud burst. The three or more unstoppered sample v i a l s from each plant were stood i n separate racks which were placed beneath fans i n the programmed freezer. Replicate sample racks were removed from the fr e e z e r (fans off) at s u c c e s s i v e l y lower 3°C temperature i n t e r v a l s chosen to include the l e t h a l temperature. They were allowed to thaw i n precooled polystyrene containers placed i n a cold room at 1°C. Temperature was recorded continuously, by thermocouples i n various v i a l s , on a multi-channel s t r i p chart recorder. The range of temperature at any given time during cooling, among large numbers of samples, seldom exceeded 0.5°C i f v i a l s were spaced to permit free a i r flow and r a i s e d above the fr e e z e r f l o o r . Water i n v i a l s was seeded by a shower of f i n e i c e c r y s t a l s at -3°C to equalize supercooling i n the leaves. Assessment of Injury Injury to f o l i a g e was estimated v i s u a l l y and, i n some cases, by both v i s u a l and e l e c t r o l y t i c methods (the l a t t e r method i s described below). V i s i b l e i n j u r y to f o l i a g e of whole plants, a f t e r a 4-day recovery period under a 16-hour photoperiod at 21°C and 1000 f t - c was given a value of 0 to 10 based on the f r a c t i o n of needles completely brown or more than one-half discoloured. V i s i b l e i n j u r y to each needle of excised-needle samples was assessed as number of tenths discoloured or brown a f t e r a 7-day dark incubation period at 21°C i n the stoppered v i a l s . The t o t a l f o r 10 needles was expressed as a percentage. The e l e c t r o l y t i c method records the ease with which e l e c -t r o l y t e s d i f f u s e from the c e l l - 1 i n t o surrounding water as a r e s u l t of i n j u r y to the c e l l membrane, and was o r i g i n a l l y 8 developed by Dexter et a_l. (4). In d i l u t e s o l u t i o n s at constant temperature c o n d u c t i v i t y i s pr o p o r t i o n a l to i o n i c concentration. The r a t i o of c o n d u c t i v i t y of the bathing s o l u t i o n a f t e r f r e e z i n g to that a f t e r complete k i l l i n g (the r e l a t i v e c o n d u c t i v i t y , RC) thus q u a n t i f i e s f r e e z i n g i n j u r y independently of i n i t i a l t i s s u e e l e c t r o l y t e concentration, mass of t i s s u e or volume of bathing s o l u t i o n . For e l e c t r o l y t i c determination of f o l i a r i n j u r y to whole plants, a v e r t i c a l transect sample of 7 to 10 needles was taken from each, with clean forceps immediately a f t e r thawing. Samples were shaken with 3ml d i s t i l l e d water (conductivity 3i0.5 umho) i n clean, stoppered v i a l s f o r 12 hr at room temperature, and measured with the CDC 104 c o n d u c t i v i t y c e l l of a Radiometer, Copenhagen, c o n d u c t i v i t y meter at 21±0.5°C. Tissue was then k i l l e d by immersing v i a l s i n a water bath at 95°C (under pressure to prevent evaporation) or i n l i q u i d nitrogen, f o r 5 min (both methods are compared below). F i n a l c o n d u c t i v i t i e s were measured a f t e r shaking f o r a f u r t h e r 6 hr. For determinations of i n j u r y i n frozen excised-needle samples, v i a l s containing the needles were made up to 3 ml with d i s t i l l e d water and the above pro-cedure followed. Ultimate recovery of whole plants i n a c o n t r o l l e d environment favourable to growth was recorded a f t e r 3 months. They were c l a s s i f i e d as recovered (new growth), indeterminate (partly green but quiescent), or dead. RESULTS & DISCUSSION  Comparison of Injury C r i t e r i a Figure 1 shows that both e l e c t r o l y t i c and v i s u a l measures record an increasing degree of i n j u r y with lower f r e e z i n g tempera-ture. But the e l e c t r o l y t i c measure has both a smaller range, and a greater variance among i n d i v i d u a l plants. The use of l i q u i d N rather than hot water f o r k i l l i n g d i d not a l t e r t h i s variance s i g n i f i c a n t l y , contrary to Sukumaran and Weiser's f i n d i n g s (19). The following changes i n procedure a l s o f a i l e d to m a t e r i a l l y reduce variance i n RC: lengthening or decreasing e l u t i o n time, using purer d i s t i l l e d water, surface washing leaves, and increas-ing the number of needles per sample. This r e l a t i v e i n s e n s i t i v i t y of RC was confirmed during other experiments, and suggests that the c e l l membrane i s perhaps not the primary s i t e of i n j u r y i n Douglas-fir. A s i m i l a r r e s u l t has been reported f o r apple shoots (11) but not f o r Scots pine (2). I t was a l s o noticed during the present i n v e s t i g a t i o n that e l e c t r o l y t i c measurement became more s e n s i t i v e i f an incubation period was allowed, as i n the pro-cedure f o r v i s u a l estimation. However, the e l e c t r o l y t i c method, which i s more laborious, then loses i t s p r a c t i c a l advantage of providing e a r l i e r r e s u l t s . Although v i s u a l estimation i s more subjective, a second assessment of one experiment a f t e r re-randomization scored whole plants at t h e i r o r i g i n a l value with occasional 1-unit d e v i a t i o n s ; Fig. 1. Sensitivity comparison of electrolytic and visual measures of freezing injury to excised needles. Visual estimates of browning are fittable by smooth curves and readily distinguished between two different groups differing slightly in hardiness (solid line is relatively hardy). Conductivity of bathing solution, expressed as a percentage of that after k i l l ing with liquid nitrogen (N) or boiling water (B), has a smaller range and does not clearly differ-entiate hardiness groups. Six plants comprised a hardiness group. Each point is the mean of 10-needle samples from the six plants. excised samples were wit h i n 5%, and conclusions were unchanged. The amount of p o s s i b l e bias i s discussed below. C o r r e l a t i o n of mean e l e c t r o l y t i c and v i s u a l i n j u r y values from a s e r i e s of acclimation treatments was high (Fig. 2), but f o r i n d i v i d u a l plants c o r r e l a t i o n was low due mainly to the variance i n RC. E x c e p t i o n a l l y good i n d i v i d u a l c o r r e l a t i o n , however, was found f o r an i n t e r i o r provenance (Fig. 3). Zehnder and Lanphear (22) reported a 0.97 c o r r e l a t i o n f o r leaves of Japanese yew. Injury by e i t h e r v i s u a l or e l e c t r o l y t i c measures was r e l a t e d to f i n a l s u r v i v a l of whole plants (Fig. 2). The average l e v e l of v i s i b l e f o l i a r i n j u r y corresponding to a 50% s u r v i v a l of whole plant"? was only about 20% i n Figure 2, due to root damage under the a r t i f i c i a l conditions used to freeze whole plants i n these experiments, and due to an i n s u f f i c i e n c y of mature l a t e r a l buds to resume growth. Under na t u r a l conditions roots are protected from the a i r temperature minima, and therefore s u r v i v a l s corre-sponding to a given l e v e l of f o l i a r i n j u r y would be higher. Alden (1) found that the leaves of t h i s species were the parts a c t u a l l y l i m i t i n g winter s u r v i v a l . Consequently, i n the absence of b e t t e r data, the 50% l e v e l of v i s i b l e f o l i a r i n j u r y was considered to define the l e t h a l temperature. This i s a l s o i n accordance with other studies (17, 19) . Figure 4 shows that v i s i b l e i n j u r y to attached f o l i a g e 50 40 o 2 N —I UJ cr u, cr K- 30 r u. < Id < to | 20 r-Q i X UJ 10 50 40 30 6 20 10 • F U L L Y R E C O V E R E D HH I N D E T E R M I N A T E I DEAD B 2 4 6 VISUAL OAMAGE S C O R E 10 2 4 6 V I S U A L DAMAGE S C O R E 10 Fig. 2. Correlation between mean electrolytic and visual values for freezing injury, and ultimate survival of whole plants. Data are from a total of 90 6-month-old plants subjected to a range of acclimation treatments and freezing temperatures. Plants were frozen in insulated boxes i n a cold room and needle samples excised for con-ductivity measurements afterwards. The visual injury score refers to attached foliage. In A, cir c l e s represent groups defined by 5% intervals across the range of conductivity values, the area being proportional to the number in the group (minimum, 2). In B, circles represent groups of 11 plants taken in order across this range. Jl I I 1 I 2 4 6 8 10 V I S U A L D A M A G E S C O R E Fig. 3. Correlation between individual electrolytic and visual injury values for freezing injury to whole plants. Data are from an interior B.C. provenance (800 m, Prince George, B.C.) subjected to a range of acclimation and freezing treatments. Each point represents one plant, assessed as described in Figure 2. The two uppermost points close to the ordinate have been omitted from the calculated regression: y=10.30+2.94x; R=0.86. could, w i t h i n the l i m i t s of error, be measured d i r e c t l y as that i n f l i c t e d upon excised samples under the described conditions (regression c o e f f i c i e n t — 1). Subsequent exothermal measurements of the f r e e z i n g process (20) showed that the rate and pattern of i c e formation was a l s o s i m i l a r i n excised needles providing that an external contact with water permitted entry of i c e through the vascular system as i n the present case. Agreement between i n j u r y to excised shoots and whole plants of Scots pine has been reported (2 ) . The foregoing data show that measurements of hardiness of excised needles are s u i t a b l e not only f o r s p e c i f i c comparisons of f o l i a r hardiness under laboratory conditions, but a l s o have meaning i n terms of some intermediate s u r v i v a l i n populations of i n t a c t p lants. Uncontrolled V a r i a t i o n i n V i s i b l e Injury Anatomical v a r i a t i o n i n i n j u r y to a s i n g l e needle has been described by Alden (1) with reference to an 8-point scale. The c l a r i t y with which browning could be c l a s s i f i e d macroscopically on the present 10-unit scale varied according to the provenance, age and h i s t o r y of treatment. In the best cases, the d i v i d i n g l i n e between brown and green was abrupt and transverse, demar-cating an e a s i l y measurable length of dead t i s s u e . In the worst cases, notably old needles, i n t e r i o r provenances, and deeply acclimated leaves, a general d i s c o l o r a t i o n pervaded the e n t i r e A T T A C H E D N E E D L E INJURY Fig. 4 . Correlation between freezing injury to excised and attached needles. Each point represents one one-year-old plant. 10-needle samples, frozen before or after excision, were assessed for percent browning after 7 days. The Mt. Prevost provenance (•) and a less hardy coastal provenance of uncertain origin (0) had been subjected to a range of acclimation treatments. Digits indicate number of coincident points. R i s the correlation co-eff i c i e n t . Intercept and slope coefficients do not d i f f e r significantly from zero and one respectively. 12 l e a f . Injury could be estimated to 1 unit and 3 un i t s r e s p e c t i v e l y . A photographic record of groups c l a s s i f i e d to a 2-unit accuracy was used to standardize i n j u r y estimation i n d i f f e r e n t experiments. This i s shown i n Figure 8 together with a photograph i l l u s t r a t i n g the v a r i a t i o n i n i n j u r y within and between whole plants at d i f f e r e n t l e v e l s of hardiness. In the worst and hig h l y improbable case, a subjective 3-unit bias i n the excised-needle i n j u r y estimates of high l y hardy plants could lead to an e r r o r i n mean l e t h a l temperature of 4°C (Fig. 6). Normally, however, u n i d i r e c t i o n a l 2-unit m i s c l a s s i -f i c a t i o n s of two or three needles at only some of the t e s t temperatures would d i s p l a c e the l e t h a l temperature of an i n d i v i d u a l plant by less than 0.5°C. V a r i a t i o n w i t h i n a plant i s shown i n Figure 5. Needles of intermediate age were hardiest; those at the t i p l e a s t hardy. The i n c l u s i o n of young needles i n the transect sample was advantageous i n a f f o r d i n g some measurable i n j u r y even when selected t e s t tem-peratures f a i l e d to include the l e t h a l l e v e l . V a r i a t i o n i n i n j u r y curves between plants of uniform age, treatment and appearance i s shown i n r e l a t i o n to the mean curve B, i n Figure 6. The i n d i v i d u a l i n j u r y curves tend to be p a r a l l e l at low to moderate hardiness l e v e l s , so that hardiness of i n d i v i d u a l s can be r e l i a b l y ranked. A 92% d i f f e r e n c e i n i n j u r y at 11°C (between extreme curves of the B population) represented a d i f f e r e n c e i n l e t h a l temperature of 6°C. Mean of the i n t e r p o l a t e d l e t h a l O 2 4 V I S I B L E INJURY Fig. 5. Age variation of v i s i b l e freezing injury within a plant. Bold curve i s the mean of the individual curves from 10 6-month-old seedlings. Each point on an individual curve is the v i s i b l e injury score to a single needle removed from that position on the stem 4 days after the whole plant had been uniformly frozen and thawed. Similar variation was exhibited by needles ex-cised before freezing. O -4 -8 -12 -16 -20 -24 -28 -32 -36 T E M P E R A T U R E ° C Fig. 6. Variation in v i s i b l e freezing injury between plants, and at different hardiness levels. Plants were of the Mt. Benson provenance. Populations A, B, C and D were the result of i n -creasingly effective acclimation treatments of similar duration. Some individual injury curves contributing to the mean curve (bold line) are shown for B. Each of the 3 points on an i n d i v i -dual curve represents injury to a 10-needle excised sample. 95% confidence limits on the hardi-ness (defined as temperature corresponding to 50% injury) are shown for each population. The effect of a 3-unit overestimating bias on apparent hardiness i s shown bv c'. 13 temperatures was 11.2±1.3""C (0.05 p r o b a b i l i t y l e v e l ) i n t h i s example; l e t h a l temperature from the mean curve was 11.3°C. The l a t t e r d e r i v a t i o n u s u a l l y d i f f e r e d by 0.2 to 1.0°C, and was used only when i n d i v i d u a l curves could not be r e l i a b l y extrapolated. The slope of the mean i n j u r y curve decreased with i n c r e a s i n g hardiness, leading to greater p o s s i b l e bias errors (Fig. 6). Associated between-tree v a r i a t i o n a l s o increased (95% confidence l i m i t s are given), but remained r e l a t i v e l y constant when expressed as a percentage of hardiness. This behaviour was exhibited by both c o a s t a l and i n t e r i o r provenances. The minimum s i z e of sample needed to detect a given hardiness d i f f e r e n c e between populations therefore depends on the average hardiness l e v e l . An approximate r u l e i s that 10% hardiness d i f f e r e n c e s (e.g., 1 degree i n 10, 3 i n 30) between populations w i l l be detectable with a sample of 15 plants from each. Figure 7 shows d i f f e r e n c e s of equally treated i n d i v i d u a l s from the same provenance with respect to time. Shape of acclima-t i o n curves was s i m i l a r . Seedlings destined to become r e l a t i v e l y hardy g e n e r a l l y exhibited t h i s advantage at an e a r l y stage of acclimation. The time of sampling to detect i n d i v i d u a l d i f f e r e n c e s , for example i n genetic studies, would therefore not be c r u c i a l . CONCLUSIONS Freezing excised needles, followed by v i s u a l estimation of degree of browning as described, provides a v a l i d , absolute 13a - 4 I I 1 1 O 5 IO 15 WEEKS A C C L I M A T I O N Fig. 7. Variation in acclimation rates of individual seedlings, Mt. Benson provenance. Hardiness was assessed visually on excised-needle samples. Points on a curve represent successive determinations on the same plant during the course of progressively more severe acclimation treatment. Superiority tends to appear early and be maintained. 1 3 b B 95 85 67 19 Fig. 8. Classification of visible injury to excised needles and to foliage of whole plants. A illustrates classification of individual needles 7 days after freezing in vials by the standard procedure (see materials and methods). Each needle in a group had the number of tenths of brown tissue indicated by the figure beneath. For comparison, B shows actual injury to 5 groups of 8 3-month-old seedlings 4 days after freezing at -8 °C . Groups were acclimated at 1°C under an 8-hr photoperiod for (from left to right) 0, 2, 3, 4 and 5 weeks. In this case the average of the 8 whole-plant injury scores (see materials and methods) is given as a percentage beneath each group. rather than comparative measure of foliar hardiness for physio-logical studies. Variation in test stresses is minimized by accurate control of cooling rates (any of several published or commercial methods could be used), prior water storage and early ice seeding. Unwanted variation in visible injury is reduced by sampling technique, comparison with defined standards, repeated use of the same individuals and selection of appropriate sample size. Greater objectivity and earlier results (desirable for nursery work) could be obtained by the electrolytic method at the expense of heavier sampling and more work per sample. Estimates are applicable specifically to attached foliage; they are related to winter survival of whole plants, and they are reproducible. REFERENCES Alden, J. N. 1971. Freezing r e s i s t a n c e of t i s s u e s i n the twig of Douglas-fir. Ph.D. t h e s i s . Oregon State U n i v e r s i t y , C o r v a l l i s , Oregon. Aronsson, A. and Elia s s o n , L. 1970. Frost hardiness i n Scots pine (Pinus s i l v e s t r i s L.) I. Conditions f o r t e s t on hardy plant t i s s u e s and f o r evaluation of i n j u r i e s by c o n d u c t i v i t y measurements. Stud. F o r e s t a l Suecica No. 77, pp. 28, Royal C o l l . Forestry, Stockholm. Cooper, W. c , Gorton, B. S. and Tayloe, S. D. 1954. Freezing t e s t s with small trees and detached leaves of g r a p e f r u i t . Proc. Am. Soc. Hort. S c i . 63:167-172. Dexter, S. T., Tottingham, W. E. and Graber, L. F. 1932. Investigations on the hardiness of plants by measurement of e l e c t r i c a l c o n d u c t i v i t y . Plant P h y s i o l . 7:63-78. Driessche, R. van den, 1969. Measurement of f r o s t hardiness i n two-year-old Douglas-fir seedlings. Can. J. Plant S c i . 40:159-172. Evert, D. R. and Weiser, C. J . 1971. Rel a t i o n s h i p of e l e c t r i c a l conductance at two frequencies to col d i n j u r y and acc l i m a t i o n i n Cornus s t o l o n i f e r a . Plant P h y s i o l . 47:204-208. Goodman, R. N., K i r a l y , Z. and Z a i t l i n , M. 1967. The bio-chemistry and physiology of i n f e c t i o u s plant disease. Pp. 154-155. D. Van Nostrand Co., Toronto, Ontario. 16 8. Hudson, M. A. 1961. The l i m i t a t i o n s of a cut le a f t e s t f o r assessing the f r o s t r e s i s t a n c e of the tuber-bearing Solanums. Euphytica 10:169-179. 9. Hutcheson, C. E. and Wiltbank, W. J. 1970. Cold hardiness o f selected C i t r u s v a r i e t i e s as determined by f r e e z i n g detached leaves. Proc. F l a . State Hort. Soc. 83:95-98. 10. Kitaura, K. 1967. Supercooling and i c e formation i n mulberry leaves. In: C e l l u l a r i n j u r y and r e s i s t a n c e i n f r e e z i n g organisms. Internat. Conf. Low Temp. S c i . , Proceedings. Sappora, Japan. 143-156. 11. Lapins, J . 1962. A r t i f i c i a l f r e e z i n g as a routine t e s t o f cold hardiness i n young apple seedlings. Proc. Amer. Soc. Hort. S c i . , 81:26-34. 12. L e v i t t , J . 1956. The hardiness of plants. Academic Press, New York. 13. Lucas, J . W. 1954. Subcooling and i c e nucleation i n lemons. Plant P h y s i o l . 29:245-251. 14. Parker, J . 1963. Cold r e s i s t a n c e i n woody plants. Bot. Rev. 29:123-201. 15. Scheumann, W. 1962. The development of quick s e l e c t i o n methods for breeding of f r o s t hardy f o r e s t trees. Abstr. of t h e s i s i n Wissenschaftliche Z e i t s c h r i f t der U n i v e r s i t a t Rostock. 11:366-367. 17 16. Siminovitch, D. and Briggs, D. R. 1953. Studies on the chemistry of the l i v i n g bark of black locust and i t s r e l a t i o n to f r o s t hardiness. I l l The v a l i d i t y of plasmolysis and d e s i c c a t i o n t e s t s f o r determining the f r o s t hardiness of bark t i s s u e . Plant P h y s i o l . 28:15-34. 17. Steponkus, P. L. and Lanphear, F. O. 1967. Refinement of the t r i p h e n y l t e t r a z o l i u m c h l o r i d e method of determining cold i n j u r y . Plant P h y s i o l . 42:1423-1427. 18. Stushnoff, C. 1972. Breeding and s e l e c t i o n methods f o r c o l d hardiness i n deciduous f r u i t crops. HortSci. 7:10-13. 19. Sukumaran, N. P. and Weiser, C. J . 1972. An excised l e a f l e t t e s t f o r evaluating potato f r o s t r e s i s t a n c e . HortSci. i n press. 20. Timmis, R. 1972. E l e c t r i c a l and thermal records of f r e e z i n g i n Douglas-fir needles. Chapter 4 of t h i s t h e s i s . 21. Wilner, J . 1967. Changes i n e l e c t r i c a l r e s i s t a n c e of l i v i n g and i n j u r e d t i s s u e s of apply shoots during winter and spring. Can. J . Plant S c i . 47:469-475. 22. Zehnder, L. R. and Lanphear, F. O. 1966. The infl u e n c e of temperature and l i g h t on the cold hardiness of Taxus  cuspidata. Proc. Amer. Soc. Hort. S c i . 89:706-713. 23. Zsoldos, F. 1972. Isotope technique f o r i n v e s t i g a t i o n of cold r e s i s t a n c e i n r i c e and Sorghum v a r i e t i e s . Plant and S o i l 34:659-663. CHAPTER 2 ENVIRONMENTAL CONTROL OF COLD ACCLIMATION IN DOUGLAS-FIR DURING GERMINATION, ACTIVE GROWTH AND REST ABSTRACT Con t r o l l e d environment experiments were conducted on Pseudotsuga menziesii (Mirb.) Franco seedlings during t h e i r f i r s t year. Hardiness of f o l i a g e was assessed by v i s u a l l y estimating i n j u r y a f t e r f r e e z i n g t e s t s . Germinants (1 week) were unable to a t t a i n any f r e e z i n g tolerance under 8-hr days at 2°C even a f t e r 9 weeks, but were k i l l e d whenever i c e formed. Their a b i l i t y to supercool increased by 5°C during t h i s treatment. However, seedlings older than 3 weeks (1 to 2 cm of e p i c o t y l ) could develop true hardiness under the i n f l u e n c e of e i t h e r short days (less e f f e c t i v e ) or low p o s i t i v e temperatures, independently of l i g n i f i c a t i o n , bud s e t t i n g or entry i n t o r e s t . A b i l i t y to acclimate increased g r a d u a l l y with age, and was i n v e r s e l y r e l a t e d to growth and maturation, apparently because the l a t t e r processes had higher temperature optima, photo-period a f f e c t e d growth and bud formation only above about 15°C, but influenced hardiness at 1°C with a longer i n d u c t i v e day-length at low than at high l i g h t i n t e n s i t i e s (12 and 8 hours r e s p e c t i v e l y ) . I n t e r r u p t i o n of the long i n d u c t i v e dark period with 15 min of red l i g h t (650 nm) caused a decrease i n hardiness and bud set, and an increase i n growth. Far-red i n t e r r u p t i o n s (750 nm) alone had no e f f e c t , but enhanced the red l i g h t e f f e c t when applied immediately afterwards. Night f r o s t s (-7°C) caused s i g n i f i c a n t dehydration, and rapidly increased hardiness, only i f both th warm short-day, and chill ing "stages" had been supplied f irst and the daily supply of light continued. INTRODUCTION The environmental c o n t r o l of cold a c c l i m a t i o n i n coniferous evergreens i n r e l a t i o n to t h e i r e a r l y growth and development has received l i t t l e study i n s p i t e of i t s obvious relevance to f o r e s t nursery work. Most i n v e s t i g a t i o n s have been c a r r i e d out at the end of a growing season on broadleaved woody species which, u n l i k e c o n i f e r s , cannot acclimate s u b s t a n t i a l l y during t h e i r period of a c t i v e growth, nor i n most cases, a t t a i n maximum hardiness while s t i l l bearing f u n c t i o n a l leaves. Several workers have found that two or three sequential stages must be followed f o r maximum and most e f f i c i e n t a c c l i m a t i o n i n the broadleaved group (9, 16, 22). These stages have been summarized by Weiser (24) as occurring i n response to short days (and entry i n t o winter dormancy or "rest") cool temperatures (and l e a f a b s c i s s i o n ) , and prolonged sub-zero temperatures (lower than -30°C). Their independence of each other and of associated phenology, however, appears to vary considerably between species (7, 8, 10). In c o n i f e r s , t h i s pattern of change has not been described, although there i s scattered evidence f o r some sequential processes. The separate e f f e c t i v e n e s s of short days and low temperatures i n inducing hardiness has been widely reported (5, 13, 26)> but maxi-mum hardiness has not been shown to depend on the i n i t i a l exposure to short days when long acclimation periods are considered; nor has the r o l e of rest, e i t h e r i n a c c l i m a t i o n or loss of hardiness, been 22 established. In f a c t , preliminary experiments i n the present study i n d i c a t e d that s u b s t a n t i a l a c c l i m a t i o n can occur during the temporary suspension of a c t i v e growth at low temperatures. Cabbages apparently acclimate best while continuing growth (2), which i n d i c a t e s that, i n some species at l e a s t , the two processes are not biochemically opposed. Scheumann and B o r t i t z (18) observed a rapid a c c l i m a t i o n response to temperatures a few degrees below the f r e e z i n g point as opposed to acclimating temperatures j u s t above zero, thus i n d i c a t i n g a f u r t h e r d e v i a t i o n from the broad-leaved model. Others have f a i l e d to detect a sub-freezing tempera-ture e f f e c t (5, 26). The following i n v e s t i g a t i o n s were begun i n 1968 to more c l o s e l y define the environment, growth and hardiness r e l a t i o n s h i p s i n c o n i f e r seedlings during the f i r s t seasonal c y c l e from germination to winter r e s t . GENERAL METHODS  Plants A t o t a l of f i v e experiments was conducted on Pseudotsuga menziesii (Mirb.) Franco seedlings during or immediately a f t e r t h e i r f i r s t season's growth. Four B r i t i s h Columbia provenances were used: (1) southern Vancouver Island, 500 m (Mt. Prevost, l a t . 48°52' x long. 123°45'), (2) southern Vancouver i s l a n d , 800 m (Mt. Benson, 49°09' x 124°04'), (3) Prince George, 800 m (54°15' x 122 ,50") and (4) Chil l i w a c k , 1050m (Mt. Thurston, 49°07' x 121°04'). The lower e l e v a t i o n Vancouver Island provenance was used i n a l l 23 experiments, and d e s c r i p t i o n s r e f e r to t h i s unless otherwise i n d i c a t e d . Plants were r a i s e d i n 2 x 11 cm pots i n peat-sand or peat-vermiculite mixtures under favourable conditions, e i t h e r i n a greenhouse or under p a r t i a l shade outdoors. They were f e r t i -l i z e d weekly with a complete n u t r i e n t s o l u t i o n . In a l l experiments, controls and treatments at warm temperatures f*15°C) continued to receive f e r t i l i z e r s , though les s frequently, i n order to maintain health. Subsequently i t was shown (21) that hardiness was not s i g n i f i -c a n t l y a f f e c t e d by t h i s extra feeding. Evaluation of Hardiness Hardiness was measured e i t h e r by f r e e z i n g whole plants i n an i n s u l a t e d box i n a cold room (subsequently a programmed f r e e z e r was used), or by f r e e z i n g samples of 7 to 10 excised needles i n v i a l s according to the methods pr e v i o u s l y described (20). Cooling rates d i d not exceed 7°C/hr, nor warming rates 20°C/hr, so that i n a l l cases i n j u r y depended only on the lowest temperature reached. Injury was assessed e i t h e r as degree of browning of f o l i a g e on a 10-unit scale, or as the f r a c t i o n of e l e c t r o l y t e leached from the t i s s u e according to the method o r i g i n a l l y developed by Dexter ^ t al (3). Where excised-needle samples were frozen, determinations were repeated at s u c c e s s i v e l y lower 3°C i n t e r v a l s on the same group of plants and a " l e t h a l temperature", corresponding to 50% v i s i b l e i n j u r y , was i n t e r p o l a t e d . This provided an absolute rather than comparative measure of hardiness r e l a t e d to ultimate s u r v i v a l of whole plants (20). Lethal temperature was analysed by a n a l y s i s of variance and means were tested by Duncan's M u l t i p l e Range Test. V i s u a l i n j u r y scores (at a s i n g l e f r e e z i n g temperature), and percentage c o n d u c t i v i t y data, were f i r s t "normalized" by ar c s i n e transformation. EXPERIMENTS AND RESULTS  Experiment 1 - Growth, Temperature and Photoperiod This was c a r r i e d out between J u l y and November, 1968, i n three cold rooms maintained at 1, 6 and 11°C (±1.3°C). In each col d room were three photoperiod treatments (8, 12 and 16 hr) at an incan-descent l i g h t i n t e n s i t y of 100 f t - c . Temperatures during the l i g h t period exceeded the cold room temperature by 1.5°C. Six plants from each of Mt. Prevost and Prince George provenances were placed under each of these nine treatment combinations 6, 11, and 15 weeks a f t e r late-May germination. Control groups were placed i n 75% shade outdoors at the same time, and f o r the e a r l i e s t treatment an a d d i t i o n a l c o n t r o l was placed i n a 12-hr, 21-15°C day-night, c o n t r o l l e d environment. Height, number of l a t e r a l buds and s i z e of terminal bud ( i f present), were recorded before and a f t e r the 8-week treatment. R e l a t i v e hardiness was measured by f r e e z i n g whole plants at one or two temperatures and v i s u a l l y estimating i n j u r y a f t e r 4 days as described p r e v i o u s l y (20). R e l a t i v e hardiness i s shown i n Figure 1. There were s u b s t a n t i a l (and s t a t i s t i c a l l y s i g n i f i c a n t ) d i f f e r e n c e s i n h a r d i -ness, f o r both i n t e r i o r and c o a s t a l provenances, associated with temperature, photoperiod and the date on which treatments were begun. Even plants at an e a r l y stage of a c t i v e growth (2 cm of e p i c o t y l ) had acclimated under short days and c h i l l i n g to a hardiness l e v e l below -8°C. Acclimation under 12-hr days was greatest a t a l l stages of development at the r e l a t i v e l y low l i g h t i n t e n s i t y of t h i s experiment. This photoperiod e f f e c t was r e l a -t i v e l y unimportant a t low temperatures; i t operated both on plants i n a c t i v e growth and those with buds set and i n r e s t . Under the cool temperature (*12°C), and at l i g h t i n t e n s i t y around the photo-sy n t h e t i c compensation point, photoperiod d i d not i n f l u e n c e bud development or extension growth (data not shown). Growth and maturation were dependent only on temperature; both decreased under the low temperatures which favoured a c c l i m a t i o n (Fig. 2). Outdoor controls (which form the basis f o r percentage values i n Figure 2) underwent normal completion of growth, l i g n i f i c a t i o n and bud development between the time of beginning f i r s t treatments TREATMENT BEGUN MID-JULY FROST AT -8.5°C (I7°F) n = l8 TREATMENT BEGUN EARLY AUGUST FROST AT -8.5°C (I7°F) n = 9 FROST AT •I6.5°C (2°F) n = 9 TEMPERATURE °C HOURS PHOTOPERIOD-AT IOO FT CANDLES TREATMENT BEGUN MID-SEPTEMBER 8 12 16 FROST AT -I6.5°C(2°F) n = 4 FROST AT -25°C(-I3°F) n=4 (COASTAL DOUGLAS-FIR ONLY) 8 12 I 6 \ CONTROL LODGEPOLE PINE, PRINCE GEORGE, SOWN MAY DOUGLAS-FIR .PRINCE GEORGE, SOWN MAY DOUGLAS-FIR, EAST VANCOUVER ISLAND , SOWN MAY Fig. 1. Injury to seedlings after 8 weeks' acclimation under low light intensity (Experiment 1). Injury to whole plants was scored visually between 0 (undamaged) and 10 (completely killed) 4 days after freez-ing to the indicated temperature. The Mt. Prevost provenance occurs throughout a l l treatments. Pinus  contorta Dougl. i s also shown for comparison in July and August treatments. Each column i s the mean of n seedlings, n/3 of each species or provenance. C O A S T A L D O U G L A S - F I R INTERIOR D O U G L A S - F I R I 3 5 7 9 II 3 5 7 9 II CONDITIONING T E M P E R A T U R E ° C F i g . 2. R e l a t i o n s h i p b e t w e e n d e v e l o p m e n t and h a r d i n e s s i n c o a s t a l ( M t . P r e v o s t ) and i n t e r i o r p r o v e n a n c e s ( E x p e r i m e n t 1). L e a s t s i g n i -f i c a n t d i f f e r e n c e a t 5% l e v e l i s i n d i c a t e d , a s s u m i n g common v a r i a n c e among p r o v e n a n c e s . 26 (July) and ending the l a s t (mid-November). They acclimated gr a d u a l l y during t h i s period, p a r t i c u l a r l y the i n t e r i o r provenance which a l s o began maturation e a r l i e r (Fig. 2) i n the cold rooms regardless of photoperiod. The data i l l u s t r a t e d i n Figure 1 do not show t h i s a c c l i m a t i o n i n the case of the c o a s t a l provenance due to a c c i d e n t a l loss of r e p l i c a t e s , but subsequent measurements confirmed i t . Experiment 2 - Acclimation of Germinants Two types of seedlings were i n v e s t i g a t e d i n t h i s experiment. Seedlings 1 to 2 weeks a f t e r germination possessed only hypocotyl cotyledons and a t u f t of new needles; seedlings 2 to 3 weeks a f t e r germination had developed at l e a s t a centimeter of stem above the cotyledons and were p h y s i o l o g i c a l l y independent of them. Seedlings of both types were given a 10-hr photoperiod with 300 f t - c mixed flu o r e s c e n t and incandescent l i g h t , i n a cold room at 1±1.5°C. Samples were removed at i n t e r v a l s of 1, 2, 3, 4 and 9 weeks. They were tested i n a f r e e z i n g chamber designed to freeze only the above-ground parts, and to permit a continuous record to be made of e l e c t r i c a l c o n d u c t i v i t y of stem and l e a f t i s s u e during f r e e z i n g (Fig. 3). The purpose of t h i s arrangement was to determine the temperature and approximate rate of i c e formation i n the t i s s u e i n accordance with Olien's method (14) which assumes that the amount of l i q u i d i n t e r c e l l u l a r water i s p r o p o r t i o n a l to the current flowing at low voltages. Gold-plated copper electrodes were attached to stems, Z6a Fig. 3. Freezing chamber for measuring hardiness and rate of ice formation i n aerial parts of very young seedlings (Experiment 2). Electrodes pa r t i a l l y encircled the stem (A) and a cotyledon (B) (or new needle). Tissue was held in non-injurious contact by a small amount of foam rubber within the electrode loop and a film of elec-troconductive paste between tissue and metal. Thermocouple.(T) recorded ambient temperature. Layer of vermiculite (C) insulated the root system against freezing. The freezing chamber actually contained 8 plants, among which a total of 4 electrode pairs could be distributed. cotyledons or needles of four plants during each f r e e z i n g t e s t , a f i l m of electroconductive paste (Burton Parsons Co. "EK.G SOL") ensuring good e l e c t r i c a l contact between metal and t i s s u e (Fig. 3). Each c i r c u i t contained a 3 v dry c e l l , and a s t r i p chart recorder s e n s i t i v e to a 10-microamp range of current. Six runs were made at each sampling date, each succeeding run being cooled to a lower temperature so that range from completely safe to completely l e t h a l was obtained. Each run used eight plants of which four were e l e c -t r i c a l l y monitored. The more developed seedling type was tested only a f t e r the 4-week treatment period. The r e s u l t s are shown i n Figure 4. P r i o r to elongation of the e p i c o t y l , seedlings were not capable of developing any tolerance of f r e e z i n g . Measurements of current showed that when i c e formed, germinants were i n v a r i a b l y k i l l e d i n s p i t e of short-day, low tem-perature treatment f o r as long as 9 weeks. A f t e r a s i m i l a r period, seedlings with 1 t o 2 cm of new shoot t o l e r a t e d f r e e z i n g , which took place at a more gradual rate. During the course of c h i l l i n g treatment, however, germinants acquired a greater capacity f o r supercooling (Fig. 4) and could thus avoid l i g h t f r o s t s of short duration. Neither the permanance of t h i s condition, nor the extent to which i t might develop outdoors was i n v e s t i g a t e d . Summary of Growth, Development and Temperature Relationships The observations of Experiments 1 and 2 f o r c o a s t a l Douglas-fi are summarized i n Figure 5. According to t h i s representation, the 27 a W E E K S C O N D I T I O N I N G Fig. 4. The cold-acclimation and freezing pattern of 1-week (—0—) and 3-week-old (—0—) seedlings (Experiment 2). The lethal temperature (It.) i s that at which 50% v i s i b l e injury occurred after freezing. Each point i s the mean of 10 to 40 seedlings and the upward trends are highly significant. Inset diagrams represent the relative mobility of liquid water (M) in two seedling types, as a function of temperature during uniform cooling at 7°C/hr. M i s the ratio of e l e c t r i c a l conductance at temperature T to that at 0°C, multiplied by V /V , the ratio of v i s -cosities of liquid water at the two temperatures (14). fp i s the freez-ing point after i n i t i a l supercooling. 8-WK. TREATMENT, BEGUN JULY ( j ) B>-^8-WK. TREATMENT, BEGUN AUGUST d> • SOWING I MAY JUNE JULY AUG. SEPT. 8-WK. TREATMENT , BEGUN SEPT. OCT. T T 20° rn o m 2 O 5 12° m 2 m 70 GROWTH ROOM 8a BV)0 c 70 m NOV. II°C TREATMENT 6°C TREATMENT 1° C TREATMENT Fig. 5 . Schematic relationship of seedling development and hardiness to timing, duration and severity of cold treatments (Experiments 1 and 2 ) . Circled numbers indicate the succession of timing treatments in their i n i t i a l developmental positions. Arrowheads indicate the growth status at the end of their ^ respective temperature treatments or under outdoor conditions in Victoria, B.C. The dotted portion of xr the treatment lines represents the 2-week period during which no change in hardiness could be detected (Fig. 4 ) . 28 f i n a l c o n d i t i o n of the plant depends p r i n c i p a l l y upon the amount of time spent wi t h i n the su c c e s s i v e l y lower temperature ranges f o r extension growth, maturation (bud set, l i g n i f i c a t i o n ) , and cold acclimation. Thus, f o r example, seedlings exposed to 1°C i n J u l y spent no time w i t h i n the temperature range f o r completing height growth or maturation, but instead developed hardiness while s t i l l small, pale green and succulent i n appearance. The 11°C treatment, on the other hand, allowed maturation to proceed but the plants did not enter the c o l d - a c c l i m a t i o n range. September treatments began with f a i r l y mature plants, which under 6°C a t t a i n e d a hardiness equivalent to that induced by the P C treatment begun i n J u l y ; 11°C at t h i s time caused some loss of hardiness by mid-November compared with the outdoor plant. The slope of the a r b i t r a r i l y p o s itioned growth-stage boundaries allows f o r a l i m i t e d endogenous c o n t r o l of development which was observed under constant conditions. Experiment 3 - Photoperiod and Light I n t e n s i t y The e f f e c t of photoperiod under the low l i g h t i n t e n s i t y of Experiment 1 was to increase hardiness only at the intermediate, 12-hr l e v e l . Differences between 8 and 16-hr treatments were not s i g n i f i c a n t , suggesting that a photosynthetic d e f i c i e n c y was l i m i t i n g a c c l i m a t i o n under the normally favourable 8-hr day. This experiment was designed to c l a r i f y these r e l a t i o n s h i p s . 29 Twelve plants each of the Mt. Prevost and Prince George provenances were placed under photoperiods of 0, 1, 2, 4, 8, 12, 16, 20 and 24 hr i n a cold room at 1+1.5'C f o r 6 weeks. A l i g h t i n t e n s i t y of 800 f t - c was provided i n each l i g h t - t i g h t compartment by two 40 w fluorescent tubes (one "cool white" and one Sylvania E l e c t r i c "gro-lux") 20 cm above the plant tops. A i r was c i r c u l a t e d with fans so that average temperatures around the plants remained uniform i r r e s p e c t i v e of photoperiod. Single and double layers of shading c l o t h reduced l i g h t i n t e n s i t i e s to 200 and 50 f t - c respec-t i v e l y i n subsections of the 8-hr and 16-hr compartments. Plants were 10 weeks old when treatments began and had not formed terminal buds. Controls, under a 12-hr, 21-15°C day-night regime continued to receive a p p l i c a t i o n s of f e r t i l i z e r . Hardiness was assessed by f r e e z i n g whole plants, then sampling f o l i a g e f o r r e l a t i v e conduc-t i v i t y determinations (20), and a l s o recording v i s i b l e i n j u r y a f t e r a f u r t h e r 4-day period. Figure 6 shows that 16-hr days were i n h i b i t o r y to a c c l i m a t i o n at each of three l i g h t i n t e n s i t i e s according to two i n j u r y c r i t e r i a a f t e r two f r e e z i n g tests, r e l a t i v e to the 8-hr day. Light i n t e n s i t y became severely l i m i t i n g below 200 f t - c and increased a c c l i m a t i o n at l e a s t up to 800 f t - c . The greater acclimating e f f e c t of 8-hr days was exhibited under the red-emitting fluorescent tubes of t h i s experiment even at l i g h t i n t e n s i t i e s h a l f those of the non-effective 8-hr incandescent treatments of Experiment 1. This may be associated OAY LENGTH 16 HOURS OAY LENGTH 8 HOURS CONTROL FROST AT -8°C FROST AT -I2°C Fig. 6. Effect of light intensity and photoperiod on hardi-ness (Experiment 3 ) . Controls remained in an environment favourable to growth, while a l l others experienced 1°C for 6 weeks. Different plants were used in each of the two freezing tests. Numbers on columns indicate average visual score (out of 10) or conductivity percent. Four plants from each treatment were sampled for conductivity, of which two were subsequently assessed visually. Z9b LU 4 0 -h-< cn z> LL. 3 0 -U . o -EX-2 0 -K - -Z LU 1 0 -O CC L U 0-Fig 7. Freezing injury to foliage after conditioning at 1 C under a range of photoperiods (Experiment 3). Each point is the mean of obser-vations on four plants. The increased hardiness at intermediate day-lengths is highly significant. 30 with the fluorescent emission having (1) a greater proportion of p h o t o s y n t h e t i c a l l y u s e f u l l i g h t or (2) a greater proportion of red to f a r - r e d l i g h t . A comparison of the whole range of b r i g h t photoperiods f o r hardiness i n d u c t i o n i s shown i n Figure 7. The trough corresponding to minimum i n j u r y under 8-hr photoperiods i s h i g h l y s i g n i f i c a n t . This optimum was a l s o e x h i b i t e d by the i n t e r i o r provenance of n o r t h e r l y l a t i t u d e . Experiment 4 - Red and Far-Red Night-Interruptions The aim was to demonstrate the mediation of the phytochrome system i n the photoperiod response of growth and hardiness studied e a r l i e r . This has been postulated by various workers (e.g., 5, 24), and r e c e n t l y shown by experiment i n the broadleaved species Cornus  s t o l o n i f e r a Michx. (25). A unique e f f e c t of red l i g h t (650 nm) which can be reversed by f a r - r e d l i g h t (730 nm) c o n s i t i t u e s strong evidence f o r the p a r t i c i p a t i o n of t h i s pigment system i n a p a r t i c u l a r phenological process. In the present experiment, as i n work on flower i n i t i a t i o n (17), the long inductive dark period was b r i e f l y i n t e r r u p t e d by red (R) and f a r - r e d l i g h t (FR). Five treatments were ap p l i e d by the apparatus i l l u s t r a t e d i n Figure 8. This consisted of f i v e l i g h t - p r o o f compartments each with i n t e r i o r incandescent l i g h t i n g at approximately 800 f t - c . Four 500 w Sylvania E l e c t r i c "cool beam" r e f l e c t o r f l o o d lamps mounted e x t e r n a l l y under a l i g h t - p r o o f cover and above a system of colour f i l t e r windows, provided d i r e c t i o n a l beams of R and FR Fig. 8. Apparatus for night-interruption treatments during hardening (Experi-ment 4). Plants (G) in cooled chambers received long or short days from incandes-cent lamps (F). 500 w flood lamps (A), with reflectors transparent to infra-red, provided a 15-min burst of red or far-red light. B and C respectively are the liquid and solid f i l ter components for red; D and E, for far-red. Numbers beneath designate the treatment combinations (see text). Air exit vents (H) were light-proof. 30b 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 W A V E L E N G T H nm Fig. 9. The spectral distribution of energy from red and far-red sources (Experiment 4). The integral energy (= "intensity") in each case is about 60 uw. 31 l i g h t to the plants. The colour f i l t e r s consisted of red or f a r - r e d Carolina Monochromatic Light F i l t e r s (Carolina B i o l o g i c a l Supply Co.) i n combination with a d d i t i o n a l components of other CBS F i l t e r s , placed beneath a " p l e x i g l a s s " tank containing i n f r a - r e d absorbing s o l u t i o n s , i n accordance with the system developed by Poff'and Norris (15). The treatments were given on a 24-hr c y c l e f o r 9 weeks. Treatments 1 to 4 consisted of an 8-hr l i g h t period, and a 16-hr dark period which was e i t h e r (1) continuous, (2) i n t e r r u p t e d i n the middle by 15 min of R l i g h t , (3) i n t e r r u p t e d by 15 min of R followed by 15 min of FR, or (4) i n t e r r u p t e d by 15 min of FR. Treatment 5 was a 16-hr l i g h t period with an 8-hr continuous dark period. The t o t a l energy of R and FR bursts, measured as the area beneath intensity/wavelength curves (Fig. 9), was equalized by adj u s t i n g the distance of the lamps. The spectrum of r a d i a t i o n i n t e n s i t i e s was measured at plant height with an ISCO model SRR spectroradiometer (Instrument S p e c i a l i t i e s Co., L i n c o l n , Nebraska) and remote probe. The long-day treatment received i t s a d d i t i o n a l 8 hours of l i g h t from a low i n t e n s i t y (25 w) incandescent source so as not to introduce s i g n i f i c a n t photosynthetic or a i r temperature d i f f e r e n c e s . Flood lamps were switched on by an automatic timing system f o r the 15-min period every night. Each compartment was cooled by a i r conditioners under thermostatic c o n t r o l . The experiment was repeated three times under d i f f e r e n t conditions i n an e f f o r t to obtain c l e a r - c u t r e s u l t s , observations of bud development and measurements of height growth were made before and a f t e r treatments i n each case. I n i t i a l l y , groups of 20 3.5-month-old plants, nearing the end of extension growth and bearing i n c i p i e n t buds i n some cases, were given the f i v e t r e a t -2 ments. R and FR r a d i a t i o n i n t e n s i t i e s were 50 and 60 uw/cm res p e c t i v e l y , with a 10% v a r i a t i o n between chambers and across a beam. Day and night temperatures were r e s p e c t i v e l y 21±4 and 10±4°C, the v a r i a t i o n c y c l i n g with a 1-hr period. In the second t r i a l , i n t e n s i t i e s were increased, and the proportion of FR was increased f u r t h e r to compensate f o r i t s greater r e f l e c t a n c e from 2 the f o l i a g e . R and FR had i n t e n s i t i e s of 570 and 3500 uw/cm r e s p e c t i v e l y i n t h i s case. Three-month-old plants of the C h i l l i w a c k provenance were a l s o treated. In the t h i r d t r i a l , seedlings at the stage of rapid extension growth (2 months) were treated so that e f f e c t s on growth and development might be more obvious. The high i n t e n s i t i e s , which had produced no s i g n i f i c a n t l y c l e a r e r response, 2 were reduced to almost h a l f t h e i r o r i g i n a l l e v e l at 30 ^iw/cm f o r both wavelengths. The temperature c o n t r o l was improved to e l i m i -nate small d i f f e r e n c e s between chambers, and the average night temperature was r a i s e d to 16±1'C so that a c c l i m a t i o n could not be a t t r i b u t e d to cool temperatures. In a l l cases, at the end of the treatment period needle samples were taken from 10 to 12 plants to determine hardiness by f r e e z i n g at 3 3C temperature i n t e r v a l s and v i s u a l l y assessing i n j u r y as described. 33 Results of moderate and high i n t e n s i t y i n t e r r u p t i o n s of the long i n d u c t i v e dark period are shown i n Figure 10. A c t i v e l y growing 3.5-month-old seedlings under short days had ceased height growth and set buds a f t e r 9 weeks, and were about 4'C hardier. Red l i g h t i n t e r r u p t i o n s caused a s i g n i f i c a n t decrease i n both maturation and hardiness («&1.5°C) i n accordance with the hypothesis that phytochrome i s involved. Far-red i n t e r r u p t i o n s alone had no e f f e c t , but when applied immediately a f t e r the red caused a f u r t h e r , and s i g n i f i c a n t , extension of the red l i g h t e f f e c t — that i s , more growth and lower hardiness. No r e v e r s a l of the red l i g h t e f f e c t was demonstrated. Seedlings beginning treatment i n October ( T r i a l 2) were equa l l y t a l l and dormant a f t e r 9 weeks whatever the treatment. In the case of the c o a s t a l provenance t h i s was expected because the seedlings were older, with buds set, when treatments began. The high eleva-t i o n C h i l l i w a c k provenance probably completes seasonal growth i n response to longer photoperiods because f r o s t occurs e a r l i e r i n i t s native s i t e , and presumably i t had received t h i s stimulus outdoors before treatments s t a r t e d . However, i n both cases, the e f f e c t of n i g h t - i n t e r r u p t i o n s on cold a c c l i m a t i o n was s i m i l a r to that already described. Reversal by f a r - r e d was not demonstrated even at a three-times greater "absorbed" energy l e v e l than pre-ceding red ("absorbed" denotes i n c i d e n t minus r e f l e c t e d r a d i a t i o n ) . I n t e r r u p t i o n treatments i n the t h i r d t r i a l produced plants which were not s i g n i f i c a n t l y less hardy nor f a s t e r growing than 33a DUNCAN 5 0 0 m trial I lOOr - 6 - 8 - IO -12 -14 4 0 2 0 O - 4 CHILLIWACK 1 0 5 0 m - I O -12 - 1 4 T E M P E R A T U R E f ract ion of buds set dur ing t rea tment 5 0 % height growth -16 °C Fig. 10. Effect of night interruptions on hardiness of Duncan (Mt. Prevost) and Chilliwack provenances (Experiment 4). 0 short day, 0 long day, uninterrupted night night-interruption by 15 min red lig h t , by 15 min far-red l i g h t , or by 15 min R followed by 15 min FR. Each curve represents eight plants, each point on a curve i s the mean of 10-needle samples taken from the eight plants. The effect of R light i n reducing hardiness, and of FR i n further reducing hardiness were significant, as were the differences i n development i n T r i a l 1. 34 plants r e c e i v i n g the short-day (uninterrupted night) treatment, presumably because energy l e v e l s were below the threshold f or response (data are not shown). The d i f f e r e n c e s i n hardiness and growth between long- and short-day treatments at the higher average temperature of t h i s t r i a l , however, were greater than i n the two e a r l i e r t r i a l s : 116% height growth, no buds, versus 25% growth, a l l buds set, f o r long and short days r e s p e c t i v e l y , and a hardiness d i f f e r e n c e of 5°C. Experiment 5 - Sequential Acclimation Treatments Three treatments, p o s s i b l y responsible f o r d i f f e r e n t stages of acclimation, were applied i n various sequences to groups of twelve 3 .5-month or 5-month-old seedlings. Treatments were: (0) a greenhouse c o n t r o l with night temperature at 1 5 . 5 i 2 ° C and day temperatures i n the range 17 to 2 5 °C, provided with supplementary incandescent and fluorescent l i g h t (800 f t - c ) to extend the photo-period to 16 hr; (1) short days (8 hr, 800 f t - c mixed a r t i f i c i a l l i g h t ) , with day temperature 1 6 i o . 5°C and night temperature 1 4 ± 1 . 5 ° C ; (2) short days as i n (1) but with 2 2 5 ± 2 5 f t - c , and a constant c h i l l i n g temperature of l.S^O.S'C (2'C with l i g h t s on); (3) a short-day c h i l l i n g treatment as i n ( 2 ) , but with a 6-hr f r o s t at -7+l°C i n the middle of the dark period. An a d d i t i o n a l twelve plants i n the t h i r d a c climation treatment were kept i n continuous darkness (treat-ment 3D). Temperatures during the n i g h t l y f r o s t descended at approximately 10'C/hr and caused i c e to form i n the leaves. Plants from the r e l a t i v e l y high e l e v a t i o n Mt. Benson provenance were the p r i n c i p a l subjects. Treatments began on J u l y 6, 1971, 5 months a f t e r germination, at which time height growth had ceased (at about 15 cm) and small terminal buds were set. The t o t a l a c c l i m a t i o n period of 18 weeks was subdivided i n t o three consecu-t i v e i n t e r v a l s of 5, 8 and 5 weeks and the following sequences of treatments defined r e s p e c t i v e l y : 0-0-0, 1-1-1, 2-2-2, 1-2-3, 1-2-3D and 2-2-3. Thus, f o r example, the 1-2-3 designation means that plants were given 5 weeks of treatment 1 (short days), followed by 8 weeks of treatment 2 (SD + c h i l l i n g ) , then 5 weeks under treatment 3 (SD + c h i l l i n g + night f r o s t ) . For two addi-t i o n a l sequences, 1-2 and 1-3, the ac c l i m a t i o n period was considered as being i n two parts: 8 and 10 weeks. Excised-needle samples from a l l p l a n ts of the Mt. Benson provenance were measured f o r hardiness by f r e e z i n g t e s t s at the end of the 5th, 13th and 18th weeks, and a l e t h a l temperature was estimated f o r each plant. An a d d i t i o n a l sample of 5 needles was removed from each plant a f t e r the 18th week. These were weighed fresh (±0.05 mg) and a f t e r oven-drying at 105°C fo r 24 hr (±0.02 mg) to determine water content. Smaller, 3.5-month-old Mt. Prevost seedlings (averaging 8 cm i n height and with buds not set) were placed under some of these treatment sequences, t o t a l l i n g only 12.5 weeks duration and beginning 21 July. In t h i s case the i n t e r v a l s were 5, 5 and 2.5 weeks f o r sequences 0-0-0, 1-1-1, 2-2-2, and 2-2-3; and 7.5 and 5 36 weeks f o r the 1-2 treatment sequence. Hardiness of these plants was measured only at the end of the 12.5-week period. A l l plants were returned to the c o n t r o l environment (treatment 0) for a 2-month period a f t e r f i n a l hardiness sampling, to assess the extent to which treatments had induced or broken r e s t . The e f f e c t of sequences i n which short-day, c h i l l i n g , and night f r o s t treatments were applied i s shown i n Figure 11. Plants under warm long days (0-0-0) d i d not f l u s h , thus i n d i c a t i n g that a l l plants had entered r e s t p r i o r to treatment. Growth t e s t s afterwards showed that a l l c h i l l i n g treatments had broken r e s t . C h i l l i n g temperatures i n conjunction with short days (2-2-2) caused most rapid acclimation, and t h i s developed mainly w i t h i n the f i r s t 5 weeks. P r i o r exposure to warm short days (1-2) d i d not increase the e f f e c t i v e n e s s of the c h i l l i n g treatment i n s p i t e of the f a c t that the short-day treatment alone caused a change i n appearance (darker, r i g i d and more spreading leaves, woodier stem) and a moderate increase i n hardiness of 5'C. Night f r o s t treatments caused considerable deepening of hardiness i f plants had f i r s t received a period of c h i l l i n g , and i f l i g h t continued to be supplied (1-2-3, 2-2-3). Frosts applied d i r e c t l y a f t e r the warm short-day treatment (1-3) caused death of most i n d i v i d u a l s , although r e s u l t i n g i n f u r t h e r a c c l i m a t i o n of those which survived. This acclimation, however, was not s i g n i f i c a n t l y greater than that of plants only c h i l l e d during the same period (1-2). Stage 3 treatment i n 36a - 4 I i i i i 1 --1 J i i O 2 4 6 8 IO 12 14 16 18 WEEKS CONDITIONING Fig. 11. Effect of treatment sequence on hardiness of the Mt. Benson provenance (Experiment 5). Number of plants i n each treatment combina-tion at 18 weeks was 12, except in the 1-3 combination (which was omitted from the analysis at this stage due to low survival). Points bearing the same letter do not d i f f e r significantly at the 5% probabil-i t y level (this analysis was conducted for each treatment duration independently). Numbers denote foliage water content (fresh weight basis, and where sufficient foliage remained for sampling) at the end of treatment, and common subscript letters indicate the lack of s i g -nificant difference between them. Additional points, •, represent hardiness of Mt. Prevost plants at 13 weeks, after corresponding treat-ments . 37 continuous darkness (1-2-3D) increased hardiness to a much l e s s e r extent and caused the f o l i a g e to abscise. Maximum hardiness was attained under the conditions most re cembling those i n nature (1-2-3). This was s i g n i f i c a n t l y greater than hardiness under 2-2-3 and the plants were acclimating at a f a s t e r average rate when the experiment ended. Water content, on a f r e s h or dry weight ba s i s , of f o l i a g e subjected to these two f r e e z i n g treatments was s i g n i f i c a n t l y lower, whereas among most other treatments water contents were s i m i l a r (Fig. 11). Yet i f these leaves were rehy-drated to "normal" water l e v e l s by a 14-hr immersion i n cold water, t h e i r hardiness remained unchanged (data not shown). The younger and r e l a t i v e l y low e l e v a t i o n Mt. Prevost provenance responded i n a s i m i l a r way (Fig. 11) with the exception that night f r o s t s i n f l i c t e d g radually i n c r e a s i n g i n j u r y , so that the e f f e c t of t h i s f a c t o r on hardiness could not be evaluated. DISCUSSION The r e l a t i v e e f f e c t s of temperature, photoperiod and l i g h t i n t e n s i t y on hardiness reported above are i n close agreement with those found i n older seedlings of thi= species a f t e r c e s s a t i o n of growth (5, 6). Maximum ac c l i m a t i o n under a s i n g l e treatment occurred at low p o s i t i v e temperatures and short (8-hr) days. Low l i g h t i n t e n s i t i e s had a longer optimum photoperiod (12-hr), and very short days (less than 6 hr) reduced hardiness whatever the l i g h t i n t e n s i t y — a l l i n accordance with the accepted view that 38 there i s both photoperiodic and photosynthetic c o n t r o l of acclimation. Upper and lower threshold l e v e l s of l i g h t i n t e n s i t y are a l s o i n agreement with reported values (6, 26). Long-day i n h i b i t i o n was important only at higher temperatures. The i n a b i l i t y of germinants to acquire f r e e z i n g tolerance (Fig. 4) does not appear to have been pr e v i o u s l y noted. The a c q u i s i t i o n of f r e e z i n g avoidance i n them i s most probably an a r t i f a c t of reduced water f l u x through the plant and s l i g h t dehy-dra t i o n , which occurred i n the cold room — water content and supercooling are frequently r e l a t e d . The true a c c l i m a t i o n at s l i g h t l y l a t e r stages of development (Fig. 1) c l a r i f i e s s e v e r a l points. (a) Since both non-dormant (July) and dormant (September) plants acclimated a t low temperature, then the process does not depend on entry i n t o r e s t . This q u a l i t a t i v e independence has been demonstrated (10) i n viburnum plicaturn Miq. (b) The short-day treatment was equally e f f e c t i v e on both a c t i v e l y growing plants, and those with buds set and i n r e s t . Moreover t h i s treatment, while markedly a f f e c t i n g hardiness, had no e f f e c t on development under low l i g h t i n t e n s i t y and cool conditions. There-fore, the short day does not increase hardiness by inducing r e s t or promoting bud development and l i g n i f i c a t i o n , but does so independently. (c) The s i g n i f i c a n t l y greater hardiness induced at l a t e r stages of seasonal development (Fig. 4) appears to be endogenously superimposed upon environmental e f f e c t s . That i t 39 i s not a r e s u l t of exposure to shortening days outdoors (before treatments began) i s shown by the f a i l u r e of warm, short-day treatment f o r 5 or 13 weeks to increase the a c c l i m a t i o n during subsequent c h i l l i n g (Fig. 11). An endogenous seasonal rhythm of hardiness, ( i . e . , under constant conditions) has been demonstrated i n Pinus cembra L. (19) and was a l s o i n d i c a t e d by observations on red o s i e r dogwood (7) and Haralson apple (8). Such rhythms can be ones of i n d u c i b i l i t y rather than hardiness per se. (d) The apparent opposition between development and c o l d - a c c l i m a t i o n (Fig. 2) i s most simply viewed as a consequence of d i f f e r i n g temperature optima rather than a production of hardiness i n h i b i -t o r s during growth. Figure 5 incorporates only the endogenous rhythm and temperature e f f e c t s because no photoperiodic c o n t r o l of development was observed i n Experiment 1, although t h i s became s i g n i f i c a n t at higher l i g h t i n t e n s i t i e s and temperatures (Fig. 10), as found by others (11, 17, 23). The e f f e c t of night i n t e r r u p t i o n s by red l i g h t i n b r inging about a s i g n i f i c a n t reduction i n hardiness suggests, but does not prove, a phytochrome c o n t r o l because the red l i g h t e f f e c t was not shown to be unique among v i s i b l e wavelengths, nor reversed by f a r -red. The t o t a l energy supplied was two or three orders of magnitude above the threshold f o r the flowering response i n other species. In the second t r i a l the FR i n t e n s i t y was s u f f i c i e n t l y high (3500 2 uw/cm ) to compensate for i t s two times greater r e f l e c t a n c e from the leaves (measured with the ISCO), the lower absorption by P as opposed to P (x 1.5), and the lower quantum e f f i c i e n c y of (17). I t therefore could not simply have been e n e r g e t i c a l l y i n s u f f i c i e n t to reverse the e f f e c t of red. In f a c t the t o t a l FR energy supplied i n T r i a l 2 was close to the l e v e l of the "high energy r e a c t i o n " (HER) i n which FR or blue l i g h t can produce e f f e c t s s i m i l a r to lower i n t e n s i t i e s of red (17). However, the s i m i l a r FR enhancement of hardiness at 60 uw l e v e l s i n T r i a l 1, and t y p i c a l r e v e r s a l e f f e c t s on hardiness shown at much higher energies i n red o s i e r dogwood (25). both suggest that the HER was not a complicating f a c t o r here. Prolonged treatment of plants with FR of low i n t e n s i t y sometimes causes e f f e c t s i d e n t i c a l with those of red (17) and Dinus' data f o r Douglas-fir seedlings provide such an example (4) — a l b e i t at 30-min exposures rather than the 15-min exposures used i n the present study. So i t appears l i k e l y that the present FR enhancement might be a f u r t h e r case of t h i s long-exposure phenomenon, which i s , however, s t i l l explainable i n terms of the phytochrome system (17). The greatest a c c l i m a t i o n under a sequence of treatments (Fig. 11) occurred when these corresponded roughly with the three stages summarized by Weiser (24). When only short-day and c h i l l i n g treatments are considered, there was more or le s s complete inde-pendence: c h i l l i n g f o r 11 weeks brought hardiness to a c e r t a i n P fr conversion (x 1.5) compared with the reverse process \ l e v e l regardless of preceding warm short days with t h e i r accompanying morphological changes; short days induced a d i s -t i n c t , constant but lower l e v e l of hardiness which was not a d d i t i v e to subsequent c h i l l i n g e f f e c t s — at l e a s t i n the balance of the 4.5-month treatment period. Both treatments, however, appeared to be necessary f o r ac c l i m a t i o n at f r e e z i n g temperatures because hardiness under 1-2-3 exceeded both 2-2-3 and survivors of 1-3 (which were no hardier than plants given 1-2). I t i s thought that the be n e f i t of warm short days was connected with t h e i r obvious e f f e c t on morphology, which seemed to confer better long-term p r o t e c t i o n against repeated f r e e z i n g , whether the furth e r a c c l i m a t i o n of p e r i o d i c a l l y frozen t i s s u e c o n s t i t u t e s a t h i r d "stage" i n the p h y s i o l o g i c a l or biochemical sense, as opposed to a c l o s e r approach to the temperature optimum f o r a s i n g l e cold-induced process, was not d e f i n i t e l y e s t a b l i s h e d . Three points favour the hypothesis of a t h i r d stage: (a) the dual short day and c h i l l i n g p r e r e q u i s i t e s , (b) the somewhat f a s t e r response, and (c) the concurrent dehydration as a po s s i b l e induc-t i v e f a c t o r d i s t i n c t from temperature (12). Alden's data (1) a l s o i n d i c a t e that the sub-freezing response of cut Douglas-fir twigs i n the dark was associated with dehydration. A separate and r e l a t i v e l y short-term p h y s i o l o g i c a l response to sub-zero tempera-tures, which requires l i g h t , has al s o been i n d i c a t e d by hardiness and gas exchange measurements i n t h i s species (18). The i n i t i a l independence and eventual reinforcement of the hardening f a c t o r s (endogenous, short-day, c h i l l i n g and freezing) must provide a valuable adaptive f l e x i b i l i t y to the plant i n nature, p a r t i c u l a r l y at higher a l t i t u d e s and in l a n d . In t h i s respect there i s good agreement with the a c c l i m a t i o n process i n apple (8). From a p r a c t i c a l viewpoint, i t i s more important that hardiness can be induced by a s i n g l e treatment at any stage a f t e r i n i t i a l e p i c o t y l growth. Such a "minimal a c c l i m a t i o n " might allow overwintering of plants soon a f t e r greenhouse germination, thereby permitting a more continuous schedule of sowing and other nursery operations. I t would a l s o permit pre-adaption of groups of mature seedlings destined f o r r e l a t i v e l y cold p l a n t i n g s i t e s . 43 REFERENCES 1. Alden, J . N. 1971. Freezing r e s i s t a n c e of t i s s u e s i n the twig of Douglas-fir. Ph.D. t h e s i s , Oregon State Univ. C o r v a l l i s , Oregon. 2. Cox. W. and L e v i t t , J . 1969. D i r e c t r e l a t i o n between growth and f r o s t hardening i n cabbage leaves. Plant P h y s i o l . 44:923-928. 3. Dexter, S. T., Tottingham, W. E. and Graber, L. F. 1932. Investigations on the hardiness of plants by measurement of e l e c t r i c a l c o n d u c t i v i t y . Plant P h y s i o l . 7:63-78. 4. Dinus, R. J. 1968. E f f e c t of red and f a r - r e d l i g h t upon growth of D o u g l a s - f i r (Pseudotsuga menziesii (Mirb.) Franco seedlings. Ph.D. t h e s i s , Oregon State Univ., C o r v a l l i s , Oregon, D i s s e r t . Abstr. 29 B (6). 5. Driessche, R. van den 1969. Influence of moisture supply, temperature and l i g h t on f r o s t hardiness changes i n Douglas-fir seedlings. Can. J . Bot. 47:1765-1772. 6. 1970. Influence of l i g h t i n t e n s i t y and photoperiod on f r o s t hardiness development i n Douglas-fir seedlings. Can. J . Bot. 48:2129-2134. 7. Fuchigami, L. H., Weiser, C. J. and Evert, D. R. 1971. Induction of cold a c c l i m a t i o n i n Cornus s t o l o n i f e r a Michx. Plant P h y s i o l . 47:98-103. 44 8. Howell, G. S. and Weiser, C. J . 1970. The environmental c o n t r o l of cold acclimation i n apple. Plant P h y s i o l . 45:390-394. 9. Huystee, R. B. van, Weiser, C. J. and L i , P. H. 1967. Cold a c c l i m a t i o n i n Cornus s t o l o n i f e r a under natural and c o n t r o l l e d photoperiod and temperatures. Bot. Gaz. 128:200-205. 10. I r v i n g , R. M. and Lanphear, F. O. 1967. Environmental c o n t r o l of cold hardiness i n woody plants. Plant P h y s i o l . 42:1191-1196. 11. Lavender, D. P., Ching, K. K. and Hermann, R. K. 1968. E f f e c t of environment of the development of dormancy and growth of Douglas-fir seedlings. Bot. Gaz. 129:70-83. 12. L i , P. H. and Weiser, C. J . 1970. Increasing cold r e s i s t a n c e of Cornus s t o l o n i f e r a by a r t i f i c i a l dehydration. C r y o b i o l . 8:108-111. 13. McGuire, J. J . and F l i n t , H. L. 1962. E f f e c t s of temperature and l i g h t on f r o s t hardiness of c o n i f e r s . Proc. Am. Soc. Hort. S c i . 80:630-635. 14. Olien, C. R. 1961. A method of studying stresses occurring i n plant t i s s u e during f r e e z i n g . Crop S c i . 1:26-34. 15. Poff, K. L. and Norris, K. H. 1967. Four low-cost monochromatic sources of known equal i n t e n s i t y . Plant P h y s i o l . 42:1155-1157. 16. Sakai, A. 1955. The r e l a t i o n s h i p between the process of development and the f r o s t hardiness of the mulberry tree. Low Temp. S c i . , Ser.B 13:21-31. 45 17. Salisbury, F. B. and Ross, C. 1969. Plant Physiology. Wadsworth Publ. Co., Belmont, C a l i f o r n i a . 18. Scheumann, W. and B o r t i t z , S. 1965. Studien zur physiologie der Frosthartung b e i Koniferen. I. Die Rolle des Lich t e s bei der Frosthartung und Verwohnung. B i o l . Zbl. 84:489-500. 19. Schwarz, W. 1968. Der e i n f l u s s der Temperatur und Tageslange auf d i e Frostharte der Zirbe. In H. P o l s t e r , ed. Klimaresistenz Photosynthese und Stoffproduktion (pp 55-63) Deut. Akad. Landwirtsch. B e r l i n . . 20. Timmis, R. 1972. An excised-needle f r e e z i n g t e s t of cold hardiness i n Douglas-fir. Chapter 1 of t h i s t h e s i s . 21. Timmis, R. 1972. The hardening of one-season Douglas-fir and lodgepole pine under a r t i f i c i a l conditions. P a c i f i c For. Res. Cent., Can. For. Serv., I n t e r n a l Report BC-35. 22. Tumanov, I. I., Kuzina, G. C. and Karnikova, L. D. 1964. Dormancy and f r o s t r e s i s t a n c e i n Betula verrucosa and Robinia pseudoacacia. Sov. Plant P h y s i o l . 11:592-601. 23. Vegis, A. 1953. The s i g n i f i c a n c e of temperature and the d a i l y l i g h t / d a r k period i n the formation of r e s t i n g buds. Experientia 9:462-463. 24. Weiser, C. J . 1970. Cold r e s i s t a n c e and i n j u r y i n woody pl a n t s . Science 169:1269-1278. 46 25. Williams, B. J., J r . , P e l l e t , N. E. and K l e i n , R. M. 1972. phytochrome c o n t r o l of growth cessation and i n i t i a t i o n of cold a c c l i m a t i o n i n selected woody pl a n t s . Plant P h y s i o l . 50:262-265. 26. Zehnder, L. R. and Lanphear, F. O. 1966. The in f l u e n c e of temperature and l i g h t on the cold hardiness of Taxus cuspidata. Proc. Amer. Soc. Hort. S c i . 89:706-713. CHAPTER 3 TRANSLOCATION OF DEHARDENING AND BUD-BREAK PROMOTERS IN CLIMATICALLY "SPLIT" DOUGLAS-FIR ABSTRACT Forked Douglas-fir seedlings were exposed to warm (20°C) and cold (2°C) environments simultaneously, by i n s e r t i n g one branch i n each environment. A l l received the same l i g h t conditions. Hardiness of f o l i a g e a f t e r one to f i v e months was measured as the f r e e z i n g temperature causing 50% v i s i b l e i n j u r y i n excised needles. The c h i l l i n g stimulus f o r breaking r e s t and inducing hardiness was confined to the c h i l l e d branch, but the warm branch apparently transmitted a f a c t o r which prevented f u l l hardening i n the c h i l l e d one. A f a c t o r moving i n the same d i r e c t i o n a l s o promoted f l u s h i n g i n branches c h i l l e d only at night from December to June (and r e c e i v i n g greenhouse tempera-tures and natural photoperiods by day). This was not replaceable by a s i n g l e i n j e c t i o n of g i b b e r e l l i c a c i d . Factors from the expanding shoot caused loss of short-day-induced hardiness i n previous year's f o l i a g e and stimulated cambial d i v i s i o n . C h i l l i n g at night prevented the dehardening but d i d not prevent cambial a c t i v i t y . The dehardening f a c t o r was translocated to an opposite branch whereas movement of cambium stimulator was s t r i c t l y b a s i p e t a l . Tt i s suggested that promoter-inhibitor l e v e l s c o n t r o l l i n g dormancy are independently regulated, and that a two-stage 49 dehardening process might protect against premature loss of hardiness in nature. The simultaneous induction of cold acclimation and dehardening in the same plant provides material for comparing properties of. hardy and nonhardy tissue. INTRODUCTION Recent studies have demonstrated that, whereas the short day (SD) induction and long day (LD) i n h i b i t i o n of hardiness are tr a n s l o c a t a b l e i n broadleaved woody plants (6, 7, 8, 10), the cold stimulus f o r deeper a c c l i m a t i o n i s not (8). The loss of hardiness i n Douglas-fir has been a t t r i b u t e d only to r i s i n g temperature (4), but i n other species i t a l s o depends on the sta t e of winter dormancy or " r e s t " (11) — although r e s t has l i t t l e i n f l u e n c e on the a c q u i s i t i o n of hardiness (18). Questions thus a r i s e as to whether there are other dehardening s t i m u l i i n co n i f e r s , whether warm temperature e f f e c t s are translocated, and what might the pr e c i s e r e l a t i o n s h i p s be between environment, shoot growth phenology and hardiness i n spring. Such r e l a t i o n -ships have p r a c t i c a l importance i n f o r e s t nurseries where provenances are being r a i s e d away from the environment to which they are s p e c i f i c a l l y adapted. The o v e r a l l aim of the fol l o w i n g i n v e s t i g a t i o n was to confirm the suspected (and, a t that time, unreported) l o c a l i z e d nature of the c h i l l i n g Ftimulus, with a view to obtaining nonhardy and hig h l y hardy leaves on the same plant f o r comparative studies. The a c t u a l r e s u l t s , however, provide some i n s i g h t i n t o the r e l a t i o n s h i p s discussed above. GENERAL METHODS Two-year-old plants were pruned at the beginning of t h e i r second growing season to produce forked tops. When the branch p a i r s had attained a Length of 5 to 10 cm i n a greenhouse and entered rest under natural photoperiods (November), the plants were incorporated i n t o one of three experiments i n v o l v i n g exposure of each member of a branch p a i r to d i f f e r e n t tempera-tures under the same l i g h t conditions (Fig. 1). In two of the experiments, pots were arranged with respect to the two environ-ments so as to have equal numbers of " s p l i t " plants with warm as with c h i l l e d root systems and stem. Possible e f f e c t s of root and stem temperature on hardiness and f l u s h i n g could then be assessed. Controls, with a l l parts c h i l l e d or a l l parts warmed, were a l s o included. Foam rubber ensured a non-injurious and convection-free s e a l between bark and p l e x i g l a s s at the j u n c t i o n between environments. A i r was c i r c u l a t e d both i n t e r n a l l y and e x t e r n a l l y by fans so that temperature gradients d i d not develop. Hardiness was evaluated by a s e r i e s of f r e e z i n g t e s t s , on samples of 10 excised needles, to temperatures spaced at 3°C i n t e r v a l s across the expected l e t h a l range. Injury was assessed v i s u a l l y a f t e r 7 days and hardiness i n t e r p o l a t e d as the tempera-ture corresponding to 50% i n j u r y as previously described (17). A 1-cm length of branch immediately on each side of the p l e x i g l a s s was excluded from the sampling operation because of p o s s i b l e e f f e c t s of heat conduction along the branch t i s s u e s , where both branches received i d e n t i c a l treatment, only one branch was sampled for hardiness. Analysis of variance was performed assuming that a l l observations were independent (although greater s p e c i f i c s e n s i t i v i t y would have been permitted by t r e a t i n g the "divided p l a n t " observations i n p a i r s ) . Duncan's M u l t i p l e Range t e s t was used to d i s t i n g u i s h i n d i v i d u a l means. EXPERIMENTS AND RESULTS The e s s e n t i a l features of the three experiments are i l l u s t r a t e d i n Figure 1. A l l plants had entered r e s t p r i o r to beginning treatments. Experiment 1 In the f i r s t experiment, conducted on plants of a southeast Vancouver Island provenance, a c h i l l i n g treatment was a p p l i e d to one branch (1.5±0.5°C) while the other was kept warm (21±l°c). Mixed incandescent and fluor e s c e n t l i g h t (400 f t - c ) was supplied during an 8-hr day. Temperatures were 0.5°C higher i n the l i g h t . A f t e r 8 weeks, the c h i l l e d branch was about 9°C h a r d i e r on average than the warm one on the same plant (curves C and D i n F i g . 2). Warm branches had acquired the same moderate degree of hardiness (-1VC) under short days whether or not they had cold partners — showing that the e f f e c t of c h i l l i n g was not trans-located. But c h i l l e d branches with warm partners were s i g n i f i -c a n tly l e s s hardy (by about 5°C) than branches of wholly c h i l l e d plants (curve E, F i g . 2), showing that some translocated f a c t o r was involved. 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 hardiness E X P T ! E X P T 2 E X P T 3 C O L D ROOM 8-hr daylength fo r 6 weeks Fig. 1 . Experimental treatments and their effects on flushing. A plexiglass chamber separates the two temperature treatments under a common light source. Shading represents chilling applied con-tinuously (uniform shading) or at night only (banded shading). Temperatures to the right of oblique stroke are dark-period temperatures. GREENHOUSE COLD ROOM f o r 6 weeks f o r 5 weeks 52-b Fig. 2. Hardiness of foliage on climatically split plants. Letters identify treatments shown in Figure 1. Injury curves B and E each represent 10 branches; C and D each represent 20 branches because data for plants having warm roots and chilled roots (Table I) were pooled. A and F are means of 7 branches. Each point on a curve is the mean of 10-needle samples from each branch. Only treatments B and C did not differ significantly at the 5% l e v e l . Table I. E f f e c t of Root and Stem Temperature on the F o l i a r Hardiness of Warm and C h i l l e d Branches i n Experiment 1. Roots and Stem C h i l l e d Roots ; and Stem Warm Seedling No. Hardine ss °C Seedling No. Hardiness °C C h i l l e d Branch Warm Branch C h i l l e d Branch Warm Branch 1 -32.0 -24.5 11 -22.0 -15.0 2 -19.5 -14.5 12 -28.0 -15.5 3 -26.0 -24.0 13 -23.5 -15.0 4 -31.0 -15.5 14 -22.5 -16.0 5 -21.5 -15.5 15 -26.5 -16.5 6 -27.0 -20.5 16 -28.5 -18.0 7 -32.0 -22.0 17 -22.5 -18.0 8 -19.5 -14.0 18 -31.5 -18.0 9 -25.0 -17.5 19 -29.0 -19.0 10 -31.5 -20.0 20 -29.0 -17.5 Mean -26.5 -18.8 -26. 3 -16.8 associated with the p o s i t i o n of the root system and stem with respect to the two environments (Table 1 ) . Experiment 2 Plants of an i n t e r i o r B r i t i s h Columbia provenance (Cranbrook) were exposed to the natural photoperiods of Vancouver, B.C. i n a greenhouse during the winter hardening and spring dehardening periods of 1970-71. C h i l l i n g was c a r r i e d out only during a 15-hr period which coincided approximately with the nat u r a l night. Under t h i s arrangement the warm branch (and the " a l l warm" controls) received a night temperature of 16.5±2°C, while the c h i l l e d branch (and corresponding controls) experienced 3±2°C during the same period. A i r temperatures around a l l branches during the day depended on the i n t e n s i t y and duration of sunlight, and increased gradually during the period December to June (Fig. 3). Swelling of buds was observed ( A p r i l 9) only on those branches which had been c h i l l e d and had a warm partner. R e l a t i v e hardiness of branches under the. d i f f e r e n t treatments at about t h i s time ( A p r i l 22) was s i m i l a r to that i n Experiment 1 (confirming the translocated "warm branch" e f f e c t ) : warm branches -17°C, c h i l l e d branches with warm partners ft* -25°C, c h i l l e d branches with c h i l l e d partners « -30°C (Fig. 3). Hardiness i n the f o l i a g e of all-warm plants 6 weeks l a t e r (June 8) had not s i g n i f i c a n t l y changed, but had decreased s i g n i f i c a n t l y i n warm branches with c h i l l e d fend flushing) partners. Moreover, t h i s new low l e v e l of hardiness was the same as that of old f o l i a g e on outdoor plants (same provenance) which had flushed at about the same time outdoors. F i n a l l y , there was no s i g n i f i c a n t loss of hardiness i n the (old) f o l i a g e of c h i l l e d branches bearing a new f l u s h . By June 8, only one t h i r d of the wholly c h i l l e d plants had flushed, and these shoots f a i l e d to elongate by more than 1 or 2 cm. These r e l a t i o n s h i p s are shown i n Figure 3, and provide evidence f o r the following hypotheses. (1) Flushing out i s dependent not only on the breakage of re s t by c h i l l i n g , but a l s o upon a t r a n s l o c a t a b l e f a c t o r ( s ) produced during warm nights. Only the c h i l l e d branch with the warm-night partner received both. (2) A second f a c t o r i s produced during the expansion of new shoots, which can cause loss of hardiness i n older f o l i a g e to which i t i s tran s l o c a t e d . This accounts f o r some of the dehardening of outdoor p l a n t s i n spring, and the loss of SD-induced hardiness i n warm branches with c h i l l e d (and flushing) partners. (3) The e f f e c t of t h i s second f a c t o r i s i n h i b i t e d by low night temperatures, because o l d f o l i a g e on the c h i l l e d branch beneath a new f l u s h f a i l e d to deharden. Under Hypothesis (1) i t was expected that "warm-factor" l e v e l s i n branches of wholly c h i l l e d plants would eventually b u i l d up and permit f l u s h i n g , as i n f a c t occurred to a l i m i t e d extent (Fig. 3). In j e c t i o n s of 200 ug g i b b e r e l l i c a c i d (GA 3) i n 10 u l ethanol 1 cm below the buds of three trees on June 26, however, f a i l e d to bring about any advance i n f l u s h i n g date when compared with c o n t r o l s r e c e i v i n g ethanol only. F i g . 3 . H a r d i n e s s and f l u s h i n g o f an i n t e r i o r p r o v e n a n c e u n d e r n a t u r a l p h o t o p e r i o d s i n a g r e e n h o u s e ( E x p e r i m e n t 2 ) . N i g h t t e m p e r a t u r e r a n g e s and 3 - d a y a v e r a g e s o f d a i l y max ima a r e p l o t t e d i n t h e u p p e r a x e s , t o -g e t h e r w i t h p e r c e n t a g e o f p l a n t s f l u s h i n g ( 0 — ) , and t h e t o t a l l e n g t h o f new g r o w t h f r o m a l l b u d s ( v e r t i c a l a r r o w s ) . L e t t e r s i d e n t i f y t r e a t -men ts i n F i g u r e 1 . L o w e r a x e s show c o r r e s p o n d i n g f o l i a r h a r d i n e s s w i t h c o n t i g u o u s h i s t o g r a m c o l u m n s r e p r e s e n t i n g warm and c h i l l e d f o l i a g e on t h e same p l a n t . O u t d o o r ( f l u s h e d ) p l a n t s w e r e m e a s u r e d i n J u n e ( c o n t . ) . T r e a t m e n t s G and H r e p r e s e n t 18 p l a n t s ; K and J e a c h r e p r e s e n t 7 p l a n t s . A l l h a r d i n e s s d i f f e r e n c e s a r e s i g n i f i c a n t a t t h e 5% l e v e l , a s a r e d i f -f e r e n c e s i n f l u s h i n g d a t e and a v e r a g e l e n g t h o f new g r o w t h p e r p l a n t b e t w e e n H and J . £ The r e l a t i o n s h i p between dehardening (Hypothesis 2) and cambial a c t i v i t y (which i s a l s o stimulated during bud expansion) was examined by sect i o n i n g the branches and stem. These sections showed a c l e a r absence of new xylem i n the unflushed member compared with new wood i n the flushed one. In the upper part . of the stem there was cambial a c t i v i t y only on the side d i r e c t l y below the flushed branch, although t h i s xylem segment increased i n g i r t h and decreased i n r a d i a l thickness further down (Fig. 4), presumably due to slow l a t e r a l transport and d i l u t i o n of the stimulating hormone. Experiment 3 The e f f e c t of f l u s h i n g was to cause a loss of hardiness, while the e f f e c t of sub-zero temperatures i n concurrent experi-ments (18) was to increase hardiness beyon^. that obtainable by c h i l l i n g . These observations prompted the t h i r d experiment, with the aim of obtaining greater d i f f e r e n c e s i n hardiness between branches of the same plant and providing a t e s t of hypotheses (2) and (3) . Plants (of Vancouver Island o r i g i n , as i n Experiment 1) were f i r s t subjected to a 16-week c h i l l i n g treatment, under the same conditions as a l l - c o l d controls i n Experiment 1, to break r e s t i n both branches. They were then placed i n the d i f f e r e n t i a l tempera-ture apparatus under the same temperature treatments as Experiment 1 ( i . e . , continuous, uniform warm or c h i l l i n g temperatures), except that i n the c h i l l i n g treatment a 6-hr f r o s t at -7il°C Fig. 4. Stem cross sections of a climatically split seedling bearing a flushing and a dormant branch (x27). New xylen (X) formed directly beneath the flushed branch (1), and was s t i l l observable as a one-sided re- t/, sponse in both middle (2) and basal (3) sections. Distance between (1) and (3) was approximately 7 cm. {£' Stems were harvested 8 weeks after flushing. occurred i n the middle of each 16-hr dark period. Tt was established that needles were frozen rather than supercooled during t h i s period. Flushing began i n the warm branches a f t e r 3 weeks and was not a f f e c t e d by the temperature of the opposite branch or the roots. Of 20 s p l i t p l a n ts only the warm branches of three f a i l e d to f l u s h i n 5 weeks. Two of these were i n the "cold-root" group. Hardiness was evaluated only i n s p l i t - p l a n t branches due to lack of time. Hardiness of old f o l i a g e of flushed branches a f t e r 5 weeks was s l i g h t l y lower than that i n dehardened outdoor plants or dehardened warm branches (partner flushed) of the preceding experiment (-10°C). Hardiness i n c h i l l e d - f r o z e n branches was about -35°C (Fig. 2, curves A and F) . Thus, the t r a n s l o c a t a b l e dehardening f a c t o r produced during shoot expansion i s , i n t h i s case, demonstrated by a comparison of curves A and B i n Figure 2. The i n h i b i t o r y e f f e c t of l o c a l i z e d c h i l l i n g over t h i s f a c t o r was shown i n t h i s case by f a i l u r e of the cold branch to deharden. The a d d i t i o n a l hardening e f f e c t of f r e e z i n g temperatures demon-strated i n an e a r l i e r study (18) was confirmed. A d i f f e r e n c e i n hardiness of approximately 25°C was obtained between branches. DISCUSSION The l o c a l i z e d nature of c h i l l i n g i n inducing hardiness, as shown i n the three experiments, has re c e n t l y been demonstrated i n apple (8). The confinement of e f f e c t s to the c h i l L e d t i s s u e i s al s o c h a r a c t e r i s t i c of rest-breakage (3, 17) and v e r n a l i z a t i o n (15) Translocated hormones have been found to induce or i n h i b i t a c c l i m a t i o n i n several broadleaved species as c i t e d above. However, an a l t e r n a t i v e explanation of the present r e s u l t s could be based on n u t r i t i o n a l requirements. In Experiment 2, the warm branch might simply have supplied a d d i t i o n a l photosynthate to the rest-broken bud of i t s partner thereby permitting the partner to f l u s h even though the partner's own carbohydrate production was c u r t a i l e d by an i n s u f f i c i e n t l y long warm-day (8 h r ) . The expanding shoot might then continue to act as a sucrose sink, causing a n u t r i t i o n a l dehardening i n the warm donor, of the type that occurs during prolonged darkness (12). There are s e v e r a l points against t h i s i n t e r p r e t a t i o n . (1) Foliage on the c h i l l e d branch bearing the new f l u s h d i d not lose hardiness, yet there i s no reason to suppose that i t was any l e s s prone to deharden as a r e s u l t of the putative carbohydrate demand (without making further, complicating assumptions). (2) In Experiment 3, f l u s h i n g proceeded j u s t as r a p i d l y (also under an 8-hr day) i n s p i t e of the absence of a warm partner, i n d i c a t i n g that no addi-t i o n a l photosynthate was necessary. (3) The lower hardiness of c h i l l e d branches with warm partners, when there was no f l u s h i n g , i s d i f f i c u l t to ex p l a i n on any source-sink n u t r i t i o n a l basis, but on a hormonal bas i s t h i s can be a t t r i b u t e d to the same f a c t o r that the warm branch supplies to promote f l u s h i n g . (4) The photosynthetic requirement f o r maintaining hardiness i s small (sufficient was produced under a 200 ft - c 8-hr photoperiod at 2T; in earlier studies, 18), and the period of negative C0 2 uptake in flushing shoots i s relatively brief (13). Thus, photo-synthesis in old needles would probably be adequate for both. These four objections strongly favour the hormonal hypotheses. The data indicating a warm-night flushing promoter, sequential to c h i l l i n g , are not conclusive, because i t was not known for certain (1) whether the absence of a root temperature effect on hardiness (Experiment 1) and on flushing (Experiment 3) was also true for the interior provenance used i n Experiment 2; or (2) whether the 29-day continuous c h i l l i n g requirement for the Cranbrook provenance (17) had been satisfied by the 130 days of intermittant c h i l l i n g actually given, with regard to the latter uncertainty, Bennett (2) found that the c h i l l i n g require-ment of pear buds was increased by alternating warm and cold temperatures. If, as Bennett's data imply, there were a warm-day reversal of nightly c h i l l i n g , then flushing promotion i n s p l i t plants could be attributed, for example, to some continuing effect of the warm-night factor in preventing this reversal. A more probable explanation in the absence of adequate c h i l l i n g i s that the warm branch might simply have supplied a long photoperiod hormone which, in p a r t i a l l y rest-broken plants, i s often necessary to provide the added stimulus 'for growth (14). The photoperiod effect may not have been perceived by the cold-night branch due to 59 r e t a r d a t i o n of phytochrome or intermediate dark reactions; e a r l i e r r e s u l t s (18) showed an absence of SD-induced bud s e t t i n g below 15°C. However, the s u b s t a n t i a l c h i l l i n g of 15 hr/day for 130 days makes explanations based on incomplete rest-breakage seem improbable. The remaining a l t e r n a t i v e to the o r i g i n a l hypothesis, namely that long photoperiods are necessary even a f t e r r e s t has been f u l l y broken, f i n d s l i t t l e support i n the l i t e r a t u r e , and would be i n disagreement with the r e s u l t s of Experiment 3. In terms of the current idea that an inhibitor-promoter balance controls the breakage of r e s t (1, 16), the present r e s u l t s suggest that the l e v e l s of these two regulators might be c o n t r o l l e d independently. Perhaps they a l s o act at separate biochemical s i t e s rather than i n q u a n t i t a t i v e balance. The dehardening during expansion of new shoots occurred i n both provenances. I r v i n g and Lanphear (11) found that breaking r e s t i n Acer negundo and Viburnum plicatum accelerated subsequent dehardening. They reviewed s i m i l a r f i n d i n g s by others, but apparently none of these studies reported on changes associated s p e c i f i c a l l y with f l u s h i n g . In Howell and Weiser's data f o r apple (9) s i g n i f i c a n t dehardening patterns d i d accompany the phenological changes, although i t i s not p o s s i b l e to d i s t i n g u i s h these e n t i r e l y from natural environmental temperature e f f e c t s . Worrall (17) has shown (using the Cranbrook provenance and s i m i l a r temperature regimes) that f l u s h i n g of previously c h i l l e d buds leads to cambial a c t i v i t y i n lower u n c h i l l e d regions of the stem, i n accordance with the theory of polar auxin transport. However, the loss of hardiness i n warm f o l i a g e of branches with f l u s h i n g partners cannot be a t t r i b u t e d to the same f a c t o r because no new xylem was formed i n these branches. Therefore the "close coincidence" of cambial a c t i v i t y with gain and loss of hardiness i n t h i s species (5) does not have a common hormonal bas i s . The i n i t i a l dehardening i n Douglas-fir occurs i n response only to temperature (4). The present r e s u l t s suggest that f i n a l l o s s of hardiness (above the short-day induced le v e l ) i s dependent on f a c t o r s from expanding shoots. The data f o r apple (9) appear to embody both i n f l u e n c e s . Such a dual c o n t r o l of dehardening would be e c o l o g i c a l l y important. While the cold-induced (and deeper) phase, of hardiness (18) may f l u c t u a t e r a p i d l y i n response to short-term changes of temperature (16), the SD-induced phase might impose an upper l i m i t on unseasonal dehardening u n t i l , as a r e s u l t of cumulative longer-term environmental changes, the plant i s committed to i t s summer programme of growth. 61 REFERENCES 1. Amen, P. C. 1968. A model of seed dormancy. Bot. Rev. 34:1-31. 2. Bennett, J. P. 1950. Temperature and bud r e s t period. E f f e c t of temperature and exposure on the r e s t period of decidu-ous plant l e a f buds in v e s t i g a t e d . C a l i f . A g r i c . 4:11-16. 3. C o v i l l e , F. V. 1920. The in f l u e n c e of cold i n s t i m u l a t i n g the growth of plants. J. A g r i c . Res. 20:151-160. 4. Driessche, R. van den. 1969. Influence of moisture supply, temperature and l i g h t on f r o s t hardiness changes i n Douglas-fir seedlings. Can. J . Bot. 47:1765-1772. 5. . 1969. Measurement of f r o s t hardiness i n two-year-old Douglas-fir seedlings. Can. J . Plant S c i . 49:159-172. 6. Fuchigami, L. H., Weiser, C. J . and Evert, D. R. 1971. Induc-t i o n of cold a c c l i m a t i o n i n Cornus s t o l o n i f e r a Michx. Plant P h y s i o l . 47:98-103. 7. Fuchigami, L. H., Evert, D. R. and Weiser, C. J . 1971. A t r a n s l o c a t a b l e cold hardiness promoter. Plant P h y s i o l . 47:164-167. 8. Howell, G. and Weiser, C. J. 1970. The environmental c o n t r o l of cold a c c l i m a t i o n i n apple. Plant P h y s i o l . 45:390-394. 9. . 1970. Fl u c t u a t i o n s i n the cold r e s i s t a n c e of apple twigs during spring dehardening. J. Amer. Soc. Hort. S c i . 94:190-192. 62 10. I r v i n g , R. M. and Lanphear, F. 0 . 1967. The long-day l e a f as a source of cold hardiness i n h i b i t o r s . Plant Physiol. 42:1384-1388. 11. . 1967. Dehardening and the dormant con d i t i o n i n Acer and Viburnum. Proc. Am. Soc. Hort. S c i . 91:699-705. 12. L e v i t t , J . 1956. The hardiness of plants. Academic Press, New York. 13. Neuwirth, I. V. 1961. Der CC^-Stoffwechsel eineger Koniferen wahrend des Knospenaustriebes. B i o l . Z e n t r a l b l . 78:559-584. 14. Romberger, J. A. 1963. Meristems, growth and development i n woody plants. U.S. Dept. Ag r i c . Tech. B u l l . No. 1293. 15. Salisbury, F. B. and Ross, C. 1969. Plant Physiology. Wadsworth Publ. Co., Belmont, C a l i f o r n i a . 16. Smith, H. and Kefford, N. P. 1964. The chemical r e g u l a t i o n of the dormancy phases of bud development. Amer. J . Bot. 51:1002-1012. 17. Timmis, R. 1972. An excised-needle f r e e z i n g t e s t of cold hardiness i n Douglas-fir. Chapter 1 of t h i s t h e s i s . 18. Timmis, R. 1972. Environmental c o n t r o l of cold acclimation i n Douglas-fir during germination, a c t i v e growth and re s t . Chapter 2 of t h i s t h e s i s . 19. Weiser, C. J . 1970. Cold resistance and i n j u r y i n woody plants. Science 169:1269-1278. 63 20. Worrall, J. 1971. Absence of " r e s t " i n the cambium of Douglas-fir. Can. J. For. Res. 1:84-89. 1 CHAPTER 4 ELECTRICAL AND THERMAL RECORDS ' FREEZING IN DOUGLAS-FIR NEEDLES ABSTRACT The progress of fr e e z i n g i n needles of hardy and nonhardy branches of c l i m a t i c a l l y " s p l i t " Pseudotsuga menziesii (Mirb.) Franco seedlings was recorded simultaneously by d i f f e r e n t i a l thermal a n a l y s i s and the conductance of low voltage d i r e c t e l e c t r i c current. The r e s u l t s of both methods exhibited the same major patterns. Freezing i n immature leaves was n o n e q u i l i -brium and i n t r a c e l l u l a r . Freezing i n needles cold-acclimated under short days was an e q u i l i b r i u m process preceded by a short non-equilibrium f r e e z i n g of the free i n t e r c e l l u l a r water f r a c t i o n . This pattern d i d not change i n leaves more deeply cold-acclimated by low temperatures. Thawing i n mature needles was characterized by a greater proportion of i c e (than during freezing) at a l l temperatures, with i n d i c a t i o n s that not a l l the o r i g i n a l c e l l water was reabsorbed. Freezing records are i n t e r p r e t e d as showing that the c e l l membrane became more permeable to ions a f t e r i n j u r i o u s slow f r e e z i n g but retained i t s e s s e n t i a l i n t e g r i t y , whereas rapid f r e e z i n g caused immediate membrane damage. No features of the fre e z i n g or thawing curves of f i r s t or subsequent freeze-thaw cycles were u s e f u l as p r e d i c t o r s of i n j u r y by slow f r e e z i n g . The proportion of unfrozen water, determined c a l o r i m e t r i c a l l y , was less in immature needles and did not differ between hardy a nonhardy mature ones, but the latter data are inconclusive. INTRODUCTION Most theories on cold hardiness accord some ro l e e i t h e r to changes i n c e l l membrane permeability (17, 18, 24), water binding (18, 38, 42), type of e x t r a c e l l u l a r nucleators and c r y s t a l growth (30), l o c i of i c e nucleation with respect to t i s s u e s (1, 9, 11, 31), or to other adaptations r e f l e c t e d i n the o v e r a l l k i n e t i c s of i c e formation i n whole t i s s u e s or organs. While there are d e f i -n i t e l i m i t s to the r e s o l u t i o n and i n t e r p r e t a t i o n of measurements made at t h i s l e v e l (18 27), such records nevertheless provide valuable information f o r a species under i n i t i a l study. Two main methods have been developed f o r recording i c e formation without the disturbance of microscopic or macroscopic s e c t i o n i n g . These are measurement of heat of fusion, and monitoring the conductance of weak d i r e c t or low frequency e l e c t r i c currents. There i s considerable uncertainty i n the i n t e r p r e t a t i o n of each. Exothermal p r o f i l e s tend to be d i s t o r t e d according to thermocouple placement and smoothed out due to heat conduction lags (40). I t i s always d i f f i c u l t to d i s t i n g u i s h t i s s u e - b y - t i s s u e compartmentalization of the f r e e z i n g process from the f r e e z i n g of p a r t i c u l a r water f r a c t i o n s w i t h i n a t i s s u e (1, 33). The c h a r a c t e r i s t i c double f r e e z i n g point has not been s a t i s f a c t o r i l y explained (23), and r e s p i r a t i o n during thawing may contribute s i g n i f i c a n t amounts of heat (16). I t i s g e n e r a l l y accepted that low frequency e l e c t r i c current i s conducted through the apoplast of healthy t i s s u e s because of the r e l a t i v e l y high resistance of the c e l l membrane (3, 7, 28). Thus, conductance as a function of temperature under model conditions has been considered to represent the proportion of unfrozen e x t r a c e l l u l a r water i n e q u i l i b r i u m with e x t r a c e l l u l a r i c e and with supercooled c e l l water (28). Stamm (34) notes, however, that conductance of water i n wood f i b r e walls a c t u a l l y decreases at a faste r - t h a n -l i n e a r rate with mass. In l i v i n g t i s s u e s during f r e e z i n g and thawing the conductance represents, i n a d d i t i o n , the cumulative e f f e c t s of: (1) p o s s i b l e membrane permea b i l i t y changes due to non-injurious c h i l l i n g (9, 10, 14, 18); (2) p a r t i a l i r r e v e r s i -b i l i t y of water e f f l u x (6, 35) from the c e l l s during f r e e z i n g ; and (3) i n j u r i o u s changes i n membranes during f r e e z i n g , while frozen and during thawing (5, 19). For both types of measure-ment e q u i l i b r i u m at any given temperature i s probably not at t a i n e d under simulated f r o s t s of f a i r l y short duration on i n t a c t plants (16), and random supercooling e f f e c t s determine the onset and i n i t i a l l e v e l of the "steady s t a t e " which i s recorded instead. Because of these u n c e r t a i n t i e s , both e l e c t r o p h o r e t i c and thermal records were obtained from a sub-sample of r e p l i c a t e s i n the present experiment to improve confidence i n the l a r g e l y q u a l i t a t i v e comparisons. A p p l i c a t i o n of both these techniques to potato leaves (36) showed d i f f e r e n c e s between hardy and nonhardy v a r i e t i e s which i n d i c a t e d e a r l i e r membrane i n j u r y i n the nonhardy type. In Douglas-fir seedlings d i f f e r i n g markedly i n hardiness, no c l e a r d i f f e r e n c e s i n e l e c t r o p h o r e t i c f r e e z i n g patterns (e.g., of the type reported by O l i e n i n barley crowns, 29) were observed (39). However, genotype and water content v a r i a t i o n was considerable i n these preliminary studies, and may have obscured d i f f e r e n c e s a c t u a l l y present. The aims of the present study were: (1) to e s t a b l i s h t y p i c a l f r e e z i n g patterns i n immature and i n hardy and nonhardy mature needles by the two methods; (2) to extend t h i s comparison to the thawing process and to repeated freeze-thaw cycles f o r observation of r e v e r s i b i l i t y and the e f f e c t s of i n j u r y ; and (3) to compare thermograms of excised needles obtained by d i f f e r e n t i a l scanning calorimetry (DSC) with the i n s i t u exotherm records, and to obtain q u a n t i t a t i v e estimates of the mass of i c e . MATERIALS AND METHODS  Plants Two-year-old Pseudotsuga menziesii (Mirb.) Franco seedlings of a c o a s t a l provenance (500 m, Duncan, Vancouver Island) were used i n a l l experiments except DSC, which was conducted on an i n t e r i o r provenance (Cranbrook, B.C.). Seedlings were r a i s e d i n a greenhouse and pruned at the beginning of the second growing season to produce forked tops. In August the plants were a c c l i -mated i n a col d room at 5'C with one branch i n a p l e x i g l a s s chamber at 20 3C. Night temperatures were 2°C lower i n each case. Both c h i l l e d and warm branches received 500±50 f t - c mixed incan-descent and fluorescent l i g h t 8 hr/day f o r 6 weeks. This regime produced an average d i f f e r e n c e o f 12^0 i n hardiness i n the f o l i a g e of warm and cool branches (41). One day before sampling f o r fr e e z i n g t e s t s , pots were soaked with water and the warm chamber humidified to bring f o l i a g e to a comparable state of hydration. Immature f o l i a g e was obtained from warm branches which had pro-duced a l a t e f l u s h . Measurement of Injury and Hardiness The f o l i a r hardiness of i n d i v i d u a l branches was f i r s t e s t ablished by sequential f r e e z i n g t e s t s on excised mature needles. Injury was assessed v i s u a l l y on a 10-point scale a f t e r a 7-day recovery period and hardiness was defined as the temperature corresponding to 50% i n j u r y (40). In ad d i t i o n , i n j u r y to unmonitored samples, frozen alongside monitored needles, was assessed f o r each p a r t i c u l a r run as a check on electrode i n j u r y . Freezing Methanol coolant was pumped through a dry ice/acetone bath, and temperature co n t r o l was achieved by reheating i n a Dewar f l a s k r e s e r v o i r under the co n t r o l of a commercial temperature programmer. The coolant c i r c u l a t e d slowly through v i n y l tubing surrounding a fr e e z i n g chamber equipped with an i n t e r n a l fan. Uniform cooling and warming within a wide range of rates and 71 temperatures could be achieved and duplicated i n successive cycles by t h i s arrangement. Freezing curves described below are based on a pre-determined maximum safe cooling rate of 7°C/hr, followed by warming at 20"'C/hr with a 15 °min period at the minimum tempera-ture, unless otherwise stated. Recording of Ice Formation Gold plated copper electrodes were placed approximately 1 cm apart on the needles, and p a r t i a l l y e n c i r c l i n g them. Pastes of charcoal f o r e l e c t r i c a l contact (28) proved u n r e l i a b l e on these r e l a t i v e l y xerophytic leaves and a commercial electro-conductive paste of e l e c t r o l y t e s was used sp a r i n g l y instead (EKG Sol, pH 5.6, Burton, Parsons and Co. Inc.). The l o c a l i z e d i n j u r y which r e s u l t e d caused a gradual increase i n the current flowing at constant tem-perature, but only at temperatures above f r e e z i n g . Under isothermal conditions a f t e r i n i t i a l f r e e z i n g , the current flowing at 3 v remained steady over long periods of time i n the microamp range. The paste does not freeze i n the temperature range inv e s t i g a t e d , and i t i s assumed that i t s change i n conductance with temperature i s adjustable by the same v i s c o s i t y c o r r e c t i o n f a c t o r as that f o r pure water (see below). The f r e e z i n g chamber contained s i x sample holders and electrode p a i r s , each linked i n s e r i e s with a dry c e l l and s t r i p chart recorder, permitting three hardy and nonhardy r e p l i c a t e s to be monitored at once. One of the r e p l i c a t e s from each treatment 72 was a l s o monitored by a 30-gauge thermocouple with i t s reference j u n c t i o n at ambient a i r temperature to provide a d i f f e r e n t i a l ther-mal p r o f i l e . The arrangement f o r a s i n g l e sample i s shown i n Figure 1. Subsequently, t e s t s were performed on detached needles because i t was found that these underwent f r e e z i n g i n a s i m i l a r manner and without d i f f e r e n c e i n i n j u r y , providing that water was supplied to the basal end from the small r e s e r v o i r v i a a f i l t e r paper wick. This permitted numerous comparisons w i t h i n a s i n g l e p l a n t . The time-based record of current flowing through the sample was corrected f o r temperature and i n i t i a l current, and the r e l a t i v e m o b i l i t y of l i q u i d water (28), M, p l o t t e d as a f u n c t i o n of tempera-ture, during f r e e z i n g and thawing. where I and I are current flowing at t and 0°C r e s p e c t i v e l y , and t o V and V are the v i s c o s i t i e s of l i q u i d water at these temperatures, t o V i s c o s i t y values below -10°C were extrapolated from tabulated values p/t>m (38) using the empirical r e l a t i o n v = Ae where T i s the absolute temperature, R the gas constant, and A and B constants for the f l u i d . A and B are assumed not to change with the degree of c o l l o i d a l a s s o c i a t i o n of water with e x t r a c e l l u l a r surfaces. Calorimetry The DSC c e l l of the Dupont 600 D i f f e r e n t i a l Thermal Analyser 71a Fig. 1. Arrangement for obtaining thermal and elec-trophoretic freezing records of needles. Attached needle (C) i s held into gold plated electrodes (A) by small piece of foam rubber (B). Thermocouple junction (Ti) i s held against lower surface of leaf by a light adhesive tape (D), and records temperature difference from ambient (T2). Electrode and thermocouple leads (G) are input to an external dual-channel recorder. The twig is held in an supportive framework (E) with i t s base in water (F) inside the freezing chamber. was operated at i t s slowest cooling rate of 30°C/hr with l i q u i d nitrogen as coolant. A weighed (iO.025 mg) needle was placed, p a r t l y c o i l e d up but undamaged, i n the sample pan, while the reference pan remained empty. An external time-based s t r i p chart recorder recorded the exotherm during f r e e z i n g . Oven-dry weight was obtained a f t e r 24 hr at 105°C. A t o t a l of 42 runs was made on the hardy and nonhardy needles of 10 plants. Amount of water frozen, W, at completion of detectable exotherm was c a l c u l a t e d from: W = A. A T . E AH.C o where A = area under peak (cm ) , A T = s e n s i t i v i t y of recorder s e t t i n g (°C/cm), E = c a l i b r a t i o n f a c t o r ( c a l o r i e s / 0 C-min), A H = l a t e n t heat of fu s i o n of pure water (cal/gm), and C = chart speed (cm/min). w was expressed as % of t o t a l water at f u l l turgor, measured g r a v i m e t r i c a l l y . E was determined from runs using pure water o r mercury f o r which both W and £xH were known. No attempt was made i n t h i s comparative study to co r r e c t f o r several sources of e r r o r (41), which are assumed to a f f e c t both treatments more or less equally. RESULTS  Freezing Curves Freezing of immature needles was i n v a r i a b l y a nonequilibrium and l e t h a l process characterized by a s i n g l e sharp exotherm and a sudden decrease i n M (Fig. 2). Simple hand sections made a f t e r thawing showed rupture of c e l l s and release of t h e i r contents, c l e a r l y i n d i c a t i n g i n t r a c e l l u l a r f r e e z i n g . In about h a l f of the t r i a l s , i n c l u d i n g a number made on succulent stem t i s s u e of 4-week-old seedlings i n a separate i n v e s t i g a t i o n , the decrease of current was immediately preceded by an increase of 0.5 to 2 sec duration (Fig. 2). Mature leaves exhibited, i n most cases, both nonequilibrium and e q u i l i b r i u m f r e e z i n g processes corresponding with the f i r s t dM and second exotherms r e s p e c t i v e l y . A d e r i v a t i v e p l o t , ^ (Fig. 2) i n d i c a t e s that the e l e c t r i c a l measure records the f i r s t f r e e z i n g with much greater s e n s i t i v i t y , as would be expected i f t h i s represents the free e x t r a c e l l u l a r water. No d i f f e r e n c e s were detectable between the f r e e z i n g curves of hardy and nonhardy mature needles, even though genetic v a r i a -t i o n had been avoided by the use of s p l i t p l ants. Figure 3 i l l u s t r a t e s the s c a t t e r among s i x hardy and nonhardy needles of a s i n g l e plant. Other plants were equally v a r i a b l e . Thawing A h y s t e r e s i s was c o n s i s t a n t l y observed i n both e l e c t r o -phoretic and thermal records of a freeze-thaw cycle (Fig. 4). Thawing was a gradual and continuous process producing a s i n g l e smooth endotherm. I t appeared that even under very slow c o o l i n g or warming rates, or when temperatures were held constant to 7 4 3 M 1-2 l-O 0 - 8 -07 0-4 0 - 2 O-O - R I M M A T U R E M A T U R E \ d M d T A T ° C 0 - 4 0-2 O-O 0 - 6 A T ° C 0 - 4 0-2 O-O o - 4 8 - 1 2 - 1 6 M A T U R E I M M A T U R E - 8 - 12 T E M P E R A T U R E ° C - 16 Fig. 2. Typical records of relative mobility of water, M (see materials and methods), and exotherms, during freezing of mature and immature needles. AT = elevation of leaf surface temperature above ambient. dM/ dT i s the derivative of the mature-needle M curve with respect to tem-perature (arbitrary units). Peak R was observed in about half the t r i a l s . Cooling rate was 7°C/hr. 74 b o-o' 1 1 1 1 1 1 1 1 1 - 4 - 8 -12 -16 - 2 0 T E M P E R A T U R E ° C Fig. 3. Relative mobility of water, M (see materials and methods), as a function of temperature, i n hardy and nonhardy needles of a single s p l i t plant. The observations were made under comparable conditions within a 2-day period. Cooling rate was 7°C/hr. O-O I 1 1 1 1 1 1 1 : 1 " -J O - 4 - 8 -12 -16 - 2 0 T E M P E R A T U R E ° C 020i CH Fig. 4. Relative mobility of water, M (see materials and methods), and tem-perature elevation/depression (AT) In mature needles during 3 cycles of freezing a n d thawing. The diagram Is based on average values of the various onset a n d Inflection points from 14 t r i a l s , and on actual curve shapes, fp » freezing point, mp • melting point. Cooling and warming rates were 7°C/ hr a n d 20°C/hr respectively. allow c l o s e r attainment of equilibrium, the proportion of i c e at a given temperature was higher i n the thawing cycle. However, f i n a l values of r e l a t i v e m o b i l i t y (M ) always exceeded pre-F f r e e z i n g values. M was s i g n i f i c a n t l y higher when mean values F of nonhardy needles i n a run were compared with hardy means (Table I ) , but no c o r r e l a t i o n could be established between M F and i n j u r y to i n d i v i d u a l needles assessed a f t e r 7 days. A f t e r i n j u r y by rapid f r e e z i n g to a normally safe temperature, on the other hand, or a f t e r i n j u r i o u s warming rates, M and v i s u a l F i n j u r y followed a s i m i l a r trend (Fig. 5). Repeated Freeze-Thaw Cycles T r i p l e cycles were monitored on seven r e p l i c a t e s of hardy and nonhardy needles from two seedlings. M was c a l c u l a t e d with respect to I at the beginning of the cycle i n question. Exotherms show a progressive loss of " s t r u c t u r e " and t h e i r r e l a t i v e sharpness i n d i c a t e s a more rapid i n i t i a l c r y s t a l l i z a t i o n during successive f r o s t s i n s p i t e of s i g n i f i c a n t l y reduced super-coo l i n g . These changes are a l s o noticeable i n the e l e c t r o p h o r e t i c records which, i n a d d i t i o n , show a progressive reduction i n M F and a decrease i n the upper melting point. Hysteresis tends to become less pronounced. These features are shown schematically i n Figure 4 and i n d i v i d u a l values of s i g n i f i c a n c e are given i n Table I I . Table I . Relat i v e increase i n Conductance (M ) F of Weak E l e c t r i c Current Through Douglas-fir Needles A f t e r Slow Freezing Run No. Tree NO. Hardy Branch Mean K i l l i n g Point of Needles M_ Nonhardy Branch Mean K i l l i n g M Point of Needles 1 1 -28.0 2.03 (2) -15.5 5. 51 (2) 2 1 -28.0 1.96 (3) -15.5 2. 32 (3) 3 2 -31.0 2.95 (2) -15.5 4.78 (2) 4 3 -26.5 1.48 (2) -16.5 2. 34 (2) 5 3 -26.5 1.63 (3) -16.5 1.70 (3) 6 3 -26.5 1.74 (2) -16.5 1.75 (2) Note: Numbers i n parentheses i n d i c a t e number of r e p l i c a t e s of which M i s the mean. K i l l i n g points were obtained by sequential f r e e z i n g F t e s t s i n preliminary experiments using l a r g e r samples. The low temperature was -20°C (except -12°C i n run No. 6). M values of hardy and nonhardy populations d i f f e r s i g n i f i c a n t l y a t F the 5% l e v e l . 15b 3-8 Fig. 5. Freezing injury to hardy needles as a function of cooling and warming rates (logarithmic scale). VL, «= relative mobility of water (see ma-ter i a l s and methods) after thawing. Injury was assessed visually after 24 hr as the proportion of browning i n two monitored needles (as Fig. 1) plus 5 others from the same twig. K i l l i n g temperatures for foliage of trees 1 and 2 were -21 and -25°C respectively at natural cooling and warming rates. Cooling rate t r i a l s were warmed at 20°C/hr; warming rate t r i a l s cooled at 7°C/hr. The low temperature was -18°C i n a l l cases. I t i s reasonable to suppose that changes i n successive cycles are mainly due to i n j u r y . Table II compares these parameters with respect to hardiness and v i s u a l l y assessed i n j u r y . Second and t h i r d cycle melting points were s i g n i f i c a n t l y lower i n the nonhardy group. Also, M approaches u n i t y sooner (that i s , F a f t e r fewer cycles) f o r nonhardy plants, r e f l e c t i n g the f a c t that i n these the point of maximum i n j u r y i s more r a p i d l y a ttained. The loss of the second exotherm i n subsequent cycles, however, was not c o n s i s t a n t l y associated with corresponding i n j u r y , nor even with the hardiness c l a s s , except where ra p i d f r e e z i n g (Fig. 5) had been the cause of death. Calorimetry Freezing of detached needles i n the DSC c e l l took place i n many cases at temperatures w e l l below the normal supercooling range. Supercooling was h i g h l y c o r r e l a t e d with various para-meters d e s c r i b i n g exotherm shape and with percentage of t o t a l water frozen at completion of the detectable exotherm, but was i t s e l f a l a r g e l y random event. Lower f r e e z i n g temperatures were s i g n i f i c a n t l y c o r r e l a t e d with lower t i s s u e water contents 2 to a small extent, (R =0.11). Of those runs which f e l l w i t h i n the supercooling range of f r e e z i n g chamber studies, exotherm shape resembled that recorded by a thermocouple at the surface of attached needles. The f i r s t exotherm represented between 5 and 15% of the f i n a l mass of i c e . 7&? Table II. C h a r a c t e r i s t i c s of E l e c t r i c a l Conductance Records of Douglas-fir Needles During Repeated Freeze-thaw Cycles i n Rela t i o n to Hardiness and Injury o 2 0 z cn r-4 (U c CU <u ro 0 u U « CQ Supercooling ° C. Melting point ° C. M_ V i s u a l % i n j u r y 1 Cycle 2 3 1 Cycle 2 3 1 Cycle 2 3 1 day a f t e r cycle 1 1 H 5.0 D 3.4 I 3.2 I -0.2 0.0 -1.0 1. 70 1. 33 1.12 15 NH 6.4 D 4.7 4.4 0.0 -1.2 -3.4 1. 83 1.19 1.02 100 2 1 H 4.8 D 4.2 I 4.1 -0.2 -1.0 -1.0 1. 93 1.24 1.08 18 NH 6.0 D 4.2 I 4.0 -1.2 -1.5 -2.4 1. 48 1.12 1.05 100 3 1 H 5.5 D 3.5 3.4 I -1.1 -1.0 -1.0 2. 24 1. 12 1.11 13 NH 5.3 3. 3 4.0 0.0 -1. 2 -2.7 3. 66 1.43 0.90 100 4 3 H 6.4 D 5.0 I 4.6 0.0 -0.5 -2.2 1. 48 1.33 1.11 58 NH 4.6 D 5.0 D 5.5 0.0 -1. 3 -2. 7 1. 86 1.35 1.00 100 5 3 H 8.6 I 5.6 I 4.6 -0.6 -1.0 -0.8 1. 48 1.13 1.00 62 NH 7.4 4.7 4.0 -1.0 -1.0 -1.2 2. 83 1.42 1.03 100 6 3 H 5.8 D 5.0 D 5.0 -0.5 -1.0 0.0 1. 64 1. 28 1.09 64 NH 6. 2 D 5.0 4.8 -1.2 -2.0 -1.5 1. 28 1. 21 1.00 100 7 3 H 5.8 D 5. 3 5.0 I -0.2 0.0 -1.4 1. 84 1. 24 1.20 62 NH 5.9 D 4.7 4.3 -0.2 -1.0 -1.2 2. 22 1.45 1.00 100 Mean H 6.0 4.6 4.2 -0.40 -0.64 -1.06 1. 76 1. 23 1. 10 41 NH 6.0 4.5 4.4 -0.51 -1.31 -2. 16 2. 16 1. 31 1.00 100 Note: Le t t e r s a f t e r numerical values i n d i c a t e presence of d i s t i n c t (D) or i n d i s t i n c t (I) second exotherm, i n hardy (H) and nonhardy (NH) needles. Mp i s the r e l a t i v e increase i n conductance a f t e r thawing compared with that at the beginning of the cycle. Injury values represent the mean of the monitored needle and 5 unmonitored needles. In immature needles 85 to 95% of the water froze, whereas i n mature needles 40 to 75% s o l i d i f i e d (Table H I depending p a r t l y on supercooling. When i n d i v i d u a l values were covariance-adjusted fo r supercooling, 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 between the proportions of unfrozen water i n hardy and nonhardy mature leaves. Means are shown i n Table I I I . DISCUSSION The int o l e r a n c e of i c e formation by immature t i s s u e s , due to i n t r a c e l l u l a r f r e e z i n g , has been widely reported f o r other species (8, 11, 29, 35) and appears to be the case i n the present study. I t i s improbable that 90% of the water could have moved so qui c k l y , and without any exothermal d i s c o n t i n u i t i e s , to s i t e s outside the c e l l . I t i s f u r t h e r considered that the b r i e f increases of current, r e g i s t e r e d only by recorders with a r e l a t i v e l y f a s t response time, are probably equivalent to the somewhat slower ones observed i n tender t i s s u e s of barley (28, 29). This would mean that they represent the momentary s i t u a t i o n where i c e c r y s t a l s have ruptured the c e l l membrane, but have not spread completely throughout the c e l l . The p r o b a b i l i t y of i n t r a c e l l u l a r nucleation i s greater i n immature t i s s u e s , which have a higher c e l l water content, a r e l a t i v e l y small volume of i n t e r c e l l u l a r space, a lower membrane permeability (18) and a lack of e f f e c t i v e e x t r a c e l l u l a r nucleators (12). In support of t h i s , the range of supercooling of immature versus mature Douglas-fir t i s s u e s was ge n e r a l l y f a r t h e r below C C . 11a Table m. percent Frozen Water at Completion of Detectable Exotherm i n Hardy and Nonhardy Excised Douglas-fir Needles Cooled at 30°C/hr i n a D i f f e r e n t i a l Scanning Calorimeter Mature Hardy Mature Nonhardy Immature Mean K i l l i n g Temp.= Mean K i l l i n g Temp.= Mean K i l l i n g Temp.= -24°C -11.5°C -6.5°C "ree Super- % Water Super- % Water Super- % Water l o . c o o l i n g Frozen cooling Frozen c o o l i n g Frozen 0 c °c °c 11. 2 56.8 10.0 46.4 .... 10.7 62.6 14.2 64.2 .... .... 13. 7 54.5 20.0 68.5 20.0 94.2 14.5 63.4 15.5 74.7 19.5 85.8 7.0 55.2 8.0 50.6 .... .... 14.7 53.9 11.7 51.7 .... .... 7.5 46.9 7.7 40.0 .... .... 12.2 49.9 15.5 60. 2 14.3 87.0 10.7 47.2 12.2 44.0 .... .... 13.5 46.8 13.7 56.5 • • • • !ote: Values represent the mean of two determinations made on an i n t e r i o r S r i t i s h Columbia provenance (Cranbrook, B. C.). However, a f t e r the change from nonequiLibrium to l a r g e l y e q u i l i b r i u m f r e e z i n g associated with maturation and preliminary cold a c c l i m a t i o n under short days, no f u r t h e r changes i n the e l e c t r o p h o r e t i c "freezing curve" accompanied the a d d i t i o n a l a c c l i -mation which occurs at low temperatures. McLeester e^ t a l (22) report two or three exothermal f r e e z i n g curve shapes i n red o s i e r dogwood twigs passing through a s i m i l a r hardiness range, but over a period of time. The evidence that the nonequilibrium part of the mature needle f r e e z i n g curve i s due to free e x t r a c e l l u l a r water supports conclusions made by other workers (reviewed i n 23). The h y s t e r e s i s i s considered to be a t l e a s t p a r t l y an a c t u a l c h a r a c t e r i s t i c of the l i v i n g c o l l o i d a l system rather than a simple lag e f f e c t due to f a i l u r e to a t t a i n e q u i l i b r i u m a t a given tem-perature. The evidence f o r t h i s i s that: (1) the h y s t e r e s i s i s not s e n s i t i v e to cooling and warming rates w i t h i n a wide range; and (2) i t tends to become less pronounced as i n j u r y increases i n subsequent freeze-thaw c y c l e s . Stark (33) f i r s t noted a greater amount of i c e i n the thawing part of a cycle using calorimetry. More recent data on yew and h o l l y leaves (4 2) support t h i s , and show that equilibrium, a f t e r a sudden 10°C. temperature change i n thawing, i s c l o s e l y approached wi t h i n only 8 min. The time allowed f o r an equal temperature change i n these studies was several times greater. Krasavtsev (16) has a t t r i -buted t h i s "general imbalance of heat exchange" to r e s p i r a t i o n during thawing. The present e l e c t r i c a l conductance measurements, however, may be considered i n s e n s i t i v e to r e s p i r a t i o n e f f e c t s . The f a c t that M exceeds unity, even without i n j u r y , i n d i c a t e s F that not a l l the o r i g i n a l c e l l water i s re-absorbed from the i n t e r c e l l u l a r space. This i s another aspect of the h y s t e r e s i s , but one which needs f u r t h e r v e r i f i c a t i o n due to the p o s s i b i l i t y of membrane permeability t r a n s i e n t s c i t e d e a r l i e r , and s i g n i f i c a n t electrode e f f e c t s . Features of f r e e z i n g and thawing curves o f f e r a t t r a c t i v e p o s s i b i l i t i e s f o r q u i c k l y estimating i n j u r y , and hence hardiness, i n p h y s i o l o g i c a l studies. Glerum (4) mentions many e a r l y i n v e s t i -gations showing that e l e c t r i c a l conductance increases a f t e r s t r e s s . He found that conductance of c o n i f e r twigs increased 76 to 93% a f t e r l e t h a l f r e e z i n g , and Takeda (36) obtained a c o r r e l a t i o n between the a c t u a l degree of f r o s t and subsequent low frequency conductance i n needles of Cryptomeria japonica (L.) Don. The value of M obtained i n the present study, however, while d i f f e r i n g F on average between hardy and nonhardy populations, i s not a s u i t a b l e p r e d i c t o r of p a r t i a l i n j u r y . This suggests that i n j u r y from e q u i l i b r i u m f r e e z i n g at stages of moderate a c c l i m a t i o n a r i s e s from causes other than simple membrane damage, and takes some time to develop. This i s a conclusion a l s o postulated by Evert and Weiser (2). I t i s fu r t h e r strengthened here by the observa-t i o n that i n j u r y by rapid freezing, g e n e r a l l y regarded as an i n t r a c e l l u l a r process (24), produces an immediately detectable and p r o p o r t i o n a l increase i n M (Fig. 5). I t i s a l s o i n agree-F ment with the r e s u l t s of an e a r l i e r study (40) which showed that Dexter's e l e c t r o l y t i c measurement of f r e e z i n g i n j u r y , based on the leakage of ions through injured membranes in t o an e x t e r n a l s o l u t i o n , i s le s s s e n s i t i v e than v i s u a l estimation of browning a f t e r 7 days. The u n s u i t a b i l i t y of multiple f r e e z i n g points during r e f r e e z i n g as a c r i t e r i o n of v i a b i l i t y i s a l s o i n con-t r a s t to other work (23), and p o s s i b l y f o r the same reason. However, i n t e r p r e t a t i o n i s complicated by the appearance of lower melting points i n subsequent cycles, e s p e c i a l l y of nonhardy leaves. Since these decreases i n melting point are not accom-panied by large increases i n M / e.g., between the second and t h i r d freeze-thaw cycles, then i t i s presumed that they represent a leakage of ions into the i n t e r c e l l u l a r space and a depression of f r e e z i n g point there, rather than a t r a v e r s a l of e l e c t r i c current through the r e l a t i v e l y concentrated s o l u t i o n of the c e l l i n t e r i o r . Simple osmotic pressure d i f f e r e n c e s between hardy and nonhardy c e l l s (cited by L e v i t t , 18) would tend to act i n the opposite d i r e c t i o n , and i n any case are mainly due to non-e l e c t r o l y t e s (32). Thus, the t e n t a t i v e conclusion i s that the c e l l membrane becomes l e a k i e r during i n j u r i o u s f r e e z i n g but s t i l l r e t a i n s i t s i n t e g r i t y as a b a r r i e r to e l e c t r i c current and i c e c r y s t a l growth, while c r i t i c a l i n j u r y proceeds as a comparatively slow development of some "secondary p l a s t i c s t r a i n " (20). Postulated i r r e v e r s i b l e s t r a i n s of f r e e z i n g include removal of " v i t a l " water (13, 40), s a l t - d e n a t u r a t i o n of proteins (21), and mechanical e f f e c t s f o l lowing dehydration below a c e r t a i n mini-mum volume (25, 26). These are avoided i f the c e l l i s able to r e t a i n a s u f f i c i e n t amount of water i n the l i q u i d state. However, t h i s method of avoidance by hardy t i s s u e i s not apparent from the data of Table m . Meyer (27) a l s o detected no d i f f e r e n c e i n amount of unfrozen water between hardy and nonhardy mature Pinus resinosa A i t . needles, but i n the majority of e a r l y i n v e s t i g a t i o n s on herbaceous plants, c i t e d by L e v i t t (18), the amount of i c e i n hardy t i s s u e s at a given temperature was s i g n i f i c a n t l y l e s s . More recent and p r e c i s e work (15, 16, 38) confirms, f o r apple twigs, the l a t t e r r e s u l t w i t h i n the temperature range of i n t e r e s t here. Because of the unnaturally high cooling rate, compounded by e r r a t i c and excessive supercooling, and other t e c h n i c a l l i m i t a -t i o n s of the commercial instrument used, the c a l o r i m e t r i c measurements i n t h i s work are regarded as i n c o n c l u s i v e . In those cases where abnormal supercooling, and probably extensive i n t r a c e l l u l a r f r e e z i n g , d i d not occur, the data i n d i c a t e that there were no major d i f f e r e n c e s i n the proportion of unfrozen water between hardy and nonhardy leaves. 82 REFERENCES 1. Alden, J. N. 1971. Freezing resistance of t i s s u e s i n the twig of Douglas-fir. Ph.D. t h e s i s . Oregon State U n i v e r s i t y C o r v a l l i s , Oregon. 2. Evert, D. R. and Weiser, C. J. 1971. Relationship of e l e c t r i c a l conductance at two frequencies to cold i n j u r y and ac c l i m a t i o n i n Cornus s t o l o n i f e r a . Plant P h y s i o l . 47:204-208. 3. Fensom, D. S. 1966. On measuring e l e c t r i c a l resistance in s i t u i n higher plants. Can. J. Plant S c i . 46:169-175. 4. Glerum, C. 1962. Temperature i n j u r y to c o n i f e r s measured by e l e c t r i c a l r e s i s t a n c e . For. S c i . 8:303-308. 5. Greenham, C. G. 1966. 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C r y o b i o l . 8:386. Timmis, R. 1972. An excised-needle f r e e z i n g t e s t of cold hardiness i n Douglas-fir. Chapter 1 of t h i s t h e s i s . 86 41. Timmis, R. 1972. Translocation of dehardening and bud-break promoters i n c l i m a t i c a l l y s p l i t Douglas-fir. Chapter 3 of t h i s t h e s i s . 42. Tumanov, I. I., Krasavtsev, 0. A. and Trunova, T. I. 1969. In v e s t i g a t i o n of the i c e formation process i n plants by measuring heat evolution. Sov. Plant P h y s i o l . 16:754-760. 43. Weast, R. C. 1969. Handbook of chemistry and physics. Chemical Rubber Co., Cleveland, Ohio. 44. Weiser, C. J . 1970. Cold r e s i s t a n c e and i n j u r y i n woody plants. Science 169:1269-1278. 45. Williams, R. J . and Meryman, H. T. 1965. A c a l o r i m e t r i c method f o r measuring i c e i n frozen s o l u t i o n s . C r y o b i o l . 1:318-323. 46. Yoshida, S. and Sakai, A. 1968. The e f f e c t of thawing rate on f r e e z i n g i n j u r y i n plants. I I . The change in the amount of i c e i n leaves as produced by the changes i n temperature. Low Temp. S c i . Ser. B. 26:23-31. CHAPTER 5 THE ROLE OF BOUND WATER IN HARDINESS OF DOUGLAS-FIR NEEDLES ABSTRACT Binding of water in climatically s p l i t Pseutosuga menziesii (Mirb.) Franco seedlings was compared for mature needles differing i n hardiness by 25°C. Heats of vapourization ( AHV) of weighed increments, of water, removed from excised needles under vacuum, were estimated from the calibrated vapourization endotherms recorded on a d i f f e r e n t i a l thermal analyser. In a second method, needle water contents were measured gravimetrically after equilibration with lithium chloride solutions of known desiccating energy. A R^, a proposed measure of water binding near surfaces, increased as the proportion of water remaining i n the leaf decreased. For each increment removed, AH V was significantly higher i n hardy needles, not-withstanding various possible sources of error. Hardy needles also retained more water non-osmotically (than nonhardy needles) after equi-libration with L i C l isopeistic with their f r o s t - k i l l i n g temperature. Dehydration avoidance probably accounted for 12 centigrade degrees of the hardiness difference between branches. 89 INTRODUCTION The most i n j u r i o u s s t r e s s i n hardy plant t i s s u e s during slow freez i n g i s the dehydration of c e l l s as t h e i r water moves to i n t e r -c e l l u l a r i c e c r y s t a l s (11). The dehydrating force may be expressed as the free energy d i f f e r e n c e f o r vapourization. A F = RT In P (1) v — P o where R i s the gas constant, and where P i s the vapour pressure over i c e and P q that over supercooled water, both at absolute temperature T. L e v i t t (11) has emphasized that c e l l s may survive f r e e z i n g e i t h e r by being able to t o l e r a t e the dehydration i n some manner, or by r e t a i n -ing water against the dehydrating force (dehydration avoidance). This water r e t e n t i o n or "binding" may be achieved to a l i m i t e d extent by an increase i n the solute content of the c e l l water; i t may e s p e c i a l l y be due t o an increase i n the number or nature of i n t e r n a l surfaces which impose some short range order and s t a b i l i t y on the complex water struc-ture (2,13). Osmotic r e t e n t i o n i s only able to protect against l i g h t f r o s t s because the A o f p h y s i o l o g i c a l l y high concentrations of, f o r example, sucrose (which can be c a l c u l a t e d from Equation 1 taking p as the vapour pressure over the s o l u t i o n and P q as the v.p. over pure water, both at temperature T) i s equivalent to that of e x t r a c e l l u l a r i c e at only a few degrees below 0°C. A maximum equivalence of about -15°C i s p o s s i b l e , f o r a saturated s o l u t i o n , but t h i s would be achieved only i n very 90 dehydration-tolerant c e l l s during the f r e e z i n g process. Sukumaran and Weiser (24) have shown that the osmotic p o t e n t i a l of potato l e a f c e l l s i s s u f f i c i e n t to account f o r the greater (but s t i l l r e l a t i v e l y low) hardiness of c e r t a i n v a r i e t i e s . •I Water bound at and near surfaces, on the other hand, probably has a continuous spectrum of a c t i v a t i o n energies" (13), and i s quantita-:: t i v e l y retained at the very lowest temperatures occurring i n nature. A number of i n v e s t i g a t o r s have measured t h i s f r a c t i o n , between a r b i -t r a r i l y defined binding energies i n hardy and nonhardy p l a n t s , to determine i t s r o l e i n f r e e z i n g tolerance. These studies have given d i f f e r e n t r e s u l t s according to the species and method used. The e a r l i e r i n v e s t i g a t i o n s have been c r i t i c a l l y reviewed by L e v i t t (9), and i n d i c a t e d i n most cases that there i s a greater proportion of non-osmotically bound water i n hardy c e l l s (exceptions included c o n i f e r leaves). L e v i t t (10) used a vacuum and heating method to d i f f e r e n t i a t e between free and bound water i n various c e l l f r a c t i o n s of cabbage. He found s i g n i f i c a n t l y more bound water i n hardened than i n unhardened pla n t s . However, Parker (18) detected no seasonal d i f f e r e n c e s i n white pine and eastern red cedar leaves with the same method. Studies on winter wheat (20,21) have shown that hardened t i s s u e s r e t a i n more t o t a l water than unhardened t i s s u e s when at e q u i l i b r i u m with aqueous so l u t i o n s of NaCl, but i n t h i s case the non-osmotically bound p o r t i o n i s derived by making the u n j u s t i f i e d assumption that t h i s surface-binding i s a s p e c i a l energy-requiring c h a r a c t e r i s t i c of the l i v i n g c e l l . C a l o r i m e t r i c 91 measurements of hardy and nonhardy wheat and apple (7,29) show that the proportion of "bound" ( i . e . unfrozen) water i s greater i n the hardy plants over a range of f r e e z i n g temperatures, but a l s o make the same i n v a l i d assumption about surface binding. In general, where the data permit conclusions about the temperature or dehydration-tolerance ranges over which surface binding confers pro-t e c t i o n , these conclusions are u s u a l l y that such binding i s i n s u f f i c i e n t to f u l l y account f o r the observed hardiness d i f f e r e n c e s . Nevertheless, any avoidance of dehydration stress by t h i s means would contribute q u a n t i t a t i v e l y to the o v e r a l l f r e e z i n g tolerance. The present study was undertaken to determine the r o l e of c e l l water r e t e n t i o n i n f r o s t tolerance of Douglas-fir needles. Two methods were employed to measure the energy of water i n leaves. In one method the heat absorbed during the vacuum-evaporation of a known mass of water from i n t a c t leaves was estimated by means of a d i f f e r e n t i a l scanning calorimeter (DSC). In the other, the water content of leaves at equi-l i b r i u m with l i t h i u m c h l o r i d e solutions of known dehydrating energy was determined g r a v i m e t r i c a l l y . Both methods are described below. MATERIALS AND METHODS Plants In order to avoid genotypic v a r i a t i o n and reduce v a r i a t i o n i n c a l o r i m e t r i c data associated with l e a f mass and shape, needles were taken from hardy and nonhardy branches of the same plant. To obtain these, two-year-old Pseudotsuga menziesii (Mirb.) Franco seedlings of a 92 c o a s t a l provenance were d i f f e r e n t i a l l y cold-hardened by a procedure i n which one branch was subjected to c h i l l i n g and night f r o s t , while the other was made to deharden and grow i n a warm environment, as previous-l y described (28). This treatment r e s u l t e d i n needles of warmed and c h i l l e d branches having a hardiness of approximately -10°C and -35°C r e s p e c t i v e l y . Hardiness was defined as the f r e e z i n g temperature causing 50% v i s i b l e i n j u r y to excised needles (26). Theory of the c a l o r i m e t r i c Method There i s evidence that the proportion of s t r u c t u r a l l y modified water i n l i v i n g c e l l s i s considerably more than that accounted f o r by hydrogen-bonded monolayers, and w i l l increase the average heat of vapourization by a measurable amount. The hi g h l y bound s i n g l e layers themselves may co n s t i t u t e up to 50% of the dry weight (2), yet the influence of hydrophylic surfaces g e n e r a l l y extends through two or three water molecules (2,8,15,23). Furthermore, "hydrophobic bonding" i s thought to be responsible f o r s t a b i l i z a t i o n of a large proportion of water as "icebergs" contained by non-polar surfaces (2,25). Nuclear magnetic resonance spectra have i n d i c a t e d at l e a s t two phases of "ordered" water i n some t i s s u e s (4). Drost-Hansen (2) has c i t e d evidence that nearly a l l the water i n t i s s u e s has a greater or l e s s e r degree of s t r u c -t u r a l ordering imposed upon i t . The extent to which water binding w i l l increase the l a t e n t heat of vapourization can be calculated, and compared to that of water i n bulk, i f we know how the vapour pressure of bound water v a r i e s with i t s 93 temperature. These f a c t o r s are r e l a t e d by the Clausius-Clapeyron equation, which i n i t s i n t e g r a l form i s : AI^ = R T L T 2 In P 2 (2) T 2 - T X P X where AH V i s the heat of vapourization (cal/mole), R the gas constant (cal/mole/°C), T-j_ and T 2 are two absolute temperatures, and p^ and 1?^ the respective vapour pressures. The presence of solute molecules, even at high concentrations, does not a f f e c t the slope, dP , of the vapour dT pressure/temperature curve, and therefore AtL^ i s not a f f e c t e d . But dP i s a f f e c t e d by surface binding (5,23). Vapour pressures of water dT bound i n wood f i b r e s are given by Kelsey (6). S u b s t i t u t i n g these data (for want of l i v i n g c e l l values) i n t o Equation 2 suggests that the increase i n AH V f o r the most weakly bound water (at 23% dry wt) would be about 360 cal/mole. The value f o r water i n bulk i s 10,440 cal/mole (16). I f the l a s t 1.5 mg of water evaporated from a c o n i f e r l e a f ( i . e . about 30% of the t o t a l water) had t h i s higher AH^ , then a f u r t h e r 360 x 1.5 = 30 meal would be absorbed during i t s vapourization. This 18 i s measurable with the DSC c e l l of a Dupont 600 d i f f e r e n t i a l thermal analyser (DTA), as described below. The DSC c e l l c o n s i s t s of a heating block supporting two i d e n t i c a l aluminum calorimeter pans (each about 7 mm i n diameter) i n a convection-free enclosure (Fig. 1). The c e l l i s a l s o equipped with a system per-m i t t i n g vacuum maintenance over long periods of time. The thermal behaviour of the sample under study i s recorded with respect t o a VACUUM PUMP Fig. 1. Apparatus for vacuum d i f f e r e n t i a l scan-ning calorimetry. Heating block (H) supports samples (S) and reference calorimeters i n a thermally uniform environment. Thermocouple series T R and T g senses the temperature de-pression in the sample as water i s evaporated under vacuum. R i s a rubber seal between glass cover and metal base. 94 thermally i n a c t i v e reference sample of equal heat capacity. The tem-perature d i f f e r e n c e between the two calorimeters, AT, i s measured by the s e n s i t i v e thermocouples, T and T , i n s e r i e s , and forms the input R S to a s t r i p chart recorder. The heat absorbed or evolved during a tran-s i t i o n can then be c a l c u l a t e d from the area under the exotherm i f an empirical constant f o r the instrument i s f i r s t determined, r e l a t i n g the known enthalpy change of a standard pure substance t o i t s recorded exotherm area under the same operating conditions. In the present study the block temperature was held constant by s e t t i n g the instrument to "isothermal mode", and water was removed from a sample under vacuum. This avoids errors due to p o o r l y matched thermal capacity of the reference sample, because the reference i t s e l f undergoes no appreciable temperature change. Heat of vapourization i s given by: AHy = mt AT E' (3) where VL^ i s the mass of water evaporated during the time, t (min), during which an endotherm i s recorded, AT i s the temperature depression of the sample calorimeter below the reference calorimeter, E' i s the c a l i b r a -t i o n constant (cal/°C-min), and m the molecular weight f o r water. In p r a c t i c e the endotherm i s a rough curve (Fig. 2) i n which AT varies with time. Hence AHy i s c a l c u l a t e d from-. AH V = mAE (4) Mv where A i s the area under the curve, and E i s expressed d i r e c t l y as 2 cal/cm of endotherm area. 94a - |-5 - 1 - 0 — 0 5 O O U o < - 1 - 0 - 0 - 5 O O WATER D R O P L E T > o o - 0 - 5 -W B - l - O -- 0 - 5 ' W B '»% M O I S T E N E D F ILTER PAPER O O -8 12 16 T I M E ( m i n s ) F i g . 2. Typical vaporization endotherms for water evaporated under re-duced pressure at 30°C. AT i s the temperature difference between sample and reference calorimeter pans. The rectangular part of the middle curve i s assumed to be "free water." WA and Wg are weights of samples used for c a l i b r a t i o n , immediately before and af t e r evacuation. Leaf water (lower curve) was removed i n 3 increments, with sample weights Wi to W4 being recorded before and a f t e r each. P o s i t i o n of W2 was defined by i n t e r -section of a tangent with the baseline. W3 was recorded when AT had decreased to half i t s value at the beginning of increment 2. was r e -corded when AT equaled 0.075°C with respect to the o r i g i n a l baseline. In some runs W2 was the only intermediate weighing. 95 Operating Procedure f o r Calorimetry The following procedure was c a r r i e d out at an isothermal temper-ature s e t t i n g of 30°C. A l e a f was placed, p a r t l y c o i l e d up but un-damaged, i n a weighed (Wi) calorimeter pan. Pan and sample were reweighed (W2, * 0.2 mg) and q u i c k l y t r a n s f e r r e d to the DSC c e l l . Pressure i n the c e l l was reduced w i t h i n 10 sec to 25 mm Hg by opening the tap to a vacuum pump. The endotherm was recorded on an external time-base recorder at a speed of 2.54 cm/min. Attenuation of the recorder was adjusted to accommodate AT (usually 100 mV, i . e . 0.1°c/cm). The vacuum was released at an intermediate point i n the endotherm, defined as shown i n Figure 2, to include p r i m a r i l y the free water i n the sample. The calorimeter plus sample were then reweighed (W3) and returned to the DSC c e l l . A second intermediate weight (W^ ) was obtained i n some samples by a r e p e t i t i o n of t h i s procedure. The run was terminated when AT = 0.075°c, below which the err o r i n E becomes unduly large (see below), and a f i f t h weighing (W^ ) made. An oven dry weight was recorded a f t e r 24 hr at 105°C. I t was established that there was no measurable l o s s or gain of water from the a i r during the weighing period. Throughout each run the act u a l base temperature, T, was recorded (+0.05°C), with respect to an ice-water reference, on the DTA recorder so that correc-t i o n s could subsequently be made f o r departures from the isothermal condition. Runs were conducted a l t e r n a t e l y on hardy and nonhardy leaves from the same plant. The leaves had been allowed to e q u i l i b r a t e at 100% humidity and 0°C overnight. This procedure was repeated f o r 10 needles 96 from each branch, f o r three seedlings, over a period of 10 days. Determination of E The c a l i b r a t i o n constant, E, was determined from Equation 4 by using a small droplet (3 to 5 mg) of water f o r the sample and taking the tabulated value of AHy corresponding to base temperature, T (16). was the d i f f e r e n c e between i n i t i a l and f i n a l weights of the c a l o r i -meter plus sample (WA - WB i n F i g . 2). A was determined (+ 2 meal) by planimetry. For fur t h e r determinations, water was evaporated from r o l l e d - u p pieces of f i l t e r paper to more r e a l i s t i c a l l y simulate the thermal and d i f f u s i v e c h a r a c t e r i s t i c s of leaves. In t h i s case W~ was a measured before the endotherm deviated from the rectangular form t y p i c a l of f r e e water. The value of E was found to vary i n v e r s e l y with AT, because heat t r a n s f e r between sample and block becomes les s e f f i c i e n t when the rate of vapourization i s low; a greater proportion of heat i s d i s s i p a t e d . Therefore, a ser i e s of determinations of E was made corresponding to d i f f e r e n t rates of vapourization of water, the l a t t e r quantity being v a r i e d by small changes i n vacuum pressure. (The density of a i r i n the vacuum was assumed not to d i r e c t l y a f f e c t heat exchange s i g n i f i c a n t l y . ) E was p l o t t e d as a f u n c t i o n of A T (Fig. 3), and takes the form: E = b L + b 2 (5) A T The constants b^ and were determined by the method of l e a s t squares. The three determinations of E made with a droplet l i e on the same curve Fig. 3. Calibration curve for isothermal vacuum vaporization of water in the differential scanning calorimeter. E is the heat equivalent to 1 cm^ of endotherm area (at recorder settings given in text). Shaded area represents 95% confidence limits on the regression line. Runs with leaf samples were terminated at e (AT = 0.075°C) to avoid exces-sive error in E. 97 as those made with moistened f i l t e r paper, i n d i c a t i n g that errors due to the geometrical d i s p o s i t i o n of water i n the calorimeter were n e g l i -g i b l e . E i s a l s o subject to small v a r i a t i o n s between runs because some of the heat f o r vapourization i s supplied by the sample i t s e l f (and i s not recorded by the block thermocouple, T g ) , and the sample mass va r i e s between runs. However, even with a 25% increase i n the sample's water weight (say, from 4 to 5 mg), and where AT = 1.5°C, the a d d i t i o n a l heat contributed by the sample ( i . e . the underestimate of MyAHy) w i l l be only about 1.5 x (5 - 4) = 1.5 meal. This i s close to e r r o r s i n area measurement and weighing, and can therefore be ignored. Computation of H v for Leaf Water The curved endotherms f o r l e a f water (Fig. 2) were converted to d i g i t a l form using a Gravicon D i g i t i z e r . This instrument automatically t r a n s f e r s the coordinates to punched cards as a pen i s moved manually along the curve. AH^ was then computed from: AH = £ ( aEK) (6) V W - W J_1 n n+l where a i s the area under a very small segment (= 2 mm) of the curve, assumed rectangular, E i s the c a l i b r a t i o n constant f o r the segment (calculated from Equation 5), K i s the r a t i o of AH^ f o r pure water at 30°C (the isothermal setting) to that at T°C (16), and W i s the weight of calorimeter plus sample at the s t a r t of evaporated increment, n ( = 1, 2 or 3) .. 98 Dehydration over L i C l Four groups of f i v e needles were removed from a branch, allowed to e q u i l i b r a t e at 100% humidity and 0°C overnight, and weighed (+ 0.05 mg). Groups were then suspended by f i n e wire over 2.10 molar, 3.50 M or 4.96 M L i C l or over pure water i n sealed containers at 0°C. The volume of s o l u t i o n was large r e l a t i v e to that of t i s s u e . The dehydrating energy of the solutions ( AF^ from Equation 1) was equal to that of i c e at -10°, -20° and -35°C respectively''". These four treatments were applied t o the hardy and nonhardy branches of four seedlings. The suspended samples were weighed at i n t e r v a l s u n t i l (after 1 to 3 months) there was no fur t h e r measurable weight loss over a 3-day period. I t was then assumed that the most weakly bound water i n the t i s s u e had a A F ^ equal to that of the external s o l u t i o n . Oven dry weight was obtained (+ 0.02 mg) a f t e r 24 hr at 105°C. RESULTS  Heat of Vapourization The present c a l o r i m e t r i c data must be viewed with caution because the observed d i f f e r e n c e s , although i n the expected d i r e c t i o n , are f i v e to s i x times greater i n magnitude than t h e o r e t i c a l l y predicted. Between-plant and between-leaf v a r i a t i o n are a l s o much higher than expected. Vapour pressures f o r i c e , supercooled water and aqueous solutions were obtained from I n t e r n a t i o n a l C r i t i c a l Tables (16), and f o r h i g h l y super-cooled water were ca l c u l a t e d from the r e l a t i o n s h i p s given by O l i e n (17). 99 The r e s i d u a l (between-leaf) v a r i a t i o n was l e a s t i n the seedling (No. 2) having r e l a t i v e l y large needles, presumably because errors i n E, a and W (Equation 6) were proportionately small. The data from seedling No. 2 are given i n Table I. These data i n d i c a t e (a) that the heat of vapourization increases s i g n i f i c a n t l y (p = 0.02) between successive increments, that i s as the proportion of water remaining i n the t i s s u e becomes l e s s ; and (b) that, f o r any one increment, the heat of vapourization i s s i g n i f i c a n t l y higher i n the hardy l e a f . These trends i n the data from i n d i v i d u a l needles are apparent only i f the order i n which needles were measured i s a l s o included as a source of v a r i a t i o n . Thus i t appears that s i g n i f i c a n t changes of A H occurred over time due to d r i f t i n the instrument, to d i u r n a l changes i n the plants before sampling, or to changes i n the needles themselves during storage. The data from seedlings No. 1 and 3 represent only two increments per sample and, although more v a r i a b l e , support the p r i n c i p a l obser-vations above. The mean values are p l o t t e d with seedling No. 2 means i n Figure 4. A multiple regression a n a l y s i s of these curves (with independent v a r i a b l e s "seedling", percent water, and hardiness) again shows both increment and hardiness factors to be s i g n i f i c a n t at the 0.05 p r o b a b i l i t y l e v e l . Further cause to question the v a l i d i t y of the data, however, i s given by the marked d e v i a t i o n of A for the f i r s t increment (which i s presumed to be l a r g e l y free water) from the standard value for free water (also shown i n F i g . 4). Possible sources of er r o r are discussed below. Table I. Heats of vapourization of water from hardy (H) and nonhardy (NH) needles of a Douglas-fir seedling. HEAT OF VAPOURIZATION AH kca 1/mole Water v content Leaf p a i r Branch Increment 1 Increment 2 Increment 3 % oi fresh 1 H 11.25 11.79 58.1 NH 9.42 11.59 63.1 2 H 9.14 10.40 59.4 NH 9.20 10.54 63.8 3 H 8.85 9.79 10.44 58.9 NH 8.28 8.60 10.67 63.0 4 H 9.94 10.36 10.07 58.9 NH 8.36 8.85 11.00 62.8 5 H 9.83 8.57 11.30 59.2 NH 9.86 10.30 10.62 65.1 6 H 11.60 13.07 22.47* 58.4 NH 8.69 10.47 12.92 65.8 7 H 9.96 10.56 12.64 58.1 NH 9179 9.40 10.87 64.8 8 H 10.63 10.46 11.34 56.6 NH 9.61 9.13 10.13 64.3 9 H 8.63 10.06 11.15 58.2 NH 5.90 8.63 9.85 64.8 10 H 7.87 9.62 11.28 57.3 NH 7.79 6.07 8.57 64.6 Mean H 9.77 10.31 12.58 58.3 NH 8.69 8.93 10.58 64.2 wt Data are from seedling No. 2. Paired H and NH values are from immediately successive runs. Needles having deviant values (*) are omitted from F i g . 3. Values of the second increment f o r leaves 1 and 2 are a l s o omitted from means of t h i s table but are included i n analyses of F i g . 4. Analysis of variance of AH f o r l e a f p a i r s 3 through 8 shows both increment and hardiness f a c t o r s s i g n i f i c a n t a t the 0.02 l e v e l . 99b 8 I 1—. 1 1 1 1 1 J 1 1 1 — O 2 0 4 0 60 8 0 IOO W °/o Fig. 4. Heats of vaporization for water removed under vacuum from excised needles. Each point i s the mean of 10 needles from the same branch. W% i s the average percentage of tissue water (dry weight basis) removed during evapora-tion, which was carried out i n 2 or 3 stages. Variation i n AH due to hardi-ness i s significant at the 5% probability level. V 100 Dehydration over L i C l The water retained by needles i n e q u i l i b r i u m with L i C l i s o p e i s t i c with i c e at -10, -20 and -35°C i s given i n Table I I . In a l l except one instance a greater proportion was retained i n the hardy needles. Needles over pure water gained i n weight, a l l hardy ones i n t h i s case e q u i l i -b r a t i n g at a lower percentage water content. These data, u n l i k e those of the previous study, a l s o r e f l e c t the osmotic component of retention, and no separate determination of t h i s was attempted. However, the maximum value which the osmotic component can assume i n hardy c o n i f e r leaves (3,12) i s about 30 atm (=-13.6 cal/mole) compared with 10 atm (=-4.36 cal/mole) i n the nonhardy condition. I t can be shown that t h i s could account f o r only a part of the d i f f e r e n c e between hardy and nonhardy needles i n Table I I . For example, i f a hardy l e a f with a c e l l sap osmotic p o t e n t i a l of -13.6 cal/mole loses 80% of i t s water while suspended over a L i C l s o l u t i o n of A F = v -190 cal/mole, the osmotic p o t e n t i a l i n i t becomes -13.6 x 100 = -68 cal/mole. Therefore, the net dehydrating energy 80 of the external s o l u t i o n , taking i n t o account the c e l l solute, A F ^ ' = -190 -(-68) = -122 cal/mole. I f percent water r e t e n t i o n i s p l o t t e d against these adjusted values of A F ^ , a new value of percent water, corresponding to -190 cal/mole can be obtained from the curve. This value represents a conservative estimate of the surface-bound water f r a c t i o n at that dehydrating s t r e s s . Such estimates are given f o r each s t r e s s l e v e l i n parentheses i n Table I I , and are s i g n i f i c a n t l y higher (p = 0.01) f o r hardy needles. Table I I . Water r e t e n t i o n by needles at eq u i l i b r i u m with L i C l at 0°C Leaf water percent of dry wt „ , at e q u i l i b r i u m with L i C l s o l u t i o n Seed- Branch l i n g 2.10 M 3.50 M 4.96 M (-10°C) - (-20°C) (-35°C) 3 H 75.3 NH 72.4 4 H 105.6 NH 55.4 5 H 91.7 NH 94.8 6 H 93.5 NH 69.5 Mean H 91.5 NH 73.0 (38.0) 75. 3. (38. 0) 35. 2 (33. 5) (61.0) 39. 1 (31. 5) 26.8 (24. 5) (68.5) 53. 3 (40. 0) 35.4 (27. 0) (48.0) 38. 2 (34. 5) 25.1 (22. 0) (66.0) 61. 2 (51. 5) 46.7 (42. 0) (65.0) 54. 5 (44. 0) 39.5 (37. 0) (75.0) 69. 0 (45. 5) 36.6 (25. 0) (56.5) 39. 1 (36. 5) 34.7 (33. 0) (61.9) 55. 8 (43. 4) 38.5 (32. 0) (57.6) 42.7 (36. 6) 31.5 (29. 1) M in d i c a t e s molarity. Equivalent i c e temperature i s given. H = hardy (average k i l l i n g temperature = -35°C), NH = nonhardy ( k i l l i n g temperature =s -10°C). Values i n parentheses are minimum estimates of the proportion retained nonosmotically (see t e x t ) . 101 DISCUSSION The data from both experiments support the hypothesis that non-osmotic binding of water contributes to a greater dehydration avoidance i n hardy Douglas-fir needles. In making t h i s statement the following p o s s i b l e sources of er r o r have been evaluated. (a) An er r o r i n the slope of the calorimeter c a l i b r a t i o n curve would lead to systematic d i f f e r e n c e s i n A H ^ between increments (because both E and Increment No. are r e l a t e d to vapourization r a t e ) . The 95% confidence l i m i t s f o r the expected value of E are given i n Figure 3. I t can be shown that i f the curve i s moved anywhere wit h i n these l i m i t s .the s i g n i f i c a n t d i f f e r e n c e between increments (Table II) i s not e l i m i -nated. Therefore, random error i n the o r i g i n a l c a l i b r a t i o n curve slope (not exceeding the 0.05 p r o b a b i l i t y ) i s not a s u f f i c i e n t cause f o r the A d i f f e r e n c e s observed. Furthermore, vapourization rate was a l s o somewhat greater i n nonhardy needles, due to t h e i r greater content of 2 water and probably a l s o to greater stomatal apertures (1), yet the nonhardy needles had higher, not lower, values f o r A H ^ . I t must be assumed that the curve has some e r r o r (perhaps associated with the n e c e s s a r i l y d i f f e r e n t type of sample used i n c a l i b r a t i o n ) because free water and Increment 1 A H ^ values do not agree. But t h i s i s a " l e v e l " rather than a "slope" error, and would not a f f e c t r e l a t i v e values of A H among treatments, v 2 Van den Dnessche, R. (1972), unpublished data. 102 (b) The presence of v o l a t i l e o i l s w i l l a f f e c t the r e s u l t s of both c a l o r i m e t r i c and dehydration studies. Parker (18) recognized t h i s d i f f i c u l t y i n applying L e v i t t ' s method f o r bound water (10) to eastern red cedar leaves, i n pines, the o i l s c o n s t i t u t e up to 0.5% of the 3 needle fresh weight (14), and have an average heat of vapourization of about 80 cal/gm. Assuming the heats of vapourization of water and o i l to be a d d i t i v e i n mixture, the e r r o r can be c a l c u l a t e d from AH = AH W + AH W w v y o o (7) W w where AH i s the heat of vapourization f o r o i l , AH the unknown value o w f o r water and AH^ the experimentally determined value f o r the oil-water mixture. w q and are the corresponding weights. This error turns out to be small, so that i n an average case AH^ f o r the f i r s t increment (assuming t h i s to contain nearly a l l the o i l ) underestimates the true value f o r t i s s u e water by only 1.7%. This c o r r e c t i o n i s i n d i c a t e d i n Figure 4 and would not a f f e c t the present conclusions unless nonhardy Douglas-fir needles contained 3 or 4 times as much o i l as found i n other co n i f e r s , while hardy needles contained l i t t l e or no o i l at a l l . I t can a l s o be seen that the amount of 1.25% of the dry weight, accounted f o r by v o l a t i l e o i l s , i s only a small part of the d i f f e r e n c e observed . between hardy and nonhardy r e t e n t i o n percentages i n Table II. This was c a l c u l a t e d from tabulated vapour pressure/tempearture r e l a t i o n -ships (30) f o r several monoterpenes t y p i c a l l y found i n pine o i l (14), using the Clausius-Clapeyron equation. 103 (c) Nonhardy needles contained up to 10% more water. I f t h i s a d d i t i o n a l 10% were a l l free water (the amount of "structured" water remaining constant), then there would be a decrease i n the average AH of nonhardy needles. Arithmetic c a l c u l a t i o n shows that t h i s decrease would be only about 1%, whereas observed d i f f e r e n c e s between hardy and nonhardy needles averaged 5%. I t may be concluded that t i s s u e water i s at a lower (more negative) p o t e n t i a l i n frost-hardy Douglas-fir needles, thereby allowing these to more e f f e c t i v e l y avoid dehydration s t r e s s . While osmotic lowering almost c e r t a i n l y plays a r o l e (11), there i s an a d d i t i o n a l lowering presumably caused by the proximity of macromolecular surfaces. Cytc— l o g i c a l changes which lead to an increase i n i n t e r n a l surfaces during hardening have been described by Pomeroy and Siminovitch (19). The magnitude of t h i s dehydration avoidance, due to the combination of osmotic and c o l l o i d a l forces can be roughly estimated from Figure 5 . This shows that the water content of hardy needles over a s o l u t i o n i s o -p e i s t i c with t h e i r k i l l i n g temperature (-35°C) averages 38% of the dry weight. Nonhardy needles, however, have at t a i n e d t h i s c r i t i c a l water content at -23°C. Therefore, there has been a 12°C gain i n hardiness as a r e s u l t of dehydration avoidance by the hardy l e a f . This assumes that dehydration by i n t e r c e l l u l a r i c e over a period of hours and over L i C l f o r several months are equally i n j u r i o u s . In f a c t , i c e may cause somewhat more i n j u r y (17), or le s s i n j u r y (22) than i s o p e i s t i c desic-c a t i o n . In the present case the prolonged d e s i c c a t i o n caused more 1033 2 0 0 r A F y F i g . 5. W a t e r c o n t e n t s o f n e e d l e s a t e q u i l i b r i u m w i t h L i C l s o l u t i o n s . E a c h p o i n t i s t h e mean o f 5 - need le s a m p l e s f r o m t h e h a r d y o r n o n h a r d y b r a n c h e s o f f o u r s e e d l i n g s . L o w e r p a i r o f c u r v e s shows t h e e f f e c t o f m a k i n g a g e n e r o u s a l l o w a n c e f o r o s m o t i c p o t e n t i a l d i f f e r e n c e s b e t w e e n h a r d y and n o n h a r d y n e e d l e s ( s e e t e x t ) . I n b o t h c a s e s t h e r e t e n t i o n c u r v e s d i f f e r s i g n i f i c a n t l y a t t h e 5% p r o b a b i l i t y l e v e l . r 104 i n j u r y than f r e e z i n g (data are not shown), and i t i s probable that the re t e n t i o n curves show smaller d i f f e r e n c e s between hardy and nonhardy samples due t o a. gradual breakdown of p h y s i c a l structure i n the t i s s u e a f t e r e x c i s i o n . I f so, 12° i s a conservative estimate of the- avoidance component. The co n t r i b u t i o n of surface binding to the t o t a l avoidance, based on the conservative estimates i n Table II (of which means are pl o t t e d i n F i g . 5), i s about 9 centigrade degrees. These non-osmotic r e t e n t i o n curves are also i n agreement with the idea reviewed e a r l i e r , and suggested by the c a l o r i m e t r i c data, that surfaces influence a r e l a t i v e l y large mass of water with a wide range of energies rather than a small f r a c t i o n a t a d i s c r e t e energy l e v e l . The remainder of the d i f f e r e n c e between hardy and nonhardy needles on a plant — about 13 centigrade degrees — must be explained by some mechanism of dehydration tolerance, f o r which sev e r a l theories have been proposed (11,31). Further study i s needed to determine whether avoidance and tolerance adaptations are associated with s p e c i f i c "stages" of cold acclimation (27,31) and dehardening (28). 1 0 5 REFERENCES 1. Christersson, L. 1972. The t r a n s p i r a t i o n rate of unhardened, hardened, and dehardened seedlings of spruce and pine. Ph y s i o l . Plant. 26(258-63). 2. Drost-Hansen, w. 1970. Structure and properties of water at bio-l o g i c a l i n t e r f a c e s . In H.D. Brown, ed. Chemistry of the c e l l i n t e r f a c e . Academic Press, New York. 3. G a i l , F.W. 1926. Osmotic pressure of c e l l sap and i t s probable r e l a t i o n to winter k i l l i n g and l e a f f a l l . Bot. Gaz. 81(434-45). 4. Hazlewood, C F . and Nichols, B.L. 1969. Evidence f o r the existence of a minimum of two phases of ordered water i n s k e l e t a l muscle. Nature 222(747-50). 5. Hori, T. 1956. On the supercooling and evaporation of t h i n water f i l m s . Low Temp. S c i . A. 15(34-42) (English T r a n s l a t i o n No. 62, U.S. Army. Snow, Ice and Permafrost Establishment, Wilnett, 111. 1960). 6. Kelsey, K.E. 1957. The sorption of water vapour by wood. Austr. J. Appl. S c i . 8(42-54). 7. Krasavtsev, O.A. 1969. Heat exchange of woody plants during gradual f r e e z i n g and thawing. Sov. Plant P h y s i o l . 16(846-51). 8. Kuntz, I.D., J r . et a l . 1969. Hydration of macromolecules. Science 163 (1929-31). 106 9. L e v i t t , J. 1956. The hardiness of plants. Academic Press, New York. 10. L e v i t t , J . 1959. Bound water and f r o s t hardiness. Plant Physiol. 34(674-7). 11. L e v i t t , J . 1972. Responses of plants to environmental s t r e s s . Academic Press, New York. 12. L e v i t t , J. and Scarth, G.W. 1936. Frost hardening studies with l i v i n g c e l l s . I. Osmotic and bound water changes i n r e l a -t i o n to f r o s t resistance and the seasonal cycle. Can. J . Res. 14c (267-84). 13. Meryman, H.T. 1966. Review of b i o l o g i c a l f r e e z i n g . In: H.T. Meryman, ed., Cryobiology. Academic Press, New York, p. 48-58. 14. Mirov, N.T. 1967. The genus Pinus. Ronald Press Co., New York. 15. M r e v l i s h v i l i , G.M. and Privalov, P.L. 1969. C a l o r i m e t r i c inves-t i g a t i o n of macromolecular hydration. In Kayushin, L.P., ed., Water i n b i o l o g i c a l systems (63-66). Translated from Russian. Consultants Bureau, New York, 1969. 16. National Research Council, U.S.A. 1933. I n t e r n a t i o n a l c r i t i c a l tables of numerical data, physics, chemistry and technology. Vol. 3. McGraw-Hill, New York. 17. Olien, CR. 1971. A comparison of d e s i c c a t i o n and fr e e z i n g as st r e s s vectors. C r y o b i o l . 8(244-8). 107 18. Parker, J. 1963. Cold r e s i s t a n c e i n woody plants. Bot. Rev. 29(123-201). 19. Pomeroy, M.K. and Siminovitch, D. 1971. Seasonal c y t o l o g i c a l changes i n secondary phloem paranchyma c e l l s i n Robinia  pseudoacacia i n r e l a t i o n to cold hardiness. Can. J. Bot. 49 (787-96). 20. Samygin, G.A. and L i v s h i n , A.Z. 1970. Water r e t a i n i n g forces i n the c e l l s of various plants i n r e l a t i o n to t h e i r r e s i s t a n c e to dehydration, and f r e e z i n g during the formation of extra-c e l l u l a r i c e . Sov. Plant P h y s i o l . 17(114-9). 21. Samygin, G.A. and L i v s h i n , A.Z. 1970. Water r e t a i n i n g forces i n the c e l l s of winter wheat leaves and t i l l e r i n g nodes i n connection with t h e i r r e s i s t a n c e to slow f r e e z i n g and d e s i c -cation. Sov. Plant P h y s i o l . 17(669-75). 22. Siminovitch, D. and Briggs, D.R. 1953. Studies on the chemistry of the l i v i n g bark of black locust and i t s r e l a t i o n to f r o s t hardiness. I l l The v a l i d i t y of plasmolysis and d e s i c c a t i o n t e s t s f or determining the f r o s t hardiness of bark t i s s u e . Plant P h y s i o l . 28(15-34). 23. Stamm, A.J. 1964. Wood and c e l l u l o s e science. Ronald Press Co., New York. > 24. Sukumaran, N.P. and Weiser, C.J. 1972. Freezing i n j u r y i n potato leaves. Plant P h y s i o l . 50(564-7). 108 25. T a i t , M.J. and Franks, F. 1971. Water i n b i o l o g i c a l systems. Nature 230 (91-94). 26. Timmis, R. 1973. An excised-needle f r e e z i n g t e s t of cold hardiness i n Douglas-fir. Chapter 1 of t h i s t h e s i s . 27. Timmis, R. 1973. Environmental c o n t r o l of cold a c c l i m a t i o n i n Douglas-fir during germination, a c t i v e growth and r e s t . Chapter 2 of t h i s t h e s i s . 28. Timmis, R. 1973. Translocation of dehardening and bud-break promoters i n c l i m a t i c a l l y s p l i t Douglas-fir. Chapter 3 of t h i s t h e s i s . 29. Tumanov, Krasavtsev, O.A. and Trunova, I . I . 1969. Inves-t i g a t i o n of the i c e formation process i n plants by measuring heat evolution. Sov. Plant P h y s i o l . 16(754-60). 30. Weast, R.C. 1969. Handbook of chemistry and physics. Cleveland, Ohio, Chemical Rubber Co. 31. Weiser, C.J. 1970. Cold r e s i s t a n c e and i n j u r y i n woody plants. Science 169(1269-78). 

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