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Vegetative storage protein accumulation and physiological changes occurring within interior spruce seedlings Binnie, Sheila Catherine 1993

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VEGETATIVE STORAGE PROTEIN ACCUMULATION AND PHYSIOLOGICAL CHANGES OCCURRING WITHIN INTERIOR SPRUCE SEEDLINGS by SHEILA CATHERINE BINNIE B.Sc., The University of British Columbia, 1989. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Botany)  We accept this thesis as conforming  THE UNIVERSITY OF BRITISH COLUMBIA April 1993 ©Sheila Catherine Binnie, 1993.  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.  (Signature)  Department of  botany  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  r1aAC1k315  ABSTRACT The appearance and accumulation of vegetative storage proteins in nursery - grown Interior spruce seedlings (Picea glauca and P. engelmanni complex) was evaluated along with changes in seedling physiology. The 30 kD and 27 kD vegetative storage proteins (VSP) appeared after vegetative maturity (budset) and accumulated during the development of rest and the acquisition of cold hardiness. This trend was observed in bud extracts of three seedlots QL, EK, and PG as well as in the ambient treatment (natural fluctuations in photoperiod and temperature) of seedlot FIN. Stem and root tissue had similar accumulation patterns. Cold hardiness levels were measured using the electrolyte leakage method. This test is used annually to predict lifting date for cold storage of seedlings. Both the LT 50 (temperature for 50% electrolyte leakage) and the index of injury at -18°C (I.I.@-18°C) were low during early fall when storage protein levels were negligible in seedlot QL extracts. Afterwards, hardiness (I.I.@-18°C) was acquired and VSP accumulated until late October. Scanning densitometry of SDS-PAGE gels indicated that 15% of total protein was VSP by early November. These changes were accompanied by a decline in photosystem II activity, mitotic index, and dormancy. In seedlot QL, vegetative storage protein patterns were correlated to LT 50 (r = -0.972), photosystem II activity (r = -0.971), index of injury at -18°C (r = -0.900). and days to budbreak (r = -0.893). Seedlings stored at 4°C showed a slight decline in VSP and a decrease in seedling quality after 6 months of storage. Fall acclimation treatments ii  long day 16 hr, 10°C days/5°C night (LD/Cold), short day <12 hr, 22°C day/ 18°C night (SD/Warm), and Ambient treatment - indicated that prolonged short days caused maximum VSP accumulation within 30 days whereas under ambient conditions it took between 80 to 100 days. Cold temperatures may help cause the normal gradual increase in VSP and cold hardiness within buds. The SD/warm root bark was not as strongly influenced by daylength as bud and stem tissue because VSP accumulation was gradual. Although SD/warm seedlings accumulated maximum VSP levels, these seedlings remained dormant and did not become cold hardy. The results indicate that VSP accumulation and cold hardiness usually develop in parallel but they are unrelated. Under normal fall acclimation conditions, VSP accumulation and cold hardiness acquisition patterns are very similar; therefore, both could be used to predict lifting date in the future, with VSP used to predict lifting date and hardiness testing used to ensure cold hardiness.  iii  TABLE OF CONTENTS Page  ABSTRACT ^ TABLE OF CONTENTS ^  iv  LIST OF TABLES ^  vi  LIST OF FIGURES ^ ACKNOWLEDGEMENTS ^ 1.  2.  vii viii  INTRODUCTION AND LITERATURE REVIEW ^ 1  1.1 Dormancy ^ 4 1.2 Food Reserves ^ 5 1.2.1 Starch ^ 9 1.2.2 Lipids ^ 11 1.2.3 Nitrogen ^ 12 1.3 Development of Cold Hardiness requires cytological and biochemical changes ^ 14 1.4 Proteins as biochemical markers ^ 15 1.4.1 Discovery of vegetative storage proteins ^ 16 1.4.2 VSP characterization and induction ^ 17 1.4.3 Linking Storage Proteins to Nursery Practices ^ 20 1.5 Monitoring Seedling Quality ^ 21 1.5.1 Fall Acclimation Conditions Influence Hardiness and Dormancy ^ 22 1.5.2 Lifting Date and Storage ^ 25 1.6 Concluding Remarks ^ 30  MATERIALS AND METHODS ^  31 2.1 Seedling Material ^ 31 2.2 Study Design ^ 32 2.3 Protein Analysis ^ 36 2.3.1 Extraction ^ 36 2.3.2 Protein Assay ^ 36 2.3.3 Electrophoresis and Characterization ^ 37 2.4 Dormancy Status and Mitotic Activity ^ 39 2.4.1 Days to Budbreak ^ 40 2.4.2 Mitotic Index ^ 41 2.5 Cold Hardiness Assessment ^ 43 2.5.1 Sample Preparation ^ 43 2.5.2 Frost Induced Electrolyte Leakage Test ^ 44 2.6 Photosystem II Activity ^ 45 iv  2.7 Root Growth Potential ^ 2.8 Colour and Chlorophyll ^  46 47  RESULTS ^  3.  48 3.1 Morphological and Biochemical Changes ^ 48 3.2 Physiological changes and vegetative storage protein accumulation during the autumn months ^ 53 3.2.1 VSP Accumulation ^ 53 3.2.2 Cold Hardiness Acquisition ^ 59 3.2.3 Photosystem II Activity and 62 Chlorophyll Measurements ^ 3.2.4 Dormancy Status and Cell Division ^ 65 3.2.5 Changes in Seedling Quality during Storage ^ 72 3.2.6 Summary of Physiological Changes ^ 73 3.3 Additional seedlots have similar patterns of VSP Accumulation ^ 74 3.4 Effect of Temperature and Photoperiod on VSP Induction and Accumulation ^ 79 3.5 PAS Staining and 2-D Electrophoresis under Reducing and Nonreducing Conditions ^ 87 3.6 Comparison of Scanning Densitometry Techniques ^ 92  4.  DISCUSSION ^ 4.1 4.2 4.3 4.4 4.5  95 Appearance of Vegetative Storage Protein ^ 95 VSPs Association to Physiological Parameters ^ 99 VSP Content, Lifting Date and Seedling Quality ^ 103 Other VSP Characteristics ^ 105 106 Conclusions ^  5. LITERATURE CITED ^  108  LIST OF TABLES Page TABLE 1: Sample Collection ^  35  TABLE 2: Post Storage Characteristics ^  73  TABLE 3: Summary of Physiological Traits of Treatments ^ 79  vi  LIST OF FIGURES Page FIGURE 1:  Height Growth of Seedlot QL ^  49  FIGURE 2:  Fall Phenology Changes ^  51  FIGURE 3:  Summer Protein Profiles ^  52  FIGURE 4:  Total Protein Increases in Autumn ^  54  FIGURE 5:  Autumn Protein Profiles ^  57  FIGURE 6:  VSP Accumulation in Seedlot QL Buds ^  60  FIGURE 7:  Acquisition of Frost Hardiness ^  61  FIGURE 8:  Photosystem II Activity ^  63  FIGURE 9:  Chlorophyll Content of Needles  ^  FIGURE 10: Dormancy Status - MI and DBB ^ FIGURE 11: Illustrations of Apical Bud Squashes  ^  64 66 68  FIGURE 12: Dormancy Release Index ^  71  FIGURE 13: Protein Profiles of Other Seedlots ^  76  FIGURE 14: VSP Accumulation in EK and PG Seedlings ^  78  FIGURE 15: Altering Total Protein Patterns of Bud Tissue ^  82  FIGURE 16: Total Protein Patterns in Stem and Root Tissues From Fall Accclimation Treatments ^  84  FIGURE 17: VSP Patterns are altered by Fall Acclimation Conditions ^  86  FIGURE 18: PAS Staining of Total Protein Samples ^  89  FIGURE 19: Disulphite Bond Analysis of Total Protein Samples. .91 FIGURE 20: Comparison of Two Different Gel Scanning Systems to Quantify VSPs ^  93  FIGURE 21: Summary of Seasonal Changes in Physiology and Biochemistry ^  94  vii  ACKNOWLEDGEMENTS  I would like to thank many people for their help and guidance during my explorations into vegetative storage proteins, seedling physiology, and seedling quality. Thanks to my committee members Dr. D. R. Roberts, Dr. Edith Camm, Dr. S. Grossnickle, and Dr. C. J. Douglas. I also greatly appreciated the financial support of the Science Council of B.C. G.R.E.A.T. scholarship program. Pelton's Reforestation in Maple Ridge and Sharon Gillies at Simon Fraser University for the seedlings and use of the fluorometer. I also enjoyed the support of my family, friends, and future husband Arthur Ma. In addition, I could not have completed this project without the use of laboratory and computer facilities at the Forest Biotechnology Centre, B.C. Research.  viii  1.0 INTRODUCTION AND LITERATURE REVIEW  All plants, including seedlings and mature trees, alter their physiology in response to changes in their environment. Water status, daylength and temperature conditions are some of the main factors which influence plant growth and development. Since plants are immobile, they have evolved with an ability to adjust temporarily to environmental changes. Woody plants are perennials and can exist vegetatively year round. Their survival in temperate climates is made possible because many woody plants have the ability to stop growing, become dormant, and develop cold hardiness. Other plants do not have the capability of adapting t o below zero temperatures; therefore, they either do not live in those habitats or overwinter in a different form such as a seed or tuber. Changes in a plant's external environment are perceived by internal receptors which in turn signal alterations in gene expression and enzyme activity often through modifications of plant growth regulator ratios.  Coniferous trees and seedlings represent one group of plants whose physiology is altered in response to seasonal climatic changes. During the spring and summer months rapid growth occurs as long as favourable conditions persist. By late summer, possible drought conditions and declining daylength generally cause growth to cease and budset to occur. Then as the cooler temperatures and shorter days of autumn appear, the trees alter their rate of photosynthesis, store reserves and develop cold hardiness. The  1  degree and time scale of these changes has been shown to vary with species, provenance, and specific environmental conditions (Neinstadt 1967, Glerum 1980, Arora et al. 1992). In this introduction and literature review, I will discuss what is known about fall acclimation processes in trees with a specific slant towards the use of seedling physiology in nursery cultural practices of conifers.  Coniferous trees, particularly spruce, are very important reforestation species in British Columbia. Nursery owners aim to produce high quality stock that will survive removal from styroblock containers during lifting and subsequent frozen storage (-2°C) for up to six months before planting. Rigorous monitoring helps accomplish this goal. Seedling quality is monitored by measuring morphological and physiological changes during the growing season and during fall acclimation. Specifically, cold hardiness is used to predict lifting date for cold storage and root growth potential is used to measure seedling viability. The Ministry of Forests requires seedlings to have less than 25% damage after a -18°C stress prior to lifting (Simpson 1990). However, nursery workers and researchers still debate over whether these are the best parameters to measure or if alternative methods would be preferrable. Certainly, cold hardiness is a reasonable measurement to do because seedlings must survive cold storage. Measurement of the decreasing mitotic activity in the terminal bud or the declining photosystem II activity in the needles have been 2  suggested as alternatives (Grob, 1990, Vidaver et al. 1989). The integrating fluorometer which measures PS II activity is currently being tested for its ability to predict lifting date (S. Gillies, personal communication). Food reserves as biochemical markers for lifting date have only been addressed recently (Omi 1990, Roberts et al. 1990). Correlations between cold hardiness, dormancy, photosystem II, mitotic activity, and root growth potential have been gradually defined, but the linkages between these fall acclimation processes has not yet been fully explored or understood (Burr 1990, Ritchie and Tanaka 1990).  My thesis research attempted to establish the relationships between the appearance and accumulation of a specific vegetative storage protein and seedling physiology of Interior spruce throughout the growing season and especially during fall acclimation. The study was also done in a nursery context. I wanted to see if the pattern of vegetative storage protein (VSP) accumulation was related to the lifting date, cold hardiness, PSII slowdown, or to the dormancy characteristics of the seedlings. Experiments were also designed to see whether an abundance of VSP at lifting could increase survivability in storage and after storage root growth potential. In addition, a small experiment assessed the influence of temperature and photoperiod on VSP accumulation patterns. Once these relationships were defined, the data was analyzed to see whether VSP was correlated and could predict optimal lifting date or hardiness level. 3  1.1 Dormancy A plant is said to be dormant if it will not grow under favourable environmental conditions. Development of a dormant state begins when growth ceases and buds form. In addition, leaves of deciduous species senesce, while the wax coating on needles of conifers thickens. Nutrients from senescing leaves are recovered and transported to stem tissues (Kramer and Kozlowski 1979). Since conifer current year needles are retained for several years, fewer nutrients are available for other tissues. These changes are often initiated by a shortening daylength and cooler nights which trigger an internal shift in hormone ratios (Kramer and Kozlowski 1979, Rodriquez et al. 1991). For example, Qamaruddin et al. (1990) documented that cytokinins were low in buds and needles of dormant Scots Pine. There is also thought to be an increase in abscisic acid (ABA) during the cessation of growth in apple trees (Mousdale 1983). Other internal changes also occur during dormancy. While the metabolic pace is greatly reduced, starch and sugar ratios are fluctuating; cytological features are changing; lipid composition is altered; and proteins accumulate (Pomeroy and Siminovitch 1971, Kramer and Kozlowski 1979). These activities indicate that dormancy is not a simple on/off state of growth versus no growth because internal changes are still occurring.  The recognition that dormancy is not a static state has led to the definition of three stages - correlated inhibition, rest, quiescence (Lavender 1991). Correlated inhibition is the first 4  level of dormancy that occurs during early stages of bud formation. At this point, a seedling will easily flush if climatic conditions are favourable. Rest is true dormancy which was defined as an inability to grow regardless of environmental conditions. The period of rest is the longest phase. During this period, the seedling is incapable of resuming growth because the appropriate internal signals are not present. Theories of the induction and release of dormancy suggest that phytochrome is involved as well as several different plant growth regulators (Vince-Prue 1984, Qamaruddin et al. 1990). However, the plant is capable of sensing temperature and daylength because research has shown that dormancy release requires the accumulation of chilling hours (Nienstadt 1967, Kramer and Kozlowski 1979). Once a plant has received its chilling requirement, the rest phase is completed. The seedling then enters the last dormant state, quiescence, in which growth is inhibited by the environment and not by physiology. When spring temperatures or daylengths return, dormancy ends and growth resumes.  1.2 Food Reserves Food reserves are generally comprised of carbohydrates (starch), lipids, and nitrogen containing compounds (amino acids and proteins). Temporary storage of reserves can occur during the growing season; this mainly involves carbohydrate stores. Plants are actively growing, respiring, and photosynthesizing. Excess photosynthate is stored as starch within amyloplasts or as starch 5  granules often during the night. This starch is then converted to simple sugars and transported to areas of high sink strength during the day. Lipids do not accumulate during the growing season because their breakdown products are being utilized as a base for numerous secondary substances and the cell membranes are continually expanding. Nitrogenous compounds are acting as building materials and not as storage compounds during the growing season. Long term storage of food reserves occurs predominantly during the autumn and winter months.  In the leguminous annual soybean, a special case of temporary nitrogen (N) storage exists where specific proteins serve as middlemen by storing excess N until it is required. This vegetative storage protein (VSP) is a dimer of 29 and 27 KD subunits which is localized in the bundle sheath and paraveinal mesophyll cells in the stem and leaves (Wittenbach, 1983). The VSP first appears in the cotyledons during germination as cotyledonary reserve proteins are being degraded (Staswick, 1990). Toxic levels of free amino acids are not accumulated because excess amino acids are readily converted into vegetative storage proteins. The levels of vegetative storage proteins and their mRNA have been shown to remain constant until after flowering occurs and seeds are developing. Then this vegetative storage protein declines presumably because sink strength is changing; therefore, its amino acids are released and translocated via the phloem. The amino acids are then reassembled into new seed specific storage proteins within 6  the developing seed (Staswick 1988, 1989). Soybean vegetative storage protein gene transcription and translation is enhanced by removing seedpods, increasing level of N fertilization, wounding leaves, inducing drought, or by adding Jasmonic acid (Anderson et al. 1989, Mason et al. 1990, Surowy and Bayer 1991, Huang et al. 1991). The general storage pattern can be described as follows: if the plant contains excess N and no strong sink exists then vegetative storage proteins are formed; these storage proteins are then localized within vacuoles; when sink strength increases, these proteins are degraded and their amino acids are transported via the phloem so that they can be utilized by the sink. Thus soybean VSP increase N reserves within the plant throughout the growing season and helps cycle N throughout the plant as needed. Soybean, as an annual, possibly synthesizes this VSP in order to maximize uptake and utilization of all available nitrogen during its only growing season. No such equivalent protein has been found to exist during the summer growing season for woody plants and perennials. Perhaps since the growing season is limited and the plant structure was developed to last longer than one season, C and N storage reserve strategies are different because woody and herbaceous perennials' physiology has to be more adaptive.  At the same time that the plant is dormant and metabolism is slowing down, food reserves are accumulating. Perhaps this activity is possible because the energetic needs of the plant are also changing. Instead of focusing on construction and expansion of new 7  tissues as in growth mode, the plant survives the winter by entering a storage mode. Increases in protein levels and changes in carbohydrates suggest raw materials are available for synthesis of reserves despite the slower rate of photosynthesis and changes in enzyme activities. Evidence indicates that transcription of new enzymes can occur and these changes in gene expression are triggered early in dormancy before air temperature and thus plant temperature decline significantly (Kramer and Kozlowski 1979, Tseng and Li 1987). Chemical reactions work more slowly at lower temperature which suggest that different isozymes may be activated (Kramer and Kozlowski 1979). General metabolites such as fatty acids, amino acids, glucose, and sucrose are converted into the molecules lipid, protein, and starch as part of the fall acclimation process. These reserves are stored in living parenchymous cells such as the pith cells of seedlings, the ray parenchyma cells of the xylem and phloem (Berggren 1985, Bonicel et al. 1990, Greenwood et al. 1986). The general accumulation of lipids, soluble sugars, and proteins most likely plays a role in the development of cold hardiness.  Most of the original work with storage compounds was done with fruit trees (Kang and Titus 1980), but in recent years the storage patterns in commercial forest trees such as poplar, willow, spruce, and Douglas fir has been investigated (Bonicel et al. 1990, Green et al. 1986, Roberts et al. 1991). Early results showed that certain tree species store different proportions of carbohydrates  8  versus lipids during wintertime. Specific protein fractions appear in apple trees during the development of cold hardiness (Kang and Titus 1987). Most conifers and diffuse porous hardwood species store a greater porportion of fats versus carbohydrates; while ring porous hardwoods preferentially accumulate starch (Glerum 1980).  Previous studies have also noted that in the spring, accumulated food reserves are assimilated for construction of tissue and respired to produce energy (Glerum 1980, Kang and Titus 1980). When required, their assimilates are recycled and transported to the area of greatest sink strength. Reserves can provide energy for budburst and for biochemical activities until efficient photosynthetic, transpirational and nutrient uptake mechanisms become reestablished (Mousdale 1983). Early spring physiology is comparable to early seedling development of most higher plants. So the presence of storage compounds appears to be an important strategy for maintaining a surplus food supply as well as for surviving the winter months.  1.2.1 Starch Starch is the predominant form of carbon storage, although hemicellulose can also serve a reserve as well as a structural role. The starch cycle is well documented in the literature (Pomeroy and Simnovitch 1970, Glerum 1980, Omi 1990). Maximal levels of starch are present in early spring and fall prior to and just after the growing season. Generally most of the starch is 9  stored in amyloplasts inside parenchymatous cells the roots (Cyr and Bewley 1990a) or the buds (Berggren 1985). Monterey pine (Pinus radiata) needles contain high starch levels in the winter (Coker 1991). In spruce needles, raffinose and sucrose levels also increase in late autumn before maximum hardiness is achieved (Durzan 1968).^After the fall peak, starch is converted into soluble sugars. Starch to sugar conversions occur during the growing season and in late winter during acquisition of maximal hardiness. If starch and soluble sugars are measured just before and after fall and spring starch levels peak, starch concentration can indicate transition points in the cycle. In the spring, preparation for flushing is often signalled by conversion of accumulated simple sugars to starch. The total winter increase seen in soluble sugars is only partly accounted for by starch conversions. Specifically, winter conversions increase sugar levels which in turn helps cause the cells' osmotic potential to rise. With this effect, soluble sugars act as cryoprotectants and aid in the acquisition of maximal cold hardiness (Glerum 1980, Pomeroy and Siminovitch 1971, Brown 1977). Freezing tolerance of western red cedar is also achieved by changing tissue water content, symplastic volume, and passive osmotic adjustment (Grossnickle 1992). Throughout the period of starch accumulation and conversion to soluble sugars, the cells develop increased cell wall invaginations and rough endoplasmic reticulum which is consistent with increased lipid synthesis (Greenwood et al. 1990). In the spring, axial and ray parenchyma cells begin to accumulate starch as photosynthesis  10  and growth resumes (Kramer and Kozlowski 1979). In Jack pine (Pinus banksiana), carbohydrate reserves are used more slowly than in fruit trees because old needles reach photosynthetic efficiency and export food while the new needles are elongating (Glerum 1980). Since food production resumes when growth begins, the conifers' carbohydrate reserves are in less demand than in deciduous trees unless partial defoliation has occurred.  1.2.2 Lipids Lipids are the primary component of all membranes, are starting materials for secondary compounds, and provide energy when respired and are thus distributed throughout the plant. Research done up to 1970 debated over whether winter increases in lipids occurred. Early studies claimed that carbohydrates were converted into lipids in conifer needles during the winter but the dye techniques used stained both phenolic compounds and lipids which made the results misleading (Kramer and Kozlowski 1979). Although, initial extraction results from whole plants had shown that amounts of crude lipids hardly varied when compared to starch and sugar changes in conifers, later studies revealed significant lipid increases in the xylem as well as smaller increases in the bark, which had been masked in the whole plant extracts (Glerum 1980, Pomeroy 1970, Ziegler 1964). Unsaturated fatty acids such as linoleic acid increased during this fall period of cold acclimation (Kramer and Kozlowski 1979). Lipid accumulation occurred predominently in the stem tissue; this was consistent with the fact that 11  stems are fully exposed to the environment while the roots are partially insulated by the soil. The concentration in needles and roots remained steady throughout the year. Microscopy of willow (Salix sp.) buds has demonstrated that lipids increased along with tannins, starch, and phytoferritin throughout the autumn months (Berggren 1985). Although lipids comprise a small portion of the total storage compounds, they can be metabolized to provide energy and raw materials and be used for membrane alterations and tissue insulation.  1.2.3 Nitrogen  Glerum's review of food reserves in temperate trees noted that although several early reports mention that conifers probably store nitrogen as proteins, the results varied widely, possibly due to inconsistent analytical methods, a problem also encountered with early lipid studies. Variation in results may also have occurred because of the inherent problem of protein-phenolic interaction (Wetzel and Greenwood 1989, Wetzel et al. 1989). The highest concentration of nitrogenous compounds exists within meristematic cells during the growing season (Durzan 1968). Although nitrogen is an essential component of all twenty amino acids and of proteins, specific amino acids and specific proteins are usually accumulated as storage products. Frequently, glutamine and asparagine increase in concentration during the autumn months. Arginine and glutamine have been shown to increase in Scots pine (P. s_ylvestris) (Glerum 1980). The leaves and buds of white spruce (Picea glauca) synthesis 12  and store proline and arginine. In addition, arginine levels also change in the stems and leaves of apple (Malus) and yew (Taxus) species (Tromp 1970). Changes in amino acid concentrations and proportions have been shown to vary widely in the buds, apices and leaves of white spruce saplings (Durzan 1968). Radiolabelled versions of amino acids, specifically leucine, are incorporated into proteins during cold acclimation in red pine (Pinus resinosa) and black locust (Robinia pseudoacacia). Sugar and protein reserves increase simultaneously in some trees by the accumulation of glycosylated proteins. Lectins and other glycosylated proteins have recently been found in a leguminous tree (Sophora japonica) (Herman et al. 1988, Baba et al. 1991) and European elder (Sambus nigra) (Greenwood et al. 1991). Titus and Kang demonstrated that 90% of total nitrogen was stored as proteins in overwintering fruit trees (Kong and Titus 1980, Kang and Titus 1987). Another interesting experiment looked at cold hardiness and polypeptide changes within bark and xylem of sibling deciduous and evergreen peach (Arora et al. 1992). Both accumulated a 19kD bark polypeptide throughout the winter and recycled it in the spring. The more frost hardy deciduous tree accumulated higher levels and consistently accumulated an additional 16kD protein which declined to low levels in the evergreen. Xylem tissues contained mainly the 19kD VSP. Most of the proteins that increase in concentration are water soluble, and they bind free water within the cells which limits intracellular ice formation (Kramer and Kozlowski 1979). These results suggest that proteins may serve both a cryoprotective and 13  a N storage role during autumn and winter (Pomeroy et al. 1970, Pomeroy and Siminovitch 1971).  1.3 Development of Cold Hardiness Requires Cytological and Biochemical Changes  Besides the seasonal acquisition of food reserves, other biochemical and cytological changes occur during the development of cold hardiness (CH) in temperate zone plants. These changes include alterations in gene expression by increasing amounts of ribosomal RNA, certain mRNAs and nuclear DNA (Pommeroy and Siminovitch 1971, Brown 1977, Tseng and Li 1987). In addition, the surface area of membranes is increased. Electron microscopy of phloem parenchyma cells of the bark of an 8 year old black locust showed that the plasmalemma became invaginated and folded during the winter. As these trees became cold hardy total protoplasm increased; organelle and membrane structure changed; starch was converted to sugars; and lipid bodies increased. The central vacuole was being degraded when protoplasmic augmentation was occurring. Then new membrane bound vesicles were made from the vacuolar membrane material (Pommeroy and Siminovitch 1971). Radiolabelled leucine studies in 15yr old red pine showed that the maximum rate of incorporation into proteins occurred when cold hardiness was increasing (Pomeroy et al. 1970).  Attempts have been made to document the relationships between 14  the cold hardiness cycle and either protein, starch, or amino acid cycles. Carbohydrate and amino acid cycles were not correlated to the cold hardiness cycle in red pine (Pomeroy and Siminovitch 1971). Maximum hardiness was achieved when the starch was converted into soluble sugars late in the winter. Since starch reappears in the spring well before hardiness declines, the starch cycle is poorly correlated to cold hardiness (Pomeroy and Siminovitch 1971). Taken together, increased cell protoplasm, starch to sugar conversion and membrane invaginations modify the cells' water relations and enable the cells to survive effects of dehydration during freezing. Changes in membrane structure and increases in total protein appear to coincide with the maximum acquisition of cold hardiness (Kang and Titus 1987, Arora et al. 1992). This research showed that both the lipid changes or general protein accumulation could be used as biochemical markers for the acquisition of cold resistance. Proteins are easily identified and quantified by the use of immunological techniques; while lipids can be measured by gas chromatography.  1.4 Proteins as Biochemical Markers Apple trees store 90% of their total nitrogen as proteins and protein accumulates during autumn and winter months in both deciduous and coniferous species. These results suggest that N storage maybe involved in overwintering survival (Durzan 1968, Pomeroy et al. 1970, Pomeroy and Siminovitch 1971, Kang and Titus 1980, 1987). Cytological evidence indicated that new densely 15  staining vacuoles appeared within the cytoplasm during acquisition of cold hardiness (Pommeroy and Siminovitch 1971). Some interest existed as scientists postulated that proteins could be used as a biochemical markers; however, no seasonal specific proteins had been identified at that time. If these unique proteins could be found; they would be useful for studying the relationships between gene expression and seasonal developmental changes. Developmentally specific storage proteins were documented in seeds as early as 1827 by Braconnet (Kang and Titus 1980), but season specific storage proteins had yet to be demonstrated in vegetative tissues.  1.4.1 Discovery of Vegetative Storage Proteins  In the early 1980's, a number of labs found specific proteins that appeared to serve as temporary nitrogen storers (O'Kennedy and Titus 1979, Wittenbach 1983, Greenwood et al. 1986, Sauter et al. 1989, Wetzel and Greenwood 1989). These storage proteins have been called vegetative storage proteins [VSP] or bark storage proteins [BSP] (Wittenbach 1983, Staswick 1990, Greenwood et al. 1990). Soybean, chicory, leafy spurge, dandelion, poplar, willow, interior spruce, Sophora japonica, Baldcypress and Yew species have now been shown to synthesize vegetative storage proteins. Sizes and locations vary but generally they are confined to overwintering roots of weeds and the bark tissues of woody plants (Greenwood et al. 1986, Kang and Titus 1987, Cyr and Bewley 1990b). Immunofluorescence and immunogold microscopy techniques have been used to  16  show that BSPs generally accumulate within protein body-like vesicles which appear during the fall acclimation of predominantly woody plants (Sauter et al. 1988, van Cleve et al. 1988, Herman et al. 1988, Baba et al. 1991, Greenwood 1986, Greenwood et al. 1990, Wetzel et al. 1989, 1991, Harms and Sauter 1992). The storage proteins of weeds are root-specific because roots are the overwintering structure of herbaceous perennials (Cyr and Bewley 1990). In the annual soybean, plant storage proteins are stored in vegetative tissues when the sink strength changes by depodding or water deficiency (Staswick 1990, Mason et al. 1990). The seasonal increase and appearance of VSP primarily occurs in temperate zone woody plants and herbaceous weeds. During the spring budbreak, microscopy work has shown that as the protein body vacuoles are emptied, they fuse and then develop into new plastids (Sagisaka 1991). Overall, these researchers have been able to define BSP as follows: they comprise a major portion of the total extractable protein during the winter; are often sequestered in P-storing organelles; and are rapidly mobilized for new spring growth (Greenwood et al. 1990, O'Kennedy & Titus 1979).  1.4.2 VSP Characterization and Induction Researchers have currently only touched the surface with respect to studying the characterization and induction of bark storage proteins. Most studies have sampled bark from branches of mature trees in both the summer and winter (Sauter et al. 1989, Wetzel et al. 1989, 1991). Subsequently, sodium doecyl sulphate  17  polyacrylamide electrophoresis of total protein extracts and microscopy was done (Roberts et al. 1991, Wetzel et al. 1991a). Poplar, willow, European elder and Yew storage proteins were purified for production of antibodies for immunochemistry work. This confirmed that storage proteins appeared in the winter months and were often localized in protein storage vacuoles. In interior spruce seedlings, samples were taken at various timepoints prior throughout the winter and during spring budbreak; needle, bud, shoot and root bark were assessed for presence of unique storage proteins (Roberts et al. 1990). A 30 kD and a 27 kD protein appeared to accumulate in bark and bud tissue. These proteins were mobilized within 7 to 21 days after budbreak. Douglas fir seedlings also appear to have a 30 KD storage protein, but it has not yet been further characterized (Roberts et al. 1990). The Interior spruce study is the only case where storage proteins were looked for in seedlings. Since seedlings were shown to contain storage proteins, BSP accumulation may occur regardless of the age of the tree, which suggests mature "bark" is not required. A bark lectin in Sqphora japonica L. which exhibits BSP-like characteristics was shown to accumulate in young and old bark tissues, but the lectin was mobilized and utilized in the older tissues first (Baba et al., 1991). All studies to date in both coniferous and deciduous tree species indicate VSP is mainly localized in the bark tissues and is not present in the leaves or needles.  The antigenic similarity between various vegetative storage 18  proteins has only been tested in some cases. Soybean 29, 27 kD proteins, maple 24 & 16 kD, or willow 32 kD proteins do not cross react with the 18 kD chicory and dandelion protein (Cyr & Bewley 1990). But soybean VSP is related to storage proteins in other leguminous plants, while poplar's VSP reacts with willow storage protein (Wetzel & Greenwood 1991). Baldcypress contains a 35kD polypeptide that showed antigenic similarities to 32,34 kD protein of dawn redwood (Metaseguoia glyptostroboides) and 34,36 kD Yew VSP (Harms and Sauter 1991). Other combinations of storage proteins and antibodies have not been reported to date; however, the presence of antigenic similarity does suggest that VSP in trees may be closely related.  The chemical nature of bark storage proteins has been investigated for several species. Both poplar (Langheinrich & Tischner 1991) and willow (Wetzel & Greenwood 1991) contain antigenically similar glycosylated storage proteins without disulphide bonds and have several basic isoelectric isomers. The poplar native protein is a 58 kD heterodimer with a 32 and 36 kD subunit (Langheinrich & Tischner 1991). In contrast, nonglycosylated single polypeptides with a high percentage of basic amino acids are the storage forms present in chicory, dandelion (Cyr & Bewley 1990). The glycosylated nature of a winter-specific protein suggests that it could have a cryoprotective role and be related to acquisition of cold hardiness.  19  The mechanisms which trigger VSP accumulation have been investigated only in poplar (Langheinrich & Tischner 1991, Coleman et al. 1991). Short days were shown to cause accumulation of the 32 KD protein within the bark of the stems and roots. Coleman et al. 1991 measured mRNA levels and their results showed that VSP mRNA specifically increased in response to photoperiod. Preliminary experiments with far-red and red light treatments during a short photoperiod suggest that phytochrome may mediate this response (Langheinrich & Tischner 1991). Further understanding of environmentally triggered changes in gene expression such as VSP mRNA production could help explain the changes that occur in seedlings during nursery culture.  1.4.3 Linking Storage Proteins to Nursery Practices The discovery of overwintering specific proteins could provide a new tool for the nursery industry. In apple trees and radiata pine, a greater nitrogen storage capacity has been shown to reflect greater new spring growth (Tromp 1970, Coker 1991). An extra increase in VSP might explain why nitrogen fertilizer applied after budset often increases plant vigour (van den Driessche 1985). VSP or BSP level could possibly serve as a health, quality indicator. Interior spruce/white spruce is a major reforestation species in British Columbia. Researchers monitor seedling physiology in order to assess seedling health and to predict the best lifting date for storage. Currently, lifting date for cold storage is predicted by monitoring cold hardiness at -18°C and measuring root growth  20  capacity. If VSP accumulates during the acquisition of cold hardiness, it could become a likely alternative or companion test. This thesis is the first study which looks at the relationship between interior spruce VSP accumulation and changes in seedlings' overwintering physiology.  1.5 Monitoring Seedling Quality  The seasonal cycle of interior spruce like most temperate species is comprised of the phases of growth and dormancy. The growth phase is characterized by visual changes which include budbreak in April or May, shoot elongation until mid-July, and bud development accompanied by a spurt of root growth at summer's end. The dormancy phase runs from September through to April and it involves few visible changes. Therefore, during the autumn lifting period, nursery workers must evaluate seedling quality and predict lifting date by measuring internal changes within the seedlings (Duryea 1985, Burr 1990). These phases have previously been described using a Degree Growth Stage model adapted from Fuchigami which allows the researcher to visualize periods in which various physiological attributes are changing (Burr 1990). Nurseries try to maintain this natural cycle, but often use cultural practices to modify seedlings for specific needs such as summer planting, specified height range and planting schedules. Dormancy can be induced by a blackout treatment (short daylength) so that height 21  growth ceases and stress resistance increases. In addition, fertilization regimes can be changed to increase growth and size.  Generally, researchers monitor physiological parameters such as cold hardiness (Colombo and Raitanen 1991, Glerum 1985), dormancy status (Colombo 1990), root growth potential (Ritchie 1984, McKay and Mason 1991), photosynthesis (Vidaver et al. 1989) or dry weight fraction (Ritchie 1984) to determine the fall acclimation patterns of conifer seedlings. Morphological parameters and growth rate can only be measured during the summer growing season. In British Columbia, cold hardiness is monitored throughout the fall so that lifting date can be predicted. Root growth potential (RGP) measurements give additional information about seedling viability; however, RGP by itself has not been used to predict lifting date in Canada. Once the specified level of hardiness is achieved, seedlings are lifted from their styroblocks, placed in bundles, wrapped in plastic and put into wax coated boxes for cold storage. To date, biochemical indicators have not been used routinely and current research is investigating storage reserves' relationships to the physiological lifting date predictors. The discovery of overwintering storage proteins may offer the first seasonally specific biochemical marker of seedling status.  1.5.1 Fall Acclimation Conditions Influence Hardiness and Dormancy Seedling physiology can be altered by using different fall  22  acclimation treatments (Colombo et al. 1989, Simpson and Macey 1991, Burr et al. 1989, Colombo 1990). By changing the temperature and light conditions during hardening, Colombo et al. (1989) demonstrated that short days along with ambient fall temperatures (warm days, cool nights) promoted earlier acquisition of cold hardiness, quicker bud formation and a faster decline in mitotic index for black spruce seedlings. In a similar study, the influence of nightlength and temperature on the quality of interior spruce and lodgepole pine seedings was examined (Simpson and Macey 1991). For both these species, short daylength (8 hr) with 10°C day/-5°C night temperature treatment (SDC) exhibited a normal decline in mitotic activity, promoted cold hardiness and had good survivability after storage. The seedlings treated with natural or long night warm conditions attained the same quality level as the SDC treatment after a longer 10 week treatment period. Other treatment combinations indicated that short days were required for development of normal resting buds, hardiness, and post storage RGP (Simpson and Macey 1991). A study with Engelmann spruce concluded that the best fall acclimation treatment was 3 weeks of short day warm followed by short day cold conditions until lifting (Burr et al. 1989). In another black spruce trial, seedlings were acclimated under an eight hour daylength with warm day/night temperatures of 26°C/15°C. Cold hardiness developed and bud dormancy changed without exposure to low temperatures (Colombo 1990); however, black spruce roots acquire and lose hardiness only in response to temperature (Bigras and D'Aoust 1992). An analysis of cultural treatments of  23  western red cedar seedlings demonstrated that short day wet conditions during late summer followed by fall planting resulted in seedlings with the greatest cold hardiness (Folk et al. 1993, in press). The freezing tolerance and first year growth after planting was highest in these seedlings (Folk et al. 1993, in press). Hardiness development in yellow cedar and western red cedar shoots differs from spruce because temperature conditions stimulate hardening instead of photoperiod (Silim 1991).  As seen above in the cedars versus the spruce and pines, different species have different environmental triggers for processes such as cold hardiness and dormancy. Different provenances of the same species can also create variations either in the degree of change or the endpoint value reached. Interior Douglas fir has been shown to develop hardiness faster than coastal Douglas fir although by midwinter they reach the same level of hardiness (Simpson 1990). Low elevation seed sources of interior spruce are generally less hardy than northern varieties (Simpson 1990). Interior spruce and lodgepole pine are hardier species than Douglas fir. These differences may arise in part because tree species vary in their chilling requirements and timing of flushing after becoming quiescent (Nienstadt 1967, Omi 1990a). A short day fall acclimation treatment sets bud earlier than other treatments but maximum cold hardiness levels were identical (Colombo et al. 1989) which again suggests that genetic composition plays a subtle role. 24  The results of these studies suggest that changes in cold hardiness and dormancy characteristics of seedlings can be induced by daylength and/or by temperature. The dormancy process can be accelerated by short photoperiods and by warm temperatures (Colombo 1989, Simpson and Macey 1991, Burr et al. 1989). Cold hardiness acquisition appears to be triggered predominantly by short daylength in spruce species and lodgepole pine while yellow and western red cedar are influenced by temperature (Simpson and Macey 1991, Silim 1991). Peach trees also develop cold hardiness in response to low temperatures (Arora et al. 1992). The time period required for sufficient acclimation could be lengthened or shortened depending on the cultural practices used. Dormancy characteristics are often more affected by different fall acclimation conditions than the final cold hardiness levels (Colombo et al. 1989, Colombo 1990). In summary, timing and type of acclimation treatment can effect some aspects of seedling physiology, but inherent species' characteristics also influences the plant's responses (Cannell et al. 1990, Colombo et al. 1989, Bigras and D'Aoust 1992, Silim 1991).  1.5.2 Lifting Date and Storage Much of the current research on seedling quality to date centers around determining when container grown nursery stock is ready for cold storage; this is because most west coast nurseries use cold storage of seedlings and this strategy is being considered elsewhere as well. Cold storage is advantageous because frost 25  damage can be avoided and seedlings are already graded and packed for spring shipping. Cold hardiness is currently the preferred method of predicting lifting date. This parameter has been shown to increase progressively throughout the autumn months and hardiness level peaks during early winter. The Ministry of Forests in British Columbia recommends that at least 75% of tested seedlings survive a stress of -18°C before lifting is permitted. However, the exact date also varies with environmental conditions, species and provenance (Simpson 1990).  Timing of lifting and storage is key because premature storage has been shown to upset dormancy status and seasonal biochemical changes. In part, the dormancy process can be altered because early lifting may prevent the seedlings from receiving their chilling requirements. Storage also slows dormancy release and decline in stress resistance in interior spruce and lodgepole pine (Ritchie et al. 1985). In a study with ponderosa pine, over winter storage (-1.5°C) failed to fulfill chilling requirements of September and October lifted seedlings which resulted in lower quality seedlings which had limited bud flushing after storage (Omi 1990b, Omi et al. 1991). Normally, starch levels should increase in the the spring but in stored seedlings this starch increase was delayed and carbohydrate levels were not equivalent to naturally grown seedlings until two months after planting. September lifted seedlings never attained comparable levels of starch after storage (Omi 1990 a,b). In another study, Sitka spruce (2+1) and Douglas 26  fir (1+1) were lifted and cold stored at 0.5°C in November, December, and January (Cannell et al. 1990). Their results indicated that some chilling hours for dormancy release could be accumulated in cold storage. Cold hardiness and total nonstructural carbohydrate stores (including starch) declined whether or not the seedlings were stored. Species differed in optimal timing of storage. The best quality Douglas fir was lifted in January, while Sitka spruce from earlier lifts maintained high poststorage RGP (Cannell et al. 1990). Interior spruce and lodgepole pine seedlings have the best storage survival if they are lifted during early November when frost hardiness, RGP and stress resistance is high (Ritchie et al. 1985). These results suggest that frozen storage can inhibit metabolism and interrupt dormancy changes, especially after early autumn lifting.  Storage survival and subsequent field performance depend on the physiological condition of the stock at lifting which in turn is determined by the lifting date (Ritchie 1989). A short day cold treatment allowed interior spruce and lodgepole pine seedlings to acquire cold hardiness faster which in turn allowed earlier lifting (Simpson and Macey 1991). Outplanting survival, pre- and poststorage RGP of bareroot Douglas fir and Sitka spruce stock was also shown to be influenced by species and lifting date (McKay and Mason 1991). Field performance and survival two years after planting was consistently better in late autumn lifts of ponderosa pine (Omi 1991). Fertilizer applied after budset and during natural cold 27  acclimation improved and maintained seedling quality during storage (Simpson 1990, Colombo 1990, Coker 1991, Burr 1990, Ritchie and Tanaka 1990). In general, seedlings that have reached near maximum cold hardiness, high RGP levels, and low mitotic activity exhibit good survival in storage and after planting (Cannell et al. 1990, Simpson 1990, Colombo 1990).  Since premature lifting and incorrect storage conditions decrease seedling quality, the relationships between frequently measured lifting date predictors has been investigated. The initial stages of cold hardiness acquisition for ponderosa pine, interior Douglas fir, and Engelmann spruce are not correlated with RGP which remains low (Burr et al. 1989). When cold hardiness was lower than -15.0°C (lethal temperature for 50% damage - LT50); RGP was also rising and seedlings were entering quiescence. At this point cold hardiness (CH) correlated closely and could be used to estimate RGP and bud dormancy (measured as days to budbreak {DBB}). Field performance and survival were also correlated with before storage CH and RGP levels (Simpson 1990). Generally correlations between DBB and CH were weak; however, during the period of bud initiation CH was linearly correlated (r2>0.999) with DBB in black spruce (Colombo 1990). This relationship existed for only over a short period, a fact which suggests that correlations are not usually noted because the data encompasses all three levels of dormancy instead of just correlated inhibition and early rest. Dormancy release in Black spruce was not related to changes in CH levels 28  because dehardening began well before warm temperatures triggered budbreak (Glerum 1985). CH is more closely related to percent damage during storage; low CH levels prior to storage are associated with greater loss of stored seedlings (Colombo 1990). During deacclimatization, CH levels decline while RGP remains high; however, once LT50 values reach -15°C both RGP and CH decrease rapidly (Burr et al. 1989). Total nonstructural carbohydrates (including starch) have shown inconsistent correlations to RGP, CH, and bud dormancy (Cannell et al. 1990, Omi 1990); therefore, starch is considered to be a poor predictor of lifting date and poststorage survival. The level of cold hardiness can be used to infer bud dormancy status and general stress resistance at lifting because all are correlated with performance and cold hardiness is easiest to measure (Burr 1990). The possibility that specific storage proteins and/or photosystem II fluorescence levels could be correlated to CH, RGP or dormancy characteristics of seedlings has not been addressed in the literature. Overall, the literature indicates that cold hardiness levels before storage can predict lifting dates that ensure good post-storage seedling quality, but RGP measurements are more benefical for predicting field performance when measured just prior to field planting (Burr 1990, Ritchie and Tanaka 1990).  From the data collected to date it appears that root growth potential measured prior to planting is a good predictor of survival; dormancy status indicates when chilling requirements are 29  being met; and cold hardiness is sometimes correlated with these two parameters as well as being useful in predicting lifting date for frozen storage. However, none of these methods is foolproof; internal biochemical changes (nitrogen stores, starch levels) may also be modified when physiological traits change. Thus biochemical changes such as specific protein synthesis could possibly be a more consistent or complementary marker. More research is required in the area of seedling quality in order to resolve ambiguities in the literature and to gain a broader understanding of interactions between physiological processes.  1.6 Concluding Remarks Both deciduous and coniferous tree species have evolved to survive seasonal changes in the environment. Woody plants adapt to the decline in light quality and temperature by altering their metabolism and physiology. Preparation for winter is a whole tree response (Perry 1971); trees enter dormancy, develop cold hardiness, cease growth and show altered enzymic activity (Lavender 1991, Guy 1990, Tseng and Li 1987). In addition, some species produce winter specific proteins (Sauter et al. 1989, Wetzel et al. 1989, Roberts et al. 1991, Greenwood et al. 1986, Kang and Titus 1980, 1987). Each of these alterations is multifaceted, so researchers have been challenged to explore the complex interactions between these changes. In turn, commercial nurseries have applied this knowledge as a tool for monitoring and improving  30  seedling quality (Burr, 1990). My thesis studies the relationship between overwintering changes and vegetative storage protein accumulation in interior spruce and assesses whether any useful correlations exist that could provide a new tool for monitoring seedling quality.  2.0 MATERIALS AND METHODS 2.1 Seedling Material Every two weeks between June lst,1990 and January 29th,1991 interior spruce (a mixture of Picea glauca and P. engelmannii) seedlings were collected from an operational nursery (Peltons' Reforestation Ltd.,Maple Ridge, B.C.). Three seedlots were sampled: QL (Cariboo-Quensel district, lot 4307, elevation 1052M, Lat. 53°13'N Long. 121°41'W), EK (East Kootenay), and PG (Prince GeorgeMacGregor region) a northern seedlot. The seedlings were germinated in saw dust based media within styroblocks (type 160, Beaver Plastics) in April and were grown as 1-0 stock. Seedlot QL was randomly sampled from containers on five randomly selected tables (each holding about 2800 seedlings) within a greenhouse. These seedlings were part of a larger operational stock; therefore, although five tables were randomly allocated, they were moved around the nursery throughout the growing season into both outdoor and open greenhouse compounds. Blackout treatments were used to induce budset: seedlot QL was treated August 2-14th; EK was blacked out from August 12-22nd; and PG was treated from August 15-22nd. Seedlings (seedlot QL) were lifted for Part II of my study design 31  on each autumn sample date and also on November 5th for the temperature experiment [see next section]. On November 5th, sample plot size was reduced to a 20 styroblock table which was composed of 4 randomly chosen blocks from each of the original 5 tables. The operational seedlot was lifted and placed into -2°C storage by Nov. 19th. Throughout this study, collected seedlings were placed in plastic bags, transported to B.C. Research in a styrofoam cooler, and processed for various tests (Table 1).  A fourth seedlot (Finlay district [FIN], lot 29135, elevation 850m, origin 56°07'124°49'W) was grown at Hybrid Nurseries in Pitt Meadows and brought to SFU three weeks before fall acclimation experiments began. Samples were collected and stored at -80°C until protein analysis could be done at B.C. Research. This seedlot was also used in a Ph.D. study done by Sharon Gilles at Simon Fraser University.  2.2 Study Design  Twenty-five seedlings were monitored for height growth and phenology changes throughout the growing season. The majority of the experiments used seedlot QL while only the storage protein content was assessed in EK and PG. In a parallel project, the two additional seedlots (ER and PG) were also being collected for cold hardiness and root growth capacity measurements.  For Part I of the study, storage protein accumulation was 32  monitored on each collection date. Seedlot QL samples were assessed for cold hardiness, dormancy status, photosystem II activity,and vegetative storage protein content (Table 1). In addition, 20 seedlings were cold stored at -2°C for Part II - quality assessment after storage. Seedlings were always selected randomly from within each styroblock. Seedlot QL was further characterized by measuring the height, caliper, number of buds, and number of branches of samples collected for dormancy analysis. Activity of photosystem II was monitored by measuring variable chlorophyll fluorescence (6 seedlings/sample date); this test was done by Sharon Gilles at Simon Fraser University.  The aim of part II of this research project was to describe the relationship between initial storage protein content and survival and quality after cold storage. Originally this included two main experiments: the effect of lifting date on survival during a six month -2°C storage period; and the effect of different storage temperatures 4°C and -2°C on root growth potential (RGP) and storage protein after 0, 2.5, 3.5 and 6.0 months. Samples for the storage temperature experiment were chosen when the QL seedlings were cold hardy to -18°C in accordance with the Ministry of Forests guidelines (November 5th). After wrapping the root plugs in plastic, seedlings were placed in waxed coated boxes within a -2°C freezer or in sealed paper bags within a refrigerator set at 4°C. The seedlings stored at 4°C were tested for RGP (20 seedlings/test) and storage protein content. Unfortunately, the effect of initial 33  lifting date and storage protein content on post storage survival could not be assessed with seedlot QL because all seedlings stored in frozen storage(-2°C) died in a frostfree freezer. However, since I analyzed storage protein content for Sharon Gilles' study, she made post-storage root growth potential data available for seedlot FIN (SFU samples).  Simon Fraser University then provided seedlings acclimated to either short daylength or low temperature conditions along with a control group (seedlot FIN). The seedlot was divided into these three treatments on August 4th,1990. The ambient control group was kept in an unheated greenhouse and experienced normal seasonal changes in daylength and temperature. The two treatments were transferred to growth chambers: the chilling treatment had 10°C day/5°C nights with a 16 hour daylength; and the short day treatment had a 12 hour daylength with a day/night temperature of 23°C/18°C. The seedlings were slowly brought down from 23°C/18°C to the chilling test temperature at a rate of 2°C every three days. Desired test conditions were reached by August 28th which allowed sampling to begin by September 6th. Seedlings were sampled approximately every two to three weeks until December 12th. In addition the control group was also collected twice in early spring. The influence of daylength and chilling on storage protein appearance and accumulation was evaluated using seedlot FIN.  34  TABLE 1 :SALBUDLE COLLECTION SEEDLOT^PERIOD^#SEEDLINGS/^ANALYSIS COLLECTED^COLLECTION QL (4307)  June - Aug.  10  Height Growth, Protein  Aug. - Feb.  10  Protein,^(FH) Cold Hardiness  15  Mitotic Index, Fall Phenology  15  Days to Budbreak (DEB),^Phenology  6 20  Fluorescence' Cold Stored* Root Growth Capacity^(RGC)  SFU (FIN)  Aug.^- Feb.  3/trt  Protein, FH, RGC, Fluorescence'  EK  June - Jan.  3  Protein, FH,^DBB, RGC2  PG  June - Jan.  3  Protein, FH, DBB, RGC2  *Seedlings were stored 4°C 1 Fluorescence measurements were done at SFU, SFU Trts physiology data is part of another study. 2 The Ecophysiology group made this data available.  35  2.3 Protein Analysis 2.3.1 Extraction Bud,shoot, and root tissue was extracted. Predominantly, bud tissue was extracted to analyze vegetative storage protein (VSP) accumulation because they contained the most protein and no dissection was necessary prior to grinding. Seedlings were removed from -80°C freezer and appropriate portions were excised from the seedlings in the 4°C room and kept on ice during the extraction. Whole buds, bark from stem segments, or root tissue were placed in a mortar with liquid nitrogen and ground to a fine powder. Approximately 20-40 mg was then placed in a preweighed eppendorf tube.  Solubilizing buffer (0.2813 M Tris-HC1 pH 6.8 containing 22.5% mercaptoethanol, 9% sodium dodecyl sulfate (SDS) and 22.5% glycerol was added to the tissue samples (5-6 ul/mg); then they were vortexed, centrifuged briefly, well vortexed and then boiled 8-10 minutes at 100°C. Samples were then centrifuged 10 minutes at 14000K prior to assaying and to storing at -80°C.  2.3.2 Protein Assay Protein concentration was determined by a modified procedure of Ghosh et al. (1988). The samples (2u1) were spotted onto Whatman #1 filter paper, allowed to air dry and then stained with Biorad Coomassie blue R250 stain (0.1% Coomassie, 20% methanol, 10% acetic acid). The filter paper was destained with 10% acetic acid/20% 36  methanol and air dried. Sample spots were then cut out and the dye eluded in 2 ml of 1% SDS. Protein concentration was determined by the Bradford assay - absorbance at 595 nm and a BSA standard curve (DU-65 Beckman Spectrophotometer).  2.3.3 Electrophoresis and Characterization  SDS-Page gel electrophoresis was used to visualize the total protein extracts. Several percentages of acrylamide gels as well as gradient gels were tried. The most consistent results were achieved with 15 or 10 well 1.5mm or 0.75mm 12% separating gels with a 4% stacking gel using a Tris-Glycine buffer system (Laemmli 1970). Running buffer was made from a 5X stock solution containing 15 g/1 Tris [Trizma Base], 72 g/1 Glycine, and 5 g/1 of SDS. A gradient from 10 to 18% acrylamide was used for most of the seedlot QL extracts; however, problems occurred when drying gradient gels so later analyses were done with the 12% gels. The Biorad minigel system was used.  Proteins were also extracted under nonreducing conditions in which the mercaptoethanol was excluded from the buffer. These samples were run on a 1.0 mm thick gel and then the sample lane was cut from the gel. The lane was then placed on parafilm within a glass petri dish and soaked in normal solubilizing buffer for 10-20 minutes. Then the lane was run in the second dimension (1.5 mm thick gel) along with molecular weight markers and a control sample. 37  After electrophoresis, gels were first fixed in a 40% methanol/l0% acetic acid solution for an hour. This was followed by a Coomassie blue R-250 staining for 30 minutes and destaining until background staining was minimum. Gels were then rinsed in distilled water and soaked in drying solution (20% methanol/5% glycerol) for at least 3 hours. Cellulose backing was also soaked in drying solution. The Biorad gel dryer model 583 with a Edwards two stage vacuum pump was used for drying gels. The procedure was as follows: a piece of Whatman 1.0 paper was placed on the dryer and this was covered with another piece of Whatman paper that has been soaked in drying solution, a piece of cellulose backing, the gel, cellulose, and then finally saran wrap. All the air bubbles were removed between layers and vacuum was applied. The drying unit was programmed in normal mode except for gradient gels. Drying time at  50°C maximum temperature ranged from 3.0 to 6.0 hours depending on the number and thickness of the gels.  PAS staining was used to assess whether interior spruce VSP were glycosylated. The procedure used was a modified protocol from McGuckin and McKenize 1958 and Zacharius et al. 1969. Electrophoresis gels were placed in 5% methanol, 7% acetic acid solution on a shaker for 30 minutes. Then they were rinsed with distilled deionized water [DDW] 5 minutes X 3 times. Gels were then incubated in 1%(w/v) periodic acid for 50 minutes and rinsed with 200 ml DDW for 10 minutes [X6]. Afterwards, gels were covered with Schiff's reagent - [(BDH,Fisher Scientific) or freshly made 38  solution (1 gram of basic fuchsin dissolved in 200 ml of hot water, filtered into a brown bottle, and then 20 ml of 1N HC1, 3 drops concentrated HC1, 20 grams of sodium metabisulfite were added yielding a straw yellow solution, if not 1N HC1 was added as needed, the reagent is stored at 4°C in the dark for 24 hours before using). Gels were placed on a shaker in the dark for 50 minutes at room temperature or overnight at 4°C. Colour was developed by washing the gels with 0.5% (w/v) sodium metabisulphite for 10 minutes [X3], then rinsed with DDW to remove excess stain. Gels were then dried as described in the above section. All electrophoresis materials were from Sigma, Biorad, BRL, or BDH.  2.4 Dormancy Status and Mitotic Activity Once the seedlings (Seedlot QL) ceased height growth and began bud development, dormancy status was assessed. Seedlot QL was given a blackout shortday treatment from August 2nd through to August 14th (12 days) to induce dormancy because the desired height growth (18-24cm) had occurred. In addition, the PG seedlot received this treatment from August 15-22nd (7 days) and the EK seedlot experienced short days from August 12-22nd (10 days). Days to budbreak was used to assess dormancy and mitotic index evaluated the rate of cell division within the terminal bud apex.  39  2.4.1 Days to Budbreak The Days to Budbreak (DBB) was measured as in Ritchie (1984) and Lavender (1984). Fifteen seedlings were potted in 6"x7" 1 litre black plastic pots (Listo Products) containing a 3:1 peat:vermiculite mixture with 3.6 g/1 Osmocoat, 0.5 g/1 micronutrients, and 2.4 g/1 dolomite. Then they were grown with a 16 hour photoperiod {black plastic curtains surrounded the table}, a 20-25°C temperature and relative humidity of approximately 70%. The light intensity varied from 200 to 250 umol Budbreak status and general health was checked every two days. The number of days for both terminal and lateral budbreak were both counted. Visual clues were bud swelling, thinning of the bud scales, and an increase in shininess. The general flushing pattern was as follows: the lower lateral buds opened first, then flushing occurred towards the apical bud which invariably opened last. Seedlings from early collections often had a lateral shoot take over apical dominance because the terminal bud was not capable of flushing.  The dormancy release index was calculated as follows: DRI = DBBx/DBB; the DBBx is the number of days to budbreak in fully chilled seedlings (Ritchie 1984). DBBx is approximately 16 days for the terminal buds and 13 days for lateral buds.  40  2.4.2 Mitotic Index  At the same time dormancy of the whole plant was being assessed, mitotic index (MI) of the buds from ten to fifteen seedlings was tested. MI measured the rate of cell division within the terminal bud apex. The squash preparation procedure was adapted from Grob (1990). The bud along with part of the shoot was sampled to facilitate handling. Shoots were then dissected down to the last 3-4 primordia over the dome with the aid of a dissecting microscope, pins, and a scalpel. The primordia helped protect the bud apex during processing. The sample was then fixed in 10% Neutral Formalin (Sigma) for 24 hours at 4°C. This solution was replaced with 95% ethanol which allowed the tissue to be stored for three to six months before processing.  The sample was then quickly rinsed three to four times with cold deionized distilled water (dDH20); this was followed by cold dDH20 washes (3x8hr) for 24 hours. Afterwards the tissue was rinsed for 10 minutes in 20-25°C distilled water prior to hydrolysis in 5M hydrochloric acid in a temperature controlled room (22°C) for 40-60 minutes. Following a distilled water wash, samples were stained with Schiff's reagent (recipe as above or Sigma) for a maximum of two hours. Excess was then removed with SO2 water (lxlmin, 3X10min). Several 4°C dDH20 washes removed excess SO2 and the tissue was stored in fresh water at 4°C for 2 to 7 days.  The final bud dissection was done as follows: the bud 41  primordia and leaf primordia were removed by a horizontal motion of a pin tip against the cells; apices were then cut at the base of the dome; a microscalpel was then used to pick up the apex and place it on a slide containing a drop of 45% acetic acid; a coverslip was placed on top and then squashed directly on top of the apex using a pencil eraser which usually created a monolayer of cells. Slides were then placed face up on a block of dry ice and allowed to freeze for 30-60 seconds. A razor blade was then used to pry apart the coverslip and the slide. The slide immediately underwent ethanol washes - 95% ethanol for 3-4 minutes and then 100% ethanol for 3-4 minutes. While the slide was still wet, it was wet mounted and sealed with clear nailpolish. Slides were dried in the dark at room temperature for 2 days and then refrigerated for storage.  The mitotic index of individual buds was assessed by counting the number of dividing cells in a field of view and expressing it as a percentage. An average of 10 fields or 300 to 500 cells were counted per apex; in order to prevent bias the number of dividing cells within a field were counted prior to counting the total number of cells. When a large gap of time between counting sessions occurred, I checked my technique by recounting a previously analyzed slide and reviewing photos of different phases. Apical height and width were measured during the initial dissection because these traits have previously been shown to fluctuate with mitotic index (Owens and Molder 1973). 42  2.5 Cold Hardiness Assessment Nurseries currently use cold hardiness testing to determine optimal lifting date. Cold acclimation gives the seedlings the ability to survive overwintering in frozen storage or outdoors. Hardiness was measured by frost induced electrolyte leakage [FIEL] (Levitt 1980, Burr et al. 1989).  2.5.1 Sample Preparation On each sample date, needle tissue was removed from a 3-6cm region below the terminal bud on the ten seedlings that were also collected for protein analysis. These undamaged needles were pooled together in a petri dish containing a paper towel moistened with dDH20. Needles were cut perpendicularly at both ends with a doubleedged scalpel which gave a 0.5cm segment size. Segments were transferred from the alcohol cleaned cutting board to a petri dish containing dDH20. Then 0.5 ml of dDH20 was added to clean labelled test tubes. Fine forceps and a glass rod were used to transfer 24 segments to each tube. The number of segments required had been empirically determined by placing varying amounts of needles in tubes, boiling at 100°C for 20 minutes, and then reading the conductivity. The number of boiled segments giving a conductivity of 100 micro mhos (24 in this case) was used for each test. Five test temperatures each with four sample tubes were chosen to hopefully bracket the predicted lethal temperature for 50% damage (LT50) and a set of control tubes (+1°C) was also prepared.  43  2.5.2 Frost Induced Electrolyte Leakage Test  The FIEL test was performed in an ethanol bath that was kept at a constant level and cooling was programmed by a LFE Temperature Controller. The test samples were first supercooled to -2°C and then nucleated and stoppered. Ice crystals for nucleation were made by dipping test tube brushes into distilled H20 and then liquid nitrogen and shaking their ice crystals over the test tubes.  For the freezing test, the microprocessor was programmed to cool the bath at 4°C per hour and a timer was set for each test temperature. The control samples were kept in an ice slurry bath (1°C) in the cold room. At the appropriate points, samples were removed and thawed in the ice slurry bath for at least one hour. Once all samples had thawed 5.5 mls of dDH20 was added to each tube. Tubes were agitated on a shaker at 100 rpm for 20 hours in a growth chamber set at 23-24°C. The next morning the conductivity meter was stabilized to give a reading of 2.0 micro mhos for dDH20 when set at mhos and 200X scale. The samples were briefly vortexed and conductivity before boiling (CBB) readings were recorded. Unstoppered samples were then covered with foil and boiled at 100°C for 15-20 minutes. After restoppering the tubes, they were returned to the shaker for another 20 hours prior to the conductivity after boiling (CAB) reading.  The relative conductivity was calculated by dividing CBB by CAB and subtracting the average control values. The index of injury 44  (1.1.) at each temperature was then calculated by the following formula: = 1 - 1 - (Ti/T21 X 100 1 - (C1/C2) where Tl and  T2  were the conductivity of tubes from each of the  treatment temperatures after freezing and after boiling, respectively, and C1 and  C2  were the conductivity of control tubes  before and after boiling respectively. Test temperature versus 1.1. was plotted and the temperature at which 50% damage occurred was found by extrapolation. This LT50 value represents the temperature at which 50% of the needles' electrolytes leaked from the tissue.  2.6 Photosystem II Activity Photosystem II status was monitored by chlorophyll fluorescence during overwintering. Photosystem II is the first part of the light reaction and traps energy for the dark reaction of photosynthesis (Kramer and Kozlowski 1979). Seedlings were placed in styroblock containers and watered to field capacity and placed under 450 umol  In-2 S-1  photon flux density at room temperature (22 +  1°C) for 45 minutes. Plants were then dark pretreated at room temperature for 20 minutes before measurements were taken.  The whole plant shoot was placed in an integrating sphere and chlorophyll a fluorescence was monitored for five minutes using an integrating fluorometer as described by Toivonen and Vidaver (1984) that was interfaced to a personal computer for data acquisition and 45  processing (Dube and Vidaver,1990). Raw fluorescence data (F,) were normalized to compensate for plant size. Normalization assigned the unit of 1.0 to the instantaneous value, F„ the nonphotochemical component of chlorophyll fluorescence (Papagiorgiou,1975). The relative value of  Fvar  was calculated by:  Fvar = Fv^F F, Fvar = normalized variable fluorescence at time t F,^= non-normalized fluorescence at time t F.^= 0-level fluorescence  The F„rcurves were averaged by the computer, n=6. Data collection of the Fwu time course was taken with four rates for the five minute time course: rate 1^5000 Hz for 10 ms 2^120 Hz for is 3^30 Hz for 99s 4^3 Hz for 200 s; for a total of 3740 data points over the 300 seconds. Light source within the integrating sphere was provided by a Sylvania EJL 200 W 24V quartz-iodine projection lamp, the light level was 115 umol m-2 s-lphoton flux density as measured by a LICOR Model LI-185.  2.7 Root Growth Potential Root growth potential is a seedling quality test that has been correlated to seedling vigour and outplanting success. It basically is a general indicator that all systems in a seedling are functioning properly (Ritchie 1984) and a measure of seedling performance potential (Burdett 1987).  46  The root growth capacity of freezer stored seedlings from the November 5th lifting date was measured. The samples were removed from storage (4°C) after 2.5, 3.5, and 6 months. Soil was washed from the root plugs and the seedlings were placed in a 10 gallon aquarium hydroponic system. The light conditions (200-250 umol  M-2S-1  and 16 hr photoperiod) and temperature conditions were the same as in the DBB test. DBB was concurrently measured. The seedlings were allowed to growth for 14 days and then the number of white roots >0.5 cm was counted. In addition, a subset of stored seedlings (N = 4) was removed at specific points and stored in -80°C freezer until protein could be extracted. The RGP results are expressed as the mean of 21 samples.  2.8 Colour and Chlorophyll: Any visible changes in seedling colour was monitored throughout the summer and winter collection period. Some early researchers speculated that chlorophyll could indicate N status of seedlings (Linder 1980). Chlorophyll content was assessed in the autumn samples to see whether it was related to observed colour changes and photosystem II shutdown.  The amount of chlorophyll was determined by the modified dimethyl sulphoxide (DMSO) method by Eze (1980) as well as by the 100% acetone method (Lichtenthaler and Wellburn 1983). For the DMSO method, a known weight of 0.5 cm needle segments were added to DMSO at 65°C and incubated for 4 hours. Then the optical density at 663 47  and 645nm was measured against a DMSO blank. For the acetone method 2x1 ml aliquots of 100% acetone were added to a known weight of finely ground spruce needles which were collected from the upper stem of the seedlings. Extractions were done on ice and under low light conditions. Absorbances of the 4 reps were read at 662, 645, and 470 nm. Concentrations of chlorophyll a, chlorophyll b and their ratio was calculated using equations in Eze or Lichtenthaler, respectively.  3.0 RESULTS 3.1 Visual and Biochemical Changes The visual and morphological changes in seedling growth were assessed throughout the sampling period. Figure 1A shows that container grown seedling stock grew from about 4 centimetres in early June to an average height of 18 centimetres by the end of the growing season. A cutworm and caterpillar outbreak in June did not effect the flagged seedlings and only had a minor effect on the whole population. Height increased linearly from mid-June until early August. Then growth ceased during the twelve day blackout treatment which began August 2nd; the start of blackout was indicated by an arrow (Figure 1A). The seedlings were light green throughout the summer, but needle colour darkened during the blackout treatment; they were a dark blue-green in the upper half of the seedling while the lower needles were lighter green. During August, I noticed that the lateral buds began forming during the 48  ^  ^al  A) 25  20  15  10  5  0  6/4^6/19^7/4^7/16^7/30 SAMPLE DATE  8/10  ^  8/27  B) 25  20 15 10  6/4  Table  ^  1  6/19  ^  7/4^7/16 SAMPLE DATE  ^  Table 4  ^  Table 2  ^  ^  Table 5  ^  7/30  ^  8/10  1-1 Table 3  I^1 Mean Height (N=25)  FIGURE 1: Height Growth of Seedlot QL(4307) 41 1  first week of the blackout treatment. The terminal apical bud was not fully formed until the August 27th sampling date. Figure 1B indicates that no significant difference in height growth patterns was seen between sampling tables. This suggests that watering and fertilizing regimes were uniform.  Additional morphological measurements were done throughout the autumn and winter sampling period. Height, caliper, bud length, and bud width were measured on the seedlings that were collected for days to budbreak (DBB) and mitotic index testing. The results are summarized in Figure 2. Sample height (Y1 axis) was in the 20-25 cm range throughout the remaining sampling time. Caliper and bud data are shown on the Y2 axis. Caliper was 2.8 to 4.0 mm. Bud morphology was comprised of a 3-4 mm width and a 4-6 mm length. No significant change in these morphological parameters was seen between samples throughout the dormancy period. In conclusion, this data indicates that it is likely that no measurable growth or expansion is occurring within the sample population.  An example of a summer protein profile is shown in Figure 3. Both the upper and lower stem segments contained numerous high and low molecular weight proteins. The protein pattern remains fairly consistent throughout the growing season and no new bands are visible on this coomaissie stained gel by August 10th. The 30 and 27 kd bark storage proteins are not detected in stem or root tissues during the summer or after a week of blackout conditions. 50'  30 25  Vi  m  H E 20 I G H^15 T  m  c^10 m  S  8/27 9/17 10/1 10/8 10/22 11/5 11/19 12/3 12/16 1/29 SAMPLE DATE x HEIGHT N=30 111.1 BUD LENGTH N=30  M:  CALIPER N=30 BUD WIDTH N=30  FIGURE 2: FALL PHENOLOGY CHANGES  UPPER STEM^LOWER STEM -ct^cy,^-0-^(.0^c:,^c ^0) -4- (0^0^0  0 ..— 0 .— re)^.— 0 ..— 0 ,— re)^•— ■.,^....,^■...,^,-..,^....,^■.,^■...., •..., ......., -...,^.........^■., MW^(.0 CO^N.^r--. N.^CO tO CO N. N. N.^CO 0 0 0 0 0 0 0 0 0 0 0 0  FIGURE 3: Summer Protein Profiles of Seedlot 4307 Each lane contains the same amount of total protein. Gels were stained with Coomassies Blue R-250. Arrows indicate where 30 and 27 KD VSP bands are normally found.  5a  Throughout the sampling period, total protein levels in tissues were calculated. Stem bark contained about 6ug protein/mg tissue throughout the summer; this declined slightly during bud formation (Figure 4A). Levels then rose to Bug protein/mg tissue throughout the winter. Figure 43 also demonstrated that buds contained more protein and accumulated more protein (maximum 15 ug/mg) than stems during dormancy. This trend of greater amounts of protein in buds was generally maintained. When proteins from bud, stem, and root tissues were extracted from the same seedling, roots contained less protein than stem and buds respectively (data not shown). Accumulation of protein was greatest in buds.  3.2 Physiological changes and vegetative storage protein accumulation during the autumn months  3.2.1 VSP Accumulation As seen above, the total protein levels increased most significantly in the newly formed apical bud. I postulated that bud tissue would be the best tissue to use for analyzing accumulation patterns of VSP. Several reasons for this decision existed: 1) roots contained more phenolics which could bind with proteins; 2) although both bud and stem extracts solubilized easily, buds contained more protein; 3) bud samples were more uniform because the tissue was clearly defined. The extraction procedure was also tested to see if modifications could improve yields. 53  A) Total Protein in Stem Bark 10 ^ U  g P  r o t e i n / m 9  4  T i 8 S U  e  I^I^l ^t^l^t^I^I^I^l^I  06/19^07/30^09/17^10/22  Sample Date  1^l  12;03 I  01/29  B) Total Protein in Bud Tissue  la ^  U  U  P  15  r o t e i n / m 9  12  t i S S U e 09/10^10/08^11/05^12/03^12;31^01)28  Sample Date  FIGURE 4: Total Protein Increases in Stem and Bud Tissue A) Stem Bark Extract n=7 stem segments with 2 reps each, B) Whole Bud Extracts n=10 with 2 reps each, total protein was calculated from tissue weight, amount buffer, and protein concentration of each solution which was determined by a modified Bradford Assay.  6q  Attempted modifications included the addition of unhydrated and hydrated 1.5% polyvinyl-polypyrrolidone (PVPP), 0.5M NaC1 or reducing agents such as sodium metabisulfite. Acetone precipitation was also tried to help concentrate samples and reduce possible phenolic interference. Results seemed to vary depending on the specific sample. Quality of summer extracts was less predictable than fall and winter samples possibly because more proteases were active or greater phenolic interference existed. I also tried changing the ratio of buffer to sample. The range of 1 ul/mg to 8 ul/mg was tried. This mini-experiment indicated that 1-2 ul buffer /mg resulted in poorer extracts and increased the chance of tissue carryover when pipetting. Higher volumes 7-8u1/mg tissue were more dilute. Efficiency and yield were not improved. In the end, I used basically the same procedure as done in the original study of spruce VSP (Roberts et al. 1991). Six microlitres of 4XSDS buffer were used per milligram tissue and 1.5% PVPP was added during grinding to limit the possibility of phenolic interference.  The autumn protein profile of bud, stem, and root tissue is pictured in Figure 5. All electrophoresis gels were loaded on a total protein basis. The VSPs, indicated by the arrows, were either absent or present in bud tissues at very low concentration in late August and little increase had occurred by the middle of September. However, by October the two densely staining 30 and 27 kilodalton bands were major proteins within the total protein extract. These levels were maintained and increased throughout December. In stem 55  tissues, a weakly staining 30kd band was present in some Aug. 27th samples and by Sept. 17th, both VSP bands had appeared in equal porportions. The overall amount of VSP did not change much throughout early October, but by October 22nd the amount increased (Figure 5B). This level appeared to be maintained for the duration of the experiment. In the Dec. 16th and Jan. 29th samples, a visual assessment suggested three times more 30kd protein than 27kd protein existed. Root extracts showed similar trends (Figure 5C). Storage protein was present in roots by Sept. 17th and the 30 kD dominated. The 27 kD band is minor and its solubility was different from the stem and bud samples. Perhaps additional chemicals, possibly phenolics, were extracted from the root that could interfere with the protein solubility.  The point at which maximum protein has accumulated was hard to pinpoint visually; therefore, scanning densitometry was used to quantify the amount of storage protein relative to total protein present in the bud extracts. The data shown in Figure 6 represents the average percentage of VSP found in bud tissue extracts; ten buds were extracted in duplicate on each sample date. VSP comprised 7% of total protein at the end of August. This percentage increased steadily until the October 22nd sample when it reached 15%. After this date, no significant increase in storage protein was seen. Duncan's multiple range test separated the samples into three groups: mid-August to mid-September, early October, and midOctober to the end of January. The concentration of total protein 56  FIGURE 5: Autumn Protein Profiles of Seedlot 4307 SDS— PAGE gels of total Bud proteins (A), Stem bark proteins (B), and Root bark proteins (C) are seen. Molecular weight markers are indicated. The samples are in the following order: L1 — Aug. 27, L2 — Sept. 17, L3 — Oct. 1, L4 — Oct. 8, L5 — Oct. 22, L6 — Nov. 5, L7 — Nov. 19, L8 — Dec. 3, L9 — Dec. 16, and L10 — Jan.29. All gels are stained with Coomassie Blue. lane C in gel (A) is a control winter bud sample from a 1989 study.  57  A) BUD  97.4KD 4656.02KKDD  -14114#  41 ii  31 .0KD^3;^7 -^=. 1W■ -•^  44. 1141  21 .5KD^OM OS IS abwe 041 MS alk ^000 SOP 1 4.4KD MW^C^1^2^3^4^5^6^7^8^9^10 B) STEM 97.4KD 6 6.2K D 45.0KD^  _ .41^  •  31 .0KD •111.,11.^  21 .5KD NM OW Om  1 4.4KD  11■••^  -  =--ar WWII Mgt star. am NM tir  MW 1^2^3^4 C) ROOT 97.4KD _^_ 66.2KD  5^6^7^8^9^10  1^lir ,  45.0KD — .... 31 .0KD —10.—  1  1110-  1  21 .5KD qi■va, 1 4.4KD  1■1-L-MW  1^2^3^4  1  5^6^7^8^9^10  65'  in bud tissue also did not increase past late October (Figure 4). This fact means that the actual amount of VSP in the cells was indee8d constant and maintained by November.  3.2.2 Cold Hardiness Acquisition The acquisition of cold hardiness was monitored in seedlot QL after budset and until the end of January (Figure 7). The frost induced electrolyte leakage (FIEL) method was used and data was collected so that index of injury (%) at -18°C and LT50 (temperature for 50% electrolyte leakage) could be calculated. The minimum temperature for our system was -80°C. In September, the needle samples released almost 80% of their electrolytes after experiencing a -18°C stress. The corresponding LT50 value was -10°C. Since FIEL measurements are comparable to whole plant methods (Burr et al. 1990), the results (Figure 7) indicate that the seedlings were not very hardy during September or during early stages of fall acclimation. Hardiness increases steadily after this point. A Tukey's mean separation test was done on the -18°C data and this analysis indicated that after October 22nd no significant change in damage occurred at this temperature. However, overall hardiness was still being acquired because the LT50 value was changing. The rate of increase plateaued briefly in October and early November, but then increased rapidly to reach maximum hardiness of approximately -80°C in December. The seedlings remained within a greenhouse that was kept above 0°C; six weeks later the seedlings had dehardened to the October LT50 of -40°C. 59  20  17.5  ^  % V S 15 P  i n 12 . 5  P  r o t e i n  -  -  7.5  -  2.5  _  , I^  ,  09/15 10/05 10/2511/14 12/04 12/24 01/13  Date Sampled FIGURE 6: VSP Accumulation in Seedlot QL (4307) Buds  Scanning densitometry of coomassie blue stained gels was used to quantify the amount of vegetative storage protein in terminal bud extracts. Buds from ten seedlings were extracted in duplicate on each sample date. Mean and S.E. of the mean is shown.  Go  0  100 - 90  - 20  - 80 - 70 ok 60 D a  -40  - 50 a -60  - 40 - 30  - 80  -18oC  - 20  - 10 v^  rrI -100^ 08/27 10/01 10/22 11/19 ^12/16 Sample Date v  0  FIGURE 7: Acquisition of Frost Hardiness -18oC^LT50  H = Index of Injury, LT50 = Temperature for 50% Electrolyte Lea-<age 01  3.2.3 Photosystem II Activity and Chlorophyll Measurements Chlorophyll fluorescence measurements indicated that photosystem II activity declined gradually throughout the sampling period (Figure 8). A fluorescence level of less than 0.7 is thought to represent minimal PSII activity (S. Gillies, personal communication). This activity level was reached by late October with a further drop in December and January. The minimum fluorescence level recorded was 0.5 units. These results suggest that if photosynthesis itself had been measured during the winter, levels would be minimal because photosythetically active radiation (PAR) is low and plant activity is slowed.  Total chlorophyll was extracted from needles using both the DMSO and acetone methods. The DMSO results were not consistent; therefore, only the acetone results are summarized in Figure 9. Total chlorophyll levels increased slightly between the August and September sample, but amounts returned to the August range of 2300 - 1700 mg/g needles for the remaining samples. The chlorophyll A/B ratio did not change significantly over the sample period. A ratio of 2.3 was maintained throughout the experiment. Therefore, significant colour changes did not result from chlorophyll changes during the fall acclimation processes. Increased needle wax could alter the colour seen.  62  1 F I u o 0.8 r e S 0.6 C e n 0.4 C e F 0.2 V a r  0  rp rffi r,,, V/  ,P rer  .. A AA  •  r  r  AA  A  A A  8/27 9/17 10/1 10/8 10/22 11/6 11/19 12/3 1/29  Collection Date FIGURE 8: PHOTOSYSTEM II ACTIVITY Chlorophyll fluorescence was measured using an integrating fluorometer system, n=6. The average and its standard error is shown above.  43  A) T o t a I C h I m 2000 0 / g moo  n  1000 e d I^500 e $ 8  Sept.22  ^  ^ ^ Oct.22 Nov.21 Dec21 Sample Date  B) 3  C  2.5  h I^  2  R  a^1 t I o 0.5  oAug.22^Sept22^Oct.22  Nov.21  ^  Sample Date  FIGURE 9: Chlorophyll Content of Needles The amount of total chlorophyll (A) and the ratio of chlorophyll a/ chlorophyll b (B) was measured by th acetone method. Two replicates were done that contained three and four replications each.  En  Dec.21  3.2.4 Dormancy Status and Cell Division  The dormancy status of the seedlings was assessed by days to budbreak while the ability of bud cells to grow was measured with mitotic index (Figure 10). At the beginning of the sampling period, all fifteen seedlings tested did not flush. Only a few seedlings flushed, but their growth pattern was abnormal. This was possible because seedlings in very early stages of dormancy were still influenced by the environment. Seedlings in deep rest did not resume growth. Thus the sample population initially contained a mixture of seedlings in both the correlated inhibition and rest stages of dormancy respectively. By October 1st, budbreak within the seedling population was more synchronous, but breaking bud still required an average of 80 days exposure to optimum conditions. The number of days dropped sharply by October 8th to an average of 38 days. Rate of change slowed for the rest of the season and days to budbreak levelled off at 14 days. Both the terminal and lateral buds were observed and the results show that lateral buds take fewer days to break bud than terminal buds throughout all stages of dormancy. In the latter portion of the experiment the variation in the population's ability to break bud declined significantly. Overall the results show that all chilling requirements had been met by early November and the seedlings had become quiescent.  65  100 # D A Y S T 0 B U D B R E A K  80  60  DBB(L)  PP21SII  40  10/08^11/06^12/03^01/29  SAMPLE DATE  20  o  8/27 9/17 10/1 10/8 10/22 11/5 11/19 12/3 12/16 1/29  SAMPLE DATE  FIGURE 10: DORMANCY STATUS - MI AND DBB The number of days to budbreak (DDB) for terminal and lateral bucs and mitotic index for terminal apices is shown above. For procedures see materials and methods N=15 for each sample cate, N.F.> 50% of reps. never flushed.  66  The inset graph in Figure 10 displays the mitotic index data. The bud's dormancy status is seen with the days to budbreak (DEB) data. Meanwhile, mitotic index was used to specifically measure cell division within the apical bud. The apices had an average 6% division rate until the beginning of October. Then mitotic index declined to 3.5% in November and two weeks later it reached almost zero percent. Tukey's mean separation test results divided the MI data into three stages: September - early October, mid-October November, mid-November - January. When these results are compared to DBB data, they indicated that mitotic activity in the buds became negligible just after the seedlings became quiescent.  Figure 11 illustrates various features of the apical bud squashes used for enumerating mitotic index. An overview of a bud squash is seen in picture 11-A; chromatin material is stained pink and tannin deposits are brown. Tannins present within the apex could obscure cells which sometimes made counting impossible (Picture 11-B). The rest of the photos (Figure 11 C-F) are examples of some of the different views of the cells and phases of mitosis that were encountered.  Dormancy characteristics was also expressed as a dormancy release index which was a conversion of DBB data (Figure 12). The index showed that both the lateral and terminal buds have fulfilled their dormancy requirements by November and the seedlings are ready to grow by January as the index is equal to one. Resumption of 67  FIGURE 11: ILLUSTRATIONS OF APICAL BUD SQUASHES A) Overview of a Nov. 19th Bud Squash magnification 41X B) Tannin deposits obscuring cells mag. 512X C) Anaphase (A) and early Telophase (ET) cells mag. 1008X 0) Cells at various stages of mitosis — Metaphase (M), Prophase (P), and Anaphase (A) mag. 410X E,F) Closeup of dividing cells within apex mag. 1024X.  66  e  6/  ^  1.6 1.4  I  0.8  D 0.4 R I 0.2 0 A SOOONNDDJ uecccooeea gp t t t v v ccn 2^1^1^8^2^5^1^3^1^2 7 7^2^9^6 9  Sample Date FIGURE 12: Dormancy Release Index The dormancy release index was calculated as in ( Ritchie, 1984). The general formula is DBB( t) / DBB( t) of a fully chilled seedling. The three lines represent indices for: Terminal Bud A^Lateral Bud X Lateral Bud/DBB(I) •  14  growth was being inhibited by the environmental conditions. This index system was originally developed for terminal bud data. I have also applied the principles of this index [ index = # DBB(t)/ # DBB(t) of a fully chilled seedling ] to describe the dormancy release characteristics of the lateral buds. The third line on the graph was lateral bud break converted with the terminal DBB of a fully chilled seedling. This calculated index overestimated the fulfilment of dormancy requirements because the value was greater than one. This suggests that if lateral bud data is converted, its index must be based on lateral bud data only.  3.2.5 Changes in seedling quality during storage  According to the cold hardiness data (Figure 7), the seedlings had met lifting criteria for storage by the October 22nd sample date; therefore, a subset was stored on the November 5th sample date. Seedlings were then sampled after being stored in the dark at 4°C for up to six months. Table 2 summarizes the post storage characteristics of seedlings stored for 2.5, 3.5, 6.0 months. Root capacity and the number of days to budbreak declined in storage, in addition, protein gels (data not shown) indicated that VSP levels declined marginally in storage but this change was not quantified. Overall, it appears that seedling quality as predicted by RGP decreased after long term storage at 4°C and this may be associated with decline of storage reserves including VSP.  72  TABLE 2: POST STORAGE CHARACTERISTICS OF SEEDLINGS LIFTED ON NOVEMBER 5TH. Storage  RGP  Lateral Bud  Duration  DBB  Terminal Bud DBB  2.5 Months  192.7^+^16.0  11.6^+^0.2  16.5^+^0.8  3.5 Months  105.0^+^8.9  13.2^+^0.7  16.1^+^0.2  6.0 Months  75.1^+^10.6  7.3^+^0.7  10.5^+^0.7  Note:^RGP = Root Growth Potential DBB = Number of Days to Budbreak All samples were stored at 4°C. All data is reported + standard error of the mean (n=21).  3.2.6 Summary of Physiological Changes Together, the results indicated that the QL seedlings were fully acclimatized by early November and had undergone the required physiological changes. PSII was inactive by mid-October and shortly afterwards index of injury at -18°C (I.I.) and VSP accumulation levelled off. Dormancy assessment showed that seedlings were quiescent by late October as mitotic activity became negligible. Neither chlorophyll content nor ratio changed during the fall. Correlation analysis between PSII activity, FH acquisition, VSP 73  accumulation, and DBB status was done using the Pearson correlation matrix. The VSP patterns had greatest similarity to LT50 changes with r= -0.972. Vegetative storage protein accumulation also correlated well with PSII activity (r = -0.971) and index of injury [1.I.] (r = -0.900). Days to budbreak correlation with VSP (r = 0.893) was weaker. Data suggested that the lifting date predictors cold hardiness and PSII both exhibited seasonal patterns that were similar to VSP accumulation trends during fall acclimation. All the measured physiological changes were summarized in a timeline which is located on page 94 (Figure 21).  3.3 Additional seedlots have similar patterns of VSP accumulation In order to see if there was an influence of seed origin on VSP accumulation patterns a northern seedlot PG and eastern seedlot EK were collected. Bud extracts were analyzed by SDS-PAGE and densitometry (Figure 13, Figure 14). Storage protein was first visible in the August 10th sample for EK and the Sept. 12th PG sample (Figure 13,A,B). Subsequently, densitometry indicated that during the summer EK total protein extracts contained about 7% VSP while PG extracts had 4% VSP. Both visual assessment and densitometry results indicate that maximum accumulation occurred during September and October. The proportions of 30 and 27 KD proteins varied; EK extracts generally contained more of the 30 KD protein within six weeks of its appearance while about five weeks after VSP was readily visible, PG extracts had more 30 KD protein than the 27 KD band. Maximal levels of VSP were around 15% of total extracted 74  protein for both seedlots and were achieved by the middle of October (Figure 14a,b). Duncan's multiple range test divided VSP levels into three groups for PG and four groups for EK. The PG seedlings' data grouped into August - early September, September early October, and mid October to the end of January divisions. -  EK's VSP levels were statistically different in August, September, early October, and October to January. Statistical analysis of the three seedlots (QL, EK, PG) confirmed that their general accumumulation patterns were not significantly different during the August to January time period; therefore, all interior spruce seedlots tested are likely to have similar strategies for accumulating VSPs (Figure 6, 14). The EK and PG seedlots were lifted in the nursery on December 3 rd and placed in -2°C storage. Lanes 11, 12 and 13 contained protein extracts of seedlings that have been stored and lifted (Figure 13). Visually, both VSP bands appeared to remain close to their prestorage concentrations and ratios. However, quantification indicates that storage protein levels remained constant after 5.5 months in storage, but then declined slightly after this point (Figure 14). The exact percentages present during the summer must be regarded with caution because other proteins of similar size may exist and bands were barely detectable on coomassie blue stained gels at this time. Antibodies and western blotting would be required to confirm whether very low levels of VSP really existed during the summertime.  75  FIGURE 13: Protein Profile of Other Seedlots SOS — PAGE of total bud protein extracts from both EK (A) and PG (B) is shown. Molecular weights are in lane 1. The arrow points to the VSP bands. Sample dates are as follows: L2 — June 21, L3 — Aug. 10, L4 — Aug. 27, L5 — Sept. 12, L6 — Oct. 1, L7 — Oct. 15, L8 — Oct. 29, L9 — Nov. 12, L10 — Nov. 26, L11 — Jan. 21, L12 — May 15, L13 — May 28.  A) EK Seedlot Buds — Total Protein Extracts  B1 PG Seedlot Buds — Total Protein Fytroots  20 % V S P 15 i n T o 10 t a I P r^5 o t e  i  n 0  07/20 08/29 10/08 11/17 12/27 02/04 03/16 04/25  Date Sampled  FIGURE 14: VSP Accumulation in EK and PG Interior Spruce Seedlots Scanning densitometry of coomassie blue stained gels was used to quantify the amount of vegetative storage protein in terminal bud extracts. Three seedlings were extracted in duplicate for each seedlot on each collection date.  3.4 Effect of Temperature and Photoperiod on VSP Induction and Accumulation A study was started on August 4th with another seedlot FIN which originated from the Finlay region. Different fall acclimation treatments were applied in order to assess the influence of temperature and photoperiod on seedling physiology and specifically on VSP accumulation. The physiological characteristics of the three treatments were examined by S. Gillies at Simon Fraser University (Table 3). My thesis discusses the protein analysis of the SD/Warm, LD/Cold and Ambient treatments (Figure 15, 16, 17). TABLE 3: SUMMARY OF PHYSIOLOGICAL ATTRIBUTES OF TREATMENTS SD/Warm <12 hr photoperiod 22°C/18°C  LD/Cold 16 hr photoperiod 10°C/ 5°C  Ambient = natural seasonal decline in photoperiod and temp.  Frost hardiness does not change LT50 -10 to -15°C Sept. thru Dec.  Max FH achieved by November with an LT50 of -28°C.  Max. FH achieved by January with LT50 of 33°C (Nov.^LT50 = -27°C)  PSII inactive by Sept.,^shows normal pattern.  PSII is inactive by late August.  PSII inactive by October.  RGP after 4 months @ -2°C storage was excellent at all lifting dates.  RGP after storage was best for Nov. lifted seedlings. Earlier samples Aug.,^Sept. had lower RGP.  Root Growth Potential(RGP)^is negligible, Seedlings never flushed.  -  Unstored seedlings flushed in late December. Results summarized with permission from .^ies PhD research, SFU. 79  The summary of physiological characteristics (Table 3) indicates that normal seasonal patterns were altered by the fall acclimation treatments. Only the LD/Cold and the ambient treatments became sufficiently cold hardy for lifting. Photosystem II activity declined more quickly in both the LD/Cold and SD/warm treatments. SD/Warm seedlings survived poorly in storage and remained dormant; however, seedling quality after storage was excellent for LD/Cold treated seedlings regardless of lifting date. In contrast, ambient seedlings lifted by early November were of better quality than earlier lifts.  The total protein profiles of bud, stem, and root extracts are shown in figures 15 and 16. Most of the proteins remained at a constant level throughout the September to December sampling period; however, the amount of VSP varied quantitatively between treatments. SD/Warm bud extracts contained a large amount of 30 kd VSP and some 27 kd VSP in every sample. In contrast, both the ambient and LD/Cold bud extracts had a smaller percentage of VSP in September samples and accumulated VSP after this point. Levels of 27 kd VSP appeared constant. The pattern of VSP accumulation in seedlings from the chilling treatment were similar to seedlings under ambient conditions except levels declined in December. Some of this seedling group had begun to flush by this time. Stem extracts also varied between treatments. As seen in buds, SD/Warm extracts contained high levels of VSP. The ambient seedlings accumulated storage proteins throughout the sample period. 80  Meanwhile, LD/Cold stem extracts increased VSP until late October afterwhich levels declined. The ambient treatment exhibited the same VSP accumulation pattern in whole bud, stem bark and root bark extracts. However, SD/warm and LD/Cold root collar bark extracts showed different responses than their corresponding above ground portions of the seedlings. SD/warm root extracts did not contain VSP until early October while SD/warm bud and stem extracts had contained maximal amounts of VSP by September. In fact VSP accumulation in SD/day root was gradual throughout October afterwhich it increased slightly followed by a slight decline in December. This was similar to the usual accumulation pattern seen in the ambient extracts. The results suggest that daylength did not influence roots as much as bud and shoot tissues. As in LD/Cold bud and stem extracts, VSPs were also present in LD/Cold root extracts by October. Compared to SD/warm root extracts, LD/Cold root samples contained a greater percentage of VSP within total protein (Figure 16). VSP levels were steady within LD/Cold roots until levels declined slightly in November and then increased marginally in early December. This November-December period was the same time in which LD/Cold bud tissue was preparing for flushing. This evidence tentatively suggested that reserves were reallocated when the seedlings prepared for flushing.  81  FIGURE 15: Variations in Total Protein Patterns Of Bud Tissue A) Ambient — experienced natural changes in daylength and temperature B) Short Day/Warm 12hr photoperiod with 25 C day, 18 C night, C) Long Day/Cold 16 hr photoperiod with 10 C day, 5 C night Lane 1 MW markers, L2 Sept. 6th, L3 Oct. 7th, L4 Oct. 21st, LS Nov. 20th, L6 Dec. 12th. Lanes were loaded with the same amount of total protein. Arrow indicates storage protein bands.  sa  A: AMBIENT TREATMENT  B: SD/WARM  VSF  MW 9/6 10/7 10/21 1 1 /20 12/7 12/12  koN 9/6 10/7 10/21 11/20 12/7 12/12  '0  FIGURE 16: Total Protein Patterns in Stem and Root Collar Bark From Three Fall Acclimation Treatments For stem (A) , ambient treatment is in lanes 2-5, short day is in lanes 6-9, and chilling treatment is in lanes 10-13, MW markers are in lanes 1 and 14. Sample dates are as follows: L2,6,10 — Sept.6; L3,7,11 — Oct.21; L4,8,12 — Nov. 20; L5,9,13 — Dec. 12. Only the short day (L1-6) and the chilling (L8-13) treatment extracts are shown for the root collar bark (B) because the ambient pattern was the same as in stem extracts. Sample dates from left to right are Sept.6, Oct.7, Oct.21, Nov.20, Dec.3, and Dec. 12. Lane 7 contains the molecular weight markers. Arrows point to the 30 and 27 KD vegetative storage protein bands.  €8  A) Stem Bark Extracts from three acclimation treatments 97.4KD –■ — I alq 45.0KD  aloe  31.0KD  ■■■■••■  14.4KD -"Nub  ■••••••^Ay&  opal ormili  211516"'  [ Ambient 1 [ SD/Warm ] [ LD/Cold ^]  2^3^4^5^6^7^8^9 10^11^12 13 14  B) Root Collar Bark Extracts from SD and Cold treatments  85  20  P e r C e 15 n t o f T10 o t a I P r o t e i n  5  0 0^20^40^60^80^100 120  Days after Start of Treatment I Day 0 • Aug.4th I  FIGURE 17: VSP Patterns Are Altered By Fall Acclimation Conditions The percent of vegetative storage protein in total protein extracts from buds was quantified by using scanning densitometry. Three buds were extracted and analyzed in duplicate for each sample date.  $6  140  Scanning densitometry using the whole band analysis system was used to quantify the bud protein gels and to confirm observed differences (Figure 17). While both the ambient and chilling treatments gradually accumulated VSP in their bud tissue; the SD/Warm seedlings had very high levels by the first collection date which was 30 days after the treatment began. These initial levels of approximately 15% VSP were maintained in the SD/Warm extracts throughout the collection period. Ambient and chilling patterns were closely matched until late November; when ambient VSP plateaued and chilling VSP peaked at 17% of total protein. The LD/Cold seedling extracts then quickly began to decrease VSP levels as the seedlings prepared to break bud.  3.5 PAS staining and 2-D Electrophoresis under Reducing and nonreducing conditions Protein samples were also analyzed by PAS staining and by 2-D reduced/nonreducedelectrophoresis. PAS staining tested whether the storage proteins were glycosylated. Electrophoresis under reducing and nonreducing conditions was done to assess whether any disulphide bonds or various forms of the storage proteins existed.  Only one protein band was stained by PAS staining (Figure 18). This protein was the positive control Ovalbumin and it only stained at higher concentrations (Figure 18a). Incubation and developing time were varied during subsequent attempts, but no additional 87  glycosylated proteins could be detected despite the large amounts of protein in each lane (Figure 18b).  Summer and winter extracts were electrophoresed under nonreducing and then reducing conditions (Figure 19a,b). Lane C was a control winter sample which was used to better estimate where the storage proteins migrated. With this technique, proteins without intersubunit disulfide bonds would migrate according to size which would create a diagonal line of high to low molecular weight proteins. If the reducing agent opened disulphide bridges, protein subunits would migrate to two lower molecular weight locations below the diagonal. Four unique bands were present in the nonreduced and then reduced winter sample (Figure 19b). These bands were present as two pairs of 30 and 27 kD proteins. One pair unique to the winter sample migrated on the diagonal line. This data provided evidence that disulphide bridges were absent and the reducing agent did not influence the mobility of this 30 - 27 kD protein pair. The other 30 - 27 kD pair is located directly across from the other unique proteins, but migrated away from the diagonal. This suggested that either their size increased or their solubility was changed by the reducing agent. Solubility characteristics could have been altered by reduction of disulphide bonds within the storage proteins themselves. In summary, intersubunit disulphide linkages did not exist because no large molecular weight proteins divided into individual subunits, but two different forms of the 30 and 27 kD VSPs were seen. 88  FIGURE 18: PAS Staining of Total Protein Samples An acrylamide gel was first stained by A) the PAS (icteriodic acid—Schiff's reagent) method and then with B) coomassie blue. Lanes 1-4- contained 10 ul of sample (1.8 ug/ul) and lanes 5-7 were loaded with 20 ul each. Lane 1,5 — MW markers, Lane 2,6 — August 27th sample, Lane 3,7 — January 29th, Lane 4 — Pool of October and November samples. The * beside the arrow indicates the location of the positive control Ovalbumin (45 kD) in the MW lanes. The other arrow shows the location of the 30 and 27 kD storage proteins in the winter samples.  SI  B: COOMASSIE BLUE COUNTERSTAIN 97.210)^V  66.2K  * 45.01(D"" 31 .0KD Z 1 .5KD mom. 1 4.4KD—•..  1  2^3^4^5^6^7  41 0  —BME  A) SUMMER  41111.,  1C B) WINTER  VSP  1C  FIGURE 19: Disulphide Bond Analysis of Total Protein Samples The two samples used are from (A) Aug. 27 and (B) Jan. 29 bud total protein extracts. Lane 1 contains the MW markers from 97.4 KD size at the top of the gel followed by the 66.2, 45.0, 31.0, 21.5, and 14.4 KD markers. The arrows highlight the area in which VSP is usually found. Lane C shows a normal summer (A) and late fall (B) SDS buffer protein extract.  RI  3.6 Comparison of Scanning Densitometry Techniques During the course of my thesis, our lab group acquired a Millipore-Bioimage scanning system while I was in the midst of doing densitometry measurements with the Beckman-Gelscan module. Originally, I used the Beckman gelscan module for the DU-65 spectrometer which gave the relative peak area and number for individual lanes on a gel. Colour slides (4X4) of gels were scanned in duplicate for three extracts for bud tissue. This gave the curve seen in Figure 20. Most means have a large standard error especially in later winter samples which may indicate that the program has difficulty distinguishing the band boundaries when a protein was present in high proportions within a complex mixture. The same gels were later analyzed with the Millipore scanner. The main advantage of this newer system was that the lanes and bands can be precisely defined and visualized on a computer screen. This feature gave me greater confidence that I was actually quantifying the VSPs' bands. Figure 20 indicates that the general pattern of the curve was similar for both methods. However, the standard errors of the means was much smaller for the Millipore data, regardless of the sample date. From these results, the Millipore system may slightly underestimate the proportion of a protein band especially if present at higher concentrations, but overall the data collected were more precise. From experimental results seen in Figures 6, 14, and 17, the Millipore scanner system was capable of assessing possible differences between treatments and seedlots, as well as adjusting for loading differences within lanes. 92  30  25 0 20 0 A  0  15  10  *^ Beckman GelScan^0^ Millipore Bioimage  09/16 10)06 10)26 11)15 12)05 12)25 01%14  Sample Date  FIGURE 20: COMPARISON OF TWO DIFFERENT GEL SCANNING SYSTEMS TO QUANTIFY VSPs Three buds were extracted and analyzed in duplicate for both the Beckman and Millipore densitometry systems. Average percent of VSP within total protein extract is shown with its standard error.  911  VSP  None Increasing Constant  10"1""""Ow 46111+ 4'1*  FH^Low^Increasing Sm. Rise Falls  4'1* 410•■••••■ 4*** *-  II 18C High^Decreasing Constant -  PS 11^Active^Inactive MI^High^Falls^MI=0  imm"90' +1°"1"1+ 10""'"I■  DBB  Low High Decreases Quiescence  4•Nme+ 40-* ■—■■••••■••••=÷  May July Sept Nov^Jan Mar May  FIGURE 21: Summary of Seasonal Changes in Physiology and Biochemistry The abbreviations are defined as follows: VSP = vegetative storage protein, FH = frost hardiness, 14-180 = index of injury at -180. PS II = photosystem II activity, MI = mitotic index, DBB = number of days to budbreak.  4.0 DISCUSSION 4.1 Appearance of Vegetative Storage Protein (VSP)  Under naturally occurring conditions, vegetative storage protein (VSP) in interior spruce appeared after budset and accumulated during fall acclimation. This general trend was observed in three seedlots QL, ER, and PG as well as in seedlot FIN (29135 - ambient treatment). Densitometry results of late summer ER and PG bud extracts indicated that VSP comprised about 4-7% of total protein in the newly formed bud tissue. Since identification of the protein depended solely on protein size and the total protein extract was quite complex, more sensitive and specific assay methods (eg. immunological) maybe required to confirm the exact time in which VSP first appears. Analysis of coomassie blue stained SDS-PAGE gels in this study indicated that QL buds contained VSP on August 27th; ER bud extracts contained detectable VSP as early as August 10th, while PG extracts did not contain visible VSP until early September. These dates were earlier than previously reported by Roberts et al. (1991) for interior spruce (seedlot 8524, 2+0 container stock) which emphasized that the cultural and environmental differences between years influence the initiation of fall acclimation including the appearance of VSP.  The staggered appearance of VSP between different seedlots could possibly be accounted for by seedlot effects on bud formation. Both ER and PG had formed buds prior to the late August blackout treatments that were employed by the nursery to obtain similar height ranges between seedlots. In early August, only the ER seedlings contained a significant amount of VSP. Differences in 95  VSP levels between ER and PG seedlots could be attributed to genetic control of fall acclimation. Since ER is primarily Engelmann spruce and from a high elevation area (East Kootenays), it may begin to acclimate earlier in the fall. In Simpson's study seedlot differences were also seen during fall acclimation, higher elevation Engelmann spruce initially hardened at the same rate as the northern white spruce, but ultimately the white spruce were more hardy (Simpson 1990). From these results, it was apparent that acclimation occurred during the same time frame for all spruce species tested but the final level of hardiness was genetically determined. During my sampling season, ER seedlings were the least hardy (Grossnickle et al., unpublished results) of all three seedlots as expected; however, initial VSP content was higher than VSP levels in PG and QL seedlings. Visual buds formed on QL seedlings 10-14 days later than the other seedlots. Later bud formation did not delay the appearance of VSP in QL seedlings since VSP levels were comparable to ER & PG seedlings by late August. Bud formation appears to be essential for VSP induction because no VSP was found in growing seedlings. Despite differences in the timing of budset, all seedlings contained low levels of VSP during these early stages of fall acclimation.  Differences in accumulation of VSP were observed between seedlots (QL, ER, PG). Seedling tissues contained low levels of VSP (7% of total protein) until early September after which a six week period of VSP accumulation occurred. Then VSP content in ER seedlings increased rapidly to 10% within two weeks while QL and PG increases were more gradual. Patterns of VSP accumulation in QL 96  seedlings paralleled increases in total protein which suggests VSP contributes to these changes in total protein levels. PG continually had less VSP than QL and EK during fall acclimation in September and October, although all seedlots had comparable VSP levels (15-18%) by the end of October. Similarities in the relative maximum amount of VSP found in bud tissues from different seedlots suggest that the cells may have reached their maximum storage capacity. During this September - October period, natural daylength continued to decline and longer periods of cool temperatures occurred (Sakai and Larcher 1987, Grossnickle et al. unpublished results). These conditions appear to have stimulated VSP accumulation within the seedlings. Since all three seedlots were experiencing the same fluctuations in temperature and photoperiod during this period, genetic differences between seedlots may be contributing to these slight differences in VSP accumulation rates.  The influence of photoperiod and temperature on VSP accumulation was investigated by applying SD/warm and LD/cold temperature fall acclimation treatments after natural bud formation had occurred (see Section 3.4). SD/warm conditions caused maximum VSP levels to be attained within a month. Shortday photoperiod has previously been shown to cause accumulation of a 32 kD storage protein and mRNA in poplar bark tissues (Coleman et al. 1991, Langheinrich and Tischner 1991). Long day treatment of poplar seedlings has also shown lower levels of VSP relative to a short day treatment; in this case, lower amounts of VSP were a result of lower mRNA levels (Coleman et al. 1992). Future research may 97  confirm whether this is the case for interior spruce. Reduced temperature probably accounted for VSP accumulation seen in LD/cold tissues. The LD/cold treatment showed gradual VSP accumulation which was similar to accumulation patterns observed under ambient conditions. The cold temperature range (10°C day/5°C night) experienced by this treatment could be stimulating VSP accumulation. If temperature was indeed the main factor required to achieve maximum VSP accumulation in the LD/cold treatment, a LD/warm treatment applied after budset would be expected to contain only very low amounts of VSP formed after bud formation. LD/cold treatment differed from the ambient treatment because shortly after VSP levels peaked the LD/cold seedlings broke bud; as a result, VSP levels within tissues declined. Temperature is an important fall acclimation cue that triggers physiological changes which allow plants to become dormant and to resume growth (Lavender 1991). Budbreak occurred because chilling requirements for dormancy release had been met (Nienstadt 1967) and long daylength stimulated the seedlings to resume growth. The results of the LD/cold, SD/warm, and ambient treatments indicate that both photoperiod and temperature influence accumulation and maintenance of high VSP levels.  In addition to stimulating VSP accumulation, photoperiod is also one environmental cue that promotes the development of dormancy (Kramer and Kozlowski 1979, Lavender 1991) and cold hardiness in some species including white spruce (Silim 1991, Folk et al. unpublished data). Development of dormancy is known to be mediated via phytochrome (Vince-Prue 1984) and may involve changes 98  in the hormonal balance within the plant. Growth promoters including cytokinin decline (Qamaruddin 1990) and inhibitors such as abscisic acid and jasmonic acid increase (Mousdale 1983, Mason et al. 1990, Wareing 1982, Surowy and Bayer 1991) during this period of fall acclimation. Since the seedlings were becoming dormant when VSP first appeared, it is highly probable that hormonal changes stimulate VSP formation and later ultilization. Jasmonic acid has been shown to enhance VSP production in the annual soybean (Surowy and Bayer 1991). Buds and needles of Scots pine decrease cytokinin levels during the winters which increases the porportion of inhibitors (Quamaruddin 1990). Although currently no direct evidence is available that links VSP production in trees to changing porportions of plant growth regulators, future work in this area would be important for further understanding of the control of VSP biosynthesis.  4.2 VSPs Association to Physiological Parameters The results indicate that VSP accumulation occurred during the fall under nursery conditions and in parallel to other physiological changes associated with acclimation. VSP was detected soon after bud formation and accumulated during the same time period in which cold hardiness, dormancy characteristics, and mitotic activity were rapidly changing. These physiological traits as well as VSP content are all part of the fall acclimation process in interior spruce.  VSP was first detectable several weeks after bud formation had been initiated. The formation of overwintering buds is the visual 99  transition point that occurs between the annual phases of growth and dormancy which is referred to as vegetative maturity (Burr et al. 1990). At this point, cold hardiness levels are low; mitotic activity is beginning to fall; the rest phase of dormancy starts and photosystem II activity has begun to decline (Burr et al. 1990, Sakai and Larcher 1987). These physiological characteristics were observed in all three seedlots by early September (EK & PG physiological data, Grossnickle et al., unpublished results).  On sample dates in October, VSP levels increased as cold hardiness was acquired and photosystem II activity continued to decline. During this time, mitotic activity was also declining and the seedlings were proceeding through the rest phase of dormancy (Fig. 10, 12). These trends in cold hardiness and dormancy agree with the typical or expected patterns seen for temperate forest trees (Burr et al. 1990). Increases in total protein levels and apppearances of specific proteins have previously been associated with hardiness in other species (red pine; Pomeroy et al. 1970, apple; Kang and Titus 1987, review; Guy 1990). Prior to this study, the role of VSPs within tree species had not been studied in relation to other physiological changes. Although, the presence of VSP and their accumulation within storage paremchyma cells has been shown to occur in willow, yew, baldcypress, and poplar during the fall and winter months (Berggren 1985, Greenwood et al. 1990, Harms and Sauter 1992, Sauter and vanCleve 1990, Wetzel et al. 1991). The parallel development of VSP accumulation and cold hardiness in spruce supports a role for VSP in fall acclimation.  100  The EK seedlings contained more VSP than the PG and QL seedlings in late September. Since early fall frosts are common in the EK provenance (Stathers 1989) and VSP levels were relatively high, VSP could play a small part in frost resistance. Elevated solute levels within seedling tissues have previously been shown to increase the freezing point depression (Sakai and Larcher 1987, Guy 1990); VSP is one available solute which could contribute to this effect. This idea may be true because EK seedlings were initially more hardy than the other seedlots (Grossnickle et al.,unpublished results). However, these results may only reflect initial seedlot differences and not the actual role of VSP.  Maximum VSP content coincided with the end of the fall acclimation period. At that time, I.I. @ -18°C had reached steady levels of 10% or lower and photosystem II activity was inactive. Two weeks after maximum VSP levels were attained the QL seedlings had fulfilled requirements for dormancy release, had entered quiescence, and then mitotic activity fell below 1%. VSP accumulation paralleled changes in these characteristics especially cold hardiness measured by I.I. @ -18°C and dormancy release. The high correlation coefficients between VSP and I.I. @ -18°C (r = 0.90) emphasized this parallel development. However, maximum cold hardiness levels were not attained until mid-December which was six to seven weeks after the seedlings had become quiescent and maximal VSP was present. Therefore, the achievement of maximal hardiness is not dependent on dormancy characteristics or VSP content.  Interestingly, although VSP appears and accumulates during the 101  same time period as dormancy and hardiness characteristics were changing in the QL, EK, and PG seedlings, results of the fall acclimation experiment indicates that these processes are not directly related. The SD/Warm seedlings never had an LT  50  value  lower than -10°C (Gillies 1993), but contained large amounts of VSP from September throughout December. The presence of VSP may contribute only to the initial stages of cold acclimation (Perry 1971) which allow a low level of hardiness to be reached. Elevated solute levels increase the freezing point depression (Sakai and Larcher 1987, Guy 1990); VSP is one available solute which can contribute to this effect. In addition, this seedling group remained in deep rest because they were not exposed to lower temperatures that are required for dormancy release (Nienstadt 1967). The seedlings under ambient conditions exhibited the same patterns as the QL, EK, and PG seedlings which confirms that normally fall acclimation and VSP occur concurrently. Initially LD/Cold seedlings and ambient seedlings had similar VSP patterns, however; photosystem II activity declined faster and seedlings began to flush after becoming quiescent (Gillies 1993). This flushing caused VSP levels to decline which strongly suggests that VSP are responsive to changes in seedling physiology which are involved in budbreak. This result agrees with an earlier study which demonstrated the utilization of interior spruce VSP during budbreak (Roberts et al. 1991). Even though this data indicates that VSP, cold hardiness, and dormancy characteristics are not interdependent, under natural fall acclimation conditions, cold hardiness, dormancy, and VSP characteristics do change significantly over a similar timeperiod. 102  4.3 VSP Content, Lifting Date and Seedling Quality One of the objectives of this project was to evaluate the possible use of VSP measurement as a predictor of lifting date. Cold hardiness data from seedlot QL indicated that they had met established lifting criteria by October 22nd. This was the same time in which VSP levels reached their maximum and were maintained. A strong correlation (r=0.90) was seen between VSP and cold hardiness measured as I.I. @ -18°C, therefore, VSP could be used as an indirect predictor of cold hardiness which in turn could be used to estimate lifting date. The presence of large amounts of VSP in the nonhardy SD/warm seedlings demonstrated that factors besides VSP accumulation are required for cold hardiness. Thus, VSP accumulation as a predictor of lifting date should always be verified with cold hardiness testing.  When seedlings (seedlot FIN) were subjected to different temperature and photoperiod treatments during fall acclimation their storage protein accumulation and overall physiology including cold hardiness characteristics were altered. Additional information regarding VSP and storage survival of these treatments was available from the thesis of S. Gillies (Summary Table 3). These results gave some insight into relationships between VSP content at lifting and storage survival. The SD/warm conditions limited cold hardiness development (max. LT50 = -10°C) and prevented dormancy release; however, VSP accumulation was very rapid with maximal levels being achieved within a month. Subsequent storage survival and quality was poor. The ambient control treatment as well as the LD/cold samples were both very hardy by November and both had 103  attained high levels of VSP. Post-storage seedling quality was best in ambient (normally) treated seedlings when VSP content was high at lifting, but in contrast seedling quality of the LD/cold seedlings was excellent regardless of lift date. Since high stress resistance has been correlated to cold hardiness and dormancy (Burr 1990), if seedlings achieved maximum cold hardiness early in the season, stress resistance and subsequent quality would be expected to be good. The data also indicated that initiation of VSP was more closely associated with daylength and accumulation rate was influenced by cooler temperatures. Under normal conditions high VSP content at lifting appeared to benefit post-storage seedling quality. However, if seedlings fulfilled chilling requirements earlier under a LD/cold treatment, storage, survival and post-storage quality was not associated with a large amount of VSP. These results support the conclusion that VSP are associated temporally with the acquisition of cold hardiness in the fall but VSP alone do not provide frost protection.  One of the arguments against using frost hardiness is that the test period is long. PSII activity measurements are very quick to do; therefore, they are more responsive and can detect short term changes in the seedlings' photosynthetic system (Gillies 1993). However, PSII activity is not a good lifting date predictor because the main decrease occurs prior to fall acclimation. The degree of change seen during fall acclimation was small. VSP accumulated during the acquisition of cold hardiness. Thus rapid VSP analysis could decrease the cost and time involved in prediction of lifting date. Only a single cold hardiness test would then be required to 104  confirm that the seedlings were hardy.  4.4 Other VSP Characteristics  Although the majority of this thesis centers around VSP's relationship to physiological and endogenous changes, limited investigation into VSP's characteristics was also done. VSP in both poplar and willow are glycosylated (Langheinrich and Tischner 1991, Wetzel and Greenwood 1991b). Since evidence of glycosylation could help with a future protein purification strategy, PAS staining was attempted to test this hypothesis. No glycosylation was seen by this method and the positive control stained weakly which suggests that this technique had low sensitivity. Two dimensional electrophoresis under reduced and nonreduced conditions indicated that the 30 and 27 kD subunits were not attached by disulphide bonds. This result was not surprising because the ratio of 30 to 27 kD proteins varied between seedlings. Lack of intersubunit disulphide bonds has been previously documented for poplar and willow VSP. However, the presence of two 30 kD proteins and two 27 kD proteins exclusively within the winter extract following non-reduced/reduced 2-D analysis gave evidence for the existence of isomers (Figure 19B). Solubility differences between the two forms are possibily caused by intraprotein disulphide bonds. The 30 and 27 kD storage proteins have very similar molecular weights. Poplar VSPs also have this characteristic and their 36 and 32 kD proteins are known to be glycoforms of each other (Langheinrich and Tischner 1991). Although no evidence for glycosylation was seen in this study of interior spruce VSPs by the PAS staining method, the 30 and 27 kD proteins may actually be 105  glycoforms of each other. In future studies, more sensitive methods for detecting glycosylation should be attempted because it may help explain why the molecular weights were similar and the larger 30 kD protein was present in a higher proportion.  4.5 Conclusions Similar levels of VSP were accumulated by bud tissues of four different interior spruce seedlots despite differences with regards to provenances, bud formation, and maximum cold hardiness levels during fall acclimation.  Trends of VSP accumulation were strongly correlated with seasonal changes in cold hardiness (I.I. @ -18°C & LT50 levels) and photosystem II activity. A slightly weaker correlation was noted with dormancy levels and mitotic activity. In addition, the initiation of vegetative storage protein, cold hardiness, and dormancy were induced by short daylength. Declining daylength combined with a lower temperature range induced VSP accumulation at a slower rate and allowed high levels of cold hardiness to be obtained. Thus, under naturally changing environmental conditions, vegetative storage protein levels are correlated to changes in seedling physiology.  Vegetative storage protein accumulation occurs in parallel to the acquisition of cold hardiness which is currently used to estimate lifting date. Since VSP has been shown to be an integral part of typical fall seasonal changes and a strong correlation exists between VSP and hardiness levels, VSP could be used as an 106  indirect predictor of fall cold hardiness patterns. If a rapid and inexpensive VSP assessment method were available, nurseries would only have to conduct cold hardiness tests when seedlings contain near maximal amounts of VSP (15-18% of total protein). Seedlings should not be stored based on VSP data alone because long day cold treatment results suggested that hardiness is the primary factor involved in maintaining quality during long term frozen storage of stock.  In conclusion, the results of my study indicate that VSP measurement could provide a tool for indirectly monitoring physiological changes within seedlings during fall acclimation. Even though, the primary role of VSP is probably nitrogen and carbon storage, the correlation between VSP and cold hardiness is useful. Nurseries could use VSP as an indirect predictor of cold hardiness levels and lifting date. Once an assay kit is developed, its application may help save money and time. Alternately, VSP could be used as a biochemical marker in developmental and physiological studies. Regardless, future VSP studies both applied and academic could reveal the inductive mechanisms and roles of VSP within overwintering seedlings.  107  5.0 LITERATURE CITED  Anderson, J. M., S. R. Spilatro, S. F. Klauer, and V. R. Franceschi. 1989. Jasmonic Acid-dependent Increase in the Level of Vegetative Storage Proteins in Soybean. Plant Science. 62:45-62. Arora, R., M. E. Wisniewski, and R. Scorza. 1992. 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