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Regulation of cold hardiness in seedlings of western red cedar, yellow cedar and white spruce Silim, Salim Nahdy 1991

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REGULATION OF COLD HARDINESS IN SEEDLINGS OF WESTERN RED CEDAR, YELLOW CEDAR AND WHITE SPRUCE. by SALIM NAHDY SLUM B.Sc.(Forestry) Makerere University M.Sc.F. University of New Brunswick A THESIS SUBMITTED LN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctor of Philosophy in THE FACULTY OF GRADUATE STUDIES FOREST SCIENCES We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April 1991 © Salim Nahdy Silim, April 1991 In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. DEPARTMENT OF FOREST SCIENCES The University of British Columbia Vancouver, Canada Date: 8 AUGUST 1990 A B S T R A C T The development and nature of cold hardiness was examined in first-year seedlings of the three conifer species: western red cedar (Thuja plicata Donn) from southern Vancouver Island, yellow cedar (Chamaecyparis nootkatensis (D. Don) Spach) from northern Vancouver Island and white spruce (Picea glauCa (Moench) Voss) from northern interior of British Columbia. The relationship between free thiols and the development of cold hardiness, and the effects of mefluidide on the induction of hardiness in the three species were also examined. To permit an accurate estimation of cold hardiness, a reliable method of determining hardiness based on the electrolyte conductivity method (EC) was developed for the three species. Optimization of tissue preparation resulted in accurate and reliable estimates of cold hardiness in the three coniferous species. A comparison of visual assessment of shoot damage with the results obtained by the EC method indicated that the two methods were highly correlated although the EC method slightly overestimated the temperature at which 5 0 % of the samples were killed (LT5Q). The absolute lethal temperature ( L T ^ Q Q ) estimated by the EC method was lower ( 3 to 7 °C) than that determined by the visual method. The development of hardiness in the three species was characterized by different mechanisms: in white spruce it was initiated by short photoperiod, in red and yellow cedar it was basically regulated by low temperature. Low temperature ( 7 / 3 °C day/night) increased hardiness in all three species but subfreezing temperature (11-3 °C day/night) increased the rate of hardening only in the two cedars. Furthermore, white spruce seedlings were apparently able to attain extreme levels of hardiness (below - 6 5 °C) without exposure to temperatures below 0 °C. The ability to deharden in white spruce i i was related to the satisfaction of chilling requirements while red and yellow cedar seedlings appeared to deharden only in response to warm temperatures. Mefluidide (0.1 and 0.4 mg l"* ) applied as a root drench did not increase cold hardiness in any of the three species. Stomatal conductance was however decreased, thus resulting in increased shoot water potentials. Net photosynthesis was reduced predominantly due to stomatal limitations. Mefluidide increased synthesis of ABA in shoots of seedlings of the three species. The level of tissue ABA, like the other variables, was dependent on mefluidide concentration. Low temperature (7/3 °C day and night, 9 h photoperiod, 250 umoles m"^  s~*, 400 - 700 nm) induced an increase in free thiols (predominantly reduced glutathione) which was accompanied by an increase in hardiness. Although short photoperiod at a warm temperature (9 h, 20/15 °C day and night) increased hardiness in seedlings of white spruce, no significant increase in thiols was observed. Root application of buthionone sulfoximine and dichlormid affected tissue glutathione levels but these changes did not result in significant changes in freezing resistance. There appears to be no relationship between glutathione and freezing resistance. i i i TABLE OF CONTENTS ABSTRACT ii Table of Contents iv List of Figures vi List of Tables .viii Acknowledgements x CHAPTER 1 THE APPROACH AND AIM OF THE STUDY 1 1.1 General Introduction 1 1.2 General Seedling Culture 8 CHAPTER 2 DETERMINATION OF COLD HARDINESS USING THE ELECTROLYTE CONDUCTIVITY METHOD 9 Abstract 9 2.1 Introduction 10 2.2 Materials and Methods 14 2.2.1 Seedling Culture and Freezing Protocols 14 2.2.2 Effects of Different Tissue Sizes and Types, and Timing of Conductivity Measurements 15 2.2.3 Comparison of Whole Plant Damage to Conductivity of Diffusates 16 2.2.4 Estimation of LT50 and Data Analysis 16 2.3 Results 18 2.3.1 Effects of Tissue Preparation and Time of Conductivity Measurements 18 2.3.2 Comparison of Whole Plant Damage with Indices of Injury 24 2.4 Discussion 29 2.5 Conclusion 33 CHAPTER 3 SEASONAL VARIATIONS AND ENVIRONMENTAL REGULATION OF COLD-HARDINESS 34 Abstract 34 3.1 Introduction 35 3.2 Materials and Methods 38 3.2.1 Seasonal Variations in Frost Hardiness 38 3.2.2 Environmental Regulation of Hardiness 38 3.3 Results .40 3.3.1 Seasonal Variations .40 i v 3.3.2 Environmental Regulation 42 3.3.2.1 Hardening -42 3.3.2.2 Dehardening 47 3.4 Discussion 52 3.4.1 Seasonal Variations .52 3.4.2 Environmental Regulation 54 3.4.2.1 Hardening M 3.4.2.2 Dehardening 59 3.5 Conclusion 61 CHAPTER 4 EFFECT OF MEFLUIDIDE ON WATER RELATIONS, NET PHOTOSYNTHESIS, COLD HARDINESS AND ABA LEVELS 62 Abstract 62 4.1 Introduction : -63 4.2 Materials and Methods 67 4.2.1 Extraction, Purification and Estimation of ABA 68 4.2.2 Data Analysis 70 4.3 Results .71 4.4 Discussion 85 4.5 Conclusion 90 CHAPTER 5 THE RELATIONSHIP BETWEEN NON-PROTELN THIOLS AND FREEZING RESISTENCE 91 Abstract 91 5.1 Introduction 92 5.2 Materials and Methods 97 5.2.1 Seedling Culture and Treatments 97 5.2.2 Chemicals 98 5.2.3 Extraction and Analysis of Glutathione and Thiols 98 5.2.3.1 Tissue Extraction 98 5.2.3.2 Assays 102 5.2.4. Data Computations and Analysis 103 5.3 Results 104 5.4 Discussion 114 5.5 Conclusion 119 CHAPTER 6 GENERAL CONCLUSIONS AND RECOMENDATIONS 120 L r T E R A T U R E CITED v LIST OF FIGURES Figure 2.1. Changes in relative conductivity of diffusates with time 23 Figure 2.2. The relationship between injury estimated by the visual assessment of foliage damage and the conductivity method in seedlings of red cedar, yellow cedar and white spruce 27 Figure 3.1. Weekly maximum and minimum extreme air temperatures and seasonal changes in photoperiod at the University Forest Nursery, and cold hardiness of red cedar, yellow cedar and white spruce seedlings . 41 Figure 3.2. Patterns of frost hardening in seedlings of red cedar under different environmental conditions 43 Figure 3.3. Patterns of frost hardening in seedlings of yellow cedar under different environmental conditions 45 Figure 3.4. Patterns of frost hardening in seedlings of white spruce under different environmental conditions 46 Figure 3.5. Hardening of red cedar, yellow cedar and white spruce seedlings exposed to subfreezing temperature .49 Figure 3.6. Patterns of dehardening of red cedar and yellow cedar seedlings 50 Figure 3.7. Patterns of dehardening in white spruce seedlings .51 Figure 4.1. Effects of mefluidide on net C O o fixation, stomatal conductance and snoot water potential in seedlings of red cedar 72 Figure 4.2. Effects of mefluidide on net CO? fixation, stomatal conductance and shoot water potential in seedlings of yellow cedar 73 Figure 4.3. Effects of mefluidide on net C O o fixation, stomatal conductance and snoot water potential in seedlings of white spruce 74 v i Figure 4.4. Effect of mefluidide on Ci/Ca relative to the control treatments 78 Figure 4.5. Effect of mefluidide on levels of ABA in shoots of red cedar 79 Figure 4.6. Effect of mefluidide on levels of ABA in shoots of yellow cedar seedlings 80 Figure 4.7. Effect of mefluidide on levels of ABA in shoots of white spruce seedlings 81 Figure 4.8. Effect of mefluidide application on estimated lethal temperatures of red cedar, yellow cedar and white spruce seedlings 84 Figure 5.1. Changes in levels of thiols in leaves of acclimating seedlings of red cedar 105 Figure 5.2. Changes in levels of thiols in leaves of acclimating seedlings of yellow cedar 106 Figure 5.3. Changes in levels of thiols in leaves of acclimating seedlings of white spruce 107 v i i LIST O F T A B L E S Table 2.1. Effects of tissue size and type on indices of injury and estimated lethal temperature in red cedar seedlings 19 Table 2.2. Effects of tissue size and type on indices of injury and estimated lethal temperature in yellow cedar seedlings 20 Table 2.3. Effects of different treatment of needles on indices of injury and estimated lethal temperature in white spruce seedlings 21 Table 2.4. Effect of Agl during freezing on the indices of injury and estimated lethal temperature in red cedar seedlings 22 Table 2.5. Effect of timing of conductivity measurements on estimated lethal temperature 22 Table 2.6. Correlations between injury estimatedby the conductivity method and visual assesment of foliage damage 25 Table 2.7. A comparison of the estimated incipient killing temperature, absolute lethal temperature and LT50 obtained by the EC method and visual assesment of foliage damage 26 Table 2.8. A comparison of estimated lethal temperature using the conductivity method and visual assesment of damage 28 Table 4.1. Recovery of ABA by the extraction procedure 69 Table 4.2. Effect of mefluidide on Ci/Ca 76 Table 4.3. Short term effect of mefluidide and ABA on photosynthetic O2 evolution 77 Table 4.4. Effect of mefluidide concentration on ion leakage from needles of white spruce 83 Table 5.1. Effect of partitioning GSH standard against chloroform 100 v i i i Table 5.2. Effect of using PVpP on the absorbances of centrifuged yellow cedar and white spruce extracts 100 Table 5.3. Effect of using PVpP and chloroform on the absorbances of centrifuged red cedar extracts 101 Table 5.4. Time course of conjugation of GSH standard with 2-vinylpyridine 101 Table 5.5. Recovery of GSH by the extraction procedure 102 Table 5.6. Effects of water stress, BSO and dichlormid on glutathione levels and estimated lethal temperature in red cedar seedlings 110 Table 5.7. Effects of water stress, BSO and dichlormid on glutathione levels and estimated lethal temperature in yellow cedar seedlings I l l Table 5.8. Effects of water stress, BSO and dichlormid on glutathione levels and estimated lethal temperature in white spruce seedlings 112 i x ACKNOWLEDGEMENTS I wish to extend my appreciation and thanks to my supervisor Dr. D. Lavender for his help and guidance in the course of the study and preparation of this thesis, and to the members of my advisory committee: Dr. E. Camm and Dr. D. Lester for their advice, suggestions and invaluable help, and Dr. R. Guy for the discussions, ideas and always being available. Also greatly appreciated are the contributions by: L. Charleson for all kinds of help, John Russell for supplying yellow cedar cuttings, Dr. H. Weger for the numerous discussions and help with the O2 electrode, Dr. T. Kannangara for ABA analysis and Nathaniel Makoni for the discussions and the spirit. This research project was made possible by funding from B.C. Science Council and the Faculty of Forestry. The gifts of mefluidide from 3M Co (St. Paul MN) and R-25788 from Stauffer Chemicals Co (Richmond CA) are also appreciated. Finally, a special thanks and appreciation to Janet for her patience, support and love throughout the course of this study. x 1 CHAPTER 1. THE APPROACH AND ATM OF THE STUDY 1.1. GENERAL INTRODUCTION Forest nurseries strive to produce seedlings of high quality capable of successfully withstanding a variety of stresses and establishing when planted out in the forest. Failure to withstand stress can lead to severe seedling mortality. Low temperature and water deficit are among the major stresses to which seedlings are subjected. To withstand these stresses, seedlings must be either resistant or able to quickly develop resistance to the stresses. This ability, which is genetically controlled, can be modulated by the nursery cultural regimes the seedlings are subjected to. Little specific information exists on how hardiness is regulated or how it may be modulated by the nursery cultural regimes. Although the effects of environmental factors such as photoperiod, water stress and low temperature in the regulation of hardiness in woody species are generally known (Weiser 1970; Levitt 1980; Sakai and Larcher 1987), specific information on many commercially important species of British Columbia is still lacking. For instance,little information exists on factors regulating the development and maintenance of cold hardiness in seedlings of western red cedar, yellow cedar and white spruce. In red and yellow cedar, which are indeterminate species, it is assumed that short photoperiod induces hardiness as is generally the case with other woody species, but there are no data to substantiate this. A substantial proportion of white spruce seedlings planted in the province is raised in the lower mainland under conditions more favorable to growth than in the native habitat of this species. The effects of these regimes on the development of cold hardiness in this species have not been explored. 2 Specific information on the regulation of cold hardiness in the species is required to develop appropriate nursery cultural techniques for protection of seedlings from low temperature injury while at the same time maximizing other desired qualities. Specific information on hardiness development and regulation will also be critical in predicting the effects of global warming on species survival and possible redistribution. In the transition from an unhardened state to a hardened state, many biochemical, biophysical and physiological changes take place in the plant cell. A comprehensive review of this subject can be found in Burke et al. (1976), Levitt (1980), Graham and Petterson (1982), Oquist (1983), Steponkus (1984,1990), Sakai and Larcher (1987), Guy (1989,1990a,b), Kacperska (1989), Li et al. (1989), Hallgren and Oquist (1990) and Thomashaw (1990). The myriad of changes that take place in the plant cell during acclimation can be broadly grouped into; i) processes that are involved in the induction of the physiological and biochemical changes, ii) adjustment of metabolic activities to permit functioning and possible growth of the plant at low temperature, and iii) processes that are involved in survival and recovery from freezing temperatures. Although it was recognized by Weiser (1970) that acclimation to low temperature involves changes in gene expression, studies on the molecular basis of cold hardiness have just begun (for reviews see Singh and Laroche 1988; Guy 1989, 1990a,b; Thomashaw 1990). Although much information has been gained (cold acclimation proteins [CAPs] have been isolated and a few cold regulated genes [cor] cloned), the work is in its infancy and the significance of the observed changes has not been elucidated. The changes in gene expression have so far not been linked to biochemical changes associated with hardiness. Moreover, unlike heat shock proteins, the variety of proteins produced under low temperature conditions in different species does not permit general conclusions (Guy 1990a; Thomashaw 1990). 3 Actions of plant growth regulators, particularly abscisic acid (ABA) as messengers or modulators, are also important in plant stress responses. Abscisic acid levels increase in winter, and ABA has been shown to induce hardiness in a number of species (Chen and Gusta 1983; Reaney and Gusta 1987; Reaney et al. 1989; Morgan 1990) and to be involved in responses to other stresses such as water deficits and high temperature (Levitt 1980; Morgan 1990). ABA also induces expression of some proteins which are similar to those synthesized under other stresses such as water deficits (Thomashaw 1990; Guy 1990a). However, the elucidation of the role of ABA is hampered by the limited knowledge about the mode of action of the plant growth regulator. Very little is known about the receptors of ABA, and until this information is available, the elucidation of the mechanism will remain elusive and speculative at best. The biochemical adjustments associated with maintenance of primary processes in plants and those associated with survival at low temperatures have been extensively reviewed (Levitt 1980; Graham and Petterson 1982; Oquist 1983; Sakai and Larcher 1987; Kacperska 1989; Guy 1990b; Hallgren and Oquist 1990). The effect of low temperature and cold acclimation on photosynthesis, particularly the Calvin cycle, has been extensively studied. Studies by Huner and co-workers (Huner and McDowall 1976, 1978,1979; Huner et al. 1981) have demonstrated changes in substrate binding, reaction kinetics, sensitivity to freezing, net charge and conformation of ribulose bisposphate carboxylase (RuBPCase) from 'Puma' rye. These changes are related to the improvement of the enzyme activity at low temperatures thus improving overall photosynthetic efficiency at low temperature. Other changes related to the protection of the photosynthetic system from photodamage have also been consistently observed during acclimation (Hallgren and Oquist 1990). These changes are involved in the dissipation of excess excitons when 4 carbon fixation is either inhibited or slowed down by the low temperatures. Thus an increase in ascorbate, glutathione, activities of glutathione reductase, and pigment systems such as carotenes and a-tocopherol have been observed in many plant species (Guy and Carter 1984; Wise and Naylor 1987a,b; Schoner and Krause 1990; Schoner et al. 1990). Induction of pathways and accumulation of substances believed to act as cryoprotectants has been amply documented (Levitt 1980; Sakai and Larcher 1987; Hallgren and Oquist 1990). Increases in the amounts of soluble sugars and polyhydric alcohols, proline, glycine betaine and polyamines have been shown to protect membranes from freeze damage, and also act in adjusting the osmotic potential of the cytoplasm to avoid extreme freeze dehydration. Activities of the enzymes of carbohydrate catabolism are also affected by low temperature (Sakai 1987; Kacperska 1989). Changes in the kinetics and activities of glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phospate dehydrogenase, transketolase, transaldolase, isocitrate dehydrogenase and pyruvate kinase have been documented (Sobczyk and Kacperska-Palacz 1980; Sadakane and Hatano 1982; Sakai and Larcher 1987). Some of these changes are associated with a shift in the pathway of sugar oxidation from glycolysis to the pentose phosphate pathway. Changes in the plasma membrane proteins including activities of ATPase (Uemura and Yoshida 1984; Jian et al. 1982) are related to increased permeability of the plasma membrane at low temperature. Other changes associated with cold hardiness involve changes in membrane properties and composition (Steponkus 1984,1990). These changes involve the augmentation of membrane material and an increase in the phospholipid content upon 5 exposure to low temperature. Increases in digalactosyl diglyceride (DGDG) at the expense of monogalactosyl diglyceride (MGDG) have been observed. The studies that constitute this dissertation were undertaken to develop practical ways of improving cold hardiness specifically, and resistance to water deficits in seedlings of three commercially important tree species of British Columbia. Theoretically, cultural improvement of hardiness or stress resistance in forest seedlings in the nursery can be achieved by either modifying the growth environment (photoperiod, moisture availability or temperature) or application of chemicals thought to enhance resistance to stress. Modification of the nursery environment except for moisture availability is difficult and can be expensive. Even in cases where moisture stress is used as a means of hardening, no specific definitive data are available and the nursery growers generally depend on intuition. It is likely that there will be differences between species and even within species. Modulation of seedling stress resistance by chemical means could therefore provide a convenient and inexpensive alternative to the manipulation of the growth environment, if the responses to the chemicals can be characterized. There have been many attempts at chemically modifying stress responses in plants in agricultural species but not in forest species. Most of the chemicals used are artificial growth regulators (Howell and Dennis 1981; Asare-Boamah and Fletcher 1986; Asare-Boamah et al. 1986; Zhang et al. 1986, 1987; Coleman and Estabrooks 1988; Li 1989). Recent reports (Fletcher and Hofstra 1985; Asare-Boamah and Fletcher 1986; Li et al. 1989; Li 1989) indicate that synthetic plant growth regulators such as mefluidide and paclobutrazol may modulate plant responses to stress. The effects of these compounds on conifer seedlings have not been explored. 6 The tripeptide glutathione, is involved in many stress responses including low temperature and water stress (Rennenberg 1982,1988; Alscher 1989; Smith et al. 1990; Steffens 1990b). The relationship between plant tissue glutathione and environmental factors that are known to improve cold hardiness has not been explored in detail in conifer seedlings. Before any study on cold hardiness can be considered, a reliable method of determining hardiness in the specific material and species is required. Although several methods of determining cold hardiness in plants are available (Timmis 1976; Ritchie 1991), constraints related to suitability and simplicity of the method, nature of the data generated and availability of equipment usually dictate the method to be used. On this basis, the electrolyte conductivity method was chosen for this study. However, due to some contradictory data in the literature regarding the method (Stergios and Howell 1973; Zhang and Willison 1987; De Hayes and Williams 1989; Murray et al. 1989; Burr et al. 1990), a reexamination and refinement of the technique had to be undertaken. The objectives of this study were therefore: (i) to develop an appropriate and reliable method of determining cold hardiness using the electrolyte conductivity method, (ii) to examine the natural progression and environmental regulation of the development and loss of cold hardiness, (iii) to examine the effects of the artificial growth retardant, mefluidide, on cold hardiness and other physiological processes, and (iv) to examine the relationship between plant tissue glutathione and thiol content and cold hardiness in seedlings of western red cedar (Thuja plicata Donn), yellow cedar (Chamaecyparis nootkatensis (D. Don) Spach) and white spruce (Ticea glauca (Moench) Voss). 7 This dissertation is divided into four parts. In the first part, the development of an appropriate cold hardiness testing technique to be used in the rest of the study is described. The second part of the study examines the seasonal changes, and environmental factors controlling cold hardiness in the species. The third part reports the effect of mefluidide on seedling physiology, while the last part examines the relationship between thiols and glutathione, and cold hardiness. 8 1.2. GENERAL SEEDLING CULTURE Seedlings for the study were raised in an open growing compound at the University of British Columbia Forest Nursery (49° 15.5* N, 123° 13.8' W, 40 m altitude) during the 1988 and 1989 growing seasons. They were from the same seed sources and subjected to similar cultural regimes during both growing seasons. One year-old seedlings of white spruce (seedlot 3956, 55° 43' N, 123° 57' W, 777 m altitude), western red cedar (seedlot 3533, 48° 51' N, 124° 18' W, 600 m altitude, British Columbia) and yellow cedar rooted-cuttings from two-year old donors (seedlot 7721, 50° 15' N, 127° 5' W, 775 m altitude, British Columbia) were grown in a peat-vermiculite (3:1) mixture in Styrofoam containers. The mixture contained 11 kg m"* Nutricote, 1 kg m"^  Nutritrace fertilizers and 5 kg m"^  dolomite. During the period of active growth, the seedlings were fertilized twice a week with 0.5 g 1~* (20:8:20 w/w) N:P:K, 0.005 g I"* micronutrient mix, and 0.05 g 1"* potassium sulfate commercial fertilizers (high N fertilizer). Some seedlings were left outdoors during the winter of 1988/89 (used for part I and sections of part II of the study) and fertilized with 0.5 g 1"* (8:20:30 w/w) N:P:K and potassium sulfate and micronutrients as above (low N fertilizer), from August to the end of fall. The rest were transferred to growth chambers during the growing season and used for parts II, Ul and IV of the study. Specific growth conditions for each study are given under the respective Materials and Methods sections. 9 CHAPTER 2. DETERMINATION OF COLD HARDINESS USING THE ELECTROLYTE CONDUCTIVITY METHOD. ABSTRACT A method to determine cold hardiness in seedlings of western red cedar (Thuja plicata Donn), yellow cedar (Chamaecyparis nootkatensis D. Don) and white spruce (Picea  glauca (Moench) Voss.) using electrolyte conductivity (EC) is described. The effects of tissue preparation and selection, and timing of conductivity measurements on the temperature that injures or kills 5 0 % of the samples (LT50) were examined; and the method was compared with visual assessment of tissue damage. Tissue size and preparation affected the estimated LT50 and the associated confidence intervals. Yellow cedar secondary leaves and long stem sections and whole spruce needles gave either very variable readings or an over-estimation of hardiness. Chlorophyllous stem segments, 5 to 1 0 cm from the shoot tips and cut into 0 . 5 cm sections, were the most suitable for the determination of hardiness in red and yellow cedar. In white spruce, 0 . 5 cm long needle segments cut at both ends, were the most suitable. Ion leakage from frozen tissues reached a fairly steady level between 1 8 and 3 6 h after the addition of water. The visual assessment and EC methods correlated well although the latter method slightly over-estimated the LT50. The absolute lethal temperature ( L T J Q O ) estimated by the EC method was however lower ( 3 to 7 °C) than that determined by the visual method. The EC method as described here can be used for objective and accurate determination of LT50 in the three species without any need for adjustments. The most reliable conductivity measurements were obtained between 2 0 and 2 6 h after the addition of water. 10 2.1. INTRODUCTION The ability to measure cold hardiness accurately and promptly is an important factor in making decisions regarding a number of operations related to forest regeneration. It provides a basis for developing nursery techniques to protect and raise high quality seedlings (Warrington and Rook 1980; Glerum 1985; Burr et al. 1986). Essential information for decisions on lifting, storage, handling, outplanting bareroot stock, and protection of seedlings from frost damage is provided by the frost hardiness test. Laboratory measurements of cold hardiness involve artificial freezing of plants or plant tissues at set temperatures followed by the determination of the degree of injury to the plant or plant tissues. Most frequently this degree of injury is expressed as LT5Q which is generally taken to mean the temperature that injures or kills 50% of the samples. Numerous methods of determining plant or tissue damage or viability following or during freezing have been proposed. These include: visual assessment of damage, the electrical impedance method, the electrolyte conductivity test (EC), differential thermal analysis (DTA), infrared thermography, chlorophyll fluorescence, fluorescein diacetate staining (FD), the triphenyl tetrazolium chloride (TTC) test, the ninhydrin test, water potential measurement, and the quantification of ethane and ethylene evolution (Timmis 1976; Kobayashi et al. 1981; Colombo et al. 1984; Glerum 1985; Tinus et al. 1985; Harber and Fuchigami 1986; Laacke et al. 1986; MacRae et al. 1986; Zhang and Willison 1987; Sunblad et al. 1990; Ritchie 1991). Most of these methods have limitations such as technical difficulty, difficulty in interpretation of results, high costs and subjectivity. The choice of a method will therefore depend on the overall objective of the study, plant species, plant material, time, available funds and equipment and skills of the user. For the 1 1 purposes of this study, a reliable test was required that was fast, objective, inexpensive, relatively easy to perform and interpret, and permitted use of limited amounts of tissues. Of the available methods, only visual assessment, and to a limited extent, the conductivity methods are used on an operational basis in forest nurseries. The visual assessment of damage though highly reliable, is time consuming, subjective and may require extensive plant material. In addition, the results are not available for at least 7 to 14 days, which may not be as fast as is usually needed. The electrolyte conductivity test satisfied our criteria as the most suitable method of assessing viability of plant tissues after freezing; it was inexpensive, fast (results within 48 h), used a small amount of tissue, and gave objective results. However, the validity of these "objective" results is established by correlating them with "subjective" estimations. Measurement of electrolyte leakage from plant tissues to assess injury following freezing was originally developed by Dexter et al. (1930,1932) and later improved by Wilner (1959,1960). Freezing results in irreversible changes to cell membranes through rupture or loss of the ability to maintain intracellular concentrations (Steponkus 1984). When such tissues are placed in deionized water, ions leak into the water and the conductivity of the water is proportional to the amount of damage to the tissues (Levitt 1980). The data obtained by the conductivity method can be expressed as relative conductivity, which is the percentage of total ions leaked (Wilner 1959, 1960). However, a more appropriate way is to express it as index of injury (I), as described by Flint et al. (1967). Unlike relative conductivity, computations of I adjust for tissue ion leakage unrelated to freezing injury. Thus, unfrozen controls would have 1 = 0 while killed tissues would have I = 100. 12 Although there are reports that plant damage estimated by the EC method does not compare well with whole plant damage (e.g., Stergios and Howell 1973; Burr et al. 1986,1990; Zhang and Willison 1987), several other studies have also reported that estimates of hardiness using the two methods compared well (Ketchie et al. 1972; Blazich et al. 1974; Timmis 1976; van den Driesche 1976; Green and Warrington 1978; Warrington and Rook 1980; Colombo et al. 1984; Hallam and Tibbits 1988). Stergios and Howell (1973), who expressed their results as relative conductivity, reported that EC estimates did not correspond well with tissue browning in cherry, raspberry and strawberry. Using a cell suspension culture of brome grass (Bromus inermis), Zhang and Willison (1987) reported that the EC method significantly under-estimated hardiness levels (by as much as 10 °C in some cases) when compared with fluorescein diacetate vital staining (stains for esterase activity). Working with Douglas-fir, lodgepole pine and Engelmann spruce needles, Burr et al. (1990) reported that the LT^Qfor tissue leakage corresponded to the temperature that produced about 10% damage (assessed visually) to the whole shoots. These observations indicate there were potential problems with the EC method. It was therefore necessary to re-examine and refine the method to suit the material to be used before proceeding with the technique. Although no clear indications of the possible causes of the above results were obvious from the studies, it appeared that some of the problems were associated with data computations, interpretations, sample preparation (including tissue sizes) and freezing protocols. If indeed the LT5QS obtained by the EC method do not correspond to that of whole shoots (estimated visually) as reported in the above studies, but remain strongly correlated, this relationship should be examined and established. Under such circumstances, the EC results must be calibrated to shoot damage. 13 The objectives of this study were: (i) to improve the electrolyte conductivity method of determination of cold hardiness, and (ii) to compare visual assessment of whole plant damage to the conductivity method, in seedlings of western red cedar (Thuja pjicata), yellow cedar (Chamaecyparis nootkatensis) and white spruce (Picea glauca). The results obtained from this part of the study were utilized to determine cold hardiness in studies reported in the other sections of the thesis. 14 2.2. MATERIALS AND METHODS 2.2.1. Seedling Culture and Freezing Protocols Seedlings used for this study were raised and left to overwinter outdoors as described in section 1 . 2 . Plant tissues ( 5 0 to 1 0 0 mg) were placed in 2 0 ml scintillation vials with 0 . 5 ml of glass distilled water. Approximately 1 mg of silver iodide was added as a nucleator to initiate freezing. The tissues were frozen in a programmable freezer to a range of temperatures at a rate of 5 °C per h. The temperatures in the freezer were within + 0 . 5 °C of the set point. After one hour at the set temperature, the vials were removed from the freezer, quickly transferred in the dark to a cooler ( 0 to 3 °C) and left to thaw overnight. Unfrozen controls were kept in the cooler. The next day 1 5 ml of glass-distilled water was added to all the vials. The vials were left at room temperature until the conductivity of the added water was measured (C c o n t r o j and Cf r o z e n ) . The vials were then placed in a hot water bath ( 7 0 "C for 2 0 min) to kill the tissues, and the conductivity (C^JJJ) was read 1 2 h later. The amounts of ions that had leaked from the tissues were calculated as relative conductivities according to Wilner ( 1 9 5 9 , 1 9 6 0 ) as follows: RCcontrol - Ccontrol/Cckill x 1 0 0 RCfrozen = Cfrozen/Cfkill x 1 0 0 where; ^control = m e conductivity of the controls, ^ckill = t n e conductivity of the killed controls, 1 5 ^frozen = the conductivity of the frozen material, CfkJU = the conductivity of the killed frozen material, ^^control = relative conductivity of the control, and R C ^ o z e n = relative conductivity of the frozen material. The unfrozen relative conductivity (RC c o n t r o i) was used to correct for apoplastic ion concentrations or ions leaking out of tissues which had not been injured. The results were expressed as indices of injury (I) and computed according to Flint et al. (1967) as follows: I = (RCfr 0 2 e n • RCcontrol)/(100 - R C c o n t r o l ) x 100. The temperature that gave an I of 50 was considered to be the temperature that killed 50% of sample (LT5 0). 2.2.2. Effects of Different Tissue Sizes and Types, and Timing of Conductivity Measurement To examine the effects of different tissue sizes and types on the estimate of hardiness in seedlings of red cedar and yellow cedar, chlorophyllous stem segments up to 10 cm from the top were cut to 1, 0.5 and 0.25 cm lengths, and 1 and 0.5 cm segments of a composite of secondary leaves and whole primary leaves were compared. For white spruce seedlings, whole needles as well as 0.5 and 0.25 cm needle sections cut at both ends, were compared for their suitability as experimental material. The effect of using a nucleator (Agl, in this case) on estimated LT50 was also examined. To determine the optimum time for conductivity readings, a measurement of conductivity over time was made from 0 to 122 h after the addition of water. 16 2.23. Comparison of Whole Plant Damage to Conductivity of the Diffusates This experiment was conducted when the seedlings had become fairly hardy. Seedlings from each species were potted in groups of ten while another set of seedlings was used to prepare material for conductivity measurements. Half centimeter chlorophyllous stem (red cedar and yellow cedar) or needle (white spruce) segments were used. The seedlings and the materials (in vials) were frozen, to temperatures between -7.5 and -25.0 °C for red cedar, -12.5 and -32.5 °C for yellow cedar both at 2.5 °C intervals, and -20 and -41 °C at 3 "C intervals for white spruce. To protect the seedling roots from low temperature damage, the seedlings were potted in Styrofoam boxes and dry perlite placed around the roots. The amount of damage to the plants (indicated by tissue browning of the foliage and the cambium)was determined as percentages, and on terminal buds (for spruce) as counts after 2 weeks. Damage to the cambium was determined on the main stem and reported as percentages (of 10s) from the top downwards i.e.; top 10%, 20% up to 100%. Conductivity of the diffusates was measured between 0 and 60 h after the addition of water. The amount of damage to the tissues was expressed as indices of injury (I) as described earlier (section 2.2.1). 2.2.4. Estimation of LT5Q and Data Analysis Since the relationship between freezing temperature and injury is asymptotic between 0 and 100%, injury from the conductivity measurements was determined by a linear regression of I values between 15 and 85% against temperature. This range is generally the linear portion of the graph. LT50 was then determined by inverse prediction according to Zar (1984). In instances where the slope of the graph was so steep that there were only two test temperatures between I values of 15 and 85% (e.g., when the 17 seedlings were either not hardy or had not developed sufficient hardiness), the LT50 was estimated graphically. In such cases, the confidence intervals could not be estimated. To illustrate the relationship between freezing temperature and tissue damage, a nonlinear regression of I values and visual damage was performed based on the logistic model using SYSTAT (Wilkinson 1989): Y=l/(l+(b*exp(-rX))) where: Y = the estimated amount of injury at temperature X, r = the rate of increase in injury associated with a decrease in freezing temperature, b = the estimated injury when no freezing is applied (X = 0 °), and X = the freezing (test) temperature. Correlation analyses between the I values measured at 12, 24 and 36 h after the addition of water, and visual damage were performed to compare the indices of injury with visual damage of foliage using SYSTAT (Wilkinson 1989). 18 23. RESULTS 2.3.1. Effects of Tissue Preparation and Time of Conductivity Measurements Tissue sizes and preparation affected the estimated LT50 and the associated confidence intervals in all three species. Other than the 0.25 cm stem sections which gave a higher estimated LT5Q, the use of either primary leaves or secondary leaves gave similar estimates of LT50 in red cedar (Table 2.1). Similar results were obtained with yellow cedar tissues. However, the confidence intervals associated with the estimated LT5QS from primary leaves and a composite of secondary leaves were quite high (Table 2.2), reflecting the high variability associated with conductivity measurements from these tissues. The use of whole white spruce needles gave an unacceptably low estimated LT5Q although the needles were visibly damaged at a much higher temperature (Table 2.3). Cutting both ends of the needles gave more acceptable estimates, regardless of the lengths we used. It was however, a lot easier to work with 0.5 cm sections, as in the other two species. Table 2.1. Effects of tissue size and type on indices of injury, I values, and estimated lethal temperature, L T C Q (95% confidence limits) in red cedar seedlings (n=6). The seedlings were raised outdoors and foliage sampled on Dec 10 1988. Stem Sections Leaves Primary Secondary* lcm 0.5cm 0.25cm 1.0cm 0.5cm Test I values (standard deviations) Temp. °C -14 24.8(3.1) 27.7(2.9) 49.8(2.2) 29.2(4.8) 22.9(4.2) 30.1(5.1) -17 49.1(3.5) 50.1(3.2) 62.2(2.5) 47.8(3.4) 44.1(4.2) 50.9(2.8) -20 61.5(3.2) 66.4(3.1) 73.8(1.9) 62.5(5.8) 58.6(6.0) 69.5(3.6) L T 5 0 e C -17.8(0.9) -17.3(0.6) -14.3(0.9) -17.7(1.1) -18.3(1.2) -17.0(0.7) * - secondary needles together with branches cut into 1 and 0.5cm sections. Table 2.2. Effects of tissue size and type on indices of injury, I values, and estimated lethal temperature, (95% confidence limits) in yellow cedar seedlings (n=6). The seedlings were raised outdoors and sampled on Nov 20 1988. Primary Secondary* Test £ I values (standard deviations) Temp. °C -9 13.4(4.2) 16.2(3.2) 21.2(3.4) 12.1(4.7) 13.1(2.2) 13.9(5.6) -12 40.0(6.8) 45.4(4.1) 52.1(3.5) 39.2(15.6) 35.6(14.9) 38.8(5.6) -15 65.7(9.1) 74.9(5.2) 85.1(4.3) 65.3(16.9) 56.6(16.2) 60.4(16.5) L T 5 0 * C -13.4(1.1) -12.5(0.6) -11.7(0.4) -13.7(2.3) -14.8(2.1) -14.2(2.9) * - secondary needles together with branches cut into 1 and 0.5cm sections. 21 Table 2.3. Effect of different treatment of needles on indices of injury, I values, (standard deviations) and estimated LT50 (95% confidence limits) in seedlings of white spruce (n=5). The needle sections were cut at both ends. The seedlings were raised outdoors and foliage sampled on Oct 20 1988. Whole Needle sections needles 0.5cm 0.25cm Test Temp. °C I values (standard deviations) -26 3.81(1.45) 12.8(2.0) 12.4(1.7) -29 26.6(3.5) 47.6(4.9) 52.6(2.3) -32 30.2(3.9) 68.3(6.1) 76.4(1.5) -35 36.0(5.8) 77.3(5.9) 83.9(1.8) Estimated LT50 -38.8(4.2) -29.7(1.7) -29.5(1.5) The presence of an ice nucleator (Agl) affected estimated LT50 only at high temperatures where supercooling would be a factor (Table 2.4). When the tissues were not very hardy the absence of Agl as an ice nucleator resulted in an over estimation of the LT5Q. However, when the tissue hardiness was lower than -10 °C, the presence of the nucleator had no effect on estimated LT5Q. Examination of changes in relative conductivity over time indicated that the values reached a relatively steady state between 18 and 36 h after the addition of water (Figure 2.1). After this period, diffusates from the tissues which were not completely damaged (including the controls) undergo a second increase in conductivity at a fairly constant rate. The differences in estimated LT50S for the period (18 to 36 h) are about 1 °C for red and yellow cedar tissues and 3 °C for white spruce tissues (Table 2.5). 22 Table 2.4. Effect of Agl during freezing on the indices of injury, I values, and estimated lethal temperature, LT50 (95% confidence limits) of red cedar tissues. (n=5). With Agl Without Agl Temp. °C I values (standard deviations) -4 -6 -8 16.0(2.3) 77.4(4.5) 91.2(5.3) 3.84(4.0) 6.86(8.7) 92.2(3.3) * Estimated LT50 -4.8 -7.0 Temp. °C I values (standard deviations) -14 -17 -20 27.7(2.9) 50.0(3.2) 66.4(3.1) 28.1(2.1) 48.4(2.7) 67.9(2.9) Estimated LT50 -17.3(0.8) -17.3(0.6) * - estimated graphically. Table 2.5. Effect of timing of conductivity measurements on estimated lethal temperature, LT50 (95% confidence limits). (n=5). * Species Time of Measurements 18 h 24 h 36 h Estimated LT50 °C Red Cedar -18.9(0.5) -18.5(0.5) -17.8(0.5) Yellow Cedar -23.5(0.9) -22.9(0.9) -21.6(1.1) White Spruce -33.0(0.6) -32.4(0.6) -31.5(2.5) * - time since addition of water. < _l 20 40 60 80 100 120 TIME (HRS) AFTER ADDITION OF WATER 140 Figure 2.1. Changes in relative conductivity of diffusates with time. The graphs are for tissues from seedlings of red cedar (A), yellow cedar (B) and white spruce (C). The lines represent freezing temperatures of -17 (1), -14 (2), -11 (3) and -8 (4) °C for red cedar (A), -23 (1), -20 (2), -17 (3), -14 (4) and -11 (5) °C for yellow cedar (B), and -24 (1), -20 (2), -16 (3) and -12 (4) °C for white spruce (C). 24 2.3.2. Comparison of Whole Plant Damage with Indices of Injury In all three species, estimated damage assessed by the whole plant visual method was significantly (p<0.001) correlated with the conductivity method (Table 2.6). Although the correlations were high, conductivity measurements made more than six hours before or after the selected 24 h measurement time resulted in either an overestimation or underestimation of the LT5Q. In all three species, estimated LT5Q obtained by conductivity measurements at 24 h was slightly lower than that obtained by the whole plant visual assessment (Table 2.7 and Figure 2.2). This could be partly accounted for by the subjectivity of the visual assessment technique. The absolute lethal temperature (LT^QQ , temperature at which all plants or plant materials are killed) obtained by the conductivity measurements was also lower than that obtained by whole plant injury (Table 2.7, Figure 2.2). This difference was about 4 ° C in red cedar, about 7 ° C in yellow cedar and about 4 ° C in white spruce seedlings. Except in yellow cedar seedlings, the incipient killing temperatures (LTj, temperature of initial injury) for both methods were similar. In yellow cedar this value was about 4 ° C higher by the conductivity method than by the visual assessment of the whole shoot damage. At the stage of acclimation when the comparisons between the two methods were performed, the cambium was more hardy than the foliage (estimated by visual assessment) in all three species (Table 2.8). In white spruce seedlings, the terminal buds were much less hardy (by as much as 14 ° C ) in comparison to the plant. The subterminal buds, however, appeared to be almost as hardy as the shoot. In all cases where the terminal buds were killed while the rest of the plant survived, the subterminal buds (usually in groups of 2 to 4) replaced the terminal buds giving rise to a multi-leadered seedling. 25 Table 2.6. Correlations between injury estimated by the conductivity method and visual assessment of foliage damage. (n=10). Conductivity method Leaching Time (h) 12 18 24 36 Visual Assessment Correlation coefficients Red Cedar 0.906 0.947 0.941 0.950 Yellow Cedar 0.894 0.913 0.916 0.925 White Spruce 0.860 0.894 0.896 0.892 * - all the values are significant at p < 0.001 Table 2.7. A comparison of the estimated incipient killing temperature (LTj), estimated lethal temperature (LT50) and absolute lethal temperature ( L T J O Q ) (95% confidence intervals in parenthesis, n=10) obtained by the E C methodand visual assessment of foliage damage. Species L T J °c Conductivity Method £50 L T 1 0 0 L T J °C Visual Assessment £50 L T 1 0 0 Red Cedar -11.1 -18.5 -27.9 -9.7 -16.2 -23.4 (0.7) (0.5) (1.0) (1.4) (1.1) (1.4) Yellow Cedar -12.7 -22.9 -35.0 -16.1 -20.2 -28.2 (1.3) (0.9) (1-3) . (1.4) (1.1) (1.4) White Spruce -23.2 -32.4 -42.5 -20.4 -29.5 -38.5 (0.5) (0.6) (0.8) (2.5) (2.5) (2.4) 27 T — 1 — i — • — i — 1 — i — 1 — I — • — i — • — i — ' — i — 1 — i — • — i — • — I — • — i — ' — i — ' — i — 1 — r -30 -25 -20 -16 -10 -35 -30 -25 -20 -15 -40 -35 -30 -25 -20 -15 TEMPERATURE C TEMPERATURE C TEMPERATURE C Figure 2.2. The relationship between injury estimated by the visual assessment of foliage damage (1) and the conductivity method (2) in seedlings of red cedar (A), yellow cedar (B) and white spruce (Q. Table 2.8. A comparison of estimated LT50 (95%, confidence interval) using the conductivity method and visual assessment of damage. (n=10) Conductivity Visual Assessment Species Foliage Cambium T. Buds Sb. Buds L T 5 0 ' C Red Cedar -18.5(0.5) -16.0(1.1) -18.9(1.2) Yellow Cedar -22.9(0.9) -20.2(1.1) -23.6(1.2) White Spruce -32.4(0.6) -29.5(2.5) -34.3(2.9) -18.0(2.2) -31.0(2.9) * - T. buds and Sb. buds refer to terminal buds and subterminal buds respectively. 29 2.4. DISCUSSION The observed differences between I values in the different tissue sizes and types were likely the result of a combination of effects of the ratio of cut surfaces to the volume of tissue, the distance travelled by ions leaking out of the tissue and various cuticular properties of the different tissues and species. Hallam and Tibbits (1988) obtained similar results with different leaf sizes of eucalyptus. The smaller tissue sizes generally had higher control relative conductivity and since computations of I adjust for the controls, the resultant values would end up being lower in the smaller tissues despite the fact that the amount of damage may be the same. The lower conductivity values from larger tissues probably arose because of the slower rate of leakage from the tissues. Cuticular impediment to electrolyte leakage was probably the cause of the lower values with yellow cedar secondary leaves and whole white spruce needles. The higher conductivity values obtained from needles of white spruce with both ends cut indicate that this was indeed the major cause. A constant level of ion concentration in the water was reached after 22 h which probably corresponded to the level of irreversible membrane damage as caused by the freezing temperature. In tissues not completely injured by the freezing temperature, a second increase in ion leakage occurred which was probably a result of tissue death unrelated to the freezing damage. Results which do not indicate this steady level of ion concentration (e.g., Murray et al. 1989) probably reflect an incomplete electrolyte leakage from the tissue due to the utilization of either whole shoots or needles. In such cases the conductivity values would be well below those expected, leading to an under-estimation of the lethal temperature. Since ion leakage from tissues may be fast, as in protoplasts (Palta et al. 1977) or small tissues (Hallam and Tibbits 1988), or slow, as in 30 our case, timing is very critical in the determination of the lethal temperature. It is therefore necessary to determine the appropriate time to perform the measurements before proceeding with the technique. For the whole plant, the estimated LT50 is the temperature that kills 50% of the plants, whereas for conductivity measurements it is described chemically as the point where 50% of the symplastic ions leak from the tissue as a result of the freezing injury. It is important to note that the LT50 values may not correspond and indeed the results of the conductivity measurements may have little physiological relevance. As a result, rate of leakage (Murray et al. 1989), differential percent leakage (Zhang and Willison 1987), LTjQ (Burr et al. 1990) and other experimental measurements such as critical temperature (De Hayes and Williams 1989) have been preferred when either the results did not compare well or poor correlations were observed. In our situation, indices of injury of 50% generally corresponded with visual estimates for LT50 of whole plants. Although minor differences in estimated LT50 by the two methods were observed, which may have been either real or a result of inconsistent visual determination of whole plant damage, the use of LT5Q obtained by the EC method was considered acceptable for the estimation of damage to the plant. The bigger differences in the absolute lethal temperatures obtained by the two methods may be because the lethal point in acclimated tissue may not necessarily correspond to the point where all ions leak out the tissues. It is likely that irreversible tissue damage leading to death occurs at a point well before all the ions leak out of the tissues. The reported lack of correspondence and correlations in literature can be largely explained by either an inappropriate tissue preparation (Murray et al. 1989) or problems with freezing protocols. The disparity between the electrolyte leakage and FD staining 31 reported by Zhang and Willison (1987) can partly be accounted for by the different mechanisms of membrane damage caused by freezing temperatures. Hardened plant cells can be injured by freezing temperatures without being ruptured but will display loss of osmotic responsiveness (Steponkus 1984). Cells injured in this manner can still stain, but only ruptured cells fail to stain (Palta et al. 1977). Freezing injury to unhardened plant cells involves cell rupture upon rethawing (Steponkus 1984). A comparison of low temperature damage by vital staining and electrolyte conductivity (e.g., Zhang and Willison 1987) may, therefore, not be appropriate especially when the cells are hardened. One of the problems associated with determination of hardiness using electrolyte conductivity measurements of selected tissues is the differences in hardiness of different tissues. Whereas in red and yellow cedar no major differences in hardiness of different shoot tissues were observed when the whole plant was frozen, in white spruce there were significant differences between the terminal buds and the rest of the shoot, a situation similar to other woody species that form terminal buds (Sakai and Larcher 1987). Under such circumstances, it was necessary to decide whether to use the terminal bud as an indicator of mortality when the rest of the seedling was healthy or to use the foliage when the terminal bud was definitely killed by the test temperature. We decided to use the foliage on a biological basis, i.e., a purely survival basis. A major limitation of the technique (also applicable to visual analysis of plant damage) is the statistical estimation of the LT50. Several techniques have been used ranging from a simple graphical determination (Glerum 1985; Rietveld and Tinus 1987) to sophisticated curve fitting techniques (e.g., Rehfeldt 1979,1980; Burr et al. 1990). I used a linear regression of the linear portion of the graph followed by inverse prediction to determine the selected level of lethality. This decision was based on two factors; 32 requirement for fewer test temperatures, and an observed change in the shape and slope of the sigmoid curve as the plants became more hardy. At a nonacclimated stage or less hardy stages, the tissues are killed at a very narrow temperature range (usually 2 °C or less) resulting in very steep curves. As the plants become hardier, however, the temperature range at which the tissues are killed becomes broader thus changing the slope and shape of the curve, a phenomenon also observed by Burr et al. (1990) and Tibbits and Reid (1987). Although the basis of this apparent increase in the temperature window over which the tissues are damaged is not clear, it is probably related to the different mechanisms of damage to the plasma membrane in nonacclimated and acclimated tissues. In unhardened tissues, damage results from expansion induced cell lysis during rethawing whereas in the hardened tissues, although the mechanism is not clearly understood, it involves loss of osmotic responsiveness or altered osmotic behavior (Steponkus 1984). Damage as a result of expansion-induced lysis would then tend to result in a rather sudden cell rupture thus giving a large increase in conductivity readings over a narrow temperature range. Loss of osmotic responsiveness, on the other hand, is probably not a sudden event. It probably occurs over a wide range of temperature resulting in a lower increase in conductivity readings over a wider range of temperatures. 33 2.5. CONCLUSION The results presented here indicate that the electrolyte leakage technique can be used to determine cold hardiness in seedlings of the three conifer species examined. The use of an established conductivity method after optimizing tissue preparation, made it possible to obtain high and significant correlations between the indices of injury and visual assessment of whole plant tissue damage. Furthermore, the estimated LT50 obtained by the EC and visual methods were fairly close implying that an accurate prediction of frost damage in these seedlings could be made by the direct use of LT50S obtained from these measurements without any need to calibrate the conductivity measurements to visual damage. It should be possible to extend the results obtained from this study to other conifer species without having to perform similar comprehensive comparisons. Only tissue size may require optimization although 0.5 cm needle or stem sections may suffice. A comparison with whole shoot injury may not be necessary since damaged tissues can be easily identified by their water soaked or soggy appearance. 34 CHAPTER 3. SEASONAL VARIATIONS AND ENVIRONMENTAL REGULATION OF COLD HARDINESS. ABSTRACT The seasonal patterns of frost hardening and dehardening at Vancouver, British Columbia, were examined in first-year seedlings of western red cedar (Thuja plicata Donn) from southern Vancouver Island, yellow cedar (Chamaecyparis nootkatensis D. Don) from northern Vancouver Island and white spruce (Picea glauca (Moench) Voss.) from the northern interior of British Columbia. The effects of photoperiod, water stress and low temperature on the regulation of hardiness were also examined. Under the ambient conditions in 1988 and 1989, the spruce seedlings started to harden during mid to late summer in two stages; first in response to shortening photoperiod, then to decreasing temperatures. Red and yellow cedar seedlings started hardening in late fall when photoperiod was already less than 11 h and the daily maximum temperature around 10 °C. The seedlings of these two species displayed a second stage of hardening only in response to persistent subfreezing temperatures. Short photoperiod (9 h, 20/15 °C) had little effect on hardiness of the two cedars but increased hardiness in spruce to about -15 "C Water stress significantly increased hardiness in spruce under long photoperiod (18 h, 20/15 °C) but only marginally in red and yellow cedar. Low temperature (7/3 °C) increased hardiness in all species. Short photoperiod predisposed seedlings of yellow cedar and spruce but not red cedar, to start hardening in response to low temperature exposure without going through a lag phase. Subfreezing temperature (2/-3 °C) was necessary to attain maximal hardiness in red and yellow cedar but not in white spruce. The ability to deharden in white spruce was apparently related to the satisfaction of chilling requirements and not necessarily exposure to warm temperatures. d 35 3.1. INTRODUCTION Woody plants native to temperate regions go through an annual cycle of frost hardiness with changes in season. Seasonal development of frost hardiness is believed to take place in distinct stages (Weiser 1970; Levitt 1980; Sakai and Eiga 1985; Sakai and Larcher 1987; Li et al. 1989). The first stage, induced by shortening daylengths at fairly warm temperatures (10 to 20 °C), results in moderate levels of hardiness. The subsequent stage(s) is induced by low temperature with subzero temperature believed to be the most effective. The timing of initiation of hardiness and the actual level of hardiness attained by a plant in response to given environmental stimuli is under strong genetic control and varies both within and between species (Rehfeldt 1979,1980; Sakai and Larcher 1987). Ecotypes from a more northerly latitude or higher altitude commence hardening earlier than those from a southerly latitude or lower elevations when raised at the same outdoor environment (Sakai and Larcher 1987). Races of species which are distributed in widely ranging climatic regions will harden to different levels (Sakai and Larcher 1987). Species from boreal and sub-boreal regions can attain extremely high levels of hardiness, easily surviving exposure to -70 °C or even liquid nitrogen (-196 °C) in midwinter (Sakai 1983; Sakai and Larcher 1987), while those from cool temperate zones are able to attain only moderate levels of hardiness. Factors regulating hardiness have been studied extensively in many temperate perennial woody plant species. Photoperiod and temperature are believed to play major roles in these species, but water stress also induces some hardiness (van den Driesche 1969; Weiser 1970; Chen et al. 1975; Timmis and Tanaka 1976; Chen and Li 1978; McCreary et al. 1978; Blake et al. 1979; Levitt 1980; Sakai and Larcher 1987). The induction of moderate levels of hardiness by short photoperiod at relatively warm temperatures has been observed in many woody plants (Irving and Lanphear 1967, 1968; 36 Howell and Weiser 1970a; Williams et al. 1972; Berveas et al. 1978; Lavender and Silim, unpublished). Initiation of acclimation in fall can be delayed if the plants are kept artificially under long photoperiods late in summer. Plants exposed to short photoperiods during the growing season, initiate hardening sooner than similar plants kept under natural conditions. Moderate desiccation stimulates increased hardiness in many plant species including mulberry (Sakai 1962), cereals (Cloutier and Siminovitch 1982), Douglas-fir seedlings (Timmis and Tanaka 1976; Blake et al. 1979) and red osier dogwood (Chen et al. 1975; Chen and Li 1978). However, low temperature is considered to be the environmental factor responsible for hardening development. The most effective temperature to induce hardiness depends on the species, the plant organ and the developmental stage of the plant (Sakai and Larcher 1987). It is believed that subfreezing temperatures are the most effective in promoting maximum levels of hardiness (Weiser 1970; Levitt 1980; Sakai and Larcher 1987). A decrease in frost hardiness takes place after exposure to warm conditions (Howell and Weiser 1970b; Weiser 1970; Aronsson 1975; Gusta and Fowler 1976; Kobayashi et al. 1983; Greer and Stanley 1985; Burr et al. 1989). However, the rate and extent of dehardening appears to depend on duration and temperature of exposure, and probably the developmental stage of the plant. Two strategies of seasonal transition to the hardened state have been recognized in perennial plants (Sakai and Larcher 1987; Guy 1989). The first strategy, usually found in species from moderate temperate regions, involves acclimation as a direct response to low temperature exposure. The second strategy is found in species which exhibit inherent annual growth periodicity (Sakai and Larcher 1987; Guy 1989). These plants have 3 7 evolved to adjust their growth and development well in advance of the onset of the unfavorable temperature conditions. The strictly photoperiod-regulated development of bud set and dormancy found in most temperate woody species is a typical example of this strategy. However, most woody plants probably utilize both mechanisms; requiring low temperatures for the development of hardiness but developmental change (e.g., bud set) to permit it to happen. The regulation and control of the development and loss of hardiness in red cedar, yellow cedar and white spruce seedlings have not been explored in detail. White spruce is a boreal species known to respond to photoperiod, therefore the development of hardiness in this species should be controlled at least in part by photoperiod. Both red cedar and yellow cedar are from a more moderate climate and are both indeterminate species. The effect of photoperiod on cold hardiness development in these species is therefore probably not strong. The objectives of this study were therefore to examine: (i) the natural progression of frost hardiness, (ii) the effects of photoperiod, water stress and low temperature on hardiness, and (iii) dehardening patterns in seedlings of western red cedar (Thuja plica ta), yellow cedar (Chamaecyparis nootkatensis ) and white spruce (Ticea glauca ). Specific information on frost hardiness of particular species is important to the regeneration forester to aid in the development of appropriate nursery techniques by providing a guide to the timing of lifting and providing ways of protecting nursery stock. The information is also useful in determining limits of seed transfer between geographic areas. 3 8 3.2. MATERIALS AND METHODS Seedlings of western red cedar, yellow cedar, and white spruce were raised outdoors during the period of active growth as previously described in section 1.2. The outdoor temperature varied between 10 and 24 °C during this period. Until the end of July, photoperiod for white spruce seedlings was extended to 18 h with sodium lamps. 3.2.1. Seasonal Variations in Frost Hardiness Seedlings used to examine seasonal variations in hardiness were left outdoors to overwinter and levels of frost hardiness were determined at two to four-week intervals. Hardiness was determined using the conductivity method as described earlier (section 2.2.1). In red and yellow cedar, 0.5 cm stem segments not more than 10 cm from the tip were used, while 0.5 cm needle segments were used for white spruce. Tissues from each treatment were frozen to a range of three to five temperatures (2 to 4 "C apart) around the predicted index of injury of 50%, and LT50 estimated as previously described (section 2.2.4). The conductivity measurements were done 22+2 h after the addition of water. 3.2.2. Environmental Regulation of Hardiness For this experiment, seedlings raised outdoors were transferred to a controlled environment chamber during active growth, watered every other day and fertilized once a week with the high N fertilizer regime as described in section 1.2. The growth conditions were: 18 h photoperiod, 250 pmoles m'^ s'* photon flux density (400-700 nm), day and night temperatures of 22/15 °C, and approximately 40% relative humidity. Following five weeks at these conditions, seedlings from each species were then subjected to two photoperiod treatments: short (9 h, SP) and long (18 h, LP) for four weeks under the same light conditions as before and 22/15 °C day and night temperature. Under each 39 photoperiod treatment, half of the seedlings were stressed by watering every five days (WS) while the other half was watered every other day (NS). In the WS seedlings, pre-dawn shoot water potentials of about -0.9 MPa for spruce, and -1.2 MPa for red cedar and yellow cedar were attained before rewatering. Levels of frost hardiness were monitored after 3 and 4 weeks of treatment. Following the above (4 weeks), all the seedlings were subjected to a low temperature (7/3 °C day/night, 9 h photoperiod) treatment for eight weeks. All seedlings were watered to saturation every five days and fertilized every two weeks with the low N fertilizer regime as described in section 1.2. No water stress was induced by this watering regime. Frost hardiness was determined every week. To examine the effects of subfreezing temperature on hardening of the three species, a group of seedlings from the short photoperiod non-stressed (SPNS) seedlings were exposed to a low temperature regime of 2/-3 °C day/night temperatures, 9 h photoperiod and about 15 umoles m"^  s'* photon flux density. Cold hardiness was monitored for 21 days. Dehardening patterns were examined on seedlings which were subjected to the short photoperiod but not stressed (SPNS). The seedlings were exposed to 20/15 °C day/night temperatures and 13 h of photoperiod, following 0, 3, 5 and 9 (white spruce) and 1 and 5 (yellow and red cedar) weeks of low temperature (7/3 °C) exposure. Level of hardiness was monitored at different intervals up to a maximum of thirty-five days. 40 33. RESULTS 3.3.1. Seasonal Variations The hardening patterns of seedlings of one provenance each of red cedar, yellow cedar and white spruce under the ambient temperature and photoperiod conditions at the University of British Columbia Forest Nursery (Vancouver, British Columbia), are depicted in Figure 3.1. The hardening pattern in white spruce seedlings was quite different from those of red cedar and yellow cedar seedlings, both of which started hardening late (late October to early November) and rather slowly, at a rate of about 1.5 8 C per week. The initiation of hardening in seedlings from the two species coincided with the drop in ambient temperatures in fall. In both years (1988 and 1989), the seedlings of red cedar and yellow cedar started hardening approximately when the maximum daily temperatures fell below 15 °C and the minimum below 10 °C. Daylength at this time was already short (between 11 and 10 h). Seedlings from this yellow cedar provenance (from the northern tip of Vancouver Island) started hardening about two weeks earlier than those of red cedar from southern Vancouver Island. Mid-winter hardiness peaks of about -24 °C for red cedar and about -30 °C for yellow cedar were reached by the first week of January 1989. A second peak of about -30 °C for red cedar and -33 °C for yellow cedar were attained following an unusual episode of low temperatures in late January to early February of 1989 (Figure 3.1). Dehardening at ambient conditions in late winter and early spring in both species was slow at first followed by a fast and almost complete loss of hardiness by the beginning of April 1989. 41 Figure 3.1. Weekly maximum and minimum extreme air temperatures ( ) and seasonal changes in photoperiod ( ) at the University Forest Nursery (A), and cold hardiness (B) of red cedar (RC), yellow cedar (YC) and white spruce (WS) seedlings. Maximum level of hardiness in white spruce was below -65 °C. Week 0 corresponds to Sept 1 1988, and week 64 corresponds to Nov 23 1989. 42 White spruce seedlings, unlike seedlings of red and yellow cedar, started hardening by mid to late summer (early August in both years) when the daily maximum temperatures were 20 °C and above, and minimum temperatures of 10 °C or lower had not occurred. The daylength was around 15 h. This stage of hardening was slow however, reaching only about -12 °C by the end of summer (Figure 3.1). The rate of hardening increased slowly from early fall to a maximum of about 8 °C per week by mid fall. The maximum level of hardiness achieved was below -65 °C. This level of hardiness was achieved by mid to late December in both years (1988 and 1989). Spring dehardening was rapid, from below -60 at the end of February to about -15 °C by the end of March. 3.3.2. Environmental Regulation 3.3.2.1. Hardening Seedlings of the three species responded very differently to the photoperiod and low temperature treatments. In the red cedar seedlings, the short-photoperiod treatment at the warm temperature did not result in increased hardiness (Figure 3.2). Water stress, on the other hand, increased hardiness but by only about 1.5 °C. Exposure of red cedar seedlings to low temperature (7/3 °C), resulted in the same pattern and rate of hardening regardless of the previous photoperiod and water stress treatments. The hardiness level increased in all the treatments by about 3 °C after one week of exposure and remained unchanged or increased only slowly to the end of the third week. After this period, the seedlings commenced hardening at a fairly constant rate of about 3 °C per week reaching a maximum of about -24 °C by the 8th week of low temperature treatment. 43 Figure 3.2. Patterns of frost hardening in seedlings of red cedar under different environmental conditions. The seedlings were exposed to long, warm days (18 h photoperiod, 20/15 °C), regularly watered (JJDNS) and water stressed (LDSTR), and short warm days (9 h photoperiod, 20/15 °C), regularly watered (SDNS) and water stressed (SDSTR). The low temperature (7/3 °C, 9 h photoperiod) exposure was started at the end of the 4th week and continued for 8 weeks. 44 In the seedlings of yellow cedar, both short photoperiod at a warm temperature and water stress treatments resulted in minor increases of about 2 °C in hardiness (Figure 3.3). Differences in photoperiod pretreatment became more evident however, following exposure to low temperature (7/3 °C). The short-photoperiod-treated seedlings commenced hardening soon after exposure to low temperature whereas the Iong-photoperiod-treated seedlings had a lag phase similar to that observed in red cedar seedlings. The short-photoperiod-treated seedlings of yellow cedar had reached a peak hardiness of about -29 °C by the 8th week of exposure to low temperature whereas the long-photoperiod-treated seedlings had not reached peak hardiness by the end of the experiment Short-photoperiod (9 h) treatment at warm temperatures (20/15 °C) resulted in a substantial increase in the level of hardiness in white spruce seedlings. The short-photoperiod-treated seedlings attained a hardiness level of about -15 °C by the end of four weeks of the treatment (Figure 3.4). Water stress significantly increased hardiness of seedlings raised under the long-photoperiod conditions (to about -9 °C). Water stress had no effect on the freezing tolerance of seedlings exposed to short-photoperiod conditions. The hardening response of the white spruce seedlings to low temperature depended on the previous conditions to which the seedlings were exposed. The long-photoperiod, non-stressed seedlings responded very slowly in a manner similar to seedlings of red cedar and yellow cedar under the same conditions. The short-photoperiod-pretreated white spruce seedlings, commenced hardening at a maximum rate of about 10 °C per week more-or-less immediately upon exposure to low temperature. Maximum level of hardiness was below -65 "C by the end of the experiment in all but the long-photoperiod, non-stressed seedlings. 45 Figure 3.3. Patterns of frost hardening in seedlings of yellow cedar under different environmental conditions. The seedlings were exposed to long, warm days (18 h photoperiod, 20/15 °Q, regularly watered (LDNS) and water stressed (LDSTR), and short warm days (9 h photoperiod, 20/15 °C), regularly watered (SDNS) and water stressed (SDSTR). The low temperature (7/3 "C, 9 h photoperiod) exposure was started at the end of the 4th week and continued for 8 weeks. 46 Figure 3.4. Patterns of frost hardening in seedlings of white spruce under different environmental conditions. The seedlings were exposed to long, warm days (18 h photoperiod, 20/15 °C), regularly watered (LDNS) and water stressed (LDSTR), and short warm days (9 h photoperiod, 20/15 °C), regularly watered (SDNS) and water stressed (SDSTR). The low temperature (7/3 *C, 9 h photoperiod) exposure was started at the end of the 4th week and continued for 8 weeks. 47 Exposure to nightly subfreezing temperature (21-3 °C day and night) affected the rate of hardening in seedlings of red cedar and yellow cedar but not in seedlings of white spruce (Figure 3.5). In both red cedar and yellow cedar seedlings, the rate of hardiness development increased to about 10 °C per week within a week of exposure. Maximum hardiness was reached quickly and further exposure after the 12th day did not result in a substantial increase in frost hardiness. The rate of hardening in white spruce seedlings did not change as a result of exposure to the subfreezing temperature. It remained at a rate of about 10 °C/week throughout the period of exposure. 3.3.2.2. Dehardening Observed patterns of dehardening in seedlings of red cedar and yellow cedar were substantially different from that of white spruce seedlings. Upon exposure to warm temperatures, both red cedar and yellow cedar seedlings showed a lag period of about 5 days during which little dahardening took place (Figure 3.6). Complete loss of hardiness was achieved in about 2 weeks of exposure. Initiation of shoot elongation was evident after 10 days warming in red cedar seedlings and after 14 days in yellow cedar seedlings. In white spruce seedlings, dehardening appeared to have been influenced by the attainment of chilling requirements (Figure 3.7). Seedlings which had been exposed to short photoperiod and warm temperatures only, did not deharden even after 1 month of exposure at 16 h photoperiod at warm temperatures (20/15 °C day and night). Those which had been exposed to only two weeks of low temperature (7/3 °C) dehardened only marginally during the duration of warm temperature exposure. After five weeks of low temperature exposure however, this tendency to resist dehardening started to decline. Seedlings which had been exposed to 5 weeks of low temperature started to deharden slowly after 2 weeks of high temperature exposure. However, seedlings which had been 48 exposed to 9 weeks of low temperature started dehardening at a fast rate immediately upon high temperature exposure. This tendency to resist dehardening in white spruce seedlings was also related to the initiation of terminal bud break. In seedlings which lost their hardiness during the dehardening period, bud swell was evident usually about a week after the loss of hardiness, whereas it was not evident in the groups that did not loose their hardiness. Transfer of seedlings which had been exposed to 9 weeks of low temperature to moderate temperatures (11/9 °C day/night temperatures, 13 h photoperiod) also resulted in an immediate initiation of dehardening although the rate of dehardening declined after 10 days (Figure 3.7). Bud swell in these seedlings was evident after 25 days. In all cases where bud break was evident, complete loss of hardiness of the foliage did not take place during the period of the experiment even in seedlings dehardened at the warm temperature. Hardiness levels in the foliage of white spruce remained at about -12 °C. 4 9 1 5 9 13 17 21 25 DAYS OF EXPOSURE Figure 3.5. Hardening of red cedar (1), yellow cedar (2) and white spruce (3) seedlings exposed to subfreezing temperature (2/-3 °C). Vertical bars are 95% confidence intervals. 5 0 0 o O lO t3 Q LU 1 h -00 LU -4 --8 --12 -•16 --20 5 10 15 20 DAYS OF EXPOSURE 25 Figure 3.6. Patterns of dehardening of red cedar and yellow cedar seedlings. Red (1 and 2) and yellow (3 and 4) cedar seedlings were dehardened at 20/15 °C 13 h photoperiod following two (1 and 3) and five (2 and 4) weeks of low temperature exposure (7/3 °C day and night). Vertical bars are 95% confidence intervals. 51 Figure 3.7. Patterns of dehardening in white spruce seedlings. The seedlings were dehardened at 20/15 "C 16 h (1) and 13 h (2 to 4) photoperiod, and 11/9 °C 13 h photoperiod (5) following 0 (1), 3 (2), 5 (3) and 9 (4 and 5) weeks of low temperature exposure. Initial levels of hardiness for 4 and 5 were below -65 °C. Vertical bars are 95% confidence intervals. 52 3.4. DISCUSSION 3.4.1. Seasonal Variations The two different seasonal patterns of hardiness development between the white spruce seedlings and the two cedars, observed in this study at Vancouver, British Columbia, are probably indicative of different mechanisms of hardening induction in the three species. The early initiation of hardiness in these seedlings of white spruce indicates that initiation of their acclimation is tied to phenological development (bud set in this case). Two stages of hardening, marked by different rates, were observed in spruce seedlings. This is similar to the pattern described in many temperate woody species (Weiser 1970; Levitt 1980; Sakai and Larcher 1987). The first stage of hardening (achieved by the end of summer) was likely induced by shortening photoperiod. The photoperiod at Vancouver, at this time of the year, is sufficiently short to cause growth cessation in these first-year seedlings of white spruce. Early bud set and the accompanying development of moderate levels of hardiness at warm temperatures is a common adaptive mechanism in continental and boreal species (Sakai and Larcher 1987). Since the initiation of hardening in white spruce appears to be dependent on date of bud set (or photoperiod, for that matter), large ecotypic differentiation will exist along latitudinal and altitudinal gradients. A latitudinal differentiation in the initiation of cold hardiness related to date of bud set has been shown for Cornus stolonifera (Smithberg and Weiser 1968), Populus deltoides (Mohn and Pauley 1969), Liquidambar styraciflua (Williams and McMillan 1971), Quercus rubra (Flint 1972) and in Pseudotsuga menziesii var. glauca (Rehfeldt 1979). Recent studies with coastal and interior species and crosses 53 of British Columbia spruces have indicated a strong relationship between bud set and initiation of hardiness (Kolotelo 1991). When temperatures start declining in fall, a temperature-driven second stage of hardening is initiated in white spruce seedlings, which proceeds at a fast and fairly constant rate. The development of a high level of hardiness (<-60 °Q before any occurrence of subzero temperatures indicate that exposure to subfreezing temperatures is not necessary to achieve a high level of hardiness in white spruce. Seedlings of interior spruce from the Kootenays, British Columbia, raised under the same conditions as the seedlings in this experiment responded similarly (Kolotelo 1991). Seedlings of a provenance of white spruce from the Prince George region of British Columbia raised in the summer of 1990 were also able to achieve a high level of hardiness in the fall without exposure to subfreezing temperatures (Silim and Lavender, unpublished). Unlike white spruce seedlings, seedlings of red cedar and yellow cedar studied here did not display stages of hardening in response to photoperiod but only in response to persistent subfreezing temperatures, as in January/February 1989. Similar lack of hardening stages with shortening daylengths occurs in a wide range of provenances of both red cedar (Marilyn Cherry, pers. comm.) and yellow cedar (John Russell, pers. comm.). The mechanism of initiation of hardening in seedlings of these two species is likely temperature driven. In such species, intraspecific differentiation in cold hardiness may be either random or between climatic races (Sakai and Larcher 1987). A latitudinal differentiation based on photoperiodic differentiation would therefore be expected to be of only minor importance. Studies in progress with provenances of yellow cedar, ranging in distribution from northern California to Alaska, indicate a very weak latitudinal differentiation in the 54 initiation of hardiness in this species (John Russell, pers. comm.). Other studies, with western red cedar from coastal and interior British Columbia raised at Vancouver, also currently in progress, indicate little differentiation between the coastal and interior provenances in the initiation of hardiness, although there appears to be differences in the absolute levels of hardiness (Marilyn Cherry, pers. comm.). The pattern of hardening is the same, regardless of the geographic origin. The late initiation of hardening (when the photoperiod was already below 11 h and temperatures between 0 and 10 °C) observed in this study, may be indicative of a lack of photoperiod-involvement in frost hardiness in nature, in these two species. 3.4.2. Environmental Regulation 3.4.2.1. Hardening The results from the controlled environment studies indicate that seedlings of red cedar and yellow cedar studied here either do not have or have only a minimal ability to improve their hardiness by the short-photoperiod exposure at warm temperatures (9 h, 20/15 °C). An interior provenance of red cedar (from the Kootenays, British Columbia) and a provenance of yellow cedar from southern Vancouver Island also responded similarly (Lavender and Silim, unpublished). White spruce seedlings on the other hand, improve their hardiness significantly as a result of short-photoperiod treatment alone. The existence of a gradient of hardening response mediated by a short-photoperiod-treatment in woody plants has also been reported by Williams et al. (1972). They reported that short photoperiod had no effect on the level and initiation of cold hardiness in Pyracantha  coccinea (fire thorn). And although Weigela florida was responsive to the photoperiod treatment, it was much less responsive than Cornus stolonifera (Williams et al. 1972). Of the three species they studied, Cornus was the hardiest, followed by Weigela, and Pyracantha the least hardy. 55 The moderate level of hardiness attained here by seedlings of white spruce following a short-photoperiod treatment has also been confirmed in seedlings of Picea  engelmanni and Picea mariana (Lavender and Silim, unpublished) and is similar to observations in other temperate woody plants (Irving and Lanphear 1967,1968; van Huystee et al. 1967; Howell and Weiser 1970a; Berveas et al 1978; Ketchie 1985; Sakai and Larcher 1987; Juntilla and Kaurin 1989). Level of hardiness attained following a short photoperiod treatment at a warm temperature is dependent on the species' geographic origin and is related to the degree of absolute hardiness attained by the species. Boreal and continental woody species such as willows, birch and poplar which attain an extremely high degree of hardiness in the winter will harden extensively in response to short-photoperiod treatment, even in the absence of low temperature (Sakai and Larcher 1987). Black spruce (P. mariana) seedlings hardened to about -40° C in response to a short photoperiod (8 h) at about 22/15° C (day/night) temperatures for eight weeks (Colombo 1990). White spruce behaves in a similar manner. A recent study with a provenance of white spruce from the Prince George region of British Columbia, indicates that this species hardens to well below -60 °C, even without exposure to temperatures below 10 °C (Silim and Lavender, unpublished). The photoperiodic control mechanisms of cold hardiness initiation are not known. Phytochrome obviously plays a role as a photoreceptor, as indicated by reports that the process is reversible by R/FR exposure in Comus stolonifera, Weigela florida (Williams et a\. 1972; McKenzie et al. 1974a) and Picea mariana (D'Aoust and Hubac 1986). Although the effect of short photoperiod is related to the resulting growth cessation, not much is known about the physiological and biochemical processes that take place at this stage of hardiness. There are reports of increases in starch reserves, lipid changes and changes in water content (Williams et al. 1972; Mckenzie et al. 1974b; Levitt 1980; Sakai 56 and Larcher 1987) which may be the result of the short photoperiod exposure alone. Phytochrome induced physiological and biochemical changes in plant cells include cell membrane permeability and electrical properties (Roux 1986), induction of activity or synthesis of enzymes like nitrate reductase, phenylalanine ammonia lyase, ascorbate oxidase and glutathione reductase; inhibition of the expression of lipoxgenase activity (Schopfer 1977,1984; Hart 1988), and direct effects on gene expression (Schafter et al. 1986). It has also been suggested that phytochrome may affect plant hormone binding capacity (Hart 1988). Presumably it is these and other, as yet undocumented, changes that eventually lead to changes in hardiness in species like white spruce which respond to photoperiod. In this study, water stress resulted in only a marginal increase in hardiness in seedlings of red cedar and yellow cedar, and in white spruce under short photoperiod. The marginal effects of water stress observed could be either because the seedlings were stressed at a warm temperature or because the stress was insufficient to result in a larger increase in hardiness level. Large increases in hardiness have been reported when plants were stressed at a low temperature (Chen et al. 1975; Chen and Li 1978), and observations on two-year old white spruce seedlings indicate that the level of hardiness achieved following water stress is dependent on the stress level (Lavender and Silim, unpublished). The effect of water stress on hardiness in white spruce seedlings under long photoperiod, observed in this study, was probably more a result of growth cessation induced by the cycle of water stress rather than a direct effect of water stress on frost hardiness as observed by Blake et al. (1979) in Douglas-fir seedlings. The effect of water stress on cold hardiness is attributable to several factors: increased solute concentration (including sugars and amino acids proline and arginine), increased ABA levels, effect on 57 growth cessation and effect of hydration state of the protoplasm (Levitt 1980; Sakai and Larcher 1987). Cold hardiness is usually accompanied by a marked decrease in tissue water content (Burke et al. 1974; McKenzie et al 1974b; Cox and Levitt 1976). Sugars, proline and ABA have been shown to decrease the temperature at which protoplast membranes are damaged (Steponkus 1984,1990). Of the three factors examined in this study, the low temperature treatment was the most effective at inducing cold hardiness in the three species. However, in recent studies, Silim and Lavender (unpublished) observed that seedlings of white spruce (a provenance from Prince George, British Columbia) hardened at maximal rates at around 15 °C. Such temperatures are normally not considered to be favorable to hardening at maximal rates (Weiser 1970; Levitt 1980). It is, therefore, likely that if the white spruce seedlings in this study were maintained at the short photoperiod and warm temperatures longer than the four week period, a higher rate of hardening could have been achieved as observed by Colombo (1990) for black spruce. Two patterns of hardening response to low temperature, possibly indicating different hardening mechanisms, were recognized in this study. The first pattern had a lag phase during which little hardening took place. This response, displayed by red cedar seedlings and other seedlings kept under a long photoperiod, is a temperature driven mechanism. In this type of mechanism, the plants respond directly to the low temperature stimulus. The lag phase, in this case, was the period of metabolic adjustment in response to the low temperature exposure. The other pattern of hardening had no lag phase as displayed by short-photoperiod-treated white spruce and yellow cedar seedlings. This pattern is indicative of a short-photoperiod-induced mechanism prior to low temperature exposure. In seedlings which respond to a short photoperiod, hardening is initiated prior to the onset of winter. In effect these plants would be in an induced (or inducible) state 5 8 before any exposure to the stimuli. Thus when low temperatures are encountered the plants are already substantially hardened and may only increase their rate of hardening. The biochemical and physiological basis for these differences in response, have not been examined. Practically all the studies on the biochemical and physiological processes that take place during acclimation to low temperature have dealt with changes that occur after exposure to low temperatures only and do not address the changes that take place following a short photoperiod exposure. The biochemical and physiological changes associated with changes in cold hardiness/low temperature exposure have been a subject of much research (for reviews see: Burke et al. 1976; Kacperska-Palacz 1978; Levitt 1980; Oquist 1983; Kacperska 1985,1989; Sakai and Larcher 1987; Guy 1990b; Hallgren and Oquist 1990; Thomashaw 1990). The major changes involve dehydration, changes in membrane properties, an increase in solute concentrations (sugars, amino acids), an increase in ABA levels, an increase in systems involved in the detoxification of oxyradical species and dissipation of excess excitons, synthesis of new proteins, and adjustment of the primary processes like respiration and photosynthesis. These changes enable the plant to survive subfreezing temperatures. Seedlings of the three species examined here responded differently to the subfreezing temperature (21-3 °C). While red cedar and yellow cedar attained higher rates and new levels of maximum hardiness after exposure to subfreezing temperatures, white spruce seedlings did not require subfreezing temperatures to attain the maximum rate of hardening. The attainment of extreme levels of hardiness (below -60 °C) without exposure to subfreezing temperatures occurs in provenances of white spruce from Ontario, and in Engelmann spruce from British Columbia (Lavender and Silim, unpublished) and the Rocky Mountains in Arizona (Burr et ah 1989). In a recent study, (Silim and Lavender, unpublished), seedlings of a provenance of white spruce from the 59 Prince George region of British Columbia hardened to below -60 °C when maintained at 15/10 "C for 5 weeks following bud set. Salix dasyclados, Salix ledebouriana, Salix  sachalinsis, and Populus x euramericana attained extreme hardiness without exposure to subfreezing temperatures (Sakai 1966; Sakai and Yoshida 1968). These results confirm the suggestion by Sakai and Larcher (1987) that subzero temperatures may not always be necessary for the development of extreme levels of hardiness in very hardy species. It may, however, be important in species that do not attain very high levels of hardiness. Physiological changes associated with subfreezing temperatures generally involve cell dehydration and, changes in physical and chemical composition of cell membranes (Levitt 1980; Kacperska 1985). 3.4.2.2. Dehardening Loss of hardiness is thought to be very fast upon exposure to warm conditions (Gusta and Fowler 1976; Greer and Stanley 1985; Burr et al. 1989). Although we found this to be generally true, in all three species we observed a lag phase, during which little dehardening took place followed by a period of fast dehardening. The situation in white spruce was more complex since dehardening was apparently related to satisfaction of the chilling requirement. The degree to which dehardening is influenced by the depth of dormancy has been shown in a few angiosperm species (Irving and Lanphear 1967; Hamilton 1973; Fuchigami et al. 1982; Kobayashi et al. 1983; Ketchie 1985; Tanino et al. 1989), but similar data on coniferous species had not been available. According to the degree growth stage (°GS) model of Fuchigami et al. (1982), the rate of dehardening increases gradually when plants change from the state of maximum dormancy (which they called 270 °GS) towards the stage of spring bud break (termed 360 °GS). It should, however, be 60 noted that no direct dependence between physiological dormancy and state of hardiness necessarily exists (Lavender 1985,1991; Juntilla 1989; Kacperska 1989) since there are many species (like red cedar and western hemlock) that do not become dormant yet become hardy. The mechanism by which the physiology and biochemical processes in the whole plant are affected by the physiology of the shoot apex in species that become dormant is not known. Chilling is perceived at the shoot apex and is a property of the chilled cells only, the growth resumption signal is therefore not translocatable (Lavender and Hermann 1970; Lavender et al. 1970; Worrall 1971). The dehardening signal on the other hand is translocatable apparently from the chilled bud to the rest of the plant (Timmis and Worrall 1974). Although current thought is that plant growth regulators are involved, at the moment, very little is known about the mode of action of plant growth regulators to permit meaningful conclusions. 61 3.5. CONCLUSION Observations from this study indicate that both red cedar and yellow cedar seedlings have a less complicated mode of transition to and maintenance of the hardened state than seedlings of white spruce. Hardiness in the former two species is basically a temperature dependent process. Exposure to low temperature induces and maintains hardiness while exposure to warm temperatures results in loss of hardiness. In white spruce, hardiness is under a complex control of endogenous growth rhythms: initiation is photoperiodically regulated, while maintenance is related to the depth of dormancy in the apical bud. Conditions that lead to bud set in this species, while resulting in the development of moderate levels of hardiness, also predispose the plants to respond immediately to low temperature exposure. Continuous exposure to low temperature results in the satisfaction of the chilling requirements which paradoxically permits the plants to deharden. As a result of these two different strategies, the period of highest risk to freezing damage will not be the same in the three species. White spruce would have the highest risk from mid-winter to spring when the chilling requirements have been met. Exposure to moderate temperatures at this time would result in a substantial loss of hardiness which may not be easily regained. Red cedar and yellow cedar on the other hand would probably be at less risk throughout the year except where severe early frosts are a problem. Finally, it is evident that a blackout (short photoperiod) treatment as a nursery cultural tool in limiting height growth and improving cold hardiness may be of value in white spruce seedlings but not of much help in seedlings of red cedar and yellow cedar. 62 CHAPTER 4. EFFECT OF MEFLUIDIDE ON WATER RELATIONS, NET PHOTOSYNTHESIS, COLD HARDINESS AND ABA LEVELS ABSTRACT The short term effects of the plant growth retardant, mefluidide, on stomatal conductance, net photosynthesis, water potential, cold hardiness and tissue abscisic acid (ABA) levels were investigated in seedlings of western red cedar (Thuia plicata Donn), yellow cedar (Chamaecyparis nootkatensis D. Don) and white spruce (Picea glauca (Moench) Voss.). Mefluidide was applied once as a root drench at concentrations of 0, 0.1 and 0.4 mg 1"*. The seedlings were stressed by withholding water for a period of up to seven days and then regularly watered up to the 30th day. Stomatal conductance of the mefluidide treated seedlings decreased significantly and remained lower than the control seedlings throughout the stress period. The extent of stomatal limitation was dependent on the concentration of mefluidide applied. As a result of the decreased conductance, shoot water potentials in mefluidide treated seedlings remained higher throughout the stress period. There was a decrease in net photosynthesis which was predominantly caused by stomatal limitations. Mefluidide treatment resulted in an increased synthesis of ABA in shoots of seedlings of the three species. The levels of tissue ABA like the other variables, were also dependent on mefluidide concentration. The changes in ABA levels did not result in any significant changes in cold hardiness of the three species but may have been responsible for the effects on stomatal conductance and water relations. 6 3 4.1. I N T R O D U C T I O N Over the last three decades, much effort has been devoted in search of chemicals which could mitigate low temperature injury to crop plants. Most of the work up to 1981, has been reviewed by Howell and Dennis (1981), Levitt (1972,1980) and Proebsting (1978). The chemicals which have been investigated can be classified either as cryprotectants or growth regulators. Cryoprotectants can improve survival of tissues exposed to low temperatures without affecting growth and regardless of the developmental stage of the plant (Howell and Dennis 1981). Growth regulators, on the other hand, improve hardiness through their possible effects on plant growth and development. In contrast to animal tissues, little information exists on the use of cryoprotectants in plant tissues. The limited available information indicates that response is variable. Application of dimethylsulfoxide (DMSO), ethylene glycol, glycerol and polyvinylpyrrolidone were reported to increase hardiness in apple twigs and pear trees, but had no effect on grapevine, Citrus and various herbaceous species (Marlageon 1968, 1969; Ketchie et al. 1973; Burns 1974; Kentzer and Murren 1976). Most of the effort at improving low temperature hardiness in plants by chemical means has centered on the use of natural and artificial plant growth regulators (PGRs). The natural plant growth regulator, abscisic acid (ABA), has been the most extensively examined due to recognition of its involvement in numerous stress response including low temperature (Morgan 1990). However, due to its high cost, instability in light and variable responses when applied to whole plants (Pieniazeck and Holubawicz 1973; Lavender and Silim, unpublished), it has found little application. Much attention has 64 therefore been directed towards the use of artificial plant growth regulators, particularly inhibitors. Some of the earliest attempts at improving plant responses to low temperature with artificial PGRs utilized chemicals such as Alar [succinic acid 2,2-dimethyl hydrazide] (Granger and Hugue 1967; Marlageon 1968,1969; Freeman and Came 1970; Paquin et al. 1976), CCC [2-chloroethyl trimethylyammonium chloride] (Michienwicz and Kantzer 1965; Michienwicz et al. 1965; Marlageon 1968,1969; Irving 1969a; Chen and Li 1976), and AMO-1618 [4-hydroxy-5-isopropyl-2-methylphenyl trimethylammonium chloride 1-piperidine carboxylate] (Irving and Lanphear 1968; Irving 1969). Results were variable; the reported successes were mostly with non-woody species with much less success reported in woody species (see Howell and Dennis 1981; Levitt 1980; Proebsting 1978). More recently, attempts at improving general stress resistance in plants have focused on the more recently available PGRs such as triadimefon, paclobutrazol, thidiazuran, ancymidol and mefluidide which retard growth at extremely low concentrations. Mefluidide, paclobutrazol, triadimefon and ancymidol are plant growth retardants (Buchenauer and Grossman 1977; Buchenauer and Rohner 1981; Quinlar and Richardson 1983; Raese 1983; Tautvydas and Hargroder 1985; Wang et al. 1985; Borochov and Shahar 1989) whereas thidiazuran is an effective defoliant of cotton plants (Arndt et al. 1976; Suttle 1984). These plant growth retardants cause shifts in assimilate partitioning in favour of root growth, reduce leaf area and shoot growth, and increase chlorophyll and carotenoid contents in treated plants. They also confer protection from water deficits and low temperature exposure in a number of plant species (Howell and Denis 1981; Raese 1983; 65 Tseng and Li 1984; Tseng et al. 1984; Fletcher and Hofstra 1985; Lee et al. 1985; Wang 1985; Asare-Boamah and Fletcher 1986; Asare-Boamah et al. 1986; Zhang et al. 1987; Coleman and Estabrooks 1988; Li 1989). Treatment with mefluidide and triadimefon result in elevated levels of ABA in plant tissues (Asare-Boamah et al. 1986; Zhang et al. 1987). It has been suggested that these elevated levels of ABA are associated with subsequent stress resistance. Paclobutrazol and triadimefon specifically block the biosynthesis of gibberellic acids (GAs) (Buchenauer and Grossman 1977; Buchenauer and Rohner 1981; Izumi et al. 1985; Wang et al. 1985) whereas thiadiazuran induces ethylene production in plant tissues (Suttle 1984). There is also evidence that treatment with mefluidide affects synthesis of sugars, proteins, RNA, DNA, and membrane fatty acids in treated plants (Tautvydas and Hargroder 1985) although the significance of these changes is not known. To date no information exists on the use and effects of these plant growth regulators on conifer seedling physiology. It is possible that they may be useful in modifying seedling responses to stress, particularly low temperature and water deficits. Initial investigations indicated that growth retardation of some of the growth regulators (e.g., paclobutrazol) may persist for a long time rendering them impractical for the protection of forest seedlings. Mefluidide (N-[2,4,dimethyl-5-[[triflouromethyl)sulfonyl]-amino]phenyl]acetamide), the active ingredient in Embark, on the other hand has a very short half-life in the soil (about 2 days) and does not affect growth at extremely low concentrations. Thus, it may provide a means of improving conifer seedling stress resistance without the undesirable effects of growth retardation. 66 The objectives of this study were therefore to examine the effects of the plant growth retardant mefluidide on: (i) cold hardiness, (ii) stomatal conductance and net photosynthesis, (iii) levels of abscisic acid, and (iv) shoot water potential in seedlings of western red cedar (Thuja plicata), yellow cedar (Chamaecyparis nootkatensis) and white spruce (Picea glauca). 67 4.2. MATERIALS AND METHODS Seedlings of western red cedar, yellow cedar and white spruce were raised outdoors as previously described. During the period of active growth they were transferred to a controlled environment chamber set at 22/15 °C day and night temperature, 18 h photoperiod at 250 umoles m"^ s"* photon flux density (400-700 nm). Following 5 weeks under the above conditions, the following treatments were given: 0, 0.1 and 0.4 mg 1"* of mefluidide (3M Co., Agricultural Products Division. St. Paul Minnesota), applied once as a soil drench. Following the application, half the control seedlings (0 mg l'*) were watered every other day while the other half and the treatments were stressed by withholding water for 5 days for red cedar and yellow cedar seedlings and 7 days for white spruce seedlings. At the end of the 5th day (red and yellow cedar) and 7th day (white spruce) the seedlings were re-watered, fertilized and left in the controlled environment chamber for 4 more weeks. Mid-day shoot water potential, net CO2 fixation, stomatal conductance and cold hardiness were determined on 6 seedlings before the treatment, then at 1, 3, 5, 7, 9, 20 and 30 days after the treatment. For ABA analysis, 2 to 4 g of shoots from 12 seedlings were harvested in triplicates 3 h into the daily photoperiod and immediately frozen in liquid nitrogen and stored in a deep freezer (-75 to -85 °Q. Cold hardiness was determined by the electrolyte conductivity method as previously described. Water potential was determined using the pressure bomb. Stomatal conductance and net CO2 fixation were determined with a LI 6200 Portable Photosynthesis System (LI-COR Inc., Lincoln, Nebraska) at about 650 umoles m'^ s"* photon flux density (PFD) PAR, illuminated by a slide projector. 68 4.2.1. Extraction, Purification and Estimation of ABA Two to four grams of tissues were homogenized in cold 80% methanol containing 1 mM diethyldithiocarbamate as an antioxidant. The tissues were extracted overnight on a shaker at 4 °C, filtered and the residue re-extracted for 3 h at room temperature and refiltered. The filtrates were combined and the methanol was evaporated leaving the aqueous phase. The aqueous phase was adjusted to pH 2.8, frozen in a deep freeze then rethawed. The extract was centrifuged at 1000 g to precipitate proteins and other particulate material. The supernatant was partitioned three times against ether. The ether phase was retained and evaporated to dryness. The residue was dissolved in methanol, transferred to vials and dried under nitrogen and stored in a deep freezer (-75 to 85 8C) until further purification. The above extract was dissolved in 1 ml HPLC-grade methanol (100%), and an aliquot removed and made up to 1 ml with double-distilled filtered water to give a final methanol content of not more than 5% and less than 0.5 g fresh weight (f.wt) equivalent of plant tissue. This was centrifuged in a microfuge then filtered through 0.45 pm Durapore (Millipore, MA, USA) filters. Two hundred pi of the above extract were loaded on to a 0.25 ml bed volume immuno-affinity column at room temperature and the extract allowed to flow through the column at a rate of less than 0.1 ml/min. The column bed was washed with 50 ml of filtered double-distilled water. The ABA retained on the column was eluted with 20 ml of HPLC-grade methanol, evaporated to dryness and redissolved in methanol and transferred to vials. The purified extracts were dried under nitrogen and stored in a deep freezer until analyzed. The contents of the vials were dissolved in HPLC-grade methanol 69 and the ABA content estimated by analysis on a Hewlett Packard HP1090 liquid chromatograph at 254 nm. The immunoaffinity columns used were prepared with monoclonal antibodies according to Kannangara et al. (1989). The capacity of the columns used was 27 pg ABA ml"l bed volume. This capacity is far in excess of the amount of ABA in the plant extract loaded onto the immunoaffinity column. The recovery of cis, trans (+) ABA from the column was 96+3%. The overall recovery of the extraction and purification method was determined by the addition of an internal standard prior to the homogenization, and this was found to be about 90% (Table 4.1). Table 4.1. Recovery of ABA by the extraction procedure. One gram f.wt of red cedar leaves was used and 100 ng of (+)ABA standard was added prior to the extraction. Means (SD) of four replicates. Amount of ABA ng (SD) Detected Calculated Standard only 90.2(4.8) Sample only 123.3(13.8) 137.0(15.5) Sample plus standard 212.3(10.5) 236.8(13.3) * - based on 90% recovery. 70 4.2.2. Data Analysis For each date of measurement, a single factor analysis of variance for a completely randomized design was performed on all variables (except cold hardiness) for each species, using SYSTAT (Wilkinson 1989). All the treatment effects were presumed to be fixed. The means of variables with significant F values were compared using the Neuman-Kuels test. 71 43. RESULTS Treatment with mefluidide affected a number of physiological variables in seedlings of all three species. Water potential, stomatal conductance and ABA levels were the most affected. Cold hardiness was only marginally affected however, whereas net photosynthesis appeared to have been only indirectly affected. There was a significant (p<0.05) decrease in stomatal conductance 24 h after the application of mefluidide in seedlings of all three species (Figures 4.1, 4.2 and 4.3). The higher mefluidide concentration resulted in the larger decrease in stomatal conductance. Red cedar and yellow cedar seedlings were more affected than white spruce seedlings. The pattern was maintained up to the 5th day of stress in red and yellow cedar and the 7th day for white spruce. The changes in stomatal conductance of mefluidide treated seedlings were accompanied by an increase in water potential (Figures 4.1, 4.2 and 4.3). With increasing water stress (day 3 onwards), mefluidide treated seedlings had consistently lower stomatal conductance and higher shoot water potentials relative to the controls, however, the effect of mefluidide was only. Upon rewatering the seedlings, differences generally disappeared although there was initially an increase in stomatal conductance. The stomatal conductance in seedlings treated with the higher mefluidide (0.4 mg I'*) concentration increased significantly (p<0.05) above the rest in all the three species, before leveling off. 72 Q I i i i | i i i | i i i | i i i i i i i i i i i _<|£ I I I I 1— 1 • • • 1 • • • 1 • • • 1 • • • 2 6 10 14 18 DAYS SINCE TREATMENT Figure 4.1. Effects of mefluidide on net C C h fixation (A), stomatal conductance (B) and shoot water potential (C) in seedlings of redcedar. The seedlings were re-watered on the 5th day. (Means of six measurements). The treatments are: control watered ( ), control water stressed ( ), 0.1 mg I"* mefluidide water stressed ( ) and 0.4 mg I"* mefluidide water stressed ( ). Vertical lines are SE of the means. 73 Figure 4.2. Effects of mefluidide on net CO? fixation (A), stomatal conductance (B) and shoot water potential (C) in seedlings of yellow cedar. The seedlings were re-watered on the 5th day. (Means of six measurements). The treatments are: control watered ( ), control water stressed ( ), 0.1 mg 1"* mefluidide water stressed ( ) and 0.4 mg l " 1 mefluidide water stressed ( ). Vertical lines are SE of the means. 74 A, 8 1 • • ' 1 • • ' e 10 u DAYS SINCE TREATMENT 18 22 Figure 4.3. Effects of mefluidide on net CO2 fixation (A), stomatal conductance (B) and shoot water potential (C) in seedlings of white spruce. The seedlings were re-watered on the 7th day. (Means of six measurements). The treatments are: control watered ( ), control water stressed ( ), 0.1 mg 1"* mefluidide water stressed ( ) and 0.4 mg l " 1 mefluidide water stressed ( ). Vertical lines are SE of the means. 75 In all three species, mefluidide at the concentrations used did not affect net CO2 fixation until the third day for red and yellow cedar and the 5th day for white spruce, when stomatal limitation became significant (Figures 4.1, 4.2 and 4.3). Net CO2 fixation significantly (p<0.05) declined at this point in seedlings treated with the higher concentration of mefluidide. By the 5th day in red and yellow cedar and 7th day in white spruce however, there were no significant differences in net photosynthesis between the stressed controls and the mefluidide treated seedlings. A high level of water stress had developed by this time (see water potentials) leading to a very reduced stomatal conductance and CO2 fixation. Upon rewatering, however, there was an immediate increase in stomatal conductance accompanied by a stimulation of net photosynthesis in all three species, although the effect was greater in red and yellow cedar than in white spruce. This too, leveled off after a few days. Soon after the application of mefluidide, the ratio to internal over external CO2 (Ci/Ca) became significantly (p<0.05) lower in the mefluidide treated seedlings than in the controls (Table 4.2). This pattern was maintained throughout the stress period. The higher concentration of mefluidide generally resulted in lower Ci/Ca. In all treatments, Ci/Ca first decreased with increasing water stress but then increased when the water deficit became intense (day 5 for red and yellow cedar and day 7 for white spruce). Figure 4.4 shows the effect of mefluidide on stomatal limitations relative to the controls. It is evident that stomatal limitation was highest on the third day after the treatment in all three species. 76 To verify whether mefluidide or ABA had any immediate nonstomatal effect on net CO2 fixation, a short term experiment was performed using red cedar seedlings. Needle sections (0.5 mm) were vacuum infiltrated with mefluidide and ABA and incubated for 30 min, then photosynthetic oxygen evolution measured using an oxygen electrode. No short term-effect of mefluidide on photosynthetic oxygen evolution was observed (Table 4.3). Neither was there a short-term effect of addition of 2.64 mg 1"* of (+)ABA. Table 4.2. Effect of mefluidide on Ci/Ca . Means of six measurements. Day Control2 0.1 mgl"1 0.4 mgl Red Cedar Ci/Ca (standard deviation) 1 0.833(0.015)a 0.783(0.014)b 0.715(0.025)c 3 0.755(0.018)a 0.681(0.032)b 0.648(0.044)b 5 0.908(0.026)a 0.856(0.041)b 0.877(0.023)b 7 0.816(0.010)a 0.843(0.005)b 0.851(0.009)b 20 0.820(0.010)a 0.818(0.010)3 0.825(0.012)3 Yellow Cedar 1 0.844(0.008)a 0.764(0.030)b 0.744(0.018)b 3 0.765(0.033)a 0.676(0.039)b 0.631(0.070)b 5 0.941(0.008)a 0.887(0.009)b 0.889(0.009)b 7 0.832(0.013)a 0.839(0.009)3 0.845(0.018)3 20 0.827(0.005)a 0.827(0.012)3 0.824(0.010)3 White Spruce 1 0.838(0.009)3 0.785(0.008)b 0.772(0.008)c 3 0.793(0.016)3 0.731(0.015)b 0.714(0.025)b 5 0.775(0.016)a 0.742(0.020)b 0.725(0.019)c 7 0.860(0.014)a 0.832(0.026)3 0.799(0.024)b 9 0.823(0.009)3 0.846(0.008)b 0.844(0.009)b 20 0.820(0.010)3 0.826(0.010)3 0.829(0.009)3 1 - means in rows with the same letter are not significantly different (p<0.05) 2 - stressed control. 77 Table 4.3. Short term effect of mefluidide (O^mg l"1) and ABA (2.64 mg l"1) on photosynthesis. Means of four measurements. Control (SD) Mefluidide (SD) ABA (SD) C>2 evolution (nmoles mg' - 1 f.wt h"1). 14.448(1.294)a 14.835(1.583)a 15.826(2.229)a * - means with the same letters are not significantly different (p<0.05). Abscisic acid accumulation in tissues of seedlings of all three species was significantly (p<0.05) stimulated by the mefluidide treatment (Figures 4.5, 4.6 3nd 4.7). The increase in ABA levels occurred within 24 h of application of mefluidide and continued until soon after rewatering (day 5 for red and yellow cedar and day 7 for white spruce). The higher concentration of mefluidide resulted in a greater increase in tissue ABA. Since water stress resulted in increased accumulation of ABA, differences in tissue ABA between the control and the mefluidide treated seedlings became greater if we considered that the control seedlings were more stressed (as indicated by lower water potentials) than the mefluidide treated seedlings and yet had lower ABA levels. 78 Figure 4.4. Effect of mefluidide treatment on Ci/Ca relative to the control treatment The graphs represent red cedar (A), yellow cedar (B) and white spruce (C) seedlings. 7 9 8000 i i i — i i i i i i i i i i i i i i i i i i i 7000 6000 % 5000 •D a> 4000 < § 3000 2000 1000 0 I I I I I I JL • • ' • ® 0.4 mg/I © 0.1 mg/I • stressed O watered 2 6 10 14 18 DAYS SINCE TREATMENT 22 Figure 4.5. Effect of mefluidide on levels of ABA in shoots of red cedar seedlings. The treatments are: control watered, control water stressed, 0.1 mg 1"* and 0.4 mg 1~* mefluidide both water stressed. The seedlings were re-watered on the 5th day. Vertical lines are SE of the means. (Means of three replicates). 8 0 7000 I ' i i i i i i 1 1 1 i • • i 6000 5000 ™ 4000 cp < 3000 CD < 2000 1000 ' • • • ' 1 • » • 1 • * • ® 0.4 mg/I © 0.1 mg/I • stressed O watered 2 6 10 14 18 DAYS SINCE TREATMENT 22 Figure 4.6. Effect of mefluidide on levels of ABA in shoots of yellow cedar seedlings. The treatments are: control watered, control water stressed, 0.1 mg 1"* and 0.4 mg 1^ mefluidide both water stressed. The seedlings were re-watered on the 5th day. Vertical lines are SE of the means. (Means of three replicates). 81 5000 4000 -I 3000 CD C S 2000 < 1000 • 04 mg/l © 0.1 mg/l • stressed O watered 2 6 10 14 18 DAYS SINCE TREATMENT 22 Figure 4.7. Effect of mefluidide on levels of ABA in shoots of white spruce seedlings. The treatments are: control watered, control water stressed, 0.1 mg 1"* and 0.4 mg r 1 mefluidide both water stressed. The seedlings were re-watered on the 7th day. Vertical lines are SE of the means. (Means of three replicates). 82 In seedlings of all the three species, mefluidide treatment produced a negligible increase in cold hardiness (Figure 4.8). In red and yellow cedar seedlings, there was a transitory but non-significant increase (of about 1 °Q in cold hardiness which was lost soon after watering. In white spruce seedlings however, there was a long lasting effect on cold hardiness in all water-stressed seedlings regardless of the mefluidide application. The mefluidide concentrations used in the experiment did not have any effect on growth of the seedlings after the 30 day period. However concentrations over 1 mg 1"* when applied during active growth resulted in leaf and shoot tip necrosis within 10 days. This was particularly evident in white spruce seedlings. When applied to white spruce seedlings during the dormant phase, concentrations of up to 30 mg 1"^  had no effect on subsequent growth the following season. It was also observed that mefluidide application resulted in a leakage of ions out of the tissues (as measured by conductivity). This phenomenon was concentration dependent (Table 4.4); the lowest concentration (0.1 mg 1"*) had no effect while concentrations of 1 mg 1"* or more resulted in massive leakage. Over 50% of total electrolyte concentration in the tissues leaked out when 5 mg 1"^  of mefluidide was used. Table 4.4. Effect of mefluidide concentration on ion leakage from needles of white spruce. Conductivities were measured three days after application. Means of six measurements with SD in parenthesis. Mefluidide concentration % of ions leaked (mg I'1) 0 19.8(3.8) 0.05 16.9(3.1) 0.1 20.1(4.2) 0.5 21.3(4.1) 1.0 35.6(3.2) 3.0 40.2(4.5) 5.0 65.2(5.0) 84 Figure 4.8. Effect of mefluidide application on estimated LT50 of red cedar (A), yellow cedar (B) and white spruce (C) seedlings. The treatments are: control watered ( ), control water stressed ( ), 0.1 mg I -1 ( ) and 0.4 mg I - 1 ( ) mefluidide both stressed. The cedars were re-watered on the 5th day while spruce was re-watered on the 7th day. Vertical lines are 95% confidence intervals. 85 4.4. DISCUSSION The most dramatic effect of mefluidide observed in this study was on the water relations of seedlings of the three conifers. There was a decreased stomatal conductance accompanied by an increased water potential. Water potential of a plant is influenced by changes in the rate of water loss induced by changes in stomatal conductance. A decrease in stomatal conductance caused by factors other than drought, will therefore result in increased water potential. The effects of mefluidide on water relations observed in this study, are similar to those of Fletcher and Hofstra (1985) and Asare-Boamah et al. (1986) who reported a decrease in transpiration in bean plants treated with triadimefon; and Tseng and Li (1984), Tseng et al. (1984) and Zhang et al. (1987) who reported that mefluidide positively affected the water potentials of chilled corn and rice plants. Although the latter workers did not measure stomatal conductance, the observed positive effect of mefluidide on treated plants was likely the result of stomatal closure. One of the mechanisms of low temperature injury in chilling sensitive plants involves excessive water loss as a result of loss of stomatal function (Levitt 1980). Ordinarily, partial stomatal closure during the day-time is due to either low leaf water status or high evaporative demand. This leads to changes in the turgor balance between the guard cells and the surrounding epidermal cells (Bradford and Hsiao 1982). By an as yet unknown detection mechanism believed to involve cell volume changes (Dale and Sutcliffe 1986), the guard cells lose solutes (predominantly K + ) to the apoplast followed by water, leading to stomatal closure (Bradford and Hsiao 1982; Assman and Zeiger 1987). These changes in stomatal aperture are also believed to involve endogenous ABA (Cowan et al. 1982; Davies and Mansfield 1983; Dorfling and Tietze 1985; Raschke and Hedrich 1985; Davies 1986; Mansfield 1986; Raschke 1987; Raschke and Patzke 1988; Zeevart and Creelman 1988; Creelman 1989; Mansfield et al. 1990), 8 6 and under water stress ABA is believed to promote closure (Farquhar and Sharkey 1982; Davies and Mansfield 1983; Downton et al. 1988a,b). Asare-Boamah et al. (1986) and Li and co-workers (Zhang et al. 1986,1987; Li et al. 1989; Li 1989) postulated that observed changes in transpiration and water potential in plants treated with triadimefon and mefluidide were the result of treatment-induced increase in tissue ABA. It is likely that the decreased conductance upon mefluidide application observed in this study was primarily the result of ABA accumulation in the tissues. Although the specific mechanism by which ABA causes stomatal closure is not entirely known, it is believed to be through its effects on membrane properties (MacRobbie 1981a; Zeiger 1983; Mansfield 1986). This involves the efflux of K + and CP (MacRobbie 1981b, 1989; Raschke 1987; Zeevart and Creelman 1988; Mansfield et al. 1990) from guard cells and may involve C a 2 + as a second messenger (Schwartz 1985; De Silvia et al. 1985,1986; Mansfield et al. 1990). There is also the possibility that the observed increase in tissue ABA may be secondary. Mefluidide by itself may have directly caused stomatal closure possibly through changes in membrane permeabilities. If the observed changes in ion leakage are a primary non-phytotoxic effect of mefluidide, it is possible that this in itself could trigger stomatal closure. In this case, the observed increase in tissue ABA levels could then be indicative of a stress situation (which resulted in stomatal closure) rather than the primary cause of stomatal closure. The reduced net assimilation rate in the seedlings soon after the addition of mefluidide (before water stress set in) was probably predominantly the result of stomatal limitation. When stomatal conductance is reduced, CO2 assimilation tends to decrease because CO2 is progressively reduced at the carboxylating site (Farquhar and von 87 Caemmerer 1982; Farquhar and Sharkey 1982; Schulze and Hall 1982). As a result of this, the ratio of internal to external CO2 (Ci/Ca) should decrease with decreasing assimilation rate (Farquhar and Sharkey 1982). Results from this study indicate this was the case before severe water stress set in. Photosynthetic O2 evolution indicated further that at least on a short term basis neither the mefluidide nor applied ABA had a nonstomatal effect on photosynthesis. The general increase in Ci/Ca with reduced assimilation rate as the water stress became more severe (days 5 and 7 for red and yellow cedar and white spruce, respectively) indicates that a reduction in mesophyll capacity for assimilation had occurred at this stage (Farquhar and Sharkey 1982). The increased stomatal conductance and stimulation of photosynthesis following rewatering may have been due to osmotic adjustment as a result of mefluidide treatment and water stress. Although the stressed controls also increased, the increase was greater in seedlings treated with mefluidide. Osmotic adjustment involves increased concentration of solutes in cells leading to a decrease in osmotic potential. This may then increase water uptake upon rewatering leading to an increase in water potential. The increased water potential would permit a higher stomatal conductance resulting in increased Ci and thus stimulating CO2 fixation. Mefluidide increases the level of sugar in sugarcane (Tautvydas and Hargroader 1985), and it may have therefore stimulated the synthesis of sugars and amino acids in these seedlings. It is, however, also possible that mefluidide may have a transitory stimulation effect on photosynthesis which takes place sometime after application. Similar to the observations of Zhang et al. (1986,1987) with mefluidide I also observed an increase in tissue ABA with the mefluidide treatment. The exact mechanism by which this happens is not clear, although it definitely involves de novo synthesis. The most common trigger of ABA biosynthesis is stress in an, as yet, unknown way. It is 88 possible that mefluidide affects the plants at the same site (probably on the membranes) that triggers ABA accumulation under other stresses. No significant changes in cold hardiness were observed in seedlings treated with mefluidide beyond that expected as a result of osmotic adjustment during droughting. All the reported cases of improved low temperature tolerance by growth retardants in the literature (Asare-Boamah and Fletcher et al. 1986; Tseng and Li 1984; Zhang et al. 1987) are on chilling-sensitive species only and not freezing-tolerant species. Since one of the major causes of low temperature injury in chill-sensitive species is excessive water loss as a result of stomatal dysfunction (Levitt 1980), the retardants may mitigate the effects of low temperatures in these species by reducing water loss. In species which develop hardiness such as the ones studied here, damage by freezing temperature does not involve excessive water loss. There may be, however, osmotic effects of mefluidide which may affect the hardiness level of tissues through colligative effects in some minor way. This may result from ABA accumulation or a direct effect of mefluidide as described before. Although ABA seems to be involved in hardiness development (Lalk and Dorfling 1985; Reaney and Gusta 1987; Reaney et al. 1989; Lavender and Silim, unpublished) and may indeed serve as a signal triggering processes responsible for the increase of freezing resistance (Borman and Janson 1980; Farkas et al. 1985; Dorfling and Askman 1989; Perras and Sarhan 1989; Dorfling et al. 1990), the increased ABA accumulation observed in mefluidide treated seedlings in this study did not result in measurable increase in freezing resistance to freezing temperatures. It is possible that the seedlings were not in a physiologically inductive state and therefore did not respond to the increased ABA levels. 89 The effect of other growth inhibitors such as paclobutrazol, triadimefon and ancymidol have been attributed to their role in blocking GA biosynthesis. Although the mechanism of action of mefluidide on the molecular level is not known, it seems unlikely to depend on the inhibition of GA biosynthesis since its effects are not reversed by the addition of GA or any other plant hormone (Tautvydas and Hargroader 1985). Furthermore, although mefluidide does affect cell elongation, its effects on growth retardation are predominantly the result of a reduction in cell division. Its mode of action must therefore involve either additional or completely different biochemical events. The reported inhibition of GA biosynthesis (Wilkinson 1982) may, therefore, have very little to do with the mode of action of mefluidide. An overall change in some function of the plasma membranes appears to be a major effect of mefluidide. Changes in membrane leakiness observed in this study may be indicative of that. 90 4.5. CONCLUSION One of the objectives of this study was to attempt to improve cold hardiness of conifer seedlings using mefluidide. The results indicate that mefluidide has very little effect on hardiness in the three species. It is, however, possible that a more detailed study throughout the annual growth cycle may indicate a period of effectiveness. The biggest effect of mefluidide was on water status and photosynthesis. Mefluidide affects water relations in the three species by its effects on stomatal conductance. It may therefore be possible to use mefluidide in either protecting seedlings from transplanting shock, or to condition seedlings to withstand water deficits (i.e., induce osmotic adjustment) without exposure to water stress. This would enable the seedlings to continue CO2 assimilation without excessive water loss. At the same time, the maintenance of positive turgor (as a result of high water potential and osmotic regulation) would ensure continued growth and maintenance of biochemical processes. Further research is needed to determine the correct dosages and frequency of application. ' 91 C H A P T E R 5. T H E RELAT IONSHIP B E T W E E N NON-PROTEIN TH IOLS A N D F R E E Z I N G RES ISTANCE. A B S T R A C T The relationship between free thiols and freezing resistance was examined in foliage of seedlings of western red cedar flhuja plicata Donri), yellow cedar (Chamaecyparis nootkatensis D. Don) and white spruce (Picea glauca (Moench) Voss.). Exposure to low temperature (7/3 °C, 9 h photoperiod, 250 umoles m' 2 s"* photon flux density, 400-700 nm) resulted in an increase in free thiols which was accompanied by increased hardiness in all three species. The increased thiol content was largely the result of increased levels of reduced glutathione (GSH) in the plant tissues. The increase in GSH was higher in red and yellow cedar than in white spruce seedlings. Short photoperiod at a warm growth temperature (9 h, 20/15 °C and 250 umoles m"2 s"* photon flux density, 400-700 nm) did not result in increased levels of thiols or glutathione in all three species yet did increase hardiness in seedlings of white spruce. Root application of 50 pM buthionine sulfoximine (BSO) lowered tissue glutathione by up to 50% but did not significantly affect resistance to freezing. Application of 50 uM dichlormid (N,N-diaIlyl-2,2-dichloroacetamide) increased glutathione levels by about 100% but did not affect cold hardiness. Water stress marginally increased both tissue glutathione and hardiness levels in all three species. Accordingly, there appears to be no relationship between glutathione and freezing resistance. Glutathione accumulation under hardening conditions appears to be the result of low temperature exposure. Possible causes of GSH accumulation and its physiological implications are discussed. 92 5.1. INTRODUCTION Higher plant cells contain considerable amounts of free, low molecular weight thiols, and glutathione (y-L-glutamyl-L-cysteinylglycine) is the major component of this fraction (Rennenberg 1982,1988; Meister 1984,1988; Alscher 1989). Glutathione biosynthesis in green plants is formed by the same ATP-dependent two-step process as in bacterial and animal cells (Rennenberg 1982,1988; Meister 1981,1983,1988; Meister and Anderson 1983). In the first step, y-glutamylcysteine is formed from glutamate and cysteine in a process catalyzed by y-glutamylcysteine synthetase (EC 6.3.2.2). Glutathione is then synthesized from y-glutamylcysteine and glycine in a step catalyzed by glutathione synthetase (EC 6.3.2.3). Synthesis appears to take place in both the chloroplasts and the cytoplasm (Rennenberg 1988). The pathway of glutathione degradation in plant cells, which constitutes an essential process in sulfur nutrition, has not been fully elaborated (Rennenberg 1982, 1988). Available information suggests that degradation in plant cells follows a different pathway from that in animal and bacterial cells (Rennenberg 1988); the first degradation step involves the removal of the glycine moiety by GSH-carboxypeptidase. The remaining y-glutamylcysteine may then be further degraded by y-glutamylcyclotransferase (EC 2.3.2.4) to give cysteine and 5-oxoproline, and the 5-oxoproline is then transformed to glutamate by 5-oxoprolinase (Rennenberg 1988). Glutathione degradation appears to take place in the cytoplasm only. Cellular glutathione like other thiols, exists in various chemical forms; as a reduced tripeptide (GSH), as an oxidized form in which a disulfide bond joins two molecules forming a dimer (GSSG), and as a mixed disulfide formed by the interaction of GSH with protein or other low molecular weight compounds. In many plant cells, 9 3 GSH is the predominant form while GSSG and the other forms constitute only a small proportion of the total glutathione present. Glutathione concentration in cells ranges from 0.5 to 10 mM (Rennenberg 1982,1988). In plant cells, glutathione plays an important role in the detoxification of toxic oxygen species, hydrogen peroxide, pesticides, air pollutants, and heavy metal ions (Halliwell 1982,1984; Law et al. 1983; Hossain and Asada 1984; Smith et al. 1984; Rennenberg 1988; Alscher 1989). It also functions as a long distance transport and storage form of reduced sulfur in plants (Rennenberg 1982,1984,1988). In conjunction with ascorbate, glutathione detoxifies H2O2 in the chloroplasts (Foyer and Halliwell 1976; Rennenberg 1982,1988; Mahan and Burke 1987; Alscher 1989) in a process catalyzed by ascorbate peroxidase, glutathione dehydrogenase and glutathione reductase. A similar role is known to take place in the cytoplasm (Smith 1985; Smith et al. 1985; Alscher 1989). In doing so, glutathione protects labile macromolecules against attack by reactive oxygen species. Many plants form conjugates of glutathione with a broad range of exogenous electrophilic substances such as herbicides (Rennenberg 1988; Dean et al. 1990). Thus glutathione detoxifies atrazine, paraquat, chloroacetamides, fluorodifen and thiocarbamates (Lay and Casida 1976; Lay et al. 1976; Carringer et al. 1978; Law et al. 1983; Rennenberg 1988). Glutathione is also involved in the detoxification of SO2, NO x and O3 in plants (Tanaka et al. 1985; Alscher and Amthar 1988; Alscher 1989; Smith et al. 1990). There is evidence that these pollutants generate conditions of oxidative stress by raising cellular concentrations of peroxides and radicals (Grimes et al. 1983; Neta and Huie 1985; Smith et al. 1990). It also functions as a precursor of phytochelatins (Grill et al. 1985; Jackson 9 4 et al. 1987; Rennenberg 1988; Steffens 1990a,b), small metal binding proteins whose general structure is (Y-glutamyl-cysteinyl)n-glycine, where n=3 to 7. These compounds are known to bind A g 2 + , As0 4 2 - ,Cd 2 + , C u 2 + , Hg 2 + , Pb 2 + , Z n 2 + and Sb 3 + . Glutathione is believed to be involved in heat shock and fungal infection responses. Nieto-Sotelo and Ho (1986) observed an increased synthesis of glutathione in maize roots exposed to heat shock. Depleted glutathione levels in animal cells resulted in decreased heat tolerance (Mitchell et al. 1983). Wingate et al. (1988) reported that reduced glutathione (GSH), when supplied to bean (Phaseolus vulgaris L.) cell suspension culture, stimulated transcription of defense genes that encode for enzymes involved in lignin and phytoalexin production. There is evidence that GSH can protect membrane lipids from free radical damage (Barclay 1988; Smith et al. 1990). It is generally assumed that glutathione maintains proteins, cysteine and homocysteine in the reduced (i.e., metabolically active) form (Kosower and Kosower 1978) and may regulate the activities of enzymes of the Calvin cycle that are inactivated in the dark (Halliwell 1984). Current evidence, therefore, suggests that glutathione may play a role in mediating responses of plant cells to a variety of stresses such as low temperature and water deficits. Thus, Esterbauer and Grill (1978) reported an increase in glutathione during winter in needles of Picea abies. Guy and Carter (1982,1984) and Guy et al. (1984) observed an increase in glutathione which correlated with hardiness in Comus and Citrus. The increase was most dramatic for GSH but was also observed for GSSG. de Kok and Oosterhuis (1983) reported an increase in thiols with increased hardiness in spinach grown at low temperature. There have however, also been reports of lack of correlation between artificially elevated GSH levels and freezing tolerances of plant 95 tissues (de Kok et al. 1981; Guy et al. 1984). These studies were done under noninductive conditions (i.e., nonacclimating conditions) and the lack of correlations may be a reflection of this. The extent and exact involvement of glutathione in low temperature responses of plants is still unclear. Exposure of temperate woody plants to short photoperiod and water stress results in increased hardiness, but it is exposure to low temperature that ultimately determines the hardiness level attained by a plant (Weiser 1970). Examination of the relationship between these environmental factors and tissue glutathione levels should help elaborate the extent of involvement of thiols, and glutathione in particular, in plant cold hardiness responses. The level of glutathione in plant tissues can also be manipulated artificially by using compounds that enhance or block its biosynthesis. It should thus, be possible to elaborate further the involvement of glutathione in the development of hardiness by application of these chemicals under acclimating conditions. /V,/V-diallyl-2,2-dichloroacetamide (R-25788 or dichlormid), a herbicide antidote, is known to increase tissue glutathione content (Lay et al. 1976; Lay and Casida 1976; Carringer et al. 1978; Gronwold et al. 1987). S-n-butylmonocysteine sulfoximine (buthionine sulfoximine, BSO), is a specific inhibitor of y-glutamylcysteine synthetase, the first enzymes of glutathione biosynthesis (Griffith and Meister 1979; Griffith 1981; Steffens et al. 1986; Rennenberg 1988; Buwalda et al. 1990). Currently, the effects of these compounds on conifer seedlings are not known. The objectives of this study were therefore: (i) to examine the relationship between nonprotein thiol compounds and glutathione (reduced and oxidized), and the development of cold hardiness, and 96 (ii) to examine the effects of treatments that affect hardiness (growth conditions) and glutathione levels (chemicals; BSO and dichlormid) on glutathione content and cold hardiness, in seedlings of western red cedar (Thuja plicata), yellow cedar (Chamaecyparis nootkatensis) and white spruce (Picea glauca). The second objective of the study was designed to examine if observed correlations between glutathione contents and hardiness under hardening conditions had any causal relationship. 97 5.2. MATERIALS AND METHODS 5.2.1. Seedling Culture and Treatments Seedlings used in this study were raised outdoors as previously described and transferred to a controlled environment chamber while still actively growing. The growth conditions in the chamber were 22/15 °C day/night temperatures, 18 h photoperiod, 250 pinoles m"2 s"* photon flux density (400-700 nm). The seedlings were watered every 3 days and fertilized once a week with the high N fertilizer described in section 1.2. The seedlings were maintained in the controlled environment chamber under these conditions for 5 weeks before the initiation of the experimental treatments. To examine the relationship between free thiols and cold hardiness, the following treatments were given; 9 h photoperiod at the same photon flux density (PFD) and temperature, watered and fertilized as before for 4 weeks. Following this, the growth temperature was lowered to 7/3 °C day/night under the same PFD, watering and fertilization regime as before for 8 weeks. Once a week, cold hardiness was determined on five seedlings as previously described and foliage from four seedlings per replicate was harvested, immediately frozen in liquid nitrogen and stored in a deep freezer (-75 to • 85 *Q for later analysis. To examine the effects of modulating seedling glutathione contents on cold hardiness, seedlings raised under 9 h photoperiod, 20/15 °C and the same PFD as before were subjected to the following treatments: (i) watered every other day, (ii) watered every 5th day (stressed), (iii) soil drenched with 50 pM solution of R-25788 and (iv) 50 pM solution of buthionine sulfoximine (BSO) every other day. Levels of frost hardiness were determined and foliage samples for analysis were harvested and stored as before after 1, 3 and 4 weeks of treatment. After four weeks, the growth temperature was 98 lowered to 7/3 °C day/night temperature (low temperature treatment, LT). The seedlings were watered (water stress discontinued), root drenched with R-25788 and BSO once a week and fertilized every two weeks. They were maintained under these conditions for 4 weeks. Hardiness was determined after 1 and 4 weeks and plant tissue harvested and stored for subsequent analysis. In all cases tissues were harvested 4 h after the lights came on. 5.2.2. Chemicals Reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase (GR, type IV from spinach), NADPH, 5,5-dithiobisnitrobenzoic acid (DTNB), buthionine sulfoximine (BSO), polyvinylpolypyrrollidone (PVpP) and 2-vinylpyridine were purchased from Sigma Chemical Co. (St. Louis, MO). Dichlormid (R-25788) was provided by Stauffer Chemical Co. (Richmond, CA). The rest of the chemicals used were purchased from BDH (Vancouver, BC). 5.23. Extraction and Analysis of Glutathione and Thiols 5.23.1. Tissue extraction The method of extraction described below was developed to minimize problems with auto-oxidation, phenolic compounds and pigments. Two grams fresh weight (f.wt) of tissue was pulverized in a mortar with a small amount of sand under liquid N2 and extracted twice with 10 ml of ice cold 5% sulfosalysilic acid (SSA) containing 2 mM EDTA. The homogenates were filtered through Nitex into a capped centrifuge tube containing 6 g wet weight PVpP (equivalent to about 1.5 g dry weight). After mixing by bubbling with N2, the extracts were left on ice for about 10 min, filtered into another tube, flushed with N2 and kept capped on ice. Due to the presence of large amounts of an unknown slimy compound which prevented complete precipitation of the proteins and 99 chlorophyll, red cedar extracts were gently partitioned against an equal volume of chloroform (under N2) to remove pigments. The extracts were centrifuged for 4 min at 13,000 g to precipitate proteins and aliquots removed from the supernatant for immediate determination of total reduced thiols (RSH), and derivatization with 2-vinylpyridine. Vinylpyridine reacts with GSH rendering it unavailable to the enzymatic reaction thus permitting separate quantification of GSSG. Aliquots for the determination of total glutathione and total thiols (reduced plus oxidized) were stored in a freezer (-75 to -85 °C) until analyzed. Samples were derivatized with 2-vinylpyridine for 20 min at room temperature then extracted twice with equal amounts of ethyl acetate to remove excess vinlypyridine and the excess ethyl acetate was removed by bubbling with N2. This sample was stored frozen until analyzed. Partitioning the reduced glutathione (GSH) standard against chloroform did not result in a detectable loss or auto-oxidation (Table 5.1). The enzyme assay for glutathione was inhibited by 20 to 30% before the removal of phenolic compounds with PVpP. Without the use of PVpP (which also helped remove pigments) and chloroform (for red cedar), the free thiols were either over-estimated (probably due to some unprecipitated proteins [Table 5.2]) or, in the case of red cedar, impossible to estimate (Table 5.3). Complete conjugation of GSH with 2-vinylpyridine was possible within 15 min (Table 5.4) and no evidence of auto-oxidation was observed. The effectiveness of the extraction procedure, checked by a parallel addition of a known amount of GSH to leaves of yellow cedar prior to homogenization is presented in Table 5.5. 100 Table 5.1. Effect of partitioning GSH standard against chloroform. Absorbance (412 nm) Sample # Not partitioned Partitioned 1 0.570 0.561 2 0.569 0.575 3 0.572 0.576 4 0.569 0.562 Table 5.2. Effect of using PVpP on the absorbances (412 nm) of the centrifuged yellow cedar and white spruce extracts (0.25 ml). Before PVpP After PVpP -DTNB +DTNB -DTNB +DTNB Yellow 0.815 cedar 0.781 0.801 Estimated thiols (nM g"1 fewt) White spruce 0.196 0.192 0.209 Estimated thiols (nM g"1 f.wt) 1.203 0.122 0.370 1.160 0.121 0.362 1.207 0.122 0.388 765 356 0.421 0.042 0.249 0.430 0.043 0.263 0.439 0.043 0.258 291 249 * - DTNB is dithiobisnitrobenzoic acid, and PVpP is polyvinylpolypyrrollidone. Table 5.3. Effect of using PVpP and chloroform on the absorbances (412 nm) of centrifuged red cedar leaf extracts (0.25 ml). PVpP alone -DTNB +DTNB PVpP + Chloroform -DTNB +DTNB 1.407 1.705 0.292 0.484 1.368 1.702 0.295 0.493 1.401 1.684 0.285 0.496 Estimated thiols (nM/g f.wt) 514 206 1 - the crude extract blank (without using PVpP) was too cloudy and gave a very high absorbance (>2.000 AU). DTNB is dithiobisnitrobenzoic acid, and PVpP is polyvinylpolypyrrollidone. Table 5.4. Time course of conjugation of GSH standard with 2-vinylpyridine. Absorbance (412 nm)* Percentage Unconjugated 1.055,1.038,1.198 100 1 min 0.803,0.841, 0.826 71 3 min 0.569, 0.571, 0.566 45 5 min 0.434, 0.421, 0.418 30 10 min 0.189, 0.188, 0.187 5 15 min 0.138, 0.139, 0.136 0 Blank 0.139, 0.137, 0.138 based on three experiments. 102 Table 5.5. Recovery of GSH by the extraction procedure. The GSH standard was added to 2 g f.wt of yellow cedar leaves prior to homogenization. GSH added (nM) GSH detected (nM) Recovery Sample 1 ' 265.3 2 - 237.0 3 - 244.0 Standard 4 200 205.2 102.5% 5 200 197.1 98.5% 6 200 199.2 99.5% Sample + 1 200 453.2 97.4% standard 2 200 451.6 103.3% 3 200 441.3 99.4% 5.23.2. Assays The nonprotein reduced thiols (RSH) were assayed by a modification of the method described by Ellman (1959). To 0.5 ml plant extract was added 0.32 ml 0.2 M TrisHCl (pH 8.0), 80 pi 50% triethanolamine, and 100 pi 6 mM DTNB (in 0.02 M Na-phosphate buffer, pH 7.5). Absorbance at 412 nm was read after 10 min on a Milton Roy Spectronic 1201 spectrophotometer. For the determination of total thiols (reduced plus oxidized) the extracts were first reduced in 2% NaBH4 (Modig 1968; Habeeb 1973), acidified with SSA then assayed as reduced thiols as described above. Complete reduction, checked by the reduction of GSSG standards, was achieved by this method. The thiol contents of the plant extracts were determined from standard curves of known concentrations of GSH. Corrections were made for the absorbances due to the deproteinized supernatant and for the reagents. 103 The glutathione assay adapted here is the cyclic enzymatic method of Tietze (1968) as modified by. Anderson (1985) and Griffith (1985). Total glutathione (GSH + GSSG) and GSSG were assayed as follows: to 0.6 ml 0.125 M Na-phosphate buffer (pH 7.5, containing 6.3 mM EDTA) were added 0.1 ml 2 mM NADPH, 0.1 ml 6 mM DTNB, 20 to 50 pi sample extract and water to bring the total volume in the cuvette to 1 ml. Ten pi of glutathione reductase (GR), E.C. 1.6.4.2 (1IU and 3 IU for GSH and GSSG assays, respectively) was added to the assay mixture. All the solutions used and the enzyme were in 0.125 M Na-phosphate buffer (pH 7.5). The change in absorbance at 412 nm was monitored for up to 10 min on a Milton Roy Spectronic 1201 spectrophotometer, and the rate of change in absorbance over time (8A/5t) was computed. The amount of glutathione in the plant extracts (expressed in GSH equivalents, where GSSG=2GSH) was determined from a standard curve of a plot of the rate of change versus GSH concentration. 5.2.4. Data Computations and Analysis Total oxidized thiols (RSSR) were computed by subtracting reduced thiols (RSH) from total thiols (reduced plus oxidized, TSH). Reduced glutathione (GSH) was determined by subtracting oxidized glutathione (GSSG) from total glutathione. The nonglutathione reduced thiols (NSH), nonglutathione disulfides (NSSR), and the total nonglutathione thiols (NSH + NSSR, TNSH) were all computed from the values obtained from the thiols and enzymatic assays for glutathione. A single factor analysis of variance based on a fixed model for a completely randomized design was performed to compare treatment effects (except cold hardiness) by days, using SYSTAT (Wilkinson 1989). The means of variables with significant F values were compared using the Neuman-Kuels test. 1 0 4 5.3. RESULTS Exposure of seedlings to low temperature (7/3 °C, 9 h photoperiod) but not high temperature (20/15 °C, 9 h photoperiod) hardening conditions resulted in substantial increases in levels of thiols and glutathione in leaves of seedlings of all 3 species (Figures 5.1, 5.2 and 5.3). These changes to low temperature exposure generally coincided with the initiation of hardening at the maximal rates in seedlings of red cedar and yellow cedar and, to an extent, white spruce. In white spruce, although there was a significant increase in hardiness after 4 weeks of SD exposure at the warm temperature, no changes in thiols or glutathione (total, reduced or oxidized) levels were observed. Similar lack of increase was observed in red cedar and yellow cedar seedlings, species which either only marginally increased or did not increase their hardiness at all under the SD treatment without exposure to low temperature. The increase in thiol contents generally started within a week of low temperature exposure but reached the maximum levels in about three weeks and remained high or slightly increased up to the end of the experiment (8 weeks of low temperature exposure). 200 -35 6 8 WEEKS OF OBSERVATION 11 14 Figure 5.1. Changes in levels of thiols (means of 3 values) in leaves of acclimating seedlings of red cedar. Seedlings were exposed to low temperature (7/3 °C day/night) from the end of the 4th week. The graphs from top to bottom depict changes in thiols in general, glutathione and non-glutathione thiols, respectively. The solid lines represent the total (1), reduced (2) and oxidized (3) forms of the compounds while the dotted line represents the hardiness level. Vertical lines are SE of the means. (Means of 3 values). Figure 5.2. Changes in levels of thiols (means of 3 values) in leaves of acclimating seedlings of yellow cedar. Seedlings were exposed to low temperature (7/3 °C day/night) from the end of the 4th week. The graphs from top to bottom depict changes in thiols in general, glutathione and non-glutathione thiols, respectively. The solid lines represent the total (1), reduced (2) and oxidized (3) forms of the compounds while the dotted line represents the hardiness level. Vertical lines are SE of the means. (Means of 3 values). 107 400 -60 300 6 8 WEEKS OF OBSERVATION 11 14 Figure 5.3. Changes in levels of thiols (means of 3 values) in leaves of acclimating seedlings of white spruce. Seedlings were exposed to low temperature (7/3 °C day/night) from the end of the 4th week. The graphs from top to bottom depict changes in thiols in general, glutathione and non-glutathione thiols, respectively. The solid lines represent the total (1), reduced (2) and oxidized (3) forms of the compounds while the dotted line represents the hardiness level. Vertical lines are SE of the means. (Means of 3 values). 1 0 8 In both red and yellow cedars, there was a 2 to 3 fold increase in total thiols principally in the reduced form with continued exposure to low temperature (Figures 5.1, and 5.2). The reduced thiols constituted about 77% and 83% of total thiols in red and yellow cedar seedling foliage, respectively. There were also minor increases in the levels of oxidized thiols in the foliage of the three species with exposure to low temperature. Although the pattern of thiol changes in needles of white spruce seedlings during the acclimation period was similar to the other two species, the change in content was more gradual in white spruce than in seedlings of red and yellow cedars (Figure 5.3). As in the other two species, the reduced thiols contributed about 80% of the total free thiols and therefore accounted for most of the increase in total thiols. The increase in the amount of tissue thiols in all three species was primarily the result of increased levels of glutathione. Glutathione made up about 60% of the total thiols in red cedar and about 70% in yellow cedar and white spruce seedlings both before and after low temperature exposure. In all three species, total glutathione levels increased significantly after exposure to low temperature (Figures 5.1, 5.2, and 5.3). This was particularly prominent after two weeks of low temperature exposure. The increase in all cases was predominantly in the form of reduced glutathione (GSH) which more than doubled in 3 weeks of low temperature exposure. Reduced glutathione comprised from 84 to 90% of the total glutathione of the tissues both before and after low temperature exposure. Oxidized glutathione (GSSG) increased only slightly under low temperature exposure. There were also minor but gradual increases in non-glutathione thiols with duration of low temperature exposure in seedlings of all three species. Generally, both the reduced and oxidized forms increased (Figures 5.1, 5.2 and 5.3). 109 Buthionine sulfoximine, dichlormid (R-25788), low temperature and, to a limited extent, water stress treatment affected the levels of glutathione in leaves of seedlings of the three species in a similar manner (Tables 5.6, 5.7 and 5.8). These changes were, however, not necessarily paralleled by changes in cold hardiness. Low temperature exposure, as already reported above, resulted in significant increases in the glutathione levels, predominantly in the reduced form, and these changes were generally paralleled by increased hardiness. Short photoperiod treatment under warm temperature conditions on the other hand did not result in changed levels of tissue glutathione in all 3 species, despite the fact that there was a substantial increase in cold hardiness in white spruce seedlings with the treatment (Table 5.8). Levels of glutathione, predominantly in the reduced form, increased slightly when the seedlings were water stressed. These changes were accompanied by a slight increase in cold hardiness. Table 5.6. Effects of water stress, BSO and dichlormid on glutathione levels (n=3) and estimated LT50 (95% confidence limits in parenthesis, n=5) in red cedar seedlings.1 Treatment GSH GSSG Total GSH/GSSG L T 5 0 nM g'* f.wt (standard deviations) ACTIVELY GROWING 60.9(5.9) 14.9(2.6) 75.9(7.3) 4.09(0.29) -4.3(0.3) 4 WEEKS SD BSO 43.2(5.9)a 16.1(2.0)a 59.2(4.9)a 2.69(0.21)a -4.4(0.3) Watered 57.9(4.9)b 17.0(1.7)a 74.9(6.4)b 3.41(0.24)b -4.3(0.2) Stressed 64.9(3.2)c 15.2(2.0)a 80.0(3. l)b 3.61(0.38)b -5.2(0.5) Dichlormid 92.9(7.6)d 16.9(1.4)a 109.9(7.8)c 5.49(0.97)c -4.3(0.3) 1 WEEK SD/LT BSO 49.7(6.7)a 16.5(2.6)a 66.1(3.6)a 3.04(0.29)a -7.0(0.5) Watered 64.1(3.0)b 18.5(3.7)a 82.6(3.8)b 3.46(0.13)a -7.6(0.4) Stressed 69.5(4.6)b 18.9(2.6)a 88.1(5.3)b 3.66(0.46)a -9.6(0.6) Dichlormid 103.3(7.5)c 19.5(1.6)a 122.8(8.9)c 5.32(0.55)b -8.5(0.7) 4 WEEKS SD/LT BSO 78.9(5.7)a 21.6(3.5)a 99.6(8.9)a 3.55(0.49)a -12.0(0.9) Watered 141.7(6. l)b 18.6(1.5)a 160.3(6.5)b 7.62(0.69)b -13.9(1.3) Dichlormid 219.9(12.1)c 22.9(2.7)a 242.8(11.5)c 9.667(1.06)c -13.8(1.0) 1 - BSO is buthionine sulfoximine. Means in columns of treatment groups with the same letter are not significantly different (p<0.05). SD and LT treatments refer to short day (9 h) and low temperature (7/2 °Q, respectively. GSH and GSSG are reduced and oxidized glutathione respectively. Table 5.7. Effects of water stress, BSO and dichlormid on glutathione levels (n=3) and estimated LT50 (95% confidence limits in parenthesis, n=5) in yellow cedar seedlings.* Treatment GSH GSSG Total GSH/GSSG L T 5 0 nM g"l f.wt (standard deviations) *C ACTIVELY GROWING 112.1(6.9) 12.8(2.8) 125.1(8.3) 8.78(0.85) -4.4(0.2) 4 WEEKS SD BSO 58.1(8.9)a 15.3(3.2)a 73.4(8.7)a 3.83(0.72)a -5.5(0.3) Watered 105.1(9.6)b 13.6(2.3)b 118.7(10.8)b 7.72(0.36)b -5.7(0.6) Stressed 121.4(16.8)c 14.0(1.9)b 125.4(18.4)b 7.2(0.41)b -7.0(0.4) Dichlormid 164.1(14.6)d 16.0(2.5)c 180.5(15.7)c 10.02(0.80)c -6.2(0.5) 1 WEEK SD/LT BSO 73.2(7.7)a 18.5(2.8)a 91.7(8. l)a 3.96(0.57)a Watered 119.4(13.4)b 14.7(2.8)b 134.1(15.2)b 8.15(0.60)b Stressed 124.5(6.5) Dichlormid 173.3 '6.5)b 13.8(2.7)b 138.4(8. l)b 8.31(0.58)b !7.5)c 17.3(1.9)c 190.6(8.3)c 10.41(0.80)c 4 WEEKS SD/LT BSO 139.3(20.4)a 17.6(3.4)a 156.9(28.3)3 7.87(0.85)3 -17.0(1.4) Watered 244.9(32.2)b 26.1(2.9)b 271.1(29.9)b 9.38(0.56)b -18.2(1.7) Dichlormid 320.2(15.4)c 27.9(2.6)b 348.9(37.9)c 11.13(0.97)c -19.0(1.6) 1 - BSO is buthionine sulfoximine. Means in columns of treatment groups with the same letter are not significantly different (p<0.05). SD and LT treatments refer to short day (9 h) and low temperature (7/2 °C), respectively. GSH and GSSG are reduced and oxidized glutathione respectively. Table 5.8. Effects of water stress, B S O and dichlormid on glutathione levels (n=3) and estimated LT5Q(95% confidence limits in parenthesis, n=5) in white spruce seedlings.1 Treatment GSH GSSG Total GSH/GSSG LT 50 ACTIVELY GROWING nM g"1 f.wt (standard deviations) 119.3(17.6) 10.5(3.0) 129.8(19.6) 11.41(1.40) -4.3(0.3) 4 WEEKS SD BSO Watered Stressed Dichlormid 78.2(10.5)a 108.6(20.7)b 121.9(16.9)b 195.3(1 1.11c 12.5(2.6)a 13.1(2.74)3 12.6(2.8)a 14.6(2.7)3 90.7(11.8)a 121.3(21.0)b 124.5(17.5)b 209.9(12.8)c 6.27(0.83)a 8.19(1.21)b 8.07(1.07)b 13.40(1.28)c -13.8(0.4) -13.4(0.4) -15.6(0.5) -14.3(0.6) 1 WEEK SD/LT BSO Watered Dichlormid 4 WEEKS SD/LT BSO Watered Dichlormid 60.2(11.8)3 111.3(10.5)b 201.1(12.4)c 155.9(4.4)3 190.7(11.5)b 267.4(12.0)c 14.3(3.2)3 15.8(3.0)3 25.1(3.8)b 27.6(4.7)3 32.4(3.9)3 30.3(4.9)3 74.4(12.1)3 127.1(ll.l)b 226.2(13.5)c 183.6(13.5)3 223.0(14. l)b 297.7(12.6)c 4.20(0.69)3 7.13(0.98)b 8.24(0.78)c 5.21(0.51)3 5.99(0.60)3 8.83(0.83)b -19.6(1.3) -21.0(1.2) -22.6(1.1) -47.1(1.9) -50.1(1.9) -50.8(1.8) 1 - BSO is buthionine sulfoximine. Means in columns of treatment groups with the same letter are not significantly different (p<0.05). SD and LT treatments refer to short day (9 h) and low temperature (7/2 °C), respectively. GSH and GSSG are reduced and oxidized glutathione respectively. 113 Root drenching with BSO significantly lowered (up to 40%) tissue glutathione levels, primarily the reduced form, in seedlings of all three species (Tables 5.6, 5.7 and 5.8). Levels of oxidized glutathione were generally unaffected by BSO treatment. The decrease in the reduced form of glutathione, while the oxidized form remained more-or-less constant resulted in a smaller ratio of the oxidized to reduced glutathione (GSH/GSSG). Although treatment with BSO lowered glutathione levels, biosynthesis was not completely inhibited under our experimental conditions (Tables 5.6, 5.7 and 5.8). In all three species there was an increase in the amount of glutathione when the seedlings were exposed to low temperature even in the presence of the BSO treatment. Treatment with dichlormid significantly (p<0.05) increased the level of glutathione by up to about 40% in foliage of all three species (Tables 5.6, 5.7 and 5.8). The increase was predominantly in the form of GSH which constituted about 90% of the total glutathione. GSSG was not changed much by the treatment As a result of this, the treatment with dichlormid resulted in a large increase in the GSH/GSSG ratio. A combination of dichlormid and low temperature resulted in even larger increases of glutathione levels in all three species. Although there were changes in glutathione levels and GSSG/GSH ratios with BSO and dichlormid treatments, these changes generally did not translate into any significant changes in cold hardiness in all the three species, under either non-acclimating or acclimating conditions. 114 5.4. DISCUSSION Observations from the first part of the this study indicate that total foliar thiols increase, predominantly in the form of GSH, with hardening of seedlings at low temperature. Similar results were reported by de Kok and Oosterhuis (1983) with spinach, and Guy and Carter (1982,1984) with Cornus and Citrus under controlled conditions. Esterbauer and Grill (1978) also reported a ten-fold increase in glutathione levels in needles of field grown spruce during the winter. My results also indicate that short day exposure at a warm temperature significantly increased hardiness in white spruce seedlings and yet no detectable increases in glutathione were found in these seedlings. Furthermore, the increase in glutathione levels in seedlings exposed to low temperature was greater for red and yellow cedars than for white spruce. This indicates that the large between species differences in glutathione contents were not related to the relative hardiness levels attained. Data presented here confirm other observations (de Kok et al. 1981; Smith 1985) that the accumulation of thiols or glutathione in plant tissues during cold acclimation may be the result of low temperature exposure itself. The mechanism by which this happens and its significance are not known. The pool of glutathione in a leaf at any moment is determined by the relative rates of synthesis, degradation and export. Reduced sulfur is stored principally as glutathione in plant cells and glutathione is directly involved in sulfur nutrition of plants (Rennenberg 1982,1984; de Kok et al. 1986; de Kok 1990; Smith et al. 1989,1990). Its accumulation in plants exposed to low temperature with adequate sulfate supply may be due to storage of excess reduced sulfur when demand for sulfur containing metabolites declines (Rennenberg 1982,1984; Guy et al. 1984; de Kok et al. 1981; Smith et al. 1989, 115 1990; Rennenberg and Lamourex 1990). Although this may be the case, it does not completely explain the accumulation of glutathione under low temperature conditions observed here or elsewhere. In studies reported here, short day exposure at a warm temperature did not result in increased GSH levels yet shoot growth was affected. Sulfur demand was probably affected by the reduced shoot growth, but this was not reflected in glutathione levels. Furthermore the mechanism of glutathione accumulation, as a result of lowered sulfur demand under low temperature outlined above, ignores the possibility that the accumulation may be an active necessary process as a part of the oxygen radical scavenging system in plant cells as outlined by Foyer and Halliwell (1976), Halliwell (1982) and Winston (1990). It is therefore likely that low temperature in itself may actively enhance glutathione accumulation by a direct effect on its metabolism in a manner not related to sulfur nutrition. This would be related to the avoidance of photodamage and may therefore be light dependent (Bielawksi and Joy 1986; Hossain and Asada 1984; Gillham and Dodge 1987; Buwalda et al. 1988,1990; Schupp and Rennenberg 1988, 1990; Koike and Patterson 1988). Exposure of plants to low temperatures at moderate PFD may result in photoinhibition which can, in unhardened or chilling sensitive species, lead to photooxidation if severe enough (Wise and Naylor 1987a,b; Steffen and Palta 1986; Krause 1988; Somersalo and Krause 1989,1990). As a mechanism of avoidance of photodamage, plants capable of acclimating possess scavenging systems (in which glutathione plays a major role) which increase in activity substantially during low temperature acclimation (Gillham and Dodge 1987; Schoner and Krause 1990; Schoner et al. 1990). Unfortunately not enough information exists on the regulation of the enzymes of glutathione metabolism to permit meaningful speculation on the possible steps and 116 mechanisms of regulation of glutathione biosynthesis under low temperature conditions. The limited information available indicates that light may play a role in the regulation of activity of some of the enzymes of the biosynthetic pathway (Bielawski and Joy 1986; Gillham and Dodge 1986; Buwalda et al. 1988,1990; Schupp and Rennenberg 1988, 1990; Bergmann and Hell 1990). Chloroplast glutathione synthetase appears to be most active under conditions of pH and M g 2 + concentration which exist in the stroma in the light but not in the dark (Law and Halliwell 1986; Hell and Bergmann 1988; Klapheck et al. 1987; Rennenberg 1988). Activity of 5-oxoprolinase which catalyzes the last degradation step is reported to be inhibited by light even at low quantum flux densities of ~50 pmoles m"2 s"* (Schupp and Rennenberg 1990). Furthermore, y-glutamylcysteine synthetase (which catalyzes the first biosynthetic step) and 5-oxoprolinase are thought to be rate limiting. It is therefore possible that these two enzymes may be responsible for the accumulation of glutathione under low temperature conditions. Observations from the second part of the study indicated that BSO and dichlormid significantly affected the GSH contents of the plant foliage and changed the GSH/GSSG ratio. These changes, however, did not appear to be related to freezing resistance. Buthionine sulfoximine did not significantly reduce frost tolerance, while dichlormid did not significantly increase tolerance, de Kok et al. (1981) working with spinach and Guy and Carter (1984) working with Cornus and Citrus similarly observed that artificial increase of thiol or glutathione levels did not result in increased in frost hardiness. On the other hand, inhibition of glutathione biosynthesis by BSO results in reduced tolerance to heavy metals (Steffens et al. 1986; Steffens 1990b), high temperature (Mitchell et al. 1983) and, in animal cells, reduced viability and increased sensitivity to destruction by y-irradiation (Meister and Anderson 1983). Furthermore, treatment of plants with dichlormid increases GSH levels (Carringer et al. 1978) and 117 confers protection against herbicides such as atrazine and paraquat, herbicides which stimulate the production of oxyradicals in plant cells. This indicates that plant responses to the type of freezing damage induced in this study probably do not involve glutathione. The damaging effects of freezing, in this situation, may therefore be quite different from those resulting from stress induced by the above factors. The lack of response to lowered glutathione content upon treatment with BSO, may also be partly because only 30 to 50% inhibition was obtained. The amount of glutathione still remaining in the cells may therefore not be too low to cause any reduction in resistance even if GSH were involved. Although there are reports that water stress increasing the levels of glutathione in plant tissues (Dhindsa and Matowe 1981; Dhindsa 1991; Gamble 1984; Burke et al. 1985), I observed only a marginal increase in glutathione content with water stress in all three species. This could be partly because the water stress was not sufficiently high to induce an increase in the levels of glutathione. The mechanism by which the accumulation takes place is not known but it may involve water stress induced oxyradical accumulation. Glutathione in this case then serves as a scavenger of the radicals. According to Levitt's (1962,1980) hypothesis (the 'sulfhydryl-disulfide interchange1 hypothesis), freezing injury results from protein aggregation due to the formation of inter/intramolecular disulfide bonds during freezing dehydration of cells. The presence of high cellular levels of soluble thiols was therefore considered necessary in the prevention of protein aggregation thereby conferring protection from freezing damage. Although not much is known about the mechanisms of freezing damage in plant cells, it is generally accepted that freezing injury is the result of membrane damage (Steponkus 1984,1990) which may involve simple rupture (as in unhardened cells) or loss of semipermeability but may not involve protein aggregation. The involvement of glutathione or thiols in the prevention of freezing damage to plant tissues as envisioned in 118 this study, therefore assumes a role of thiols/glutathione in other functions such as prevention of lipid peroxidation. Results from studies presented here and other similar observations (de Kok et al. 1981; Guy and Carter 1984; Stuiver et al. 1988) appear to indicate that GSH is not directly involved in freezing resistance. However, it is likely that glutathione is involved in the prevention of photodamage when photosynthetic electron transport is inhibited as may occur under low temperatures and high PFD conditions. Damage resulting from low temperature exposure under such conditions is therefore different from damage that may occur at freezing temperatures in the absence of light. A role of glutathione in protection against photodamage at low temperatures is in accordance with its role in the ascorbate cycle involved in the detoxification of H 2 O 2 . The accumulation of glutathione in plant cells at low temperatures may therefore be the consequence of both disturbed sulfur metabolism and an active accumulation as a mechanism in the prevention of oxidative injury. The extent to which each of the mechanisms contributes to the accumulation awaits the confirmation of the existence and characterization of the two systems. 119 5.5. CONCLUSION The objectives of this study were to examine and characterize the nature of the relationship between foliar thiol and glutathione levels, and cold hardiness in seedlings of conifer species. This was done to explore the possibility of modulating plant responses to low temperature exposure by regulating tissue glutathione levels. Observations from the study indicate that, whereas glutathione levels increase with hardiness, this accumulation appears to be in response to low temperature exposure. Chemical modulation of levels of tissue glutathione did not affect freezing resistance. The relationship between freezing resistance and glutathione levels may therefore be purely correlative. Thus, temperature and photoperiod treatments may be more important in determining freezing resistance than the glutathione levels. Finally, the methods of modulating glutathione levels in plant tissues used here promise new opportunities for the study of plant responses to a variety of stresses in which glutathione is known or proposed to be involved (e.g., heavy metal toxicity, radiation, ozone and air pollutants). Such modulations may also serve as a means of protecting plants from these stresses. 120 CHAPTER 6. GENERAL CONCLUSIONS AND RECOMMENDATIONS This dissertation examined factors involved in the regulation of cold hardiness, and the effects of the plant growth retardant mefluidide on seedling physiology, in first-year seedlings of western red cedar, yellow cedar and white spruce. The project was undertaken to provide baseline information which could be used to improve seedling resistance to stress; in particular low temperature, with a minor reference to water deficits. As a pre-requisite to the cold hardiness studies, a reliable way of estimating hardiness levels (suitable for these species) based on the electrolyte leakage (EC) method was developed. Based on the information gained from this study, the following conclusions and recommendations can be made: 1. The use of the EC method: • The electrolyte leakage technique, can be used to estimate cold hardiness objectively and accurately in seedlings of the three conifer species. Some of the discrepancies found in the literature appear to arise from inappropriate tissue preparation and possibly freezing protocols. With appropriate refinements and optimization of tissue preparation, as described here, it is possible to obtain LT5QS which closely correspond to 50% actual damage of whole plants (assessed visually). In cases where freezing indices of 50% deviate significantly from the LT5QS of the plant, a calibration of the method can be easily accomplished. With experience and appreciation of the theory behind the technique, it should be fairly easy to transfer the method to other species. 121 2. The development and regulation of cold hardiness: • The red cedar and yellow cedar seedlings studied here appeared to have a less complicated mode of transition to and maintenance of the hardened state than the seedlings of white spruce. In seedlings of the two cedar species, hardiness development appeared to be basically a temperature induced process. The seedlings remained hardy so long as they were exposed to low temperature. Removal of the stimulus resulted in a loss of hardiness. In white spruce seedlings studied here, on the other hand, hardiness development and maintenance appeared to be under a complex control of exogenously cued endogenous growth rhythms. Exposure to short photoperiod induced the initiation of hardiness which proceeded even under warm temperature conditions. Exposure to temperatures of around 5 °C or lower, which was required for the satisfaction of chilling requirements, permited the plant to deharden. The mechanisms of initiation and maintenance of hardiness in the two groups; red and yellow cedar on one hand and white spruce on the other, represent different strategies. • From a practical point of view, a short photoperiod treatment as a nursery cultural tool for improving seedling resistance to stress and other morphological qualities will be of value for white spruce but not red or yellow cedar seedlings. 3. The use of mefluidide to improve seedling stress resistance: • Results from this study indicate that mefluidide may have little application for the improvement of cold hardiness in the three species. However, this may be a reflection of the timing of the chemical application in relation to the inductive status of the plants. The study on environmental regulation of hardiness indicated that hardiness in white spruce seedlings was easily induced by short photoperiod or, to an extent, water stress. The use of these factors in white spruce may 1 2 2 therefore be sufficient, and probably the best cultural techniques available. • Mefluidide application improved the water status of seedlings of all the three species as a result of reduced water loss. Although the actual basis for this response is not clear, it is apparent that some of the changes (directly or indirectly the result of the chemical) involved changes in membrane properties and possibly osmotic regulation. If, indeed, one of the benefits of mefluidide is to improve water conservation, it may be possible to use mefluidide to either help protect seedlings from transplanting shock, or to condition seedlings to withstand water deficits (i.e., induce accumulation of osmotica) without exposure to water stress. Further research is needed to determine the possibility of using mefluidide for this purpose. 4. The relationships between glutathione and cold hardiness: • Although results from this study indicate that tissue glutathione increases with hardiness, the accumulation appears to be a direct consequence of low temperature exposure with water stress playing a minor role. Short photoperiod which resulted in an increased hardiness in white spruce had no effect on glutathione levels in tissues of this species. It was therefore, apparent that tissue glutathione had very little direct relationship to freezing resistance. This however, does not preclude the role of glutathione in prevention of peroxidative processes under low temperature conditions. The observed increase in glutathione may simply reflect a biochemical adjustment that is geared towards averting damage that may result from exposure to excessive light in the presence of low temperatures. • Observations that BSO and dichlormid did not affect plant resistance to freezing (freezing in the dark, as performed in this study) also indicated that glutathione 123 levels are not directly involved in the resistance to this type of freezing stress. The use of modulators of glutathione metabolism therefore, appears to have limited application for the improvement of freezing stress. However, the observation that modulation of glutathione levels by chemical means in these species is possible, provides an opportunity to study plant responses to a variety of other stresses (such as toxic metals, radiation and air pollutants), and may eventually provide a means of protection against such stresses. 1 2 4 LITERATURE CITED Alscher, R.G. 1989. 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