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Carbohydrates in white spruce and lodgepole pine seedlings during winter : outdoors, in freezer-storage… Lévesque, Françoise 1995

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CARBOHYDRATES IN WHITE SPRUCE AND LODGEPOLE PINE SEEDLINGS DURING WINTER: OUTDOORS, IN FREEZER-STORAGE AND IN THAWING. by FRANCOISE LEVESQUE B.Sc, Universite Laval, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (DEPARTMENT OF FOREST SCIENCES) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July 1995 © Francoise Levesque, 1995 In presenting this thesis in partial fulfilment 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 by 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 fe><"esf ^C-ifcAc€-<s> The University of British Columbia Vancouver, Canada Date "3>&C. 0~O . l ^ S -- 6 (2/88) ABSTRACT Studies were conducted to examine factors associated with total non-structural carbohydrate (TNC) depletion during winter, either outdoors, in freezer-storage or in thawing. To see if TNC depletion was mainly due to respiration, two experiments were conducted. In Experiment 1, respiratory C0 2 evolution of white spruce (Picea glauca (Moench) Voss.) was monitored in -2°C storage for 4 months and in +2°C and +7°C thawing regimes for 6 weeks. In Experiment 2, white spruce and lodgepole pine (Pinus contorta Dougl.) respiration was monitored in -2°C storage for 2 months. In the following 6 weeks of +2°C and +7°C thawing regimes, only spruce respiration monitoring was considered in Experiment 2. TNC was measured every month beginning in December in white spruce and lodgepole pine for seedlings overwintered outdoors for 3 months, for seedlings left outdoors 2 months and then lifted in February for freezer-storage, and for seedlings freezer-stored for 4 months. TNC was also measured twice during the two thawing regimes for seedlings cold-stored for 2 and 4 months. Respiration rates and TNC depletion were compared. Frost hardiness was monitored for spruce and pine at months 0, 2 and 4 during freezer-storage and every two weeks during thawing. For seedlings kept outdoors, frost hardiness was monitored at months 0, 2 and 3. Respiration increased following a disturbance (e.g. lifting for storage) but stabilized at a lower steady-state rate thereafter. Measured TNC depletion matched respiration rate in the 4 months freezer-storage but not in any other cases. The Q10 calculated between -2°C and +2°C was found to be extreme for steady-state respiration rates, indicating a disproportionate increase in activity when seedlings are transferred from freezer-storage to thawing. Frost hardiness was released relatively slowly following 4 months of freezer-storage. Dehardening was faster for seedlings kept outdoors. Seedlings placed in freezer-storage in February were able to regain frost hardiness in storage. Starch synthesis occurred in -2°C storage although no clear environmental signals could have triggered this activity. Soluble sugars were analyzed by HPLC to explore sugar dynamics in relation to frost hardiness and growth resumption. Spruce and pine seedlings from outdoors, the 4 months freezer-storage treatment and the +2°C thawing regime were analyzed. Contents of individual sugars were plotted against frost hardiness to identify any relationship to dehardening. The results suggested that shoot raffinose content is well correlated with hardiness of spruce and pine. More species and conditions need to be tested to establish the strength of this relationship. To explore the possibility of root to shoot C H 2 O transport via phloem, white spruce seedlings were girdled at the collar zone. Intact and girdled seedlings were freezer-stored for 4 months and transferred to a +2°C thawing regime for 4 weeks. Thereafter, the temperature in thawing was increased to +7°C for 2 additional weeks. Shoot and root TNC content were compared for intact and girdled seedlings. The results showed that significant phloem transport was not likely in -2°C storage or when seedlings were exposed to +2°C for 4 weeks. However, once transferred to +7°C, there was a disproportionate accumulation of free sugars in roots of girdled seedlings, suggesting that normal root to shoot phloem transport was blocked. TABLE OF CONTENTS Abstract Table of Contents List of Tables List of Figures List of Appendices Acknowledgements 1.0 INTRODUCTION 2.0 LITERATURE REVIEW 2.1 Carbohydrates in storage 2.2 TNC depletion and translocation 2.3 TNC depletion and respiration 2.4 TNC depletion and disturbance 2.5 Expression of physiological status 2.5.1 Starch and sugar dynamics 2.5.2 Modification of sugar quality 2.6 Conclusion 2.7 Hypotheses 3.0 MATERIAL AND METHODS 3.1 Study 1 - Respiration and reserve depletion 3.1.1 Respiration rate measurements 3.1.2 TNC content 3.1.3 Frost hardiness measurement 3.2 Study 2 - Changes in sugar quality 3.3 Study 3 - Reserve translocation V 4.0 RESULTS 30 4.1 Frost hardiness 30 4.2 Carbohydrate reserves 35 4.2.1 Respiration 35 4.2.2 Reserve depletion 41 4.2.2.1 White spruce 41 4.2.2.2 Lodgepole pine 46 4.3 Starch and sugar dynamics 50 4.3.1 White spruce 50 4.3.2 Lodgepole pine 54 4.4 Sugar quality 58 4.4.1 White spruce 61 4.4.2 Lodgepole pine . 65 4.4.3 Relationship between sugar quality and frost hardiness 69 4.5 Translocation 70 4.5.1 Reserves depletion 70 5.0 DISCUSSION . 74 5.1 Frost hardiness 74 5.2 Carbohydrate reserves 76 5.2.1 Respiration 76 5.2.2 Reserve depletion 78 5.2.2.1 White spruce 79 5.2.2.2 Lodgepole pine 82 5.2.2.3 Starch and sugar dynamics 83 5.3 Sugar quality 86 5.3.1 Analytical methods for soluble sugars 86 5.3.2 White spruce ' 87 vi 5.3.3 Lodgepole pine 88 5.3.4 Relationship between sugar quality and frost hardiness 89 5.4 Translocation 92 6.0 CONCLUSIONS 94 7.0 RECOMMENDATIONS FOR FURTHER RESEARCH 97 8.0 REFERENCES 98 APPENDICES 106 vii LIST OF TABLES Table Page 1. Literature summary of presence of raffinose under adverse 14 conditions. 2. LT50 estimation for spruce and pine based on the linear part 32 of the Index of Injury curve. 3. Initial and steady-state respiration rate (mg C02/g SDW/day), 40 estimated from Y-axis intercept of each monitoring. 4. Q10 for initial and steady-state respiration rates over the temperature 41 ranges indicated. 5. Free sugars and starch concentration (mg/g SDW) in shoots and 52 roots of white spruce during winter. 6. Free sugars and starch concentration (mg/g SDW) in shoots and 53 roots of white spruce during thawing following 0N4S and 2N2S storage. 7. Free sugars and starch concentration (mg/g SDW) in shoots and 54 roots of lodgepole pine during winter. 8. Free sugars and starch concentration (mg/g SDW) in shoots and 56 roots of lodgepole pine during thawing following 0N4S and 2N2S storage. viii LIST OF FIGURES Figure Page 1. White spruce. Changes in the Index of Injury at -30°C (Ij -30) over 31 time: outdoors, in storage and in thawing. 2. Lodgepole pine. Changes in the Index of Injury at-20°C (Ij-20) over 33 time: outdoors, in storage and in thawing. 3. Expt. 1 - Respiration rates for white spruce seedlings as a function 36 of time in -2°C storage. 4. Expt. 1 - Respiration rates for white spruce seedlings as a function 37 of time in thawing after -2°C storage. 5. Expt. 2 - Respiration rates for white spruce seedlings as a function 38 of time in -2°C storage and in thawing. 6. Expt. 2 - Respiration rates for lodgepole pine seedlings as a 39 function of time in -2°C storage. 7. TNC content of white spruce over time for seedlings over-wintered 43 outdoors and in -2°C storage. 8. TNC content of white spruce over time in thawing. 44 9. Carbohydrate depletion for white spruce lifted in December as a 45 function of time in storage and thawing. 10. Carbohydrate depletion for white spruce lifted in February as a 46 function of time in storage and thawing. 11. TNC content of lodgepole pine over time for seedlings over- 47 wintered outdoors and in -2°C storage. 12. TNC content of lodgepole pine overtime in thawing. 49 13. Carbohydrate depletion for lodgepole pine lifted in February as a 50 function of time in storage. 14. Evaluation of white spruce TNC content using different methods for 59 sugar analysis (anthrone vs HPLC). ix Figure Page 15. Evaluation of lodgepole pine TNC content using different methods 60 for sugar analysis (anthrone vs HPLC). 16. Free sugars in white spruce. Changes in tissue concentration of 62 Peak 2, raffinose and sucrose over time. 17. Free sugars in white spruce. Changes in tissue concentration of 64 glucose, Peak 6 and fructose over time. 18. Free sugars in lodgepole pine. Changes in tissue concentration of 66 Peak 2, raffinose and sucrose over time. 19. Free sugars in lodgepole pine. Changes in tissue concentration of 68 glucose, Peak 6 and fructose over time. 20. Relationship between frost hardiness and raffinose content in 70 shoots of white spruce and lodgepole pine. 21. TNC dynamics over time for intact and girdled seedlings. 72 X Appendix 1. Appendix 2. Appendix 3. Appendix 4. Appendix 5. LIST OF APPENDICES Time-table 1. Respiration monitoring 107 Time-table 2. Total non-structural carbohydrates analysis 108 Time-table 3. Translocation 109 Protocol for carbohydrate analysis 110 Temperature of freezing exposure 116 Method for preparation of resins 117 Statistics for Index of injury 119 Statistics for TNC 124 Statistics for translocation 131 xi ACKNOWLEDGEMENTS I wish to express my profound gratitude to my supervisor Dr. Robert D. Guy for the trust and support he consistently gave me. His enthusiasm and patience every time I popped into his office without warning, his availability for constructive discussion and his precious guidance have kept me in focus. Without him, I would have had a hard time to complete this work. Thousands of thanks to Dr. Salim N. Silim for his continuous assistance in the laboratory. His diligent advice and his collaboration in setting up methods have saved me tons of hours of work. His endless determination was inspiring. The numerous questions and arguments we exchanged have helped me to develop a strong sense of confidence and curiosity. My attention to details has improved tremendously by working at his side. I would also like to thank the other members of my supervisory committee, Dr. Edith L. Camm and Dr. P.A. Jolliffe for the enthusiasm and interest they showed towards my project. I thank Sarah Lotz, David Kubien, Kathleen Hebb and many work-study students for technical assistance. There were quite a few samples to weigh and grind. Finally, thanks to Pacific Regeneration Technologies who funded part of my research and to Surrey Nursery for providing the cold-storage facility. Thanks to all the people in Ponderosa Annex B who encouraged me continuously. No one could have dreamed to be better surrounded. 1 INTRODUCTION The last two decades of research in forest tree seedling physiology have been characterized by trials that attempted to correlate physiological states of seedlings with their field survival. One objective was to find tests such as a dormancy index, a frost hardiness index, and a root growth potential index (RGP), that would enable us to evaluate the physiological quality of seedlings. The logic was that if vigorous seedlings were planted when they were in a state of maximum resistance their survival would not be compromised. A dormant frost hardy seedling, which is more resistant to adverse environmental conditions, would have a better chance of establishment. In order to keep the seedlings in this state of maximum resistance (e.g., dormant and hardy) cold storage is used (31). A resistant seedling is easy to define, but a vigorous seedling is not. Plant vigour has been defined in terms of growth, energy reserve synthesis, energy reserve use, bud flush capacity and new root growth capacity. The root system is recognized as the water and nutrient provider and its growth is imperative to insure seedling survival and establishment (55). A vigorous seedling has therefore been defined as having a high RGP. The test for RGP was used to evaluate seedling genetic vigour and effects of nursery practices on seedling quality. It was believed that high RGP was an important indicator of field survival and the key to a successful plantation (59). Over the years, in various circumstances, seedlings with high RGP have failed to survive. Rose (62) mentioned that several reports have shown that RGP does not always correlate with field survival. It is therefore in the interest of the forest industry to find causes of the poor seedling establishment often observed in the interior of British Columbia (B.C.). It has been suggested that events during cold storage may affect the physiology of seedlings (55). According to Rose (62), Dean et al. (16), Loesher et 2 a/.(49), Mattsson (53) and Ronco (61), seedlings with a low carbohydrate content have limited growth when planted in stressful conditions. The situation seems to persist in the entire plant for more than one season. Carbohydrate reserve depletion, observed during cold storage, was suggested as a potential cause of poor field survival but, to date, researchers have not been able to support this claim. It is reasonable to assume that when stress is high, seedlings limit physiological processes associated with growth and mobilize their reserves for maintenance and tolerance to stress. Among the possible stresses that may impair growth in spring, handling, drought, insolation and cold soil temperature are rather usual. If growth is related to the available reserves, then seedlings with a higher carbohydrate content may have a better chance to establish themselves on planting sites. However, if growth is limited by a disruption in physiology due to stress (in other words, due to modification of the normal metabolic sequences leading to growth) then, no matter how substantial the reserves are, growth will not be fully expressed. In any case, a limited number of disturbances may enable the seedlings to maintain their carbohydrate content at a higher level and may favour the expression of growth. The objectives of this research were to explore the physiological behavior in seedlings of two conifer species during freezing storage and in thawing. Different conditions of storage and thawing were expected to create differences in timing for the release of frost hardiness and differences in respiration rates and in carbohydrate content. Seedlings may use their reserves differently if they experience a stressful situation while in a different physiological state. There is much to be understood in this field. Seasonal variation of total non-structural carbohydrate (TNC) in terms of quality and quantity and in response to different treatments may even reflect cyclic changes in metabolism itself. 3 2. LITERATURE REVIEW 2.1 - CARBOHYDRATES IN STORAGE Carbohydrates serve two major purposes in plants. The first one is structural. When used as such, the carbohydrates become unavailable. In plants, structural carbohydrates are mainly recognized as cellulose, hemicellulose and pectin (28). The second purpose for carbohydrates is functional. In this respect, carbohydrates have several roles. As sugars, they may support or are involved in all biosynthesis and maintenance activities. If the plant photosynthesizes more than it immediately needs, the carbohydrates are stored and constitute a reserve for later use. Although sugars and other free carbohydrates may act in storage, starch is the most abundant form of carbohydrate reserve in plants and particularly in the root (25). Besides carbohydrates, proteins and lipids are also important storage forms in trees (39). Lipids in the forms of fats and fatty acids are important in coniferous species and occur mainly in the stem (82). It has long been recognized that carbohydrates are depleted during dark cold storage (11,13,16,52,58,61) and that storage at temperatures above 0°C leads to a faster rate of depletion (57). However, no report has been able to correlate TNC depletion with field survival. This fact raises the question: Is there any modification in reserve quality that might help to explain the poor field survival often reported in the interior of B.C.? 4 2.2 TNC DEPLETION AND TRANSLOCATION Several studies (2,18,53,56,62) related to TNC depletion have focused on just one component of the TNC (i.e., either starch or sugars) or on just one plant part (shoot or root). Therefore, the depletion they report could be either a conversion from starch to sugar, sugar to starch, or a translocation from root to shoot instead of an actual loss of carbohydrates. Chomba (13) monitored TNC depletion in Engelmann spruce (Picea engelmannii (Parry) Engelm.) seedling parts (roots, needles and stem) for two cold-storage durations (two and four months). He reported TNC depletion in all parts with the highest reduction in root tissues. Interestingly, while starch and sugars were depleted in the roots, soluble sugars in the needles seemed to have been maintained at the expense of starch (perhaps meeting frost hardiness requirements). Assuming that all plant parts respired equally, there should have been a decline of the needle sugar content. Chomba suggested that phloem translocation from root to shoot may occur during storage. Mattsson (53) reported a two year study where Scots pine (Pinus sylvestris L) seedlings were monitored for growth. Just before planting, seedlings were analyzed for starch content of the primary needles. The plants that had been cold-stored for 5 months had a starch content of 5% of needle dry weight whereas the content for the plants overwintered outdoors was 22%. After 1 month in the field, both seedling types had the same starch content at 22% and had the same height increment at the end of the first season. However, for cold stored seedlings, the needle length, bud diameter, shoot and root dry weights were consistently smaller. At the end of the second season, the difference in shoot growth was noticeable. From these results, it seems that the stress situation experienced in storage could result in a low starch content. 5 Unfortunately, Mattsson did not measure the root starch content nor the soluble sugar content of any tissues. The foliar starch depletion reported by the author may reflect a conversion to free sugars rather than actual carbohydrate depletion. If so, the question remains as to whether there were enough sugars remaining in the shoot to repair injuries and resume growth following storage. Alternatively, these needs may be met by sugar translocated to the shoot from the root. Several authors (11,16,25,49,61,78) have mentioned that in spring, conifer seedlings rely on their root reserves to heal winter injuries or restore their photosynthetic apparatus after storage. The results of Mattsson (53) and Chomba (13) raise the possibility of carbohydrate translocation from the root to the shoot during storage and thawing. Glerum (25) also speculated on a possible translocation from root to shoot. These speculations are based on the assumption that all tissues respire equally. To the best of my knowledge, no paper has actually reported translocation during storage and/or thawing for conifers. Fisher (21) reported that phloem transport occurs during winter at temperatures as low as -30°C in at least Salix, Tilia and Acer. 2.3 TNC DEPLETION AND RESPIRATION Although there is no doubt that reserves, especially stored carbohydrates, play essential roles in all trees, very little is known about what specific roles these compounds play in survival, growth, and development (49). It is known that by the end of a growing season tree roots will generally contain higher concentrations of carbohydrates and other reserves than any other tissues or organs (13,24). The literature suggests that winter survival depends on adequate reserves. For instance, Glerum and Balatinez (27) mentioned that when growth resumes in spring, cellular metabolism is shifted from a lower to a higher level of activity, which requires much energy, as does mobilization of reserves. In this regard, they proposed that reserves 6 play a major role in respiration, which supplies trees with the energy necessary to resume growth. Most of the time, respiration is invoked to account for the depletion of the reserves in storage (11,16,27,52,57,58,61). However, very few actual measurements of respiration rate have been conducted. Although the importance of respiration is clear (49), no paper has reported that respiration by itself may force reserves past a point of no return, not for seedlings over-wintering outdoors nor for seedlings placed into storage. In 1979, van den Driessche (76) reported respiration rates of cold-stored white spruce (Picea glauca (Moench) Voss) and red pine (Pinus resinosa Ait.) seedlings. He found variation in the respiration rate with both duration of measurement and with temperature. The measurement period varied from 5 hours to one week, after seedlings had been stored for 12 to 14 weeks. His results did not show a correlation between respiration rate and dry matter loss. The total carbohydrate content was not analyzed. The author was unable to relate poor field performance to higher respiration rate in storage. One interesting point brought out by the author is that when seedlings are disturbed, they increase their respiration at first but later stabilize at a lower rate. Ritchie (57) also reported a measured rate of respiration for Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings stored at 0°C. His findings were in concordance with those of van den Driessche (77) for the rate of respiration. However, after calculating this rate in terms of respired sugars, the trend Ritchie found fit the curve of total non-structural carbohydrate (TNC) depletion during the initial month of storage. He mentioned that if this rate had been maintained, the TNC depletion would have decreased linearly and depletion would have been complete by about the seventh month of storage. Rather, TNC decreased exponentially during storage and never got as low as predicted. On this basis, Ritchie supposed that the rate of respiration must have declined, caused by a reduction in substrate concentration or available oxygen 7 or both. So far, very little is known regarding the pattern of respiration and its relationship to reserve depletion during cold-storage. Lifting and packaging operations may stress the plant (52). If so, the respiration rate should be higher when seedlings enter into storage, and later on stabilize at a lower rate because, according to van den Driessche (76), the plant increases its respiration following a disturbance. Beyond normal maintenance costs, each disturbance applied to seedlings may increase respiration and/or inhibit photosynthesis and contribute to depletion of carbohydrate reserves. Disturbance or increased stress may be associated with lifting but also with storage, thawing, transportation, handling and temporary on-site storage at the planting site. These stresses are likely to be cumulative. By planting time, it is easy to imagine that the TNC may have been much depleted. Planting is also stressful and growth under conditions which are very often less than optimal may be quite demanding on carbohydrates. 2.4 TNC DEPLETION AND DISTURBANCE Transplanting shock in the B.C. interior is probably due to a combination of adverse environmental factors. The most common ones would be insolation, drought, soil mineral deficiency and cold soil temperature. No matter what combination, it seems to lead to either death or reduced growth (16,18,28,61) that is, by extension, diminution of the metabolic rate as shown by Ronco (61). Baliski (5) monitored the soil temperature at three planting sites in the Engelmann Spruce-Sub alpine Fir (ESSF) biogeoclimatic zone where seedling establishment is often difficult. He reported that over the course of the growing season, soil temperatures at 10 cm rarely rose above 12°C under natural conditions (i.e., without microsite alterations). The ESSF is known to have a short growing season. DeLucia (18) reported that low soil temperature delays the onset of root and 8 shoot growth and reduces dry matter production and height growth in conifers. Ronco (61) suggested that if the season is too short to develop a sufficient reserve following the first summer in the field, seedling establishment may still be compromised during the second winter. The factors affecting accumulation of reserves could lower levels to the point that planted trees enter the critical over-wintering period with insufficient reserves to maintain themselves. Deans et al. (16), DeHayes et al. (17), Loescher et al. (49), Mattsson (53) and Rose (62), also pointed out that this incapacity to either recover from a negative carbon balance or to develop a sufficient carbohydrate reserve may be a cause of mortality after the second growing season. For injury repair or osmotic adjustment due to maltreatment or drought, the plant needs carbohydrates. The plant also needs carbohydrates for early root establishment and shoot growth. There may be some "competition" for metabolites between growth and osmotic adjustment (7,28). In this case, even if the reserves were not depleted to a critical threshold, the plant would still be in "carbon starvation" (28). Therefore, to measure TNC depletion and to try and correlate TNC content with field survival may be somehow misleading in our quest to understand why seedling establishment is poor on some sites. Although hard data are rare, the literature often emphasizes the importance of more subtle changes in reserve composition, seasonal starch and sugar dynamics, and changes in sugar quality. This more functional approach may yield clues regarding the disruption in plant physiology either due to dark cold storage and/or thawing and/or stressful situations. 9 2.5 EXPRESSION OF PHYSIOLOGICAL STATUS 2.5.1 STARCH AND SUGAR DYNAMICS Despite the controversial viewpoints regarding reserve depletion and its correlation to field survival, there is common agreement that carbohydrates somehow play roles in the establishment of seedlings. Until recently, most investigations have been done on the quantity of the reserves rather than the composition (quality). A change in carbohydrate quality may indicate a modification in the physiological status of seedlings. Glerum (25) mentioned that: "It makes evolutionary sense that vital processes not be dependent on any one pathway or mechanism but that they have several possible solutions at their disposal to ensure survival during periods of stress." It would be efficient for non-structural carbohydrates to have other uses besides serving merely as fuel for maintenance respiration. Contributing to frost hardiness and to osmotic adjustment are likely roles. Guy (29) pointed out that: "All plants that are able to increase freezing tolerance during cold acclimation show significant alterations in carbohydrate levels. . . . Accumulation of free sugars, many of which are very effective cryoprotectants seems almost to be an absolute requirement for induction of freezing tolerance." Up to 1980, the literature reviewed by Levitt (46) showed no consensus in regard to sugars related to freezing tolerance. However, the author reported that many records correlated freezing tolerance with sugar content. McCracken (52) who supported the role of sugars in frost hardy tissue, mentioned that although imperfectly understood, it is obvious that sugars assist in preventing dehydration. Siminovitch (71), supported by Steponkus (75), proposed that the decreased incidence of intracellular ice formation in acclimated tissues may be a result of a lower nucleation temperature due to the 10 presence of sugars, that sugars could minimize the concentration of toxic solute and that they could reduce cell dehydration by osmotic adjustment. Siminovitch (71) reported that for black locust [Robinia pseudo-acacia L), the complete hardiness of the bark was never reached in late fall until, under the influence of persisting low temperature, all the starch was converted to sucrose. Conversely, in early spring, sucrose was converted to starch and simultaneously, an early diminution of hardiness was observed, before any irreversible loss of hardening had occurred. The conversion from starch to sugar in late fall or winter and back to starch in the spring was also reported by Asworth (3), Bonicel et al. (8), DeLucia (18), Gaudillere (24), Glerum (25), Keller and Loescher (38), Little (47), Omi et al. (56), Ritchie (57) and Sakai and Yoshida (64). So far, it seems obvious that sugars are important in the frost hardiness process. Osmotic adjustment is a requirement not only in cold acclimation but in many other stresses and particularly in drought stress. Guehl (28), working on water stress following transplanting, reported that starch concentrations were markedly reduced in the roots of transplanted seedlings showing a very low water potential. Yet, the concentration of soluble carbohydrates did not decrease, suggesting that the stability of the soluble sugars might reflect their "sequestration as osmotically active molecules." Rose (62), in a similar work observed that after being transplanted, there was no decline in starch when seedlings produced new roots and regained their fresh weight. However, seedlings that failed to produce new roots showed a great diminution in their starch content and in their fresh weight 2 weeks after planting. These results suggest that, first, there was a need for osmotic adjustment by seedlings that did not grow new roots and, second, starch most likely was depleted partly to provide the soluble sugars needed for this osmotic adjustment. As proposed by Asworth (3), there may be some physiological significance in the conversion of starch to sugar and back to starch. The shift in metabolism that 11 leads to growth in the spring could be detected through the early loss of hardiness by monitoring the new synthesis of starch. The absence of starch in a newly planted seedling could indicate that the plant is under stress generated by site conditions, instead of generated by the previous storage period, and that there is a need for continuous osmotic adjustment. If sugar and starch dynamics could serve as indicators of plant physiological status, changes in the kinds of sugars present at a given moment could contribute diagnostic information. For instance, some sugars may be more effective than others in preventing dehydration or to protect the cell from accumulation of toxic solutes during winter. Regarding the latter mechanism, Steponkus (75) mentioned that on a molar basis, usually the trisaccharides are more efficient than the disaccharides which are more efficient than the monosaccharides. In particular, the presence of sugars in the raffinose series during winter may be related to their protective qualities. 2.5.2 MODIFICATION OF SUGAR QUALITY Of the soluble carbohydrates, sucrose is the major photosynthetic product in many plants, the principal transportable carbohydrate and the main storage sugar. However, its presence is limited in woody roots. The hexose-reducing sugars, fructose and glucose, are commonly present in roots at higher concentrations than sucrose, but are usually at lower concentrations than in above-ground parts. It has been shown that carbohydrate content is modified in terms of quantity and quality when the plants are hardened off (41,43). Among the list of sugars analyzed in reports concerned with carbohydrate content, raffinose is of interest. This sugar has been identified as a storage carbohydrate in several plant species (2,6,8,12,38,41,43,44,47,51,67,68). Analysis very often indicates that raffinose increases in the fall (or at low temperature) and decreases in spring (or at warm temperature) (see Table 1). Many authors 12 suggest that this trisaccharide is somehow related to frost hardiness (2,47,67,68). Several possible roles have been proposed for raffinose such as involvement in osmotic adjustment (71), modification of lipid-membrane configuration (68,75), and synergy of sucrose action by preventing crystallization (6). Bonicel and de Medeiros (8) reported the presence of raffinose at low temperature and suggested a possible role as some kind of "buffer" to prevent early bud burst. Bernal-Lugo and Leopol (6) reported the presence of raffinose in maize seed embryos. In their study, the depletion of raffinose was associated with a decline in vigour. An increase in sucrose followed the depletion of raffinose with no appearance of galactose. This observation brought the authors to conclude that raffinose was cleaved to sucrose and galactose by an alpha-galactosidase and the galactose was immediately involved in Amadori and Maillard reactions (non-enzymatic carbonyl-amine reactions that take place preferentially in dry systems). Bernal-Lugo and Leopol (6) explained the loss of vigour by a depression of the enzymatic effectiveness of the seed embryo due to these reactions. It is known that galactose is among the most reactive of the hexose sugars with regard to the Amadori reaction (6). Although most of the studies listed in Table 1 associate raffinose accumulation with winter stress, it is possibly a response to other forms of stress. Low temperature, being a major natural stress for boreal species, may simply be the most studied. Lasheen and Chaplin (44) who reported the presence of raffinose during winter in two peach cultivars, did not observe a difference in the concentration of this or any other sugar between the two cultivars although one was known to be much hardier. Marutyan (51) made a similar observation for two grape cultivars. Although frost hardiness is a mechanism of protection important for boreal species, the main mechanism that enables the woody plant to minimize its susceptibility to environmental stress is dormancy. Besides the onset of winter, other stresses such as drought, mechanical strain, flooding, cold or hot spells, and nutrient 13 deficiencies can also adversely affect seedling growth. It is known that a sufficient intensity and duration of a stress will trigger the biochemical modifications that lead to dormancy. Keller and Loesher (38) reported the appearance of raffinose at the beginning of the dormant period and its disappearance at the end of the period. So far, there is no consensus on the role of raffinose. Its accumulation need not necessarily be associated with the development of frost hardiness. Bonicel and Medeiro (8) suggested that raffinose could maintain dormancy through a warm spell; Bernal-Lugol and Leopold (6) suggested that raffinose may maintain vigour in a case of really low osmotic potential; and Siminovitch (71) suggested that raffinose may play a role in osmotic adjustment. It is well known that a water stress applied to seedlings induces dormancy and that plants under water stress may also show osmotic adjustment. Loesher et al. (49) mentioned that drought may not be completely inhibitory to photosynthesis, but may inhibit shoot growth and actually increase carbohydrate reserves. If raffinose accumulation could also be induced by drought stress, this could then be an indication of its participation in osmotic adjustment, not unlike the accumulation of proline and other compatible solutes. 14 TABLE 1. Literature summary of presence of raffinose under adverse conditions. SPECIES TISSUES CONDITIONS AUTHORS Betula pendula Roth Xylem In spring, increase at 0°C stable at 10°C decrease at 21°C Sauter & Ambrosius(68) Prunus avium L 'Bing' Shoot, spurs, root Increase in fall, decrease in spring Keller & Loescher(38) Abies balsamea (L.) Mill. Needles Appears in fall, decreases in spring Little(47) Vitis vinifera (L) Shoot Present in winter Marutyan(51) Zea mays L. Embryo Exponential depletion when stored at 30°C Bernard-Lugo & Leopold(6) Prunus persica (L) Batsch Bark, Leaves, Buds Increase with dormancy onset, decrease with dormancy release. Lasheen & Chaplin(44) Pinus stobus L Needles Highest during winter Hinesley ef al. (34) Pinus virginiana L. Needles Highest during winter Hinesley ef al. (34) Pinus sylvestris L Needles Increase at short-day 15/10°C, decrease at short-day 20/20°C Aronsson et al.(2) Needles Low in summer, high in winter Fischer and Holl (22) Juniperus virginiana L Foliage Highest during winter Hinesley etal. (34) xCupressocyparis leylandii Dallim. Foliage Highest during winter Hinesley etal. (34) Populus trichocarpa x P. deltoides cv. Kaspalje Axillary buds Appears in fall when temperature drops to 10°C Bonicel & de Medeiro(8) Populus x canadensis « r o b u s t a » Stem In storage, apparent between -2°C and 5°C Sauter(67) Picea mariana (Mill.) B S P . Needles, root Appears in fall, decrease in spring Lambany & Langlois(43) Picea abies (L.) Karst Needles Increase at short-day 15/10°C, decrease at short-day 20/20°C Aronsson et al.(2) Root Increase slightly after exposure to short day (SD), slightly more to low temperature (LT) and much more to both SD and LT. Wiemken and Ineichen (80) Picea glauca (Moench) Voss. Needles, root Temperature air/soil =21/10°C, present in roots only. Temperature air/soil = 1/1 °C, present in roots and needles. Appears in fall, decrease in spring Chalupa & Frazer(12) Lambany & Langlois(43) 15 2.6 - CONCLUSION Based on the previous review, it appears that there is a depletion of TNC during dark cold-storage and that it may be due to normal respiration or stimulated by disturbance. Researchers report variation in carbohydrate content whether for starch or for soluble sugars or for both. There are indications that the shoot and root differ in their reserve content in terms of quantity and quality as well as in the way they use their carbohydrates. Carbohydrate dynamics could be related to the frost hardiness cycle and perhaps other physiological characteristics of the seedlings. The reports cover a large part of a year governing biological cycles of growth, development, dormancy, and hardiness. Events in these cycles are apparently cued by intrinsic and/or environmental factors (e.g., photoperiod) but there is little information explaining why and how they work. It is commonly agreed that the physiology of the seedling must be attuned with its natural environment to increase its chance of survival. The goal of this research was to verify the physiological activity of seedlings during freezer-storage and thawing by monitoring seedling carbohydrate content along with respiration and frost hardiness level. This approach may indicate metabolic activities at a higher level than presumed, and/or suggest possibilities to improve freezer-storage and handling. Conversely, it may indicate that the actual freezer-storage practices do not impair the metabolism of the seedlings and therefore do not need improvement. However, even if there is no problem with freezer-storage itself, the subsequent thawing period may be responsible for substantial carbohydrate depletion. Because the chilling requirement has been met by the time seedlings come out of storage, they are no longer in rest but are merely quiescent (i.e., on "standby"). This means that all that is needed to resume growth is a favorable temperature. The dehardening process is of interest because it may indicate when the seedlings are 16 shifting from a low maintenance metabolism to an active growth metabolism; the latter being much more costly in terms of energy use. 2.7- HYPOTHESES This review has lead to the following hypotheses: 1. Hi : Respiration rate is higher at the beginning of cold-storage and later stabilizes at a lower rate as a function of temperature. Ho: Respiration rates are constant irrespective of time in storage. 2. Hi : The temperature dependence of respiration rates in storage and thawing does not have the theoretical Q10 of between 2 and 2.5. Ho: Between storage and thawing, the respiration rate increases as a simple function of temperature (i.e. the Q10 should be near 2). 3. Hi : Rates of carbohydrate depletion and respiratory CO2 evolution are not well matched implying that maintenance respiration is not the only fate of carbohydrate reserves (e.g., incorporation into other products), or that all reserves are not necessarily carbohydrates. Ho: Rates of carbohydrate depletion and respiratory CO2 evolution are well matched. 17 4. Hi : Patterns of reserve depletion in girdled seedlings are different from intact seedlings, suggesting that reserve depletion is organ specific, with roots acting as the major storage location and providing other plant parts with carbohydrate as required through the dormant period. H 0 : Patterns of reserve depletion in girdled seedlings are the same as in intact seedlings, suggesting that there is no significant root to shoot translocation through the dormant period. The research was designed to test the stated null (Ho) hypotheses and to go beyond that by describing some internal activities in seedlings as reflected by changes in reserve composition. 18 3.0 MATERIAL AND METHODS Picea glauca (Moench) Voss. (white spruce; B.C. Ministry of Forests seedlot #29170; 690m elevation; 53°05'N; 122°05'W) and Pinus contorta Dougl. (lodgepole pine; seedlot #14387; 1050m elevation; 52°43'N; 121°39'W) were chosen as sub-boreal species for the investigation. During the first week of April 1992, the seedlings were sown at UBC Forest Nursery and grown using standard nursery practices for the interior British Columbia provenances. Three studies were undertaken. Study #1 was designed to test hypotheses 1, 2 and 3 and to describe changes in the balance between starch and sugar. The respiration rates of seedlings as well as their TNC content and degree of frost hardiness were monitored during the storage and thawing periods. Study #2 explored the dynamics of sugar composition over time. Study #3 was designed to test hypothesis 4; that is, the possible translocation of free sugars from the root to the shoot. 3.1 STUDY 1 - RESPIRATION AND RESERVE DEPLETION 3.1.1 RESPIRATION RATE MEASUREMENTS Respiration rates of stored seedlings were determined by measuring evolution of carbon dioxide (CO2) with time. The method was adapted from Clegg et al. (14), van den Driessche (76) and Ehleringer & Cook (20). Glass bottles of 1.024 L were used as individual closed systems for seedling respiration. Rubber-stoppers equipped with small glass tubes were used to seal the vessels. A septum placed at the end of the glass tube allowed removal of gas samples for injection onto an infra-red gas analyzer (IRGA) circuit (Beckman Model 215A, Fullerton CA). The carrier gas was 19 nitrogen at a flow rate of 1L/min. A standard curve was established for every day of sampling using 1 to 5 mL volumes of 1000 pL/L CO2. The bottles were covered with aluminum foil to prevent exposure to light. They were all tested for leakage with 10,000 ppm CO2 over a 15 day period prior to the beginning of storage. For gas removal, 1 mL plastic syringes were used. They were periodically tested for leakage and replaced as needed. For transportation from the storage facility to the IRGA, the needle tips were stuck into a rubber-stopper. Seedlings chosen for respiration monitoring were selected for uniformity on the basis of height and diameter. A sub-sample was used for initial dry weight estimation. Roots were carefully washed in running cold water and the fresh weight of every seedling was recorded. Seedlings for respiration monitoring had their roots wrapped in a moist paper towel to prevent desiccation. The seedlings were brought to the storage facility and were gently placed into the bottles, one seedling per bottle. All the bottles were placed in a waxed box for storage. The sub-sample was oven-dried at 60°C for 48 hours for dry weight measurements. Air samples were taken from the bottles on a daily basis whenever possible. Air was replenished when CO2 levels reached between 3000 and 6000 pL/L in the jars (about every 3 days at first and every 7 days later on). Two main experiments were conducted. Experiment 1 began in December 1992 with white spruce seedlings only, lifted from the UBC Forest Nursery (see Appendix 1 for Time-Table 1 related to respiration monitoring). Fifteen seedlings were used to investigate respiration in -2°C storage. To confirm that long-term trends in respiration of bottled seedlings were not different from those of cartoned seedlings, a comparison between the two was set up. Beginning in March, a sub-set of five seedlings from Experiment 1 were cut at the collar zone and the shoots were introduced into 5 new bottles. Five additional shoots were cut off fresh seedlings stored in December and were also placed into bottles. Respiration was measured for 20 one week. Both sets of shoots respired evenly suggesting that the respiration of the bottled seedlings was indeed representative of the normal trends in freezer-storage. Following this observation, it was decided that seedlings cold stored for four months could be used later in April for respiration monitoring in thawing so that Experiment 1 could continue. Respiration of the remaining ten seedlings was monitored until the end of March. At this time, these seedlings were removed from the bottles and grown in a mist chamber to check their vitality. At the end of April 1993, to monitor respiration during thawing, two sets of 10 new seedlings each were lifted from their cartons after 4 months of freezer-storage. The preparation of these seedlings was as above and was performed at +2°C to minimize disturbance. Thereafter, one set was kept at +2°C and the other was transferred to +7°C. For Experiment 2, 15 seedlings of white spruce and 15 of lodgepole pine were lifted from the nursery in February and prepared as above. As opposed to Expt.1, where two sets of seedlings were used, the same seedlings were monitored throughout freezing and thawing storage for respiration in Expt. 2. They were placed in the bottles and kept for two months in freezing-storage. In April they were transferred to the thawing rooms. Ten lodgepole pine seedlings were monitored for respiration at +2°C thawing while the other five were grown to check vitality (as it was not known whether pine behaved like spruce). Of the 15 white spruce seedlings, 10 were transferred to the +2°C room and the remaining five were transferred to +7°C. To calculate respiration rates, CO2 concentration in the air sample was estimated by comparison of peak heights to a standard curve. Concentrations were then converted to mols CO2 using the gas law and the known bottle volume. Mols of CO2 were further converted into mg carbohydrate equivalent per g Structural Dry Weight (SDW), where SDW was the total dry weight minus TNC content. The structural dry weight was used to minimized the variation in total dry weight due to 21 TNC depletion over time. SDW however includes components which are not strictely structural, e.g. proteins, lipids, fatty acids, ect.. Q10 ratios were calculated from : Qio = ( K 2 / K i ) 1 0 / < T 2 - T 1 > , where Ki and K2 represent respiration rates at temperatures Ti and T2, respectively. To test hypothesis 1, TableCurve software (Jandel Scientific, San Rafael, CA) was used to fit exponential decay equations (± 95% confidence intervals (c.i.)) to the respiration data. Initial and steady-state respiration rates were calculated at the intercept with the Y-axis. The rates were considered to be different when the confidence intervals did not overlap. The equations were then used in section 4.2.2 to predict TNC depletion over time and the results were compared graphically to the measured TNC depletion. Hypothesis 2 on Q10 could not be tested statistically. In every treatment-combination, except for seedlings lifted in February and transferred to the +2°C thawing regime, different seedlings were used in freezer-storage and in the +2°C and +7°C thawing regimes. Thus, Q10 values were evaluated between -2°C and +2°C, and between +2°C and +7°C, using the respiration rates obtained from the Y-axis intercepts at each temperature. 3.1.2 TNC CONTENT On December 21 1992, white spruce and lodgepole pine seedlings were randomly lifted and wrapped with cellophane around the root plugs into bundles of 10 seedlings. The bundles were then placed inside a waxed box containing a plastic-lined paper bag. They were oriented with the roots toward the ends of the box, and shoots overlapping in the middle. Boxes were taken the same day for dark freezer 22 storage at -2°C for 4 months. The storage facility was provided by Surrey Nursery (B.C. Forest Service). The air temperature inside the boxes in storage was monitored with data-loggers and reached the -2°C target temperature within the first two days of storage (although the plugs took a full week to freeze completely). Surrey Nursery constantly monitored the cold room temperature which remained at -2°C throughout the entire storage period. On February 19 1993, a second lot of seedlings was lifted for dark freezer storage at -2°C. This treatment created a second condition where the seedlings spent an additional two months in their containers outdoors at the UBC Nursery followed by two months in dark freezing storage at Surrey. On April 26th 1993, 4 months after the first lifting, seedlings from all treatments were divided into two groups and transferred for thawing under two different temperature regimes: A) +2°C for 6 weeks in a dark cold room and B) +7°C for 6 weeks in a dark growth chamber. The temperature treatments for thawing were chosen as representative of actual industrial practice. For TNC measurements, 10 seedlings of each species were harvested prior to storage in December 1992 for initial starch and sugar content determinations. Thereafter, 10 seedlings of each species were harvested every month at the nursery and in freezer-storage (with the exception that there was no harvest at month 4 for seedlings at the nursery as they had resumed growth). For the thawing regimes, seedlings were harvested two times over a 6 week period. Refer to Appendix 1 for Time-Table 2 related to total non-structural carbohydrates (TNC) analysis. Hereafter, the experimental treatments and sampling times will be designated as follows for seedlings harvested prior to thawing: 0N0S : initial sampling, no storage 0N1S : 0 months outdoors at the Nursery and 1 month in Storage 23 0N2S : 0 months outdoors at the Nursery and 2 months in Storage 0N3S : 0 months outdoors at the Nursery and 3 months in Storage 0N4S : 0 months outdoors at the Nursery and 4 months in Storage 1N0S : 1 month outdoors at the Nursery and 0 months in Storage 2N0S : 2 months outdoors at the Nursery and 0 months in Storage 3N0S : 3 months outdoors at the Nursery and 0 months in Storage 2N1S : 2 months outdoors at the Nursery and 1 month in Storage 2N2S : 2 months outdoors at the Nursery and 2 months in Storage Treatments and samples from thawing are designated as follows, where the 4S or 2S at the start of each code refers to 4 months Storage or 2 months Storage prior to the thawing periods (i.e., from 0N4S and 2N2S respectively): 4S2T2 : 2 weeks in Thawing at +2°C 4S6T2 : 6 weeks in Thawing at +2°G 4S2T7 : 2 weeks in Thawing at +7°C 4S6T7 : 6 weeks in Thawing at +7°C 2S2T2 : 2 weeks in Thawing at +2°C 2S6T2 : 6 weeks in Thawing at +2°C 2S2T7 : 2 weeks in Thawing at +7°C 2S6T7 : 6 weeks in Thawing at +7°C Seedlings were washed and separated into root and shoot components and then rapidly frozen in liquid nitrogen. After freeze-drying, dry weights were recorded and both parts were reduced to powder using a Fritsch planetary Micro Mill. Approximately 50 mg of dry shoot tissue and 35 mg of dry root tissue were used for sugar and starch extraction. Five mL of methanol:chloroform:water (M:C:W, 12:5:3, 24 v:v:v) was added to the tissue in a culture tube while vortexing. Each tube was capped and the preparation was left overnight at +2°C. The following morning, the samples were centrifuged, the supernatant was pipeted off into another test tube and the pellet was re-extracted twice with 4 and 3 mL of M:C:W. The pellet was then stored at -10°C for later starch analysis and 5 mL of deionized water was added to the combined supernatants. After phase separation, the upper aqueous layer containing the sugars was pipetted off into a 20 mL vial. The lower chloroform phase was discarded. The aqueous phase was reduced to dryness at room temperature using a Savant SpeedVac Plus SC 11 OA evaporator connected to a Savant RVT 4104 refrigerated vapor trap. The residue was redissolved in 3 mL of deionized water and frozen for later sugar analysis (for the detailed procedure of carbohydrate analysis, refer to Appendix 2). Starch content was determined enzymatically using glucose oxidase-peroxidase + o-dianisidine dihydrochloride (colour agent) in a spectrophotometric assay. The method was modified from Chomba (13), Ebell (19), Haissig and Dickson (30) and Rose et al. (63). Soluble sugar (free sugar) content was determined using the anthrone method adapted from Chomba (13), Jermyn (36) and Yemm and Willis (81). One-way ANOVA was used to test the significance of TNC depletion over time in freezer-storage and outdoors at the nursery. For thawing, Two-way ANOVA was used to test the significance of TNC depletion over time and the effect of temperature. 3.1.3 FROST HARDINESS MEASUREMENT The degree of frost hardiness was determined using the electrolytic conductivity method described by Glerum (26). Shoot material was harvested and washed thoroughly under running water, rinsed in distilled-deionized water and patted dry with a clean paper towel. Needles were removed and cut into 3 mm pieces. The tip and 25 base of each needle were discarded. The needle pieces were evenly distributed into a given number of 20 mL vials according to the number of test temperatures used plus one control. Water was added to immerse the needles (-0.5 mL). The control vial was kept in the dark at +2°C while the other vials were exposed to the test temperatures in a programmable freezer. The temperature was set to drop at a rate of 5°C per hour, dwelling for 1 hour at every target temperature. After 1 hour exposure to the test temperature, one vial was taken out of the freezer and kept with the control at +2°C for thawing. When the test was finished, 12 mL of deionized water was added to each vial and they were left overnight to allow the diffusion of electrolytes. The following morning, the electrical conductivity (EC) of each sample was recorded using a portable conductivity meter. The vials were then half-immersed in a 90°C water bath for 1 hour to kill all tissue. Thereafter, they were again left overnight in the dark at room temperature. The following morning, the electrical conductivity was again recorded. Index of Injury was calculated as follows: a. Relative conductivity of control (RC control) RC control = EC control X 100 EC control killed b. Relative conductivity of frozen samples (RC frozen) RC frozen = EC frozen X 100 EC frozen killed , 26 c. Index of Injury (Ij) Ij _ RC frozen - RC control 1 - RC control 100 Five seedlings of both white spruce and lodgepole pine were used for each frost hardiness test. The initial evaluation was done prior to freezing-storage. Thereafter, tests were done at 2 and 4 months for seedlings in storage and at 2 and 3 months for seedlings at the nursery. During thawing, seedlings were sampled at 2, 4 and 6 weeks for both +2°C and +7°C treatments. Test temperatures (see Appendix 3 for a complete listing) were chosen based on each previous set of results so that it would be possible to remain within the linear part of the Index of Injury curve, allowing the calculation of LT50 (i.e., the lethal temperature expected to cause 50% cell damage, as indicated by a 50% loss of electrolytes) One-way ANOVA was used to test the significance of changes in Ij over time for seedlings in freezer-storage and those overwintered outdoors. For thawing, Two-way ANOVA was used to test the significance of dehardening over time and the effect of temperature on dehardening rate. 3.2 STUDY 2 - CHANGES IN SUGAR QUALITY Changes in the types and amounts of major individual sugars over time were investigated using High Performance Liquid Chromatography (HPLC). Approximately 50 mg of shoot and 35 mg of root tissue were extracted with M:C:W as in Study 1. Prior to the addition of M:C:W, 0.8 mg of xylitol was added as an internal standard in 27 order to evaluate the recovery of the sugars in the extraction process. Extracts were kept frozen until used. Cation and anion exchange resins were used to clean extracts prior to injection onto the HPLC column (For details on resin preparation, see Appendix 4). The cation resin (Dowex-50W) was activated and converted into the H + form and 1.5 mL was loaded into a 3 mL syringe barrel fitted with a glass frit at the bottom. Another glass frit was placed on the top of the resin bed to prevent disturbance when loading and eluting the extract. The anion resin (Dowex-1) was activated and converted into the formate form and 1.5 mL was loaded into a 3 mL syringe barrel as above. These resin columns were then assembled in series with the Dowex-50W on top. One mL of the sugar extract was loaded onto the cation exchange resin and eluted with deionized water until a final volume of 15 mL was collected from the bottom of the anion exchange resin. The samples were then evaporated to dryness and dissolved in 1 mL of filtered (0.45 um) distilled-deionized water. They were kept frozen for later analysis. For HPLC analysis (23,37,72), the sugar samples were thawed, passed through a 0.22 (im filter and injected into the HPLC (Waters 600E, 20 uL sample loop; Waters Inc., Milford, MA) equipped with a RID-6A Differential Refractometer (Shimadzu Corp., Japan) operated at 16 X 10"6 RIU. The separation of sugars was accomplished via a 300 x 7.8 mm Phenomenex (Torrance, CA) RCM column preceded by a Waters Nova-Pak guard column. The temperature was maintained at 80°C. Distilled deionized filtered water, degassed by pre-heating and bubbling with helium, was used as the solvent-carrier at a flow rate of 0.6 mL/min. Pure standard sugar solutions were also injected to establish retention times and calibration curves (based on peak area). This information was checked daily. Some peaks could not be assigned, but were still quantifiable as the response of the detector is primarily mass dependant. 28 Descriptive statistics were used in study 2 as it was designed to be strictly a qualitative investigation. However, a paired t-test was used to test the significance of the discrepancy observed in the total sugar yield as determined by the anthrone and HPLC methods. 3.3 STUDY 3 - RESERVE TRANSLOCATION For the reserves translocation experiment (Study 3), containers of white spruce were brought from the nursery to the laboratory on December 22 1992. The containers were kept in a refrigerator at +5°C while individual seedlings were prepared by peeling off a 2 mm ring of the bark and cambium above the collar zone. To prevent both desiccation and fungal attack, the exposed xylem was wrapped with several layers of Parafilm before returning each seedling to the container. The following morning, the seedlings were bundled and packaged as in Study 1 and taken to Surrey Nursery for freezer storage. The TNC content was measured on 10 seedlings every month during the 4 month storage period and every two weeks during the thawing period. The thawing treatment used was a combination of 4 weeks at +2°C followed by 2 weeks at +7°C. Intact white spruce seedlings were also submitted to the same thawing regime. Starch and free sugar were analyzed as described above in Study 1 for both girdled and intact seedlings. Refer to Appendix 1 for Time-Table 3 related to TNC analysis -Translocation. Treatments and sampling times for seedlings harvested prior to thawing are designated as in Study 1 (i.e., 0N0S, 0N1S, 0N2S, 0N3S and 0N4S). Samples for thawing are similarly coded: 4S2T2 : 2 weeks in Thawing at +2°C 4S4T2 : 4 weeks in Thawing at +2°C 4S6T2\7 : 6 weeks in Thawing first at +2°C and then at +7°C 29 In both cases the prefix G- is added to indicated girdling. For example: G-4S4T2 : Girdled, 4 months in Storage followed by 4 weeks in Thawing at +2°C Two-Way ANOVA was used for seedlings in storage and in thawing to test differences in TNC depletion over time. Results from whole seedlings, shoots and roots were tested independently. 30 4. RESULTS 4.1 FROST HARDINESS When frost hardiness was measured, the range of test temperatures chosen was based on each previous set of results. However, in some cases, a few seedlings were hardier than expected and were not much damaged by exposure to any of the chosen temperatures. In other cases, the dehardening of the seedlings was faster than expected such that a few seedlings were excessively damaged after exposure to every temperature. As a result, estimation of the LT50 was not possible for every seedling, leading to missing values and bias in the statistical analysis. To solve this problem, statistical tests were run directly on the Index of Injury (Ij) data for one temperature only; namely -30°C for spruce (lj-30) and -20°C for pine (lj-20). These temperatures were chosen because they were tested all the way from entering storage until the end of the thawing period and there were no missing values. To give a better view of the dehardening process, the LT50 data are still presented in table form. When white spruce entered -2°C storage in December, the lj-30 was 25.8%. Over the 4 months of storage, the lj-30 changed significantly, going down after 2 months (less damage) and up again at month 4, without reaching the values recorded in December (Figure 1). Based on these results, it seems that spruce can not only maintain but may even deepen its hardiness while in storage. However, the same pattern was not clear for the LT50 data. In December, the temperature causing 50% cell damage was seen to be below -65°C (the lowest test temperature available), whereas after 4 months of storage the LT50 was -51.6°C (Table 2), suggesting some loss of hardiness. 3 1 Figure 1 White spruce. Changes in the Index of injury at -30°C (lj-30) over time: outdoors, in storage and in thawing. Error bars are for SEM. Triangle is for seedlings left to overwinter on the nursery compound. Circles represent seedlings lifted in December and freezer-stored for 4 months prior to thawing at +2 or +7°C (open and closed symbols, respectively). Squares represent seedlings lifted in February and freezer-stored for 2 months prior to thawing at +2 or +7°C (open and closed symbols, respectively). 32 Table 2 LT50 estimation for spruce and pine based on the linear part of the Index of Injury curve. Treatment Spruce Pine n LT50 (°C) n LT50 (°C) ONOS n = 5 <-65°C n = 4 -26.0 0N2S n = 5 < -65°C n = 5 -17.6 0N4S n = 5 -51.6 n = 5 -21.4 2N0S n = 3 -40.9 n = 4 -20.7 2N2S n = 5 -62.0 n = 5 -24.7 3N0S n = 5 -15.2 n = 5 -13.6 4S2T2 n = 3 -38.5 n = 5 -24.1 4S2T7 n = 5 -25.7 n = 3 -11.0 4S4T2 n = 5 -32.7 n = 5 -12.6 4S4T7 n = 5 -16.2 n = 5 -5.6 4S6T2 n = 5 -25.7 n = 5 -9.9 4S6T7 n = 5 -19.1 n = 5 -7.3 2S2T2 n = 5 -45.6 n = 5 -16.6 2S2T7 n = 5 -26.6 n = 3 -5.1 2S4T2 n = 3 -39.0 n = 5 -11.0 2S4T7 n = 5 -21.1 n = 5 -5.4 2S6T2 n = 3 -23.9 n = 5 -10.2 2S6T7 n = 5 -17.9 n = 5 -7.4 When spruce seedlings were kept outdoors at the nursery, the lj-30 increased to 48.1% after two 2 months (2N0S) while the LT5 0 reached -40.9°C. There is no doubt that the plants had begun to deharden. This time, however, the process was clearly reversible, as shown by the seedlings transferred into -2°C storage (2N2S) where they redeveloped a significant degree of hardiness (Figure 1 and Table 2). Meanwhile, the seedlings that remained outdoors (3N0S) continued to deharden and were severely damaged when exposed to -30°C (lj-30 =79.8%; LT50= -15.2°C). When pine entered -2°C storage, the lj.20 was 38.7% (Figure 2). Over the 4 months of storage, the degree of hardiness based on lj-20 changed significantly (P=0.0001). In contrast to spruce, the pine seedlings released their hardiness over the first two months in storage and regained it between months 2 and 4. 33 Figure 2 Lodgepole pine. Changes in the Index of injury at -20°C (lj-20) over time: outdoors, in storage and in thawing. Error bars are for SEM. Triangles are for seedlings left to overwinter on the nursery compound. Circles represent seedlings lifted in December and freezer-stored for 4 months prior to thawing at +2 or +7°C (open and closed symbols, respectively). Squares represent seedlings lifted in February and freezer-stored for 2 months prior to thawing at +2 or +7°C (open and closed symbols, respectively). 34 The LT50 values followed the same trend (Table 2), being -26°C in December, rising to -17.6°C after two months in storage and then falling back down to -21.4°C by 4 months. However, as previously mentioned, no statistical analyses were run on LT50. When pine seedlings were kept outdoors at the nursery for two months, although they lost hardiness, it was not as pronounced as when they were in -2°C storage (see 2N0S vs 0N2S, Figure 2 and Table 2). After being transferred to storage (2N2S), the pine seedlings, as with spruce, deepened their level of hardiness (lj-20 =30.2; LT50 = -24.7°C) while those kept outdoors (3N0S) released it (lj-20 = 75.43; LT50=-13.6°C). Irrespective of when they were placed into freezer-storage, once transferred to the +2°C and +7°C thawing regimes, both spruce (Figure 1) and pine (Figure 2) showed a significant temperature effect on their rate of dehardening, which was faster in the first two weeks of exposure to +7°C. After 6 weeks of thawing, for all storage-thawing combinations except 4S6T2 in spruce and pine, the Index of Injury in spruce (lj-30) and pine (lj-20) had reached around 80% and 95%, respectively. In the 4S6T2 storage-thawing treatment combination, the Index of Injury remained lower (lj-30 = 67% for spruce and at lj-20 = 87% for pine). Likewise, the LT50 values at the end of the thawing regimes were similar for the two previous storage conditions (2S and 4S) but varied between thawing temperatures. Whereas in the +2°C regime, the LT50 values for spruce and pine were evaluated at -24.5°C and -10°C respectively and the LT50 values for +7°C thawing were -18.5°C and -7.3°C for spruce and pine respectively. 35 4.2 CARBOHYDRATE RESERVES 4.2.1 RESPIRATION In Experiments 1 and 2, for spruce and pine, the seedlings showed a higher initial rate of respiration when first placed in bottles at -2°C and the rate declined over time to reach a steady-state after nearly 20 days. When transferred to the thawing rooms (+2°C and +7°C) a similar pattern of initial and steady-state respiration was observed. At the end of the thawing period, when the seedlings were taken out the bottles, pine had developed significant grey mold (Botrytis sp.). As it is not possible to know how much the fungi contributed to the measurements, the data for thawing in pine are omitted. Curves shown in Figures 3 to 6 represent exponential decay functions fitted to measured respiration rates for every treatment. During these experiments, it was observed that whenever seedlings were disturbed in storage (e.g., moved about or exposed to blowing air to accelerate air exchange), they increased their respiration for a few days and returned to the steady-state thereafter. The duration and degree of stimulation appeared to be related to the severity of the disturbance. For instance, one day, the seedlings were moved to room temperature to take air samples. This operation lasted not quite an hour. The respiration rate was increased over the next two days, returning to the steady-state after 4 days. Another time, to replenish the air in the bottles, a blower was used at room temperature (20°C). The increase in respiration rate noted was greater and lasted a little longer. When later on the blower was used inside the cold room at -2°C, there was also an increase in respiration but it was not as pronounced as for the two other disturbances. 36 h 4 30 40 50 Days From Lifting 100 Figure 3 Expt.1 - Respiration rates for white spruce seedlings as a function of time in -2°C storage. Seedlings were lifted Dec. 22, 1992. CO2 evolution is shown on the right axis, with CH2O equivalents on the left. General form of the exponential decay is y = a + b e ( " x ' c ) , where values and S.E.M. for a, b and c are : a = 0.3984 ± 0.0247 b = 2.5341 ± 0.2534 c = 1.4094 ± 0.2135 Dotted lines represent lower and upper limits for 95% confidence intervals. 37 Thawing at +2°C Thawing at +7°C h 8 4 H 5 2 H h 7 o h 5 - - - - - — L 10 20 4 S h 1 30 40 50 Days In Thawing F i g u r e 4 Expt .1 - R e s p i r a t i o n rates for white s p r u c e s e e d l i n g s a s a f u n c t i o n of t ime in thawing after -2°C s t o r a g e . Seedlings were lifted Dec. 1992, but are not the same as those used in Fig. 3. C O 2 evolution is shown on the right axis, with C H 2 O equivalents on the left. General form of the exponential decay is y = a + b e ' " x / c ' , where values and S .E .M. for a, b and c are : In+2°C thawing: a = 1.2296 ±0.1200 b = 2.1804 ± 0.2161 c = 8.9892 ± 2.1234 Dotted lines represent lower and upper limits for 95% confidence intervals. In+7°C thawing: a = 1.4824 ±0.0827 b = 3.1665 ± 0.2210 c = 6.1137 ± 0.8723 DastvDot4Dashed lines represent lower and upper limits for 95% confidence intervals. 3 8 Storage (-2°C) 4 H 3 H •2 2 03 Figure 5 • Thawing (+2°C) ° Thawing (+7°C) h 6 h 4 o 5 10 n r 20 30 40 50 70 ~ i i 1 1 80 90 100 110 120 Days From Lifting Expt.2 - Respiration rates for white spruce seedlings as a function of time in -2°C storage and thawing. Seedlings were lifted in Feb. 1993 and the same seedlings were monitored throughout. C O 2 evolution is shown on the right axis, with C H 2 O equivalents on the left. General form of the exponential decay is y = a + b e ' ' x , c ' , where values and S.E.M. for a, b and c are : In-2°C storage: a = 0.4567 ± 0.0635 b = 1.1531 ± 0.0967 c = 10.5831 ± 2.4210 Dashed lines represent lower and upper limits for 95% confidence intervals. In+2°C thawing: a = 1.0997 ± 0.0850 b = 1.5376 ± 0.1678 c = 8.2689 ± 2.0725 Dotted lines represent lower and upper limits for 95% confidence intervals. In+7°C thawing: a = 1.6225 ±0.1123 b = 3.2518 ± 0.3192 c = 5.7191 ± 1.1217 Dash-Dot-Dashed lines represent lower and upper limits for 95% confidence intervals. 39 Figure 6 Expt.2 - Respiration rates for lodgepole pine seedlings as a function of time in -2°C storage. Seedlings were lifted in Feb.1993. C 0 2 evolution is shown on the right axis, with CH2O equivalents on the left. General form of the exponential decay is y = a + b e ( " x ' c ) , where values and S .E .M. for a, b and c are : a = 0.6031 ± 0.0900 b = 1.4535 ± 0.1626 c = 8.6159 ± 2.5263 Dotted lines represent lower and upper limits for 95% confidence intervals. 40 The Y-axis intercepts (with 95% ci.) were used as values for initial and steady-state respiration rates. In Experiments 1 and 2, spruce respiration rates (initial and steady-state) increased with exposure to higher temperature (Table 3). However, in Experiment 1, the difference between initial rates at -2°C and +2°C might not have been significant (ci. did overlap). TABLE 3. Initial and steady-state respiration rates (mg of C02/g SDW/day), estimated from the Y-axis intercept of each monitoring. (95% confidence intervals are in brackets) SPECIES EXPT. INITIAL STEADY-STATE -2°C +2°C +7°C -2°C +2°C +7°C SPRUCE 1 4.30(0.41) 5.00 (0.49) 6.82 (0.45) 0.58 (0.04) 1.80 (0.18) 2.17(0.12) SPRUCE 2 2.36 (0.23) 3.87(0,37) 7.15 (0.63) 0.67 (0.09) 1.61 (0.12) 2.37 (0.16) PINE 2 3.06 (0.42) — — 0.88 (0.13) — — Between the species-Expt. combinations, results showed that in -2°C storage, the initial respiration rates were different for every case. Pine seedlings, lifted in February (Expt. 2), had a significantly higher steady-state rate of respiration compared to spruce lifted in December or February. In the +2°C thawing regime, spruce seedlings freshly removed from their cartons after 4 months of freezer-storage exhibited a slightly higher initial rate of respiration than already bottled seedlings transferred from storage (2N2S) to thawing. This could be due to higher disturbance from unpacking, root washing and introducing the fresh seedlings into bottles. However, when seedlings were exposed to +7°C thawing, the initial respiration rates did not differ between these two treatments. The steady-state respiration rates exhibited by spruce did not differ between Experiments 1 and 2. However, pine seedlings had a higher rate than spruce in -2°C. This could indicate that stabilized respiration rate is species-specific. 41 Using the measured respiration rates, the Q10S were calculated between -2°C and +2°C, and between +2°C and +7°C (Table 4). At the beginning of the experiments (initial respiration rates) the Q10S were around the value expected (-2.5) and remained fairly consistent between temperature ranges for each species-experiment combination. In contrast, when Q i o s were calculated for the steady-state respiration rates, there was a marked difference between the two temperature ranges. The Q10 values between -2°C and +2°C are extreme (9.0 - 16.7) whereas those between +2°C and +7°C are more nearly normal. As previously mentioned, statistical analysis on Q10 was not possible as these ratios were evaluated from averages as opposed to individual seedlings. TABLE 4. Q 1 0 for initial and steady-state respiration rates over the temperature ranges indicated. INITIAL STEADY-STATE SPECIES EXPT. -2 TO +2°C +2 TO +7°C -2 TO +2°C +2 TO +7°C SPRUCE 1 1.5 1.9 16.7 1.5 SPRUCE 2 3.5 3.4 9.0 2.2 4.2.2 RESERVE DEPLETION 4.2.2.1 WHITE SPRUCE In December, when the TNC monitoring began, white spruce seedlings had a TNC content of 229.6 mg/g structural dry weight (SDW). In seedlings that remained outdoors in their containers (3N0S), a significant TNC depletion occurred between December and January (Figure 7). When seedlings were lifted in February (2N0S) to undergo -2°C storage, the reserves had been depleted to 187.4 mg/g SDW and they barely changed from February until April (2N2S), finishing the storage period with 42 171.3 mg/g SDW. However, for the seedlings remaining outdoors (3N0S), the TNC content was regenerated between February and March to a higher level than measured in December; i.e., 244.0 mg/g SDW. Spruce seedlings that entered storage in December had their reserves depleted in a fairly similar pattern to seedlings from the 2N2S treatment, although they finished the 4 months storage (0N4S) with a lower CH20 concentration (154.7 mg/g SDW, a 33% loss). In the +2°C and +7°C thawing regimes (Figure 8), the difference between the thawing regimes and the changes in TNC over time were significant in spruce from both the 0N4S and 2N2S previous storage treatments. There was a significant interaction effect indicating that the rate of depletion varied between the thawing regimes. The differences in pattern of depletion were rather remarkable. Surprisingly, there was no apparent loss of carbohydrates in the spruce 4S6T2 thawing regime. The seedlings entered the +2°C thawing with 154.7 mg/g SDW but were found to contain 162.4 mg/g SDW after 6 weeks. In the 2N2S spruce thawed at +2°C, the reserves had not changed after two weeks but were depleted significantly by week 6, going from 171.3 mg/g SDW at the beginning of the thawing down to 120.2 mg/g SDW. In the 0N4S spruce thawed at +7°C, the reserves seem to have been depleted exponentially, as an equivalent amount was lost during the first two weeks of thawing as in the following 4 weeks. At the end of the +7°C thawing, TNC content was 94.3 mg/g SDW; an overall loss of 59% since lifting. For the 2N2S seedlings thawed at +7°C there appeared to be an important depletion of the reserves in the first two weeks and no significant changes thereafter. The TNC concentration of the 2S6T7 treatment group was 117.8 mg/g SDW indicating slightly less depletion than in the 4S6T7 regime. 43 270 -225 -180 -135 -90 -45 0 270 225 -180 -135 -90 45 H 270 -225 -180 135 -90 -45 -0 Nursery 3 mths outdoors -3=-, C Nursery 2 mths and -2°C Storage 2 mths =S-, be ^ r _ ^ be 2°C Storage 4 mths ab b r ^ n b 0 1 Dec. Jan. 2 Feb. 3 Mar. Soluble Sugars Starch Months after lifting 4 Apr. Figure 7 TNC content of white spruce over time for seedlings over-wintered outdoors and in -2°C storage. Standard error bars are for T N C content. Within each panel, different letters indicated significant differences at P < 0.05. 44 After 0N4S After 2N2S 175 -150 -125 -100 -75 50 25 0 175 -\ 150 125 -100 -75 50 25 0 Thawing +2°C Thawing +7°C I Thawing +2°C Thawing +7°C J 2 4 6 0 weeks of thawing Soluble Sugars Starch Figure 8 TNC content of white spruce over time in thawing. Standard error bars are for TNC content. The equations generated in the previous section 4.2.1 were integrated to predict TNC depletion. With the initial C H 2 O content at day 0, predicted and measured TNC depletion were plotted together to verify hypothesis 3. When the measured TNC over time was found to be within the confidence intervals of the predicted curve, it was 45 concluded that the observed TNC depletion was likely due to respiration alone. In Figure 9, for spruce placed in storage in December, one can see that from lifting (Day 0) until 0N3S (Day 90), the measured TNC corresponds fairly well with the expected levels based on measured respiration rates. However, at day 120 (0N4S) the TNC content falls much below the predicted level. At the end of storage, for spruce 0N4S, 74.9 mg/g SDW had been used. The respiration curve for spruce predicted a depletion of 51.4 mg/g SDW. During thawing, the TNC data was quite close to prediction except at Day 162 in the +2°C thawing regime (4S6T2), where the TNC pool seems to have increased. 250 - \ § 75 _ Predicted TNC c o 50 - - 2°C storage z 2 5 • +2°C thawing l ~ +7°C thawing 0 20 40 60 80 100 120 140 160 180 Days From Lifting Figure 9. Carbohydrate depletion for white spruce lifted in December as a function of time in storage and thawing. Data points are measured TNC concentration (means ± c.L); lines indicate predicted rates of TNC depletion based on measured respiration rates and initial CH2O content at day 0. 46 In the case of spruce lifted in February (Figure 10), the degree to which the measured TNC diverges from the expected curve is striking. Whereas the respiration rates predicted 39.6 mg ChbO/g SDW depletion, the actual measurements indicated that only 16.1 mg/g SDW was lost. In thawing, as in -2°C storage, the measured reserve depletion did not fit the predicted curve. Q cn O CM X o o o> E c 0) o O O 200 H 180 H 160 140 -120 -100 H 80 H 60 - \ 40 Measured TNC * ~2°C storage O + 2°C thawing A + 7°C thawing Predicted TNC 20 - \ - 2 ° C storage + 2°C thawing + 7°C thawing I 0 10 20 30 40 50 60 Days From Lifting 70 80 90 100 Figure 10. Carbohydrate depletion for white spruce lifted in February as a function of time in storage and thawing. Data points are measured TNC content (means ± ci . ) ; lines indicate predicted rates of TNC depletion based on measured respiration rates and initial CH2O content at day 0. 4.2.2.2 LODGEPOLE PINE In December, lodgepole pine seedlings had a TNC content of 156.6 mg/g structural dry weight (SDW). The TNC concentration of seedlings that remained outdoors in their containers barely changed from December to February (Figure 11). 4 7 175 -150 -125 -100 -75 50 25 H 0 175 150 125 H 100 75 H 50 25 0 175 -150 -125 100 H 75 50 25 0 " 3 H a r ^ n a Nursery 3 mths outdoors 3E-i a • • Nursery 2 mths and -2°C Storage 2 mths I 2°C Storage 4 mths -5E-b 3&n b 0 1 Dec. Jan. 2 Feb. 3 Mar. 4 Apr. Soluble Sugars Starch Months after lifting Figure 11 TNC content of lodgepole pine over time for seedlings over-wintered outdoors and in -2°C storage. Standard error bars are for T N C content. Within each panel, different letters indicated significant differences at P<0 .05 . 48 When the seedlings were lifted in February (2N0S) to undergo -2°C storage, the reserves had fallen to 146.8 mg/g SDW. Thereafter, whether they remained outdoors or were placed into dark storage, the reserves showed a significant depletion. In March, outdoors (3N0S), the reserves went down to 124.2 mg/g SDW. In storage (2N2S), the seedlings finished with a TNC concentration of 92.5 mg/g SDW. Pine seedlings that entered storage in December had the greatest depletion over the first two months, showing a TNC content of 116.6 mg/g SDW in February (0N2S) and finishing the storage period (0N4S) with 109.6 mg/g SDW. In the +2°C and +7°C thawing regimes (Figure 12), the difference between the thawing regimes and the changes in TNC over time were significant in pine from both 0N4S and 2N2S previous storage treatments. There was a significant interaction indicating that the rate of depletion varied between the thawing regimes. The depletion pattern in thawing appeared to be exponential in every treatment combination, and was more severe than in spruce (compare Fig.8). After 6 weeks, TNC concentrations had fallen to 48.2 and 43.0 mg/g SDW in 4S6T2 and 2S6T2, and to only 32.7 and 27.2 mg/g SDW in 4S6T7 and 2S6T7, respectively. These figures correspond to losses, since December, of 69% and 73% in the former, and 79% and 83% in the latter. Well over half this depletion occurred in thawing. 49 125 100 -\ 75 50 -\ 25 0 O CO O) D) E. co "c 0) C O > o" 125 CD CD g 100 o _ 3 O 75 50 25 After 0N4S Thawing +2°C Thawing +7°C After 2N2S Soluble Sugars Starch 1 4 6 0 2 weeks of thawing Thawing +2°C Thawing +7°C nr 4 Figure 12 TNC content of lodgepole pine over time in thawing. Standard error bars are for TNC content. As shown in Figure 13, although the TNC depletion for pine lifted in February is quite close to the predicted amount at the end of the storage period, at day 30 there is a major gap between the two. Either the CO2 measurements or the TNC analysis could account for this discrepancy. The TNC depletion during thawing in pine cannot be compared to the respiration curve because, as previously mentioned, these data 50 ft were discarded because of fungal contamination (although, in their cartons, the pine seedlings did not develop any mold). Figure 13. Carbohydrate depletion for lodgepole pine lifted in February as a function of time in storage. Data points are measured TNC content (means ± c i . ) ; lines indicate predicted rates of TNC depletion based on measured respiration rates and initial CH2O content at dayO. 4.3 STARCH AND SUGAR DYNAMICS. 4.3.1 WHITE SPRUCE When spruce seedlings were overwintered outdoors (3N0S), starch content went down between December and January, going from 12.4 mg/g SDW to 5.7 mg/g 51 SDW in the shoot and from 104.3 to 62.4 mg/g SDW in the root (Table 5). A significant increase in starch concentration occurred after February and by March, the respective starch concentrations were 134.5 and 186.7 mg/g SDW (representing 57.1% and 71.5% of the TNC content of shoots and roots respectively). By this time, buds were beginning to swell on the spruce seedlings. Sugar concentrations decreased from December to March, from 217.3 to 101.0 mg/g SDW in the shoot and from 128.9 to 74.4 mg/g SDW in the root. The pattern of sugar depletion was different in shoots and roots, perhaps indicating that they used their reserves differently. When the seedlings were lifted in February for cold-storage, the starch content of the roots began to decline in the following month, whereas in the shoot a slight increase preceded the decrease observed in April. By the end of storage, 17.2 and 39.3 mg/g SDW of starch remained in these plant parts (i.e., 9.3% and 28.7% of TNC in the shoot and root, respectively). Sugar concentrations on the other hand, increased slightly during storage, rising by the end of April, from 158.3 and 72.3 mg/g SDW to 167.3 and 97.5 mg/g SDW for the shoot and root, respectively. For seedlings entering storage in December, starch content was quite reduced during the first two months, going down from 12.4 to 0.5 mg/g SDW in the shoot and from 104.3 to 4.6 mg/g SDW in the root. In March (month 3), sugar to starch conversion was indicated in that the shoot and root starch concentrations reached 13.2 and 63.0 mg/g SDW, respectively. However, this regeneration of starch did not last and by the end of April, starch levels had declined again to 1.2 and 27.4 mg/g SDW for the shoot and root, respectively. The percentage of TNC in the form of starch for seedlings stored for 4 months (0N4S) was much lower (0.8% and 16.4% for the shoot and root, respectively), than in seedlings lifted in February and stored 2 months (2N2S). 52 Table 5. Free sugars and starch concentration (mg/g SDW) in shoots and roots of white spruce during winter. (Standard errors are in brackets.) SHOOTS Month in Storage Not Lifted Sugars Starch Lifted in February Sugars Starch Lifted in December Sugars Starch 0 (Dec.) 1 (Jan.) 2 (Feb.) 3 (Mar.) 4 (Apr.) 217.3(9.9) 12.4(1.3) 197.6(9.8) 5.7(1.1) 158.3 (5.8) 39.7 (2.5) 101.0(2.4) 134.5(3.0) 217.3(9.9) 12.4(1.3) 197.6(9.8) 5.7(1.1) 158.3 (5.8) 39.7(2.5) 155.2(6.6) 48.7(3.4) 167.3 (9.0) 17.2 (2.2) 217.3(9.9) 12.4(1.3) 212.8(8.3) 4.3(0.8) 203.0 (5.8) 0.5 (0.2) 194.9(7.1) 13.2(1.6) 146.9(5.1) 1.2(0.5) ROOTS Month in Storage Not Lifted Sugars Starch Lifted in February Sugars Starch Lifted in December Sugars Starch 0 (Dec.) 1 (Jan.) 2 (Feb.) 3 (Mar.) 4 (Apr.) 128.9(4.5) 104.3(1.4) 137.2(3.5) 62.4(5.6) 72.3(5.0) 92.1(1.1) 74.4(4.3) 186.7(4.9) 128.9(4.5) 104.3(1.4) 137.2 (3.5) 62.4 (5.6) 72.3 (5.0) 92.1 (1.1) 78.7 (4.0) 67.7 (3.4) 97.5 (7.3) 39.3 (2.2) 128.9(4.5) 104.3(1.4) 165.0 (7.2) 39.4 (4.0) 197.6 (7.0) 4.6 (1.5) 120.5(8.1) 63.0(4.5) 139.7(5.5) 27.4(2.6) The shoot sugar content of seedlings lifted for storage in December remained fairly high until the end of the third month, going from 217.3 down to 194.9 mg/g SDW, but was reduced to 146.9 mg/g SDW during the last month of storage. In the root, sugars increased from 128.9 mg/g SDW in December to 197.6 mg/g SDW in February. However, by the end of storage in April, the root sugar concentration was down to 139.7 mg/g SDW. When transferred to thawing (Table 6), an increase in starch content was evident at week 2 in all treatment combinations. The lowest increase in starch was in 4S2T2 but starch regeneration kept on going so that at week 6 (4S6T2), starch concentrations were 28.1 and 93.6 mg/g SDW, in the shoot and root respectively. In the other three treatment combinations (i.e., 0N4S into +7°C thawing and 2N2S into +2°C and +7°C thawing), starch levels increased after two weeks but fell off again by week 6. 53 Table 6 Free sugars and starch concentration (mg/g SDW) in shoots and roots of white spruce during thawing following 0N4S and 2N2S storage. (Standard errors are in brackets.) SHOOTS Week 0N4S previous storage 2N2S previous storage of +2°C +7°C +2°C +7°C Thawing Sugars Starch Sugars Starch Sugars Starch Sugars Starch 0 146.9(5.1) 1.2 (0.5) 146.9(5.1) 1.2 (0.5) 167.3(9.0) 17.2 (2.2) 167.3(9.0) 17 .2 (2.2) 2 153.8(7.8) 1 6 . 0 ( 1 . 9 ) 108.2(6.6) 2 9 . 0 (2.0) 136.3(9.5) 35 .8 (3.6) 8 8 . 5 (6.4) 3 9 . 0 (4.1) 6 136.5(7.5) 28.1 (4.6) 74 .4 (5.4) 19.3 (3.4) 88 .6 (4.2) 2 8 . 3 (0.3) 87 .4(9 .8) 2 8 . 4 (4.9) ROOTS Week 0N4S previous storage 2N2S previous storage of +2°C +7°C +2°C +7°C Thawing Sugars Starch Sugars Starch Sugars Starch Sugars Starch 0 139.7(5.5) 27 .4 (2.6) 139.7(5.5) 27 .4 (2.6) 9 7 . 5 (7.3) 39 .3 (6.2) 9 7 . 5 (7.3) 3 9 . 3 (6.2) 2 7 1 . 6 (4.1) 5 3 . 6 (5.8) 3 7 . 3 ( 1 . 9 ) 57 .4 (5.1) 75 .9 (2.2) 9 2 . 0 (7.7) 3 5 . 6 (3.1) 8 3 . 7 (2.6) 6 6 5 . 3 (4.4) 9 3 . 6 (1.1) 3 1 . 6 ( 1 . 3 ) 6 4 . 0 (6.5) 4 1 . 7 (1.5) 8 6 . 5 (4.4) 4 4 . 3 (2.3) 74 .8 (8.1) Sugar conversion to starch was also indicated in the roots of spruce seedlings following 2 weeks of thawing. The lowest proportion of starch was again observed in 4S2T2. Just as for the shoot, starch continued to increase for this treatment combination, but was more static in the other three. The loss or conversion of sugars in the roots was in general more pronounced than in the shoots. For seedlings stored 4 months prior to thawing, sugar concentrations after 6 weeks were down from 139.7 mg/g SDW to 65.3 and 31.6 mg/g SDW at +2°C and +7°C, respectively. Most of the depletion occurred in the first two weeks. For seedlings lifted in February (2N2S previous storage), sugar depletion in the root was not as pronounced as for seedlings lifted in Decmber (0N4S previous storage). By the end of thawing, the root sugar concentration of 2N2S seedlings fell from 97.5 mg/g SDW to 41.7 and 44.3 mg/g SDW at +2°C and +7°C, respectively. 54 In keeping with the greater loss of free sugars in the roots as compared to the shoots, the root starch concentration after 6 weeks of thawing was relatively higher and ranged between 64 and 93.6 mg/g SDW (as compared to 19.3 and 28.4 mg/g SDW in the shoot). Therefore between 58.9% and 67.5% of the root TNC was in the form of starch, whereas only 17.1% to 24.6% of the shoot TNC was. 4.3.2 LODGEPOLE PINE When pine seedlings were overwintered outdoors, the starch content went down between December and January, going from 2.2 to 0.7 mg/g SDW in the shoot and from 78.8 to 43.6 mg/g SDW in the root (Table 7). A significant increase in starch concentration occurred in February where the starch content reached 35.3 and 79.0 mg/g SDW for the shoot and root, respectively. Table 7. Free sugars and starch concentration (mg/g SDW) in shoots and roots of lodgepole pine during winter. (Standard errors are in brackets.) SHOOTS Month in Storage Not lifted Sugars Starch Lifted in February Sugars Starch Lifted in December Sugars Starch 0 (Dec.) 1 (Jan.) 2 (Feb.) 3 (Mar.) .4 (Apr.) 138.7(6.2) 2.2(0.7) 151.2(6.7) 0.7(0.3) 120.0(4.6) 35.3(1.9) 84.8(4.7) 28.6(1.7) 138.7(6.2) 2.2(0.7) 151.2(6.7) 0.7(0.3) 120.0(4.6) 35.3(1.9) 86.2(4.2) 11.3(1.8) 98.6(4.1) 1.1(0.2) 138.7(6.2) 2.2(0.7) 143.4 (5.7) 2.6 (0.4) 110.8(4.0) 0.4(0.1) 100.8(3.2) 8.8(2.6) 117.1(7.3) 0.5(0.1) ROOTS Month in Storage Not lifted Sugars Starch Lifted in February Sugars Starch Lifted in December Sugars Starch 0 (Dec.) 1 (Jan.) 2 (Feb.) 3 (Mar.) 4 (Apr.) 105.3 (8.1) 78.8(5.0) 109.7(2.6) 43.6(4.2) 57.7 (3.4) 79.0(5.1) 55.1 (4.3) 86.0 (3.6) 105.3 (8.1) 78.8(5.0) 109.7(2.6) 43.6(4.2) 57.7(3.4) 79.0 (5.1) 80.5 (2.2) 27.5 (3.7) 76.8(5.4) 8.4(1.3) 105.3(8.1) 78.8(5.0) 117.4(5.7) 21.9(3.5) 126.0(7.5) 1.2(0.8) 75.0(6.1) 14.5(1.8) 99.6(3.9) 1.0(0.4) 55 In March, while starch was apparently converted back to sugar in the shoot (resulting in a starch concentration of 28.6 mg/g SDW), starch kept increasing in the root to 86.0 mg/g SDW. Consequently, starch accounted for 25.2% and 61.0% of the TNC content of the shoot and root, respectively. The pine seedlings had also begun candle elongation at this time. The sugar content in these seedlings declined from January to March (i.e., from 151.2 to 84.8 mg/g SDW in the shoot, and from 109.7 to 55.1 mg/g SDW in the root). As for spruce, the pattern of sugar depletion was different in the shoot and root indicating that both parts used their reserves differently. When the seedlings were lifted in February for freezer-storage, the starch content began to decline the following month. By the end of storage, 1.1 and 8.4 mg/g SDW of starch remained in the shoot and root respectively (i.e., 1.1% and 7.7% of the TNC). The sugar concentration, already reduced to 120.0 and 57.7 mg/g SDW in the shoot and root in February, showed a decline in the shoot but an increase in the root during storage. By the end of April, sugar concentrations had gone down to 98.6 mg/g SDW in the shoot and up to 76.8 mg/g SDW in the root. For seedlings entering storage in December for 4 months storage, the already low shoot starch content was reduced during the first two months, going down from 2.2 to 0.4 mg/g SDW. The reduction in starch concentration was much more pronounced in the root, falling from 78.8 to 1.2 mg/g SDW. In March (month 3), sugar to starch conversion was indicated and starch concentrations reached 8.8 and 14.5 mg/g SDW, for the shoot and root respectively. However, this regeneration of starch did not last and by the end of April, starch concentrations fell to 0.5 and 1.0 mg/g SDW, respectively. The percentage of TNC in the form of starch after 4 months was fairly similar in both 0N4S and 2N2S shoots (0.5%), but not in the roots (i.e., 1.0% for 0N4S and 7% for 2N2S). 56 The sugar concentration in the shoot for seedlings lifted in December was reduced from 138.7 to 110.8 mg/g SDW at month 2. Thereafter until the end of storage the sugar concentration fluctuated very slightly. In the root, sugars increased from 105.3 mg/g SDW in December to 126.0 mg/g SDW in February. A decrease occurred in March but by the end of storage in April, the sugar concentration had increased again to 99.6 mg/g SDW. When transferred to thawing (Table 8), an increase in starch content occurred at week 2 in all treatment combinations. In the following weeks of thawing, starch was apparently re-mobilized and depleted in all cases. By the end of thawing, the starch concentration in the shoot was around 1.0 mg/g SDW for every treatment combination (slightly lower at +7°C). Table 8 Free sugars and starch concentration (mg/g SDW) in shoots and roots of lodgepole pine during thawing following 0N4S and 2N2S storage. (Standard errors are in brackets.) SHOOTS Week 0N4S previous storage 2N2S previous storage of +2°C +7°C +2°C +7°C Thawing Sugars Starch Sugars Starch Sugars Starch Sugars Starch 0 117.1(7.3) 0 .5 (0 .1 ) 117.1(7.3) 0 .5 (0 .1 ) 98.3(4.1) 1.1(0.2) 98 .3(4 .1) 1.1(0.2) 2 85 .7 (4.5) 5.3 (2.9) 5 2 . 9 ( 1 . 5 ) 7.5(1.5) 72.0(3.9) 1.8(0.5) 62 .8(5 .5) 12.0(2.7) 6 52 .8 (1.9) 1 .5 (0 .6) 42.5(2 .0) 0.5(0.2) 46.8(1 .9) 1.1(0.5) 30.9(1 .9) 0.8(0.1) ROOTS Week 0N4S previous storage 2N2S previous storage of +2°C +7°C +2°C +7°C Thawing Sugars Starch Sugars Starch Sugars Starch Sugars Starch 0 99.6(3 .9) 1.0(0.4) 99.6(3.9) 1.0(0.4) 76.8(5.4) 8.4(1.3) 76 .8(5 .4) 8.4(1.3) 2 56.9(2.4) 13.9(3.0) 26.9(2.3) 22.4(2.7) 54.6(3.0) 9.0(2.5) 38 .3(2 .2) 15.7(4.2) 6 32.1(1.2) 6.1(2.0) 14.6(1.6) 0.8 (0.3) 30.1(2.7) 7.3(2.4) 18.0(2.9) 0.4(0.1) 57 In the roots of pine seedlings, sugar conversion to starch was also indicated after 2 weeks of thawing but a much lower amount of starch was synthesized at both temperatures following the 2N2S storage treatment as compared to seedlings previously stored for 4 months. As in shoots, root starch concentrations decreased in every treatment combination during the final weeks of thawing. Overall, for December-lifted seedlings, apparent sugar to starch conversion was more important in the roots where starch concentrations after 6 weeks of thawing were 6.1 and 0.8 mg/g SDW for the +2°C and +7°C thawing regimes, respectively, as compared to 1.5 and 0.5 mg/g SDW in the shoots. Likewise, for February-lifted seedlings, the amount of starch at the end of thawing was higher in the root (7.3 mg/g SDW) than in the shoot (1.1 mg/g SDW). At +7°C, however, the starch content at week 2 was fairly similar in shoots (12.0 mg/g SDW) and roots (15.7 mg/g SDW). By week 6, starch concentrations at +7°C were so low that comparisons between shoots and roots are meaningless. Free sugar levels fell at fairly similar rates in the shoots and roots. December-lifted seedlings appeared to have a higher rate of sugar utilization but by the end of +2°C thawing, seedlings from both 4S and 2S storage treatments had similar shoot (52.8 and 46.8 mg/g SDW, respectively) and root (32.1 and 30.1 mg/g SDW, respectively) sugar concentrations. The same similarity in sugar content was observed at the end of the +7°C thawing regime where the shoots had 42.5 and 46.8 mg/g SDW and the roots had 14.6 and 18.0 mg/g SDW, for December and February-lifted seedlings, respectively. 58 4.4 SUGAR QUALITY The sugars analysis by HPLC revealed that for both spruce and pine, sugar content estimated by the anthrone method does not present a complete picture as it does not detect all compounds of interest. For instance, sugar alcohols are known to be important in tree seedlings (2,42) but are not detected by anthrone. A statistical comparison of the apparent total yield of sugar (including sugar alcohols) by paired T-tests indicated that for spruce in storage and in +2°C thawing, there was a pronounced discrepancy (P= 0.0008) only at month 4 (0N4S) in the shoot (156.1 mg by Anthrone and 229.8 by HPLC). However, in the plants overwintered outdoors, the discrepancy of sugar content in the shoot was significant at every sample date, (i.e., 1N0S, 2N0S and 3N0S) and in the root at 2N0S (Figure 14). Differences between the yield of sugars by Anthrone and by HPLC were even more pronounced in shoots of pine where HPLC detected a significantly higher content at every sampling date tested. Significant differences were also noted in the root at 4S2T2 and for seedlings overwintered outdoors (Figure 15). These results would quantitatively change the picture of TNC depletion if they could be applied to Study 2. However, because only five of the ten seedlings harvested at each date during the TNC monitoring were used for HPLC analysis, and not every treatment was considered, the data have not been corrected. Figure 14 Evaluation of white spruce TNC content using different methods for sugar analysis (anthrone vs HPLC). Standard error bars are for T N C Figure 15 Evaluation of lodgepole pine TNC content using different methods for sugar analysis (anthrone vs HPLC). Standard error bars are for T N C . 61 4.4.1 WHITE SPRUCE In the shoots of spruce seedlings (Figure 16), the first sugar to elute from the HPLC column after the solvent peak had very little importance relative to other sugars. This sugar (Peak 2, thought to be stachyose) was present in very small amounts and remained as such throughout storage and thawing. Outdoors, the content of this sugar barely changed until February (2N0S) and by March (3N0S), it had nearly disappeared. In the root (Figure 16), Peak 2 and raffinose were present in approximately equal concentration. The two oligosaccharides tended to follow the same trends over time in both storage (going up and down) and thawing (slight decline). Outdoors at the nursery, both sugars were depleted over time. Although raffinose and sucrose were present at similar concentrations in the shoot when entering storage, sucrose levels remained relatively constant over the next 4 months, whereas raffinose was depleted in storage. In thawing, raffinose, already quite low in concentration, continued to decline slightly over the 6 weeks of monitoring whereas sucrose was considerably depleted. Outdoors, shoot raffinose and sucrose concentrations peaked at similar levels in January (0N1S). Thereafter, at 2N0S, raffinose was depleted more drastically than sucrose. At 3N0S, raffinose continued to decline whereas sucrose did not change. Sucrose was by far the preferential form of sugar in the root. It showed a remarkable increase at month 2 in storage (0N2S) and an important decrease when seedlings were transferred to thawing. The pattern of depletion from month 4 (0N4S) to 6 weeks thawing (4S6T2) was quite similar to the pattern outdoors where sucrose content was quite high in January (1N0S) and very much depleted by February (2N0S), although the depletion was faster in thawing. 62 Dark storage and thawing Outdoors 100 80 60 40 20 0 Peak 2 E L r j l =p_ 100 80 60 H 40 20 Peak 2 1 J • — 100 80 60 40 20 H 0 Raffinose rti 1 1 100 -I Raffinose 80 60 40 20 rh 1 . 1 LT^L 100 80 60 40 20 0 Sucrose 0 1 I 100 - Sucrose 80 -60 40 20 0 1 *0 Sample time 0> Sample time Shoot Root Figure 16 Free sugars in white spruce. Changes in tissue concentrations of Peak 2, raffinose and sucrose over time. 63 Glucose (Figure 17) appeared to be the preferential form of sugar in the shoot. Glucose content remained twice as high as fructose content from entering storage until end of January (0N1S). At month 2, the concentrations of both sugars increased but the fructose concentration doubled. The hydrolysis of raffinose, sucrose and starch probably accounts for the increment in hexose concentration at this time. By the end of storage (0N4S), the glucose content was still high but fructose decreased in concentration again. In +2°C thawing, glucose content fell over the first two weeks with no further change over the remaining thawing period. Fructose content dropped only slightly in thawing. Outdoors, the glucose concentration peaked in January (1N0S) and declined linearly until March (3N0S), whereas the fructose concentration remained fairly stable until February (2N0S) before declining slightly in March (3N0S). Glucose and fructose were present in much smaller concentrations, and tracked each other more closely in the root than in the shoot (Figure 17). Generally speaking, root levels of these sugars remained within the same range during storage but were quite depleted during thawing. Outdoors, just as for sucrose, the two monosaccharides increased in concentration from December to January and then declined until March (3N0S). In shoots, Peak 6 (unidentified but thought to be a sugar alcohol) did not show much change over time under all conditions except for a significant increase at month 2 of storage. Root concentrations, though lower, were also fairly constant. 64 Dark storage and thawing Outdoors 100 80 60 40 20 0 Glucose rh 1 i 1 id 1 100 80 1 60 40 20 0 Glucose rh fe ! i 100 80 60 40 20 0 Peak 6 rh 100 80 60 40 20 H 0 Peak 6 100 H Fructose 80 60 40 20 0 rh rh rh Sample time Shoot 100 4, Fructose 80 60 40 20 "1 0 , , VA r& C& r& ^ # # Sample time Root Figure 17 Free sugars in white spruce - Changes in tissue concentrations of glucose, Peak 6 and fructose over time. 65 4.4.2 LODGEPOLE PINE In the shoots of pine (Figure 18), "stachyose" (i.e., Peak 2) was present at a fairly high concentration in December but declined to a very low level in January both in storage and outdoors. The content of this sugar then remained low throughout the storage and thawing periods. Outdoors, it was completely gone by March (3N0S). In the root, Peak 2 sugar level was maintained from month 0 to month 2 for outdoor and cold-stored seedlings, but was much depleted by the end of these treatments (0N4S and 3N0S). During thawing, there was a slight increment in concentration over the first two weeks followed by a depletion at week 6. Raffinose content in the shoot was much lower than the putative stachyose content when seedlings entered into storage (ONOS). However, the decline in the former was slower and more uniform during cold storage, reaching 50% of the initial content after 4 months (0N4S). In thawing, there was a slight increase in raffinose by week 2 but the sugar was depleted to its lowest value by the end of thawing (4S6T2). Outdoors, the raffinose content barely changed from December to February (2N0S). By March (3N0S), however, its concentration had declined to a level comparable to that observed at the end of thawing. In the root, raffinose was present at the same concentration as the Peak 2 sugar in December, but fell to nearly zero within 1 month of storage; whereas outdoors, the raffinose concentration was maintained from December to February (2N0S) before it declined. At month 4 of cold storage (0N4S), the raffinose content began to increase again and at week 2 of thawing, raffinose and the Peak 2 sugar were both present at nearly the same concentration. Thereafter, the two sugars decreased to less than 4 mg/g SDW at week 6 of thawing. Dark storage and thawing 66 Outdoors 100 80 60 40 20 0 Peak 2 100 -_ Peak 2 80 '-60 : 40 -20 \ 0 : 100 80 60 40 20 0 Raffinose IX- [!]*. 100 80 60 40 20 -1 Raffinose i 1*1 100 80 60 40 20 0 Sucrose J Sample time Shoot 100 80 H 60 40 20 0 Sucrose 1 *r qT ^ Sample time Root Figure 18 Free sugars in lodgepole pine - Changes in tissue concentrations of Peak 2, raffinose and sucrose over time. 67 Sucrose concentration in the shoot did not change much during the first two months of monitoring whether the plants were cold-stored or remained outdoors. The content declined to the same value for both 0N4S and 3N0S samples. During thawing, just as for raffinose, there was a slight increase in sucrose content after two weeks followed by a decrease at week 6. The maintenance of sucrose content at month 1 and 2 for seedlings in storage and outdoors may be linked to the major decrease in the Peak 2 sugar content. Sucrose content in the root was increased substantially after 1 month of -2°C storage, but fell to its lowest level at month 4 (0N4S). In thawing, just as for the oligosaccharides, sucrose concentration increased after two weeks of exposure to +2°C and then decreased. Outdoors, the sucrose content nearly doubled in January (1N0S) and remained high in February (2N0S). Thereafter in March, while the seedlings had begun candle elongation, sucrose content declined. Glucose and fructose (Figure 19) appeared to be the preferred sugars in the shoot. Their concentrations were quite similar, remaining fairly high during -2°C storage and then declining during thawing to one third of their initial value. Outdoors, the two sugars increased in concentration slightly in January (1N0S) and then decreased over time until March (3N0S), but not dropping to as low a level as seen by the end of thawing. As seen in the shoot, glucose and fructose concentrations in the root (Figure 19) were very similar and tracked each other over time. Levels were fairly constant during the first two months of storage, but peaked at month 4 (0N4S). Part of this rise was most likely provided by the decline in oligosaccharides. Root hexose concentrations dropped precipitously by nearly 60% after two weeks at +2°C thawing. The content kept decreasing over the remaining period of thawing. Outdoors, the concentration of both sugars remained quite uniform. 68 Dark storage and thawing Outdoors 100 80 60 40 20 0 Glucose rh \ rn 1 100 80 60 -1 40 20 0 Glucose rh 1 fe E L rh i 100 Peak 6 80 4 100 80 Peak 6 60 40 20 0 I rti 1 1 rfl 60 40 20 0 r±i rri 100 4 Fructose 80 60 40 H 20 0 Sample time rh rh Shoot 100 80 60 40 20 0 Fructose m Root fe cv3 cv3 cv' ^ o f # Sample time Figure 19 Free sugars in lodgepole pine - Changes in tissue concentration of glucose, Peak 6 and fructose over time. 69 Peak 6, probably a sugar alcohol, showed a much higher concentration in the shoots of pine than in spruce. However, as in spruce, the content barely changed whether the seedlings were kept outdoors or in dark cold storage. Likewise, the Peak 6 sugar content of roots remained fairly stable in storage and thawing, and outdoors, at about half the concentration seen in the shoots. 4.4.3 RELATIONSHIP BETWEEN SUGAR QUALITY AND FROST HARDINESS To see if any relationship might exist between degree of hardiness and sugar quality, changes in individual sugar contents over time (all treatments combined where LTso was evaluated) were plotted against LTso. In order to obtain an adequate number of data-points and an even sample size (i.e., n=5 for LTso and sugar analysis), results from spruce and pine seedlings were pooled together. Changes in ratios such as oligosaccharides to hexoses, sucrose to hexoses, sucrose to raffinose, and raffinose to hexose were also compared to changes in LTso over time. Based on the present study, only raffinose content (mg/g SDW) could be related to frost hardiness. The results in Figure 20 indicated that as the LTso increased, raffinose content declined. To calculate the correlation coefficient between LTso and raffinose, two data-points had to be excluded as the LTso was estimated to be somewhere below -65°C, the lower limit of the test freezer. So, based on eight means only, r = 0.94 indicating that raffinose content in this study is in fact very well correlated to frost hardiness (P < 0.0001). 70 -10 --20 --30 -o o o in 1 -40 -_i --50 --60 -<-65 • Pine B Spruce r = .942 -i 1 1 1 1 1 r 10 X i 1 1 1 1 1 1 r 20 30 I T 1—~ 1 1 40 50 Raffinose content (mg/g SDW) Figure 20 Relationship between frost hardiness level and raffinose content in shoots of white spruce and lodgepole pine. Frost hardiness is expressed in terms of LT50 . The regression line is for pooled data from both species, and does not include the data shown for spruce where the L T 5 0 was too low to be measurable (bars near base graph). 4.5 TRANSLOCATION 4.5.1 RESERVES DEPLETION In December, when spruce seedlings entered storage, the TNC content was 229.6 mg/g SDW (Figure 21). During the 4 months of -2°C storage, there was a significant depletion of carbohydrates for both intact and girdled seedlings. Although two-way ANOVA also indicated a significant difference between treatments, one can see from Figure 21- A (whole seedlings) that overall, the rate of TNC depletion was 71 the same (i.e., no significant interaction was detected). The TNC concentrations at the end of the storage period were 154.7 mg/g SDW and 148.6 mg/g SDW for intact and girdled seedlings, respectively. When transferred to thawing, the TNC depletion for the whole seedling was also significant, but this time there was no significant difference between treatments, indicating that the whole seedlings, whether they were intact or girdled, behaved similarly in regard to reserve depletion overtime. In the absence of any change in depletion rate, whole seedling analysis can not suggest any translocation pattern. It is more relevant to study both seedling parts, shoot (Figure 21 - B) and root (Figure 21 - C), independently. In -2°C storage, the shoots and roots from both treatments showed a significant depletion of TNC over time. There was no significant difference between treatments in the shoot. In the roots however, the TNC depletion was significantly different over time between intact and girdled seedlings. These seedlings entered storage with a root TNC concentration of 233.2 mg/g SDW, but by April, TNC content in the roots was 167.1 mg/g SDW for intact seedlings and 127.3 mg/g SDW for girdled seedlings. When transferred to thawing, for both shoot and root, changes in TNC concentration over time were significant. Although Figure 21 - B (thawing) suggests that shoot TNC reserves may have been depleted differently between treatments, the statistical analysis did not detect a significant,difference. As there was an interaction between time and treatments, it appears reasonable to suggest that differences between treatments might exist but cannot be detected by comparison of overall means; the trend of depletion being different between intact and girdled seedlings. There is, however, no large or obvious effect of girdling. 72 300 250 200 150 100 50 0 300 250 H 200 150 H 100 .50 0 300 A - Whole seedling Storage Thawing -2°G B - Shoot • M ~ l -2°C C - Root +2°C +2°C +7°C m +7°C Intact H Sugars Starch Girdled , I Sugars H Starch 8 12 Weeks from lifting Figure 21 TNC dynamics over time for intact and girdled seedlings. S E bars are for starch and sugar individually. 73 In the root, the significance of the interaction and the significant difference between treatments indicate that TNC reserves were being used quite differently in girdled and in intact seedlings. In the intact seedlings, root TNC content started off at 167.1 mg/g SDW and was depleted to 125.2 mg/g SDW after two weeks and fluctuated in this range over the remaining 4 weeks thawing to finish at 109.7 mg/g SDW. Meanwhile, in girdled seedlings, root TNC content was 127.3 mg/g SDW when entering thawing and remained fairly stable until week 4. By week 6, however, after 2 weeks at +7°C, the TNC content had reached 250.1 mg/g SDW. The much lower TNC content in the shoot and the much higher TNC content in the root of girdled seedlings at week 6 of thawing suggests that phloem translocation (root to shoot) occurred in the intact seedlings after being transferred to +7°C but could not occur in the girdled ones. The carbon source supporting the dramatic rise in TNC content in the roots of girdled seedlings is not known. 74 5. DISCUSSION 5.1 FROST HARDINESS It is well known that frost hardiness varies between species and between provenances. In the present study, in December, white spruce was much more hardy (LT5o<-65°C) than lodgepole pine (LT50 = -26.0°C). The results are in concordance with Simpson (73) who reported LT50 values of -60°C and -30°C for interior spruce and lodgepole pine, respectively. The very low temperature to which spruce seedlings were hardy in December, and even after 2 months of -2°C storage (0N2S), did not allow quantification of LT50. Based on the Index of Injury, however, the results suggest that spruce, unlike pine, was able to gain a certain degree of hardiness while exposed to a dark and constant -2°C environment. Similarly, Burr et al. (10) found that Engelmann spruce gained cold hardiness following an exposure to constant -2°C. The white spruce seedlings, however, did not maintain cold hardiness throughout the entire 4 months of storage. They began to deharden somewhere between February and April even though no obvious environmental signals could have triggered this activity. Ritchie et al. (60) also reported a slow rate of dehardening for lodgepole pine and interior spruce in -1°C storage. As suggested by Cannell et al. (11) who observed dehardening of Sitka spruce, there may be some seasonal endogenous rhythm involved in the dehardening process in spruce. When spruce remained outdoors, cold hardiness was diminished and in February, LT50 reached -40.9°C. In March, the seedlings outdoors continued to deharden while those that were lifted in February re-developed deep cold-hardiness during -2°C storage. Siminovitch (71) had observed a similar behavior with black locust. Following the first signs of dehardening, the author observed a simultaneous 75 sucrose to starch conversion. When transferred to a cold chamber, the plants were able to restore their original winter hardiness. A similar observation was made here although the plants did not reach the original frost hardiness level (the results will be discussed later, in section 5.2.2.3). Siminovitch (71) reported that the reversibility of dehardening was no longer possible beyond a certain point characterized by the disappearance of constituents added to the cell during frost hardiness development. In contrast to spruce, pine seedlings lost some hardiness during the first two months of freezing storage, just as they did outdoors. While the seedlings continued to deharden outdoors, those lifted for storage may have re-gained some degree of cold resistance from February to April. However, the much narrower range of freezing temperatures that pine was resistant to created difficulties in the precision of LT50 and lj-20 evaluation. Standard errors of the means were quite large. Considering the additional error of the freezer itself, it is hazardous to make any definitive statement on the reversibility of the dehardening process in lodgepole pine. Once transferred to the +2°C and +7°C thawing regimes, hardiness was released in both spruce (Figure 1) and pine (Figure 2) whether from 2 months (lifted in February) or 4 months (lifted in December) cold storage. The "thawing" temperatures had an effect on the rate of dehardening, +7°C allowing a faster rate than +2°C. These results are in agreement with Aronsson (1) who also observed a temperature effect on the dehardening rate for pine and Douglas-fir. Therefore, it appears that a long duration of thawing, even at +2°C, would lead to much higher risk of damage to plantation stock during spring cold spells, and lodgepole pine would be more susceptible to damage than white spruce, the former being less hardy in the first place. 76 5.2 CARBOHYDRATE RESERVES 5.2.1 RESPIRATION Based on the results, hypothesis H01 stipulating that "respiration rates are constant irrespective of time in storage" is rejected. It was shown quite clearly that following lifting or moving, for both spruce and pine, respiration rates were higher immediately after disturbance and stabilized over time to reach a lower steady-state respiration rate. This observation agrees with van den Driessche's work (77) who mentioned a short-term stimulation of respiration rate when stored seedlings were disturbed. However, the values for steady-state respiration are different from those reported by both van den Driessche (77) and Ritchie (57). The experimental conditions used by van den Driessche to estimate white spruce respiration rate were very different than those of the present study. Plants were removed from storage at +4.5°C for respiration monitoring over periods ranging from a few hours to a week at different temperatures. Prior to this the plants had been stored for 12 to 20 weeks. Ritchie did not describe the experimental conditions he used. The fact that the initial respiration rates for spruce seedlings lifted in December (Expt. 1) were higher than for those lifted in February (Expt. 2) for -2°C storage could be due simply to the seedling preparation done for Experiment 2. The February lifting was done more carefully and more rapidly, being aware of the effect of disturbance. Also, the temperature at which the seedlings were introduced into the bottles was cooler (+9°C). Therefore, it is not possible to ascribe the discrepancy in initial respiration rates to the difference in physiological stage between the two liftings. In the +2°C thawing treatment, seedlings freshly removed from bundles and root washed had a faster respiration rate than bottled seedlings moved directly from 77 -2°C to +2°C with no washing. This observation supports the statement that respiration rates increased with the severity of the disturbance. At +7°C, however, the intensity of the disturbance did not ap~pear to influence the initial respiration rate. A possible reason might be the existence of a physiological limit to the degree to which respiration can be stimulated following disturbance. The fact that the Q10S for initial respiration rate in white spruce remained consistent between temperature ranges also supports this suggestion. The Q10 values for initial respiration were in the same range as those reported by van den Driessche (76). Steady-state respiration rates for spruce increased with increased temperature but did not differ between pre-treatments (0N4S and 2N2S). Pine seedlings exhibited a slightly higher steady-state respiration at -2°C than spruce, regardless of pre-treatment. The data suggest that once seedlings have acclimated to their new environment, the steady-state respiration rate may be species-specific. Further species and provenances would be needed to test these ideas. In contrast to Q10S for initial respiration rates, when Qios were calculated for the steady-state respiration rates there was a marked difference between temperature ranges. The extreme Q10 values observed between -2°C and +2°C (9.0 -16.7) indicate a far greater temperature-dependence for steady-state respiration rates near freezing than between +2°C and +7°C. In contrast, van den Driessche (76) reported a Q10 of 2 between -5 and +4.5°C for white spruce. To obtain a respiration rate at -5°C, the author transferred the plant from +4.5°C storage to -5°C and monitored CO2 evolution every 2 hours for 8 hours. It is possible that under these conditions respiration was over-evaluated (due to "disturbance respiration") contributing to an under-estimation of Q10. My results indicate that the potential for reserve depletion during thawing may be far greater than what would be expected on the basis of respiration rates measured in freezer-storage. The hypothesis Ho2, that "stabilized respiration rate increases as a simple function of temperature", is rejected. 78 5.2.2 RESERVE DEPLETION In concordance with reports concerning TNC depletion during cold storage (Cannel et al. (11), Chomba (13), Dean et al. (16), Glerum (25), Lambani (42), McCracken (52), Ritchie (57 and 58), Ronco (61) and Ziegler (82)), TNC depletion occurred during -2°C storage for white spruce and lodgepole pine, in both December and February-lifted seedlings. Not surprisingly, TNC depletion was also observed during thawing for both species. The rate of depletion appeared to be faster when plants were exposed to higher temperature (+7°C). Unfortunately, no paper has reported the incidence of a higher TNC depletion in thawing nor its relationship to plant establishment. Indeed, the "thawing" period that routinely follows cold storage in silvicultural practice has not been the subject of much study. Ritchie (57) reported an equivalent TNC depletion in Douglas-fir for seedlings exposed to 2 months at +2°C and those exposed to 6 months at -1°C. This is an indication of a temperature effect on TNC depletion rate. Although support from other research is limited, results of the present study indicate that if carbohydrate reserves are important for early growth, there is no doubt that extensive thawing could be harmful to the plant, both in terms of reserve depletion and in terms of resistance to planting stress. Both spruce and pine showed visual signs of growth resumption that were more pronounced at +7°C than at +2°C but present in both thawing regimes. Some root growth could be seen on pine after 2 weeks at +7°C and after 6 weeks on spruce. Needles were developing under the bud scales after 2 weeks of thawing in spruce (both temperatures) and the candle had begun to elongate in pine. As mentioned by Deans et al. (16), disturbance of actively growing roots could jeopardize transplant survival. It is also well known that the soft elongating candle on pine is very easily broken when seedlings are manipulated. 79 5.2.2.1 WHITE SPRUCE For spruce, when predicted and measured TNC depletion were plotted together over the 4 month storage period, it was observed that the measured TNC corresponded fairly well with the expected levels from day 0 to day 90 (Figure 9). Thereafter, the tendency for the TNC content to drift away from the predicted curve, rather evident at day 120, could be due to error in respiration and/or CH2O measurements or to alternative metabolite use or inter-conversions. Referring to Figure 14, it can be seen that for 0N4S (day 120), sugar yield was significantly higher with the HPLC than with the anthrone method. Although a limited number of analyses were made by HPLC, the results suggest that sugar quality changed over winter, as already reported by Loescher et al. (48). The presence of sugar-alcohols or other carbohydrates not detectable by the anthrone method is probably the most important factor leading to the discrepancy observed between predicted and measured TNC depletion for the 0N4S seedlings (Figure 9). During the thawing period following 4 months of freezer-storage (Figure 8), results indicated that TNC depletion occurred at +7°C but not at +2°C. Knowing that TNC content at 0N4S was actually higher than reported by the anthrone method, and knowing that sugar analysis by HPLC yielded the same sugar content as anthrone during +2°C thawing (Figure 14, samples 4S2T2 and 4S6T2), it is likely that there actually was TNC depletion at +2°C and that this depletion occurred in the first two weeks of thawing. At +7°C, using the TNC content of Figure 14 at 0N4S, the depletion observed during the first two weeks would have been much higher than reported. During thawing, measured and predicted TNC depletion do not match as well as during -2°C storage (Figure 9). Although some of the TNC measurements are quite close to the predicted curve, the discrepancy at other times, particularly in the case of 4S6T2, is extremely large. In this specific case, sugars were analyzed by both the 80 anthrone method and by HPLC. These analysis were done independently using a fresh extract each time and both methods gave the same content. Starch analysis was done in two lots, using five seedlings each time. Between the two groups, starch content was consistent. The discrepancy between measured and predicted TNC is so pronounced that even if starch content were omitted, the measured sugars would still rise above the curve. To explain this discrepancy, a few factors originating from experimental errors and from the physiology of the plant, having more or less weight but acting all together, can be suggested. It was observed that the standard errors of the means for different measurements (frost hardiness, TNC and respiration), tended to be higher during thawing than during storage. The study covered a large period of time characterized by different physiological stages. During freezer storage, the plants were rather dormant (post-dormancy to quiescence) whereas during thawing, the plants were definitely shifting from maintenance metabolism to growth metabolism (bud swelling, dehardening). Some seedlings during thawing distinguished themselves by showing either higher rates in respiration, reserve utilization or dehardening. It appears that with this "awakening", expression of individuality could affect the evaluation of a population for a given physiological parameter. By diverging from the mean, these seedlings contributed to an increase in error. At the plant level, it is possible that internal activities associated with the loss of dormancy and the development of an active growth potential created conditions that affected both respiration and CH2O content. The extreme Q10 values observed when seedlings were transferred from -2°C to +2°C are indicative of a disproportionately higher level of activity. The synthesis of CH2O from fatty acids and nitrogen compounds associated with spring (82) and dehardening (71) should lead to an increase in the CH2O pool. As growth resumes in the dark (during thawing), CH2O utilization by processes other than respiration would also be on the rise. As respiration 81 is increased by these activities, there may or may not be a net depletion of TNC reserves depending on the balance between CH2O generation and utilization. It is possible that although hardiness was released quite significantly after 6 weeks of thawing at +2°C, this temperature did not allow much structural growth or diverse metabolic activities. As such, the CH2O released may not have been incorporated as fast as presumed and as a result, the CH2O pool would show either an increase or would remain stable. At +7°C, this situation may not occur as the temperature would allow more activity in the plant. Therefore at +7°C, more CH2O would be incorporated into structural components or other sinks and the increased respiration should result in a net depletion of CH2O, as observed. For seedlings lifted in February (2N0S) to undergo -2°C storage (2N2S), the degree to which the measured TNC diverges from the expected curve is striking (Figure 10). Referring to Figure 14, it can be seen that at 2N0S in February, the sugar yield by HPLC would place the initial TNC content at 227.4 mg/g SDW. Unfortunately, HPLC analysis was not performed on seedlings from 2N1S and 2N2S and it is therefore not possible to verify the anthrone data for these dates. Yet, if the initial TNC value provided by HPLC analysis was to be used for day 0 to generate the predicted curve of TNC depletion, the measured TNC at day 30 (2N1S) and day 60 (2N2S) of storage would better fit the curve, at least until the beginning of thawing. Hypothesis H03 stating that "rates of carbohydrate depletion and respiratory CO2 evolution are well matched" is accepted in the case of white spruce at -2°C storage for 0N4S. Based on the anthrone data, H03 would be rejected in every other case. However, a complete HPLC analysis might lead to the opposite conclusion. 82 5.2.2.2 LODGEPOLE PINE In pine lifted in February, as for spruce (2N0S), changes in metabolism from maintenance to growth began prior to entry into -2°C storage. Although the results indicated that pine could re-develop hardiness, their precision is weak due to the narrow range of temperatures within which LT50 fluctuated. TNC content estimated with HPLC data showed that for outdoors (3N0S), for -2°C storage (0N4S) and for +2°C thawing, reserves in pine were significantly higher than when free sugars were evaluated by the anthrone method. However, sugar analysis was not done by HPLC on 2N1S and 2N2S pine. Because pine showed different patterns in dehardening, in sugar composition, in starch interconversion and in growth resumption, it is not possible here to speculate on explanations for the discrepancy observed between predicted and measured rates of TNC depletion. Unlike spruce, pine seems to maintain a high content of an unidentified compound (thought to be a sugar alcohol) throughout winter and even during thawing. It is likely that this compound would have remained during -2°C storage. Although Lambany (42) showed that sugar alcohols are depleted during hardiness development in jack pine (Pinus banksiana), it is not clear how much of this depletion might account for the quite important discrepancy at day 30 between predicted and measured TNC; the degree of hardiness gained during this time being so small and uncertain. Thus, all together, errors in the TNC content estimation due to lack of precision with anthrone, errors in the frost hardiness evaluation due to the narrow range of temperature in which LT50 fluctuated, and possible errors in CO2 evolution monitoring, do not allow the conclusion that respiratory CO2 evolution is well matched by the rate of carbohydrate depletion. Consequently, Ho3 is neither accepted nor rejected for pine lifted in February. 83 5.2.2.3 STARCH AND SUGAR DYNAMICS. Fluctuations in starch content were observed for spruce and pine seedlings overwintered outdoors (3N0S). Starch content went down between December and January. From January on, starch was synthesized in both roots and shoots and peaked in March (end of monitoring) for spruce. For pine, starch levels peaked in February in the shoot and in March in the root. Similar results were reported in plants overwintered outdoors by Ashworth et al. (3), Buns et al. (9), Keller and Loescher (38), Krasowski and Owens (40), Little (47), Mattsson (53), Omi et al. (56), Rose (62), Sennerby-Forsse and von Fircks (69), Siminovitch (71) and Webb and Kilpatrick (79). While most of the above reports associated spring starch synthesis with the first signs of growth resumption, Sennerby-Forsse and von Fircks (69) and Siminovitch (71) related spring starch synthesis to the first signs of dehardening. In spring, both processes occur simultaneously. Based on the frost hardiness results presented in section 4.1, seedlings of spruce and pine had already lost a significant degree of hardiness in February and were most likely shifting from maintenance metabolism towards growth metabolism. Indeed, in March, one month after a significant increase in starch content, seedlings from both species had dehardened considerably (Table 3) and had begun to resume shoot growth (candle elongation on pine and swollen buds on spruce). The starch proportion had continued to increase from February to March except for pine shoots. Because pine*had begun candle elongation and was visually ahead of spruce in growth, higher structural and metabolic requirements could explain why not as much sugar was converted into starch. Starch may also increase in spring due to accumulation of new photosynthate. A 1 4 C labeling study by Webb and Kilpatrick (79) indicated that starch accumulation in spring was from current photosynthesis and, in this respect, the authors suggested that starch could be a buffer between rapidly fluctuating photosynthetic rates and 84 subsequent translocation or growth processes. Although no conclusion was expressed specifically on the matter, the results reported by Rose (62) indicated that starch accumulation in loblolly pine could indeed be due to new photosynthate. The roots of plants from cold-storage with 5% starch content reached, after 1 month outdoors, 22% starch content, which was equivalent to the root content for seedlings overwintered outdoors. Interestingly, Rose observed that only the seedlings that increased their starch content in spring were able to grow roots. Alternatively, studies on deciduous trees (3, 38, 69, 71, 74) indicated that starch accumulation in the spring is not necessarily due to new photosynthate as photosynthesis could not happen (no leaves). Therefore, as suggested by Siminovitch (71), the dehardening process may be linked to starch accumulation by contributing sugars formerly used in cold protection. Moreover, the total dehardening and growth activation processes, characterized by mobilization of various proteins, RNA and phospholipids, could directly or indirectly influence the C H 2 O pool. The results reported in section 4.3 for dark freezer storage support the hypothesis that starch synthesis is not strictly related to new photosynthate. In the first two months of storage, for both spruce and pine lifted in December, starch was considerably depleted while the soluble sugars content increased. However, without any temperature fluctuation or exposure to light, starch was re-synthesized at month 3 of the -2°C storage, more so in the root than in the shoot. However, starch content never reached the level recorded for seedlings kept outdoors. At this time, dehardening had begun in spruce (although still quite hardy) but not detectably so in pine. In the following month, most of the starch appeared to have been converted back to sugar. Therefore, starch synthesis is neither strictly due to photosynthesis nor to the dehardening process. If sugar to starch conversion has some physiological meaning, the results suggest that it might indicate phenological changes in plant 85 metabolism. This shift would be cued by a factor intrinsic to the plant considering that no obvious environmental cue could have triggered this activity during storage. Once transferred to thawing (also a dark environment), a second sugar to starch conversion was observed after 2 weeks, such that the +7°C regime overall resulted in more starch than the +2°C regime. Dehardening was proceeding in both spruce and pine and so were the first signs of growth, especially at +7°C. Thus, starch synthesis in spring cannot be associated with one specific physiological processes. However, because it can precede (to some degree) both dehardening and growth resumption and apparently occurs without environmental cues, and also because starch synthesis appears to be related to the plant health status (28, 62), it could be quite interesting to explore the possibility of using starch dynamics as an indicator in crop management to plan spring activities and to diagnose seedling quality in the field. The sugar fraction of the TNC reserves fluctuated up and down over winter. Outdoors, the sugar content peaked in January (coolest month) in both spruce and pine. In storage, it was observed that sugar content was at least maintained (considering that some was used as respiratory substrate) in spruce transferred to -2°C storage in February, but not for pine. In this treatment, spruce seedlings were able to re-develop frost hardiness, a process that is characterized by an increase in soluble sugars (3, 18, 24, 38, 71). There was little or no re-development of frost hardiness in pine. Sugar and starch interconversion did not balance in terms of mg/g SDW. This is quite understandable considering all the different pathways and fates for sugars, besides respiration. For instance, lipid synthesis is known to happen following exposure to low temperature (26, 82); sugars can also be converted into sugar-alcohols (42); or be used as building material (50) depending on the physiological stage of the seedlings and the environmental conditions they are exposed to. 86 5.3 SUGAR QUALITY 5.3.1 ANALYTICAL METHODS FOR SOLUBLE SUGARS When the anthrone and HPLC methods for sugars analysis were compared, the discrepancy in the sugar content for spruce was significant only at month 4 of -2°C storage (0N4S) in the shoot (156.1 mg against 229.8 mg, by anthrone and HPLC, respectively). As previously mentioned, this result may account for the discrepancy observed between TNC depletion predicted by the respiration rate and the measured TNC (Figure 7). Lambany (41) has also observed an increase in sugar alcohol during the last month of storage for black spruce. In the plants kept outdoors, the discrepancy in sugar content of the shoot was significant at every measurement (1N0S, 2N0S and 3N0S) and in the root at 2N0S. The possibility exists, therefore, that the starting point TNC content was underestimated in seedlings that were lifted in February for purposes of predicting TNC depletion from rates of C0 2 evolution. Based on the results, it appears that spruce seedlings overwintered outdoors tended to keep a higher level of either sugar alcohol or other substances in their shoots. The discrepancy between the two analytical methods became smaller at 2N0S and 3N0S in spruce, indicating that the additional CH2O measured by HPLC was lost over time. Lambany (41) reported that for black spruce, the sugar alcohol level was stable from November to February and then declined from February to May for seedlings overwintered outdoors. Not surprisingly, pine differed from spruce in terms of sugar content and the analytical method used. The statistical comparison between the yield of sugars by anthrone and by HPLC indicated that in the shoot, for all samples from -2°C storage, +2°C thawing and outdoors at the nursery, HPLC detected a significantly higher sugar content. The same significant differences were also noted in the roots of the 4S2T2 87 seedlings and for seedlings overwintered outdoors. Aronsson et al. (1), Lambany and Langlois (43) and Lambany (42) all reported a higher content of sugar alcohol in pine than in spruce over the winter. Although my study was not initially designed to compare analytical methods, the results support Loescher et al. (49) who mentioned that in TNC analysis, "failure to account for sugar alcohol is a major defect in research as they are known to be a major producer of CH2O". 5.3.2 WHITE SPRUCE Throughout the -2°C storage, "stachyose", raffinose and sucrose were present in higher concentration in the root than in the shoot (Figure 16). The continuous depletion of raffinose over time in the shoot, while the two other saccharides fluctuated up and down suggests that raffinose may have another purpose besides contributing to CH2O reserves. Participation in frost hardiness is a likely role and will be discussed later. During thawing, the oligosaccharides were depleted in both tissues and the distinction between shoot and root was no longer apparent in terms of content. The monosaccharides (glucose and fructose) were consistently higher in concentration in the shoot (Figure 17). This observation corroborates numerous reports suggesting that soluble sugars play a cellular role in osmotic adjustment and/or in protection against freezing (1, 8, 24, 38, 46, 47, 64, 66, 68, 71, 75). The decline of starch observed at 0N2S might explain the sudden increase in hexoses and Peak 6 in the shoot, and sucrose in the root, observed at the same time. When starch was synthesized de novo during dark thawing (4S2T2), glucose and sucrose levels in the shoot and mainly sucrose in the root were considerably reduced. Glucose depletion in the shoot during thawing and in seedlings kept outdoors is likely to be 88 associated with increased respiration and a shift in metabolism towards growth resumption, as well as some loss of frost hardiness (conversion of sugar to starch). Outdoors, shoot and root raffinose, sucrose and glucose levels dropped considerably from January (1N0S) to March (3N0S). Overall, shoots lost 141.6 mg/g SDW of sugars while gaining 128.8 mg/g SDW of starch. In the root, 124.3 mg/g SDW of starch was synthesized while the sugar content dropped by 96.1 mg/g SDW. These results suggest that sugars may have been exported from the shoot to the root via phloem. They also suggest that other carbon sources must have been used for respiration and growth resumption as, on the whole, only slightly less sugar was depleted than starch synthesized. The other sources of carbon could be new photosynthate and/or materials released by the dehardening process. 5.3.3 LODGEPOLE PINE In storage, shoots and roots showed differences in their oligosaccharide content. "Stachyose" and sucrose appeared to be the preferential forms in the root while raffinose and sucrose appeared to be favoured by the shoot. In the shoot, the presence of raffinose along with sucrose, and its dynamics, supports its possible involvement in the frost hardiness process. The high concentrations of sucrose and stachyose in the root are more consistent with their role as C H 2 O reserves per se, in agreement with Ronco (61). For the plants overwintered outdoors, raffinose and sucrose in the shoot fluctuated together, peaking in January and being quite depleted by March. The Peak 2 sugar (likely stachyose), in higher concentration than the two other saccharides, was reduced drastically in January and completely depleted by March. Its role as a C H 2 O reserve seems likely as this sugar was depleted just as drastically as starch was in January. The parallelism between starch and the Peak 2 however was not maintained 89 in time as starch was synthesized de novo in the following month and not Peak 2. Outdoors, sucrose was the dominant oligosaccharide in the root and fluctuated the most. The Peak 2 and raffinose shared the balance and barely changed until March when they were depleted, just as sucrose was. The hexoses (glucose, fructose) and Peak 6 were definitely the preferred carbohydrates in the shoot where they probably played an active role in frost protection. While Peak 6 (probably a sugar alcohol) remained stable, fructose and glucose were depleted over time during thawing and outdoors. In the root, these three compounds were in much lower concentration than in the shoot. While all of them fluctuated very slightly in storage and outdoors, fructose and glucose were drastically depleted during thawing. In contrast to spruce, there was no major increase in any sugar at month 2 in stored pine, although starch had been depleted. After 2 weeks of thawing, the depletion of fructose and glucose was markedly higher than the amount of newly synthesized starch, indicating that these sugars, among other possibilities, must have been used in respiration and perhaps also as structural substrate. As in thawing, the depletion of sugars in pine kept outdoors was much higher than the synthesis of starch. In the shoot and root respectively, 27.9 and 42.4 mg/g SDW of starch had been added to the plant between January and March while 111.5 and 61.9 mg/g SDW of sugar were lost. The fact that pine seedlings had begun candle elongation when sampled in March suggests that the difference may have been used for early growth. 5.3.4 RELATIONSHIP BETWEEN SUGAR QUALITY AND FROST HARDINESS. Overall, in both spruce and pine, the concentrations and dynamics of individual sugars were quite different between shoots and roots. The two plant parts obviously 90 have different strategies or degree of response to resist the stress imposed by winter conditions. The roots tended to keep a higher level of oligosaccharides and starch. It has been recognized (39, 52, 65, 82) that generally speaking, below ground organs (roots, tubers and bulbs) are the main storage organs in plants. The shoots, which must deal with greater exposure to cold and desiccation, tended to keep a higher level of monosaccharides. Although the total oligosaccharides concentration was higher in the roots, the shoots of both species showed an overall higher content of raffinose. Several authors have tried to relate raffinose accumulation with frost hardiness development. A few of them (1, 34, 46), have found the relationship to be strong, as is concluded here. However, in other cases (44, 64), raffinose failed to correlate with frost hardiness. In other studies concerned with raffinose, the association was made with dormancy (4, 9, 38). Because both processes occur pretty much simultaneously, it is hard to separate them. Dormancy is naturally cued by photoperiod (15, 32) and so is the initiation of frost hardiness development in several cases (35, 70). Cold temperatures are often needed for the completion of the rest period in dormancy (i.e., to meet the chilling requirement), especially in boreal species (33, 45, 54). In frost hardiness, cold temperatures hasten and deepen the development of resistance (10, 11, 70). Chalupa and Frazer (12) demonstrated that under continuous light, raffinose synthesis can be induced by a simple exposure to cold temperature. Only the organ (shoot or root) exposed to cold temperature synthesized raffinose. Wiemken and Ineichen (80), on the other hand, demonstrated that raffinose appears following exposure to either short days at warm temperature or long days at cold temperature. Thus, it seems that short photoperiod and/or cold temperature can both initiate raffinose synthesis. Although cold temperatures were more effective than short days, the latter authors observed that simultaneous exposure to these two factors lead to 91 the highest accumulation of raffinose. Interestingly, synergism between photoperiod and temperature is also characteristic of frost hardiness development. With all these apparently conflicting reports, several authors have avoided relating raffinose with either dormancy or frost hardiness and instead, simply report it as being a "winter sugar" since it is not present during the growing season but appears with the onset of fall and remains throughout winter (12, 22, 24, 41, 43, 44, 47,51,67,68). In this study, raffinose present in the shoot in December was observed to decrease in concentration along with the release of hardiness. Although the dataset is limited, the correlation between raffinose content and hardiness levels in spruce and pine was quite high (r = 0.94). Interestingly, dehardening and raffinose depletion began without exposure to warming temperature or light in both species. However, the exposure to warmer temperatures speeded up both dehardening and raffinose hydrolysis. Although in lower concentration, raffinose was also present in the roots of both species. Frost tolerance of roots was not evaluated here but Bigras and D'Aoust (7) reported that for white spruce, root frost tolerance development was dependent upon exposure to low temperatures alone and not photoperiod. Based on the results and on the literature, it would appear feasible that shoot raffinose content might be useful as an indicator of frost hardiness (development and release). The dehardening is of interest as it corresponds to a period of the year where too often plants are considered to be resistant when in fact, they are loosing their protection quite rapidly . Considering that current methods for determining frost hardiness are elaborate, very time-consuming and lacking in precision and accuracy, raffinose monitoring may offer a good alternative. Raffinose testing is easy and the variation between individual specimens is less than in frost hardiness evaluation. However, an extensive study, including a bigger sample size and more species and 92 provenances should be made prior to reaching a solid conclusion on the use of raffinose content as an indicator of the plant's frost hardiness status from fall to spring. 5.4 TRANSLOCATION In the girdling study, during freezer storage, reserve carbohydrates in shoots of treated and control seedlings were used in similar fashion. However, the roots of girdled seedlings showed greater reserve depletion than the roots of intact controls. It seems as if the roots of girdled seedlings suffered the stress of being girdled whereas the shoots did not. Increased respiration, changes in sugar quality, or conversion to materials other than carbohydrates could account for the higher TNC depletion. The higher depletion of TNC between months 3 and 4 in the shoots of the intact seedlings and the smaller depletion in their roots when compared to girdled seedlings suggest possible transport of carbohydrates from the shoot to the root which could not occur in the girdled seedlings. Transport via phloem has been shown to happen at -30°C in willow (21). The important information that came out during the thawing period was observed at week 22, two weeks after the seedlings had been transferred to +7°C. In the root of the girdled seedlings, the TNC content (primarily the free sugar fraction) was tremendously increased. This result suggests that the CH2O pool may have been augmented by conversion from other compounds. Moreover it indicates that there is a translocation of carbohydrates via the phloem from root to shoot when temperatures are high enough to initiate shoot growth. The much higher TNC depletion in the shoots of girdled seedlings at this time, when compared to intact seedlings, is consistent with this interpretation. Overall the results indicate that phloem transport (root to shoot) does not occur in -2°C storage but may commence with growth activation during thawing. Therefore, hypothesis H04 stating that "Patterns of reserve depletion in 93 girdled seedlings are the same as in intact seedlings, therefore there is no translocation through the dormant period." is accepted in the case of -2°C storage but is rejected under conditions of thawing. 94 6. CONCLUSIONS This research has revealed some interesting points regarding both silvicultural practices and particularities in seedling physiology through winter. The study on respiration demonstrated that rates of C0 2 evolution increase following a disturbance and later stabilize at a lower level. Practically, this means that each time seedlings are manipulated (e.g., boxes moved around, transportation and so on), respiration is increased and TNC is depleted, depending of course on the severity of the disturbance. The study on TNC concentrations showed that thawing accelerates TNC depletion, dehardening and growth resumption. Thus, it is likely that the longer the thawing period, the more sensitive the seedlings would be to transplanting shock and late spring frost. These observations suggest that in practical silviculture, dark cool-storage for purposes of thawing should be avoided or limited to only a few days. It was demonstrated that the Q10 for respiration is not constant. When respiration was enhanced in response to disturbance or when temperatures tested were above zero, the Q10 remained within 2 and 3.5. However, when the range of temperatures used to evaluate Q10 extended below freezing, Q10 was much higher (9.0 to 16.7). This suggests that transfers from below to above zero temperatures trigger a disproportionate rate of activity. It was observed that seedlings, at least spruce, are able to re-develop frost hardiness if re-exposed to low temperatures. Based on the literature, it appears that this ability is a function of how advanced the seedlings are in the process of dehardening. Apparently, beyond a certain point, dehardening is no longer possible (71). The rate of dehardening is faster with increases in temperature. This should be taken with some caution as it was shown by Silim and Lavender (70) that in white 9 5 spruce, temperatures had an effect on dehardening rate only after the chilling requirement (for breaking dormancy) had been satisfied. The apparent TNC content was shown to vary depending on the analytical method used to evaluate the sugar fraction. As sugar quality changes over the winter, the sugar fraction should be assessed by methods that detect all kinds of sugar without bias. An under-estimation of sugar content could lead to erroneous conclusions regarding to CH2O dynamics in plant physiology. The fact that the study covered a period of the year characterized by seasonal changes in developmental physiology, CH2O fluctuations appeared to be due not only to respiration but also to changes in the plant physiological status. The thawing and the outdoor monitoring were both more conclusive in this matter as the plants tended to be more active under these conditions. Unexpected starch synthesis occurred during dark freezing storage, suggesting some manifestation of an endogenous rhythm or pattern. Starch synthesis was observed at the same time as a loss of hardiness. It appears that starch synthesis during winter is a transitory event and does not last unless plants are exposed to light before reaching their full growth potential. Seedlings kept outdoors continued to synthesize starch whereas in cold storage a conversion back to free sugar was observed. The possibility that early starch synthesis may be under the control of an endogenous "biological clock" is very attractive but requires further study. It could be of practical significance. For example, knowing when the clock initiates various physiological events should make it easier to plan spring activities. Starch synthesis preceeding growth resumption and any real loss of hardiness could be used as an indicator of seedling physiological status, and might reflect either a seedling's vigour or its proper synchronization with the environment. 96 The changes in sugar quality reported here do not permit any specific roles to be assigned to every sugar. However, some trends were observed. Monosaccharides were favoured in the shoot and in this respect, besides serving as fuel, they most likely were involved in osmotic adjustment and/or in cellular protection against freezing temperatures. Oligosaccharides were favoured in the root indicating their participation as TNC reserves. Raffinose content in the shoot was correlated with dehardening and may be useful as a plant indicator of frost hardiness. Further research is needed in this area to extend the correlation to further species and provenances. The results imply that carbon translocation from root to shoot via the phloem can occur in spring when there are sinks in the shoot requiring CH2O. Such translocation is not likely to be important at low temperatures during winter, or in freezer storage. 97 7. RECOMMENDATIONS FOR FURTHER RESEARCH. Based on this research, the differences found between pine and spruce regarding the quality of their sugar content throughout winter are indicative of species specific strategies to cope with the environment. Therefore, research on non-structural carbohydrate should continue and include other sources of carbon, such as lipids, sugar alcohols and proteins. There are indications that fluctuations in plant carbohydrates could reflect physiological status over the year. Research should be initiated on raffinose content and on starch-sugar interconversion in association with frost hardiness, dormancy and osmotic adjustment following severe stresses. There is a lack of knowledge on how seedlings acclimate following planting. This is mainly due to the difficulty of studying plants in there natural environment as there are few appropriate tools available. The purpose of studying carbohydrates in relation to plant physiological status should be to develop plant indicators that could be used as diagnostic tools as opposed to tools which attempt to predict survival rates. These tools could then be used to investigate the health status of tree seedlings following planting. Development of reliable plant indicators and extensive field research on tree seedling physiology should be the new direction of research in forest plant physiology. 98 8. REFERENCES 1- ARONSSON A. 1975. 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The Formation of Wood in Forest Trees., Academic Press, New York and London, pp. 303-320. 106 APPENDIX 1 TIME-TABLE 1 RESPIRATION MONITORING TIME-TABLE 2 TOTAL NON-STRUCTURAL CARBOHYDRATES ANALYSIS TIME-TABLE 3 TRANSLOCATION EXPERIMENT 107 TIME-TABLE 1 RESPIRATION MONITORING DEC.21/92 Lifted for -2C Storage Experiment 1 White spruce n=10 Feb.21/93 Lifted for -2C Storage Experiment 2 White spruce n=15 Experiment 2 Lodgepole pine n=10 MAR.21/93 Harvested from -2C storage Experiment 1 White spruce n=10 APR.21/93 Removed from storage] for thawing at indicated temperature Experiment 1 White spruce +2C n=10 +7C n=10 Transferred to thawing at indicated temperature Experiment 2 White spruce Experiment 2 Lodgepole pine +2C n=10 +7C n=5 +2C n=10 JUN.04/93 End of Experiment 1 and 2 1 0 8 TIME-TABLE 2 TOTAL NON-STRUCTURAL CARBOHYDRATES ANALYSIS DEC. 19/93 TNC analysis 0N0S, n=10 DEC.21/92 FIRST LIFTING FROM NURSERY TO STORAGE i CONTAINERS PINE SPRUCE DARK BOXES OUTDOORS AT NURSERY JAN.21/93 TNC analysis 1N0S, n=10 FEB.19/93 TNC analysis 2N0S, n=10 FEB.20/93 MAR.20/93 TNC analysis 3N0S, n=10 APR.26/93 TNC analysis MAY 10/93 TNC analysis JUN.07/93 TNC analysis FREEZING STORAGE -2C| 0N1S, n=10 0N2S,n=10 SECOND LIFTING FROM NURSERY TO STORAGE DARK BOXES FREEZING STORAGE -2d 2N1S, n=10 0N3S, n=10 2N2S,n=10 0N4S, n=10 ALL BOXES INTO THAWING 2N2S 0N4S THAWING +2G THAWING +7G 2S2T2, n=10 4S2T2,n=10 2S2T7, n=10 4S2T7, n=10 2S6T2, n=10 4S6T2, n=10 2S6T7, n=10 4S6T7, n=10 109 TIME-TABLE 3 TRANSLOCATION DEC.19/92 TNC analysis 0N0S, n=10 DEC.21/92 LIFTING FROM NURSERY DEC.22/92 GIRDLING for translocation investigation in storage. SPRUCE IN -2C STORAGE GIRDLED JAN.21/93 TNC analysis G-0N1S, n=10 FEB.19/93 TNC analysis G-0N2S, n=10 MAR.20/93 TNC analysis] G-0N3S, n=10 APR.26/93 TNC analysis! G-0N4S, n=10 INTACT 0N1S, n =10 0N2S, n =10 0N3S, n= =10 0N4S, n= =10 ALL BOXES INTO THAWING THAWING +2C MAY 10/93 TNC analysis G-4S2T2, n=10 MAY 24/93 TNC analysis G-4S4T2, n=10 4S2T2, n=10 4S4T2, n=10 BOXES MOVED TO +7C AFTER 4 WKS THAWING +7C JUN.07/93 TNC analysis G-4S6T2\7 n=10 4S6T2\7 n=10 110 APPENDIX 2 PROTOCOL FOR CARBOHYDRATE ANALYSIS I. Grinding the plant material (shoot and root) Grind the freeze-dried plant material in liquid nitrogen using a mortar and pestle. A pulverizer can be used for grinding but the timing is important to avoid over-heating the tissues. The ground material can be stored in vials below 5°C until required for analysis.(May require re-drying if condensation is picked up.) II. Separating soluble sugars from starch 1. Weigh out 20 to 50 mg of the pulverized plant material and put it in a test tube. 2. To each test tube, add 4 mL of methanol.chloroform.water (M.C.W - 12:5:3, v/v/v) and leave the samples in the freezer overnight. See section VI for solutions. 3. The following morning, remove the samples from the freezer, briefly shake them on a vortex and then centrifuge for 10 minutes. Pipet the supernatant (containing soluble sugars) into vials and save. Starch will remain with the pellet. 4. Add 4 mL of M.C.W to the pellet in the test tube. Vortex briefly and then centrifuge for 10 minutes. 5. Pipet off the supernatant and combine with the one from step 3. 6. Repeat steps 4 and 5 one time. It is important to wash the pellet properly as conifers may contain enough phenolic compounds to inhibit later enzymatic action. 7. Dry the pellet and take it immediately for starch analysis or store in the freezer. III. Final extraction of soluble sugars 1. To each vial of supernatant from above, add 5 mL of distilled water. Shake the vials gently by hand and let settle (about 2 hours). There should be phase separation. If desired, centrifuge to speed this step up. 111 2. Pipet the top aqueous layer into an evaporating flask. The lower layer is waste, mostly chloroform. Flash evaporate all the solvent from the flask, while the flask rotates in a 40°C water bath. It takes 10 to 15 minutes to do one evaporation. 3. Add 1.5 mL of distilled water to the flask, swirl gently and pipet into a vial. Wash the flask with another 1.5 mL of distilled water, swirl gently and add to the vial. Store in the freezer until required for sugar analysis. IV. Starch analysis using enzymes 1. Prepare a fresh solution of acetate buffer. 2. Prepare the starch standard solution. 3. To the pellet in the test tube, add 5 mL of acetate buffer and briefly vortex. 4. Autoclave both the starch solution and the test tubes, at 120°C for 1 hour. It is important to check the volume of the solution in the test tube after autoclaving as some evaporation may occur. 5. Transfer the sample and starch STD solution from the autoclave to a 55°C water bath for about 10 to 15 minutes. 6. For the standard curve, prepare the following dilutions in test tubes: -0 mL starch solution + 5 mL acetate buffer -2 mL starch solution + 3 mL acetate buffer -3 mL starch solution + 2 mL acetate buffer -4 mL starch solution + 1 mL acetate buffer -5 mL starch solution + 0 mL acetate buffer 7. Add to all test tubes (extracts and standards): -62.5 uL alpha-amylase solution -125 uL amyloglucosidase solution 8. Gently shake the contents by hand to mix the enzymes with the solution. Incubate for 2 hours at 55°C. 9. After incubation, add 30 - 50 mg of clean PVPP to each tube and vortex well. PVPP is used to precipitate remaining phenplics. 10. Centrifuge for 10 minutes. 112 11. Aiming for a working volume of 200 uL, pipet the desired amount of aliquot and dilute it appropriately. This requires some sense of what the starch concentration in a particular plant extract might be (i.e., varies with time of year, plant part) For dormant white spruce, the following are usually reasonable: -Needles: 200 uL plant extract + 0 uL acetate buffer -Roots: 100 uL plant extract + 100 uL acetate buffer 12. For the standard curve, use 200 uL of each previous dilution. 13. Add 2 mL of combined peroxidase/glucose oxidase/colour reagent solution. 14. Incubate the samples for 30 minutes in a 37°C water bath. 15. Read the absorbance at 450 nm. V. Soluble sugars analysis using anthrone reagent 1. Prepare the following dilutions using the glucose standard solution: -0 mL glucose solution + 5 mL distilled water -2 mL glucose solution + 3 mL distilled water -3 mL glucose solution + 2 mL distilled water -4 mL glucose solution + 1 mL distilled water -5 mL glucose solution + 0 mL distilled water 2. Pipet 200 uL of each dilution into 0.5 mL centrifuge tube for the standard curve. 3. Take the sugar extracts from III and dilute them appropriately into 0.5 mL centrifuge tube. As in the starch analysis, the dilution factor will vary from one plant part to another, time of year, ect. For dormant spruce, the following are reasonable : -Needles: 25 uL plant extract + 175 uL distilled water. -Roots: 25 uL plant extract + 175 uL distilled water. 4. To deproteinize, add to each of the above (extracts and standards) 50 uL of barium hydroxide (0.3M) and 50 uL of zinc sulfate (0.3M). 5. Centrifuge for 3 minutes, then pipet off 200 uL of the supernatant into a tall test tube. Great care must be taken when pipetting. If some of the precipitate passes 113 with the supernatant, the solution will turn pink when adding the acids and dark blue with the anthrone. This will result in an overestimation of the sugar content. 6. From this step, work under a fume-hood. To each test tube, add: 200 uLof 11.6 M HCI 40 uL of 45% formic acid 1.6 mL of sulfuric acid/anthrone solution (fresh every day) 7. Close the top of the test tube with a marble. Vortex gently and briefly (watch out for bubbles). 8. Put the entire rack in a boiling water bath (~90°C) for exactly 12 minutes. 9. After 12 minutes, remove the rack and put it in an ice water bath until cold (5-10 minutes). 10. Vortex strongly (at least 30 seconds) to get rid of the bubbles and let sit in the ice water bath until it settles. 11. Read the absorbance at 630 nm. VI. Preparation of solutions used in the carbohydrate analyses 1. M:C:W - Methanol:Chloroform:Water (12:5:3) 1000 mL of M:C:W contains: -600 mL of methanol -250 mL of chloroform -150 mL of distilled water 2. Acetate buffer a. Weigh out 4.082 g of sodium acetate powder into a measuring cylinder. Add distilled water to 200 mL level and stir well. b. In a second cylinder, measure 150 mL of distilled water and add 2.58 mL of acetic acid. Then make the volume up to 300 mL with distilled "water. c. Mix a) and b) together. The pH of the mixture should be approximately 4.5. 114 3. Starch standard solution Dissolve 16.2 mg (0.1 mmol) of soluble starch powder (BDH chemicals, ACS 879) in 100 mL of acetate buffer. Autoclave for complete dissolution. 4. Alpha-amylase solution (400 units /mL) a. Weigh out 24 mg of alpha-amylase powder (E.C.3.2.1.1, from Aspergillus, Sigma A-0273) and dissolve it in 3 mL of distilled water. b. Add between 10 to 15 mg of activated charcoal, mix very well and spin. Pipet the supernatant into a test tube and add 1.268 g of granular ammonium sulfate. Stir to dissolve. Spin. c. Pipet the supernatant out and keep the pellet. Re-suspend the pellet in 3 mL of distilled water and mix. If you get some charcoal particles, re-spin and save the supernatant. 5. Amyloglucosidase (800 units/ml) a. Weigh out 444.4 mg of amyloglucosidase powder (E.C.3.2.1.3, from Rhizopus, Sigma A-7255) and dissolve it in 6 mL of distilled water. b. Add between 18 to 30 mg of activated charcoal, mix very well and spin. Pipet the supernatant into a test tube and add 2.536 g of granular ammonium sulfate. Stir to dissolve. Spin. c. Pipet the supernatant out and keep the pellet. Re-suspend the pellet in 6 mL of distilled water and mix. If you get some charcoal particles, re-spin and save the supernatant. 6. Peroxidase/glucose oxidase/colour reagent solution. a. Dissolve the contents of 1 capsule of PGO-enzymes (PGO enzymes, Sigma cat.no.510-6) in 100 mL of distilled water in an amber bottle. Mix well. b. Add 20 mL of distilled water to the 50 mg pre-weighed o-dianisidine dihydrochloride (colour reagent) powder and mix well. c. To the 100 mL PGO-enzyme solution, add 1.6 mL of the colour reagent and mix well. Keep the colour reagent solution in the fridge for future use. 115 7. Glucose standard solution Dissolve 18 mg (0.1 mmol) of D-glucose in 100 mL of distilled water. 8. Chemicals The following products come in solution. Check the molarity and adjust if necessary. Barium hydroxide (0.3 M) Zinc sulfate (0.3 M) HCI(11.6M) Formic acid (45%) 9. Sulfuric acid/Anthrone solution Weigh out 20 mg of anthrone (Sigma A-1631) in an amber bottle. Add 100 mL of 80% sulfuric acid. Mix well. It is better to prepare this solution at the very last moment to keep the pigment stable as long as possible. 116 APPENDIX 3 TEMPERATURE OF FREEZING EXPOSURE. LODGEPOLE PINE WHITE SPRUCE -20°C ; -30°C ; -40°C ; -50°C ONOS -30°C ; -40°C ; -50°C ; -60°C -10°C ; -20°C ; -30°C ; -40°C 0N2S -30°C ; -40°C ; -50°C ; -60°C -10°C ; -20°C ; -30°C ; -40°C 2N0S -30°C ; -40°C ; -50°C ; -60°C -10°C ; -20°C -30°C ; -40°C 2N2S -30°C ; -40°C ; -50°C ; -60°C -10°C ; -20°C -30°C ; -40°C 0N4S -30°C ; -40°C • -50°C ; -60°C -5°C -10°C -15°C -20°C 3N0S -15°C • -20°C -25°C ; -30°C -10°C -20°C -30°C -40°C 2S2T2 -30°C -40°C -50°C -60°C -5°C -10°C -15°C -20°C 2S4T2 -20°C -30°C -40°C -50°C -5°C -10°C -15°C -20°C 2S6T2 -20°C -30°C -40°C -50°C -5°C -10°C -15°C -20°C 2S2T7 -10°C -20°C -30°C -40°C -5°C -10°C -15°C -20°C 2S4T7 -10°C -15°C -20°C -30°C -5°C -10°C -15°C -20°C 2S6T7 -10°C -15°C -20°C -30°C -10°C -20°C -30°C -40°C 4S2T2 -30°C -40°C -50°C -60°C -5°C -10°C -15°C -20°C 4S4T2 -20°C -30°C -40°C -50°C -5°C -10°C -15°C -20°C 4S6T2 -20°C -30°C -40°C -50°C -5°C -10°C -20°C -30°C 4S2T7 -10°C -20°C -30°C -40°C -5°C -10°C -15°C -20°C 4S4T7 -10°C -15°C -20°C -30°C -5°C -10°C ; -15°C -20°C 4S6T7 -10°C -15°C -20°C -30°C 117 APPENDIX 4 METHOD FOR PREPARATION OF RESINS. A. Dowex50w-X1-16(R-SO3"H+): Activation (H+) : Wash resin with distilled deionized water 4 times to remove the fines. Add 10 fold excess of HCI 1N, allow to stand 15 minutes with occasional stirring and then filter off the acid. Wash well with distilled deionized water to the same pH and to the same E.C.. Conversion (H + to Na + form): Slurry activated resin with 1N NaOH (~2 volumes NaOH) for 15 minutes. Wash with alkali until the pH rises above 9. Wash out excess NaOH with water until the pH drops below 9. B. Dowex 1-X1 to 10 (R-CH2N+(CH3) 3Cr) Activation (OH"): Wash resin with distilled deionized water 4 times to remove the fines. Slurry with 2N HCI for 15 minutes, with occasional stirring. Wash to pH of water. Slurry with 2N NaOH for 15 minutes. Wash with distilled deionized water to the same pH and to the same E.C. (test for Cf with AgN03 acidified with HNO3). Conversion (OH" to Formate form): Wash activated resin with 1N formic acid until pH drops below 2 (~ 2 volumes). Rinse with water until pH rises about 4.5. APPENDIX 5 STATISTICS 119 INDEX OF INJURY 1 - White spruce, 4 months -2°C storage, Index of Injury -30°C. Using One Way Analysis of Variance Group N Mean Std Dev SEM ONOS 5 25.8 3.73 1.667 0N2S 5 11.6 1.90 0.851 0N4S 5 19.5 3.57 1.596 Source of Variance DF SS MS F P Between treatments 2 508.0 254.0 25.2 <0.0001 Residual 12 121.0 10.1 Total 14 629.0 Multiple Comparison Procedures (Student-Newman-Keuls Method) Different letters indicate that means are different (P < 0.05) ONOS a 0N2S b 0N4S c 2 - White spruce, 2 months -2°C storage, Index of Injury -30°C. Using One Way Analysis of Variance Group N Mean Std Dev SEM ONOS 5 25.8 3.73 1.67 2N0S 5 48.1 12.51 5.60 2N2S 5 15.8 4.25 1.90 Source of Variance DF SS MS F P Between treatments 2 2719.4 1359 .7 21.6 0.0001 Residual 12 754.0 62.8 Total 14 3473.3 Multiple Comparison Procedures (Student-Newman-Keuls Method) Different letters indicate that means are different (P < 0.05) ONOS a 2N0S b 2N2S a 120 3 - White spruce, 3 months outdoors Nursery, Index of Injury -30°C. Using One Way Analysis of Variance Group N Mean Std Dev SEM ONOS 5 25.8 3.73 1.67 2N0S 5 48.1 12.51 5.60 3N0S 5 79.8 8.85 3.96 Source of Variance DF SS MS F P Between treatments 2 7351.3 3675 .6 44.3 <0.0001 Residual 12 995.3 82.9 Total 14 8346.6 Multiple Comparison Procedures (Student-Newman-Keuls Method) Different letters indicate that means are different (P < 0.05) ONOS a 2N0S b 3N0S c 4 - Lodgepole pine, 4 months -2°C storage, Index of Injury -20°C. Using One Way Analysis of Variance Group N Mean Std Dev SEM ONOS 5 38.7 11.29 5.05 0N2S 5 70.2 6.41 2.87 0N4S 5 41.4 18.13 8.11 Source of Variance DF SS MS F P Between treatments 2 3049.6 1524.8 9.20 0.0038 Residual 12 1989.0 165.7 Total 14 5038.5 Multiple Comparison Procedures (Student-Newman-Keuls Method) Different letters indicate that means are different (P < 0.05) ONOS a 0N2S b 0N4S a 121 5 - Lodgepole pine, 2 months -2°C storage, Index of Injury -20°C. Using One Way Analysis of Variance Group N Mean Std Dev SEM ONOS 5 38.7 11.29 5.05 2N0S 5 53.6 20.16 9.02 2N2S 5 30.2 7.81 3.49 Source of Variance DF SS MS F P Between treatments 2 1402.5 701.2 3.54 0.0620 Residual 12 2379.7 198.3 Total 14 3782.2 6 -Lodgepole pine, 3 months outdoors Nursery, Index of Injury -20°C. Using One Way Analysis of Variance Group N Mean Std Dev SEM ONOS 5 38.7 11.29 5.05 2N0S 5 53.6 20.16 9.02 3N0S 5 75.4 6.00 2.68 Source of Variance DF SS MS F P Between treatments 2 3410.2 1705.1 8.98 0.0041 Residual 12 2279.5 190.0 Total 14 5689.7 Multiple Comparison Procedures (Student-Newman-Keuls Method) Different letters indicate that means are different (P < 0.05) ONOS a 2N0S a 3N0S b 122 7 - White spruce, 0N4S and +2°C or +7°C thawing, Index of Injury -30°C. Using Two Way Analysis of Variance Group N Mean SEM Group N Mean SEM 4S0T2 5 19.5 1.43 4S0T7 5 19.5 1.43 4S2T2 5 40.2 9.30 4S2T7 5 58.2 9.67 4S4T2 5 48.1 7.16 4S4T7 5 77.6 5.44 4S6T2 5 66.6 7.00 4S6T7 5 80.1 2.25 Source of Variance DF SS MS F P Time 3 16347.7 5449.2 21.88 <0.0001 Temperature 1 2320.3 2320.3 9.31 0.0045 Time x Temperature 3 1116.0 372.0 1.49 0.2350 Residual 32 7971.4 249.1 Total 39 27755.5 711.7 8 - White spruce, 2N2S and +2°C or +7°C thawing, Index of Injury -30°C. Using Two Way Analysis of Variance Group N Mean SEM Group N Mean SEM 2S0T2 5 15.8 1.70 2S0T7 5 15.8 1.70 2S2T2 5 22.7 4.01 2S2T7 5 65.5 5.55 2S4T2 5 43.9 9.40 2S4T7 5 65.8 4.11 2S6T2 5 82.7 3.83 2S6T7 5 77.3 4.78 Source of Variance DF SS MS F P Time 3 21166.0 7055.3 40.65 <0.0001 Temperature 1 2208.7 2208.7 12.73 0.0012 Time x Temperature 3 3661.9 1220.6 7.03 0.0009 Residual 32 5554.2 173.6 Total 39 32590.9 835.7 123 9 -Lodgepole pine, 0N4S and +2°C or +7°C thawing, Index of Injury -20°C. Using Two Way Analysis of Variance Group N Mean SEM Group N Mean SEM 4S0T2 5 41.4 7.25 4S0T7 5 41.4 7.25 4S2T2 5 54.8 10.5 4S2T7 5 88.5 4.15 4S4T2 5 89.3 4.32 4S4T7 5 93.6 1.94 4S6T2 5 91.1 7.34 4S6T7 5 97.0 1.06 Source of Variance DF SS MS F P Time 3 17725.7 5908.6 28.39 <0.0001 Temperature 1 1200.5 1200.5 5.77 0.0223 Time x Temperature 3 1766.7 588.9 2.83 0.0540 Residual 32 6659.5 208.1 Total 39 27352.3 701.3 10 - Lodgepole pine, 2N2S and +2°C or +7°C Using Two Way Analysis of Variance Group N Mean SEM 2S0T2 5 30.2 3.13 2S2T2 5 66.8 6.00 2S4T2 5 85.4 3.67 2S6T2 5 93.0 2.69 thawing, Index of Injury -20°C. Group N Mean SEM 2S0T7 5 30.2 3.13 2S2T7 5 84.2 4.42 2S4T7 5 91.8 1.88 2S6T7 5 95.6 1.66 Source of Variance DF SS MS F P Time 3 25528.1 8509.4 106.8 <0.0001 Temperature 1 478.1 478.1 6.0 0.0200 Time x Temperature 3 440.7 146.9 1.84 0.1593 Residual 32 2550.5 79.7 Total 39 28997.3 743.5 124 TOTAL NON-STRUCTURAL CARBOHYDRATE (TNC) 1 - White spruce whole plant, outdoors Nursery (3N0S) Using Kruskal-Wallis One Way Analysis of Variance on ranks. Group N Median 25% 75% ONOS 10 221.4 212.5 242.5 1N0S 10 196.5 188.2 220.0 2N0S 10 182.4 175.7 202.8 3N0S 10 242.4 235.2 246.5 P = <0.0001 significant difference Multiple Comparison Procedures (Student-Newman-Keuls Method) Different letters indicate that means are different (P < 0.05) ONOS a 1N0S b 2N0S c 3N0S d 2 - White spruce whole plant, 2 months Nursery + 2 months -2°C Storage (2N2S) Using One Way Analysis of Variance Group N Mean Std Dev SEM ONOS 10 229.6 23.3 7.36 1N0S 10 201.4 19.5 6.16 2N0S 10 187.4 13.9 4.39 2N1S 10 184.0 20.8 6.59 2N2S 10 171.3 16.7 5.30 Source of Variance DF SS MS F P Between treatments 4 19743.0 4935.8 13.5 O.0001 Residual 45 16464.0 365.9 Total 49 36207.0 125 Multiple Comparison Procedures (Student-Newman-Keuls Method) Different letters indicate that means are different (P < 0.05) ONOS a 1N0S b 2N0S be 2N1S be 2N2S c 3 - White spruce whole plant, 4 months -2°C Storage (0N4S) Using One Way Analysis of Variance Group N Mean Std Dev SEM ONOS 10 229.6 23.3 7.36 0N1S 10 216.8 15.5 4.89 0N2S 10 202.5 10.2 3.23 0N3S 10 200.1 21.9 6.93 0N4S 10 154.7 12.5 3.97 Source of Variance DF SS MS F P Between treatments 4 32149.0 8037.3 26.4 <0.0001 Residual 45 13701.1 304.5 Total 49 45850.1 Multiple Comparison Procedures (Student-Newman-Keuls Method) Different letters indicate that means are different (P < 0.05) ONOS a 0N1S ab 0N2S b 0N3S b 0N4S c 4 - White spruce whole plant, 0N4S and +2°C or +7°C thawing Using Two Way Analysis of Variance Group N Mean SEM Group N Mean SEM 4S0T2 10 154.7 3.8 4S0T7 10 154.7 3.9 4S2T2 10 154.2 8.1 4S2T7 10 122.8 5.9 4S6T2 10 162.4 4.9 4S6T7 10 94.3 5.8 126 Source of Variance DF SS MS F P Time 2 7060.1 3530.0 10.3 0.0002 Temperature 1 16502.3 16502.3 48.0 <0.0001 Time x Temperature 2 11617.2 5808.6 16.9 <0.0001 Residual 54 18550.4 343.5 Total 59 53730.0 910.7 5 - White spruce whole plant, 2N2S and +2°C or +7°C thawing Using Two Way Analysis of Variance Group N Mean SEM Group N Mean SEM 2S0T2 10 171.3 5.0 2S0T7 10 171.3 5.0 2S2T2 10 171.2 8.7 2S2T7 10 125.4 5.0 2S6T2 10 120.2 4.2 2S6T7 10 11.7.8 9.7 Source of Variance DF SS MS F P Time 2 27513.4 13756.7 28.26 <0.0001 Temperature 1 3877.8 3877.8 7.96 0.0067 Time x Temperature 2 6657.2 3328.6 6.84 0.0023 Residual 54 26290.0 486.9 Total 59 64338.3 1090.5 6 - Lodgepole pine whole plant, outdoors Nursery (3N0S) Using Kruskal-Wallis One Way Analysis of Variance Group N Median 25% 75% ONOS 10 160.1 141.0 176.8 1N0S 10 150.8 148.4 159.6 2N0S 10 146.8 137.9 155.5 3N0S 10 122.9 120.4 130.7 P = 0.0017 significant difference 127 Multiple Comparison Procedures (Student-Newman-Keuls Method) Different letters indicate that means are different (P < 0.05) ONOS a 1N0S a 2N0S a 3N0S b 7 - Lodgepole pine whole plant, 2 mths Nursery + 2 mths -2°C Storage (2N2S) Using Kruskal-Wallis One Way Analysis of Variance Group N Median 25% 75% ONOS 10 160.1 141.0 176.8 1N0S 10 150.8 148.4 159.6 2N0S 10 146.8 137.9 155.5 2N1S 10 97.1 85.9 104.5 2N2S 10 96.5 84.1 100.1 P < 0.0001 significant difference Multiple Comparison Procedures (Student-Newman-Keuls Method) Different letters indicate that means are different (P < 0.05) ONOS a 1N0S a 2N0S a 2N1S b 2N2S b 8 -Lodgepole pine whole plant, 4 months -2°C Storage (0N4S) Using One Way Analysis of Variance Group N Mean Std Dev SEM ONOS 10 156.6 23.8 7.52 0N1S 10 143.0 19.0 6.01 0N2S 10 116.6 15.5 4.90 0N3S 10 101.7 14.0 4.42 0N4S 10 109.6 15.6 4.92 128 Source of Variance DF SS MS F P Between treatments 4 21666.5 5416.6 16.9 <0.0001 Residual 45 14427.6 320.6 Total 49 36094.2 Multiple Comparison Procedures (Student-Newman-Keuls Method) Different letters indicate that means are different (P < 0.05) ONOS a 0N1S a 0N2S b 0N3S b 0N4S b 9 - Lodgepole pine whole plant, 0N4S and +2°C or +7°C thawing Using Two Way Analysis of Variance Group N Mean SEM Group N Mean SEM 4S0T2 10 109.6 4.7 4S0T7 10 109.6 4.7 4S2T2 10 83.4 3.7 4S2T7 10 55.8 3.0 4S6T2 10 48.2 1.9 4S6T7 10 32.7 1.6 Source of Variance DF SS MS F P Time 2 48251.4 24125.7 181.0 O.0001 Temperature 1 3107.6 3107.6 23.3 <0.0001 Time x Temperature 2 1914.8 957.4 7.2 0.0017 Residual 54 7199.3 133.3 Total 59 60473.1 1025.0 10 - Lodgepole pine whole plant, 2N2S and +2°C or +7°C thawing Using Two Way Analysis of Variance Group N Mean SEM Group N Mean SEM 2S0T2 10 92.5 3.8 2S0T7 10 92.5 3.8 2S2T2 10 69.2 3.3 2S2T7 10 65.2 4.9 2S6T2 10 43.0 2.5 2S6T7 10 27.2 1.9 Source of Variance DF SS MS F P Time 2 33052.7 16526.4 122.0 <0.0001 Temperature 1 647.5 647.5 4.8 0.0332 Time x Temperature 2 669.4 334.7 2.5 0.0941 Residual 54 7316.2 135.5 Total 59 41685.8 706.5 129 TRANSLOCATION EXPERIMENT 1 - White spruce Whole, 4 months -2°C storage, Treatments = Control vs Girdled Using Two Way Analysis of Variance Group N Mean SEM Group N Mean SEM ONOS 10 229.6 7.0 G-0N0S 10 229.6 7.0 0N1S 10 216.8 4.6 G-0N1S 10 182.8 7.9 0N2S 10 202.5 3.1 G-0N2S 10 187.4 5.9. 0N3S 10 200.1 6.6 G-0N3S 10 178.0 4.5 0N4S 10 154.7 3.8 G-0N4S 10 148.6 7.0 Source of Variance DF SS MS F P Time 4 62326.6 15581.7 39.84 <0.0001 Treatment 1 5955.8 5955.8 15.23 0.0002 Time x Treatment 4 3557.2 889.3 2.27 0.0674 Residual 90 35198.5 391.1 Total 99 107038.1 1081.2 2 - White spruce Whole, 0N4S and +2/+7°C thawing Treat. = Control vs Girdled Using Two Way Analysis of Variance Group N Mean SEM Group N Mean SEM 4S0T 10 154.7 3.8 G-4S0T 10 148.6 7.0 4S2T2 10 154.2 8.1 G-4S2T2 10 158.7 3.7 4S4T2 10 133.0 2.7 G-4S4T2 10 150.8 6.9 4S6T2/7 10 107.5 6.1 G-4S6T2/7 10 123.0 5.0 Source of Variance DF SS MS F P Time 3 20310.3 6770.1 18.79 <0.0001 Treatment 1 1257.6 1257.6 3.49 0.0658 Time x Treatment 3 1813.3 604.4 1.68 0.1794 Residual 72 25937.0 360.2 Total 79 49318.2 624,3 130 3 - White spruce Shoot, 4 months -2°C storage, Treatments = Control vs Girdled Using Two Way Analysis of Variance Group N Mean SEM Group N Mean SEM ONOS 10 229.7 9.4 G-0N0S 10 229.7 9.4 0N1S 10 217.1 8.6 G-0N1S 10 194.7 7.8 0N2S 10 203.5 5.8 G-0N2S 10 197.9 7.4 0N3S 10 208.1 7.8 G-0N3S 10 186.3 6.3 0N4S 10 148.1 5.1 G-0N4S 10 159.4 9.3 Source of Variance DF SS MS F P Time 4 60708.4 15177.1 22.27 <0.0001 Treatment 1 1471.5 1471.5 2.16 0.1452 Time x Treatment 4 4160.0 1040.0 1.53 0.2013 Residual 90 61329.8 681.4 Total 99 127669.6 1289.6 4 - White spruce Root, 4 months -2°C storage, Treatments = Control vs Girdled Using Two Way Analysis of Variance Group N Mean SEM Group N Mean SEM ONOS 10 233.2 5.4 G-0N0S 10 233.2 5.4 0N1S 10 204.4 9.1 G-0N1S 10 166.2 12.1 0N2S 10 202.2 7.6 G-0N2S 10 164.4 10.0 0N3S 10 183.5 9.1 G-0N3S 10 165.2 8.3 0N4S 10 167.1 7.0 G-0N4S 10 127.3 6.8 Source of Variance DF SS MS F P Time 4 77349.3 19337.3 25.1 <0.0001 Treatment 1 17973.2 17973.2 23.3 <0.0001 Time x Treatment 4 6059.3 1514.8 2.0 0.1063 Residual 90 69315.4 770.2 Total 99 170697.2 1724.2 131 5 - White spruce Shoot, 0N4S and +2/+7°C thawing Treat. = Control vs Girdled Using Two Way Analysis of Variance Group N Mean SEM Group N Mean SEM 4S0T 10 148.1 5.1 G-4S0T 10 159.4 9.3 4S2T2 10 169.8 8.4 G-4S2T2 10 176.6 5.3 4S4T2 10 137.1 4.1 G-4S4T2 10 157.5 8.6 4S6T2/7 10 106.9 8.0 G-4S6T2/7 10 77.3 2.1 Source of Variance DF SS MS F P Time 3 72531.2 24177.1 47.0 <0.0001 Treatment 1 95.0 95.0 0.19 0.6686 Time x Treatment 3 7254.1 2418.0 4.7 0.0047 Residual 72 37033.3 514.4 Total 79 116913.6 1479.9 6 - White spruce Root, 0N4S and +2/+7°C thawing Treat. = Control vs Girdled Using Two Way Analysis of Variance Group N Mean SEM Group N Mean SEM 4S0T 10 167.1 7.0 G-4S0T 10 127.3 6.8 4S2T2 10 125.2 8.4 G-4S2T2 10 120.1 8.7 4S4T2 10 124.1 4.5 G-4S4T2 10 139.0 6.6 4S6T2/7 10 109.7 7.3 G-4S6T2/7 10 250.1 10.9 Source of Variance DF SS MS F P Time 3 38025.8 12675.3 19.2 <0.0001 Treatment 1 15264.9 15264.9 23.1 <0.0001 Time x Treatment 3 92455.5 30818.5 46.6 <0.0001 Residual 72 47636.0 661.6 Total 79 193382.2 2447.9 

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