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The effect of cold storage duration and soil temperature on the photosynthetic ability of Picea glauca… Harper , George James 1990

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THE EFFECT OF COLD STORAGE DURATION AND SOIL TEMPERATURE ON THE PHOTOSYNTHETIC ABILITY OF PICEA GLAUCA SEEDLINGS by GEORGE JAMES HARPER B.Sc. Agr., The University of British Columbia, 1982 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF FOREST SCIENCES We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1990 @ George James Harper, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT In response to concern over the failure and poor growth of many interior and white spruce plantations in British Columbia the effect of storage duration and soil temperature on the photosynthetic ability of white spruce seedlings was explored. Seedlings of Picea glauca were dark freezer stored (-5oC) from 9.6 to 30.6 weeks, thawed and grown for 28 days in a growth chamber at three different soil temperatures (3,7,11°C). During this period gas exchange variables and chlorophyll fluorescence kinetics were followed. Seedlings stored for periods of 22 weeks or longer had significantly lower rates of photosynthesis dependent on the outplanting soil temperature. Stomatal conductance was initially low upon outplanting and showed a recovery period of 4-7 days duration. The level of stomatal conductance increased in seedlings after they were stored for 26.1 weeks or longer. Chlorophyll fluorescence measurements of seedlings stored from 22 to 30.6 weeks showed a recovery period in photosynthetic efficiency (Fy/Fp) related to changes in photosynthesis. A decrease in seedling Fy/Fp with increasing periods of storage was noted at day 5 after outplanting. A disproportionate increase in new root growth with the increasing soil temperatures, measured after the 28 day growth period, suggested a soil temperature threshold for root growth exists between the 7oC and lloC. In contrast, the stomatal conductance and photosynthesis results suggest the seedling shoots were not directly affected by the cold soil temperatures. In general, the results suggest Picea glauca seedlings stored longer than 22 weeks in freezer conditions have reduced photosynthetic ability, root growth and overall vigor. Fluorescence and bud break data suggest the reduction was possibly due to freezing damage sustained in storage affecting photosynthetic electron transport through photoinhibition upon returning seedlings to the light. ii T A B L E OF CONTENTS Abstract u Table of Contents List of Tables . v List of Figures ^ Abbreviations v111 Acknowledgement 1. Introduction 1 1.1. Cold Storage 1 1.2. Soil Temperature 6 1.3. Research Proposal 7 Hypothesis 8 Null Hypothesis 8 2. Material and Methods -9 2.1. Plant material and growth conditions 9 2.2. Measurements 13 2.2.1. Gas Exchange 13 2.2.1.1. Leaf Area 16 2.2.2. Fluorescence Induction Kinetics 17 2.2.3. Root Growth 18 2.3. Statistics 18 2.3.1. Experimental Design 18 2.3.2. Analysis 19 3. Stomatal Conductance 22 3.1. Results 22 3.1.1. Soil Temperature 22 3.1.2. Storage Duration 25 3.1.3. Interactions -28 3.2. Discussion 29 3.2.1. Days 29 3.2.2. Soil Temperature 30 3.2.3. Storage Duration 35 4. Net Photosynthesis 37 4.1. Results -37 4.1.1. Soil Temperature 37 4.1.2. Storage Duration .40 4.1.3. Interactions 43 4.1.4. Days 46 4.2. Discussion 48 4.2.1. Soil Temperature 48 4.2.2. Storage Duration and Interactions 51 5. Internal C02 / Ambient C02 Ratio 56 5.1. Introduction 56 iii Table of Contents 5.2. Results 5 7 5.3. Discussion 65 6. Photosynthetic Efficiency 7 0 6.1. Introduction 7 0 6.2. Results 7 2 6.3. Discussion 7 9 7. Seedling Morphology 86 7.1. Seedling Growth • 8 6 7.2. Terminal Bud Dormancy 8 9 7.3. Discussion 91 8. Conclusion 92 8.1. Hypothesis Test Results 9 2 8.3. Conclusions 9*> Bibliography 9 8 Appendix 1 1 0 5 Appendix 2 1 1 3 iv LIST OF TABLES Table 2.1 Ministry Lifting Standards (1+0, PSB 313A) 1989 v LIST OF FIGURES Figure 2.1 White spruce seedlings (1+0) PSB 313A after removal from the styroblocks and prior to packaging for freezer storage 10 Figure 2.2 A view of the pot arrangement in one of the water bath treatments. Thermometers were for monitoring soil temperature 12 Figure 2.3 LI-COR 6200 gas analyzer system used during gas exchange measurements. . 15 Figure 3.1 Stomatal conductance (cm s-l) changes with increasing storage duration (9.6 to 30.6 weeks) over 28 days growth at 3,7, and lloC soil temperature, n=40 23 Figure 3.2 Stomatal conductance (cm s-1) changes over 28 days growth at 3,7, and lloC soil temperature after varying storage durations (weeks), n=40 26 Figure 3.3 New root growth >10 mm after 28 days at 3,7, lloC soil temperature, and freezer storage to 30.6 weeks. Error bars are 1 SEM, n=40. (Harper et al. 1989).... 33 Figure 4.1 Net photosynthesis (umol nr 2 s-l) changes over 28 days growth at 3,7, lloC soil temperature after various storage durations, n=40 38 Figure 4.2 Net photosynthesis (umol nr 2 s-l) changes with increasing storage duration (9.6 to 30.6 weeks) over 28 days growth at 3,7, and lloC soil temperature, n=40 41 Figure 4.3 Response surface relating changes in net photosynthesis to storage duration and outplanting period at the three soil temperature treatments (3,7, and 11°C) .44 Figure 4.4 Net photosynthesis (umol nr 2 s-l) changes with increasing storage durations (9.6 to 30.6 weeks) at day 28 after outplanting and at 3,7, and lloC soil temperatures. Solid lines represent values based on first year needle area 47 Figure 4.5 Cumulative net photosynthesis values, day 1 to day 19, for 3,7, and lloC soil temperatures after varying storage durations 55 Figure 5.1 Internal stomatal CO2 / external CO2 ratio (Ci/Ca) changes over 28 days growth at 3,7, and lloC soil temperature after varying storage durations, n=40 58 Figure 5.2 Internal stomatal CO2 / external CO2 ratio (Ci/Ca) changes with increasing storage duration (9.6 to 30.6 weeks) over 28 days at 3,7, and HOC soil temperature, n=40 61 Figure 5.3 Response surface relating changes in internal stomatal C02 / external C02 ratio (Ci/Ca) to storage duration and outplanting period at the three soil temperature treatments (3,7, and 11<>C) 63 Figure 6.1 Fluorescence induction curve showing characteristic OIDPSMT transients. . . 71 v i List of Figures Figure 6.2 Changes in Fv/Fp ratio over the 28 day growth period when planted at 3,7, and HOC soil temperature after 22, 26.1 and 30.6 weeks storage, n=12. Error bars are 1 SEM 73 Figure 6.3 Day 5 changes in Fv/Fp ratio over increasing storage duration (22 to 30.6 weeks), n=12. Error bars are 1 SEM 76 Figure 6.4 Relationship between net photosynthesis and photosynthetic efficiency in white spruce seedlings stored for 26.1 weeks and outplanted. Measurements were made from 2-16 days after outplanting .77 Figure 6.5 Changes in net photosynthesis and photosynthetic efficiency for similar days (day 4 and 5, day 27 and 28) and storage durations at 3,7, and 11<>C soil temperatures, net photosynthesis, n=40. Fv/Fp ratio, n=12 78 Figure 7.1 Dry weight values for various storage duration treatments after the 28 day growth period at 3,7, and HOC soil temperatures. (A) Shoot/root ratio, (B) shoot dry weight, (C) root dry weight 87 Figure 7.2 Change in, (A) days to terminal bud break (TBB) and, (B) height increment (cm), for the various storage duration treatments after the 28 day growth period at 3,7, and HOC soil temperatures, n=40 90 vii ABBREVIATIONS A Net photosynthesis ANOVA Analysis of variance ATP Adenosine triphosphate oC degrees Celsius Ca Ambient carbon dioxide concentration Ci Intercellular carbon dioxide concentration C0 2 carbon dioxide d.f. degrees of freedom E Transpiration Fv Variable fluorescence FP Peak fluorescence gc conductance to CO2 diffusion gs Stomatal conductance (water vapour) mb millibar n sample size NADP Nicotinamide adenine dinucleotide phosphate ppm parts per million P probability RGP Root growth potential SEM Standard error of the mean wks. weeks viii ACKNOWLEDGEMENT Financial support for this study was provided by the Science Council of British Columbia. Seedlings and storage facilities were provided by the B.C. Ministry of Forests. In particular, I wish to thank Jim Sweeten, Tony Willington and Ruth Sanragret for their technical support. I also wish to thank Dr. C. Chanway for his thoughts concerning statistical analysis, Salim Silim and Dave Simpson for their helpful discussions on seedling development. Finally, many thanks to Dr. E. Camm for her guidance and enthusiastic support throughout the entire project. ix 1. INTRODUCTION Reforestation in British Columbia has increasingly come to rely on efficient mass production of container and bareroot stock. The demand for over 250 million seedlings a year, fueled by efforts to restock backlog NSR (not sufficiently restocked) areas, has caused rapid expansion of the nursery industry. Over half of the conifer seedlings produced are interior spruce (Picea glauca engelmannii complex), and white spruce (Picea glauca (Moench.) Voss.). The failure of large plantations of white spruce in B.C. (Butt 1986) has questioned our ability to produce healthy successful seedlings. Interior and northern NSR areas have been the greatest concern. The poor growth of planted interior and white spruce has reduced the ability of plantations to compete with non-crop vegetation and ultimately this affects establishment and survival. 1.1. COLD STORAGE Seedlings are produced in southern latitudes and lower elevations for planting in more northerly, high elevation sites. This with the extension of the planting season has led to widespread storage of planting stock. The simple logistics of planting several hundred million seedlings demands some kind of storage. As a result seedlings are frozen stored for 4 to 8 months, a process that is not without problems. Normally in B.C., seedlings are placed in cold storage after they have attained a minimum cold hardiness and stress resistance. The storage regimes currently in use have evolved from considerable research on storage temperature, mold prevention, and length of storage (for review see Hocking and Nyland 1971). Guidelines have been developed for maintaining proper storage conditions to ensure seedlings are healthy and vigorous upon planting. Recent research has been concerned with more extensive studies on the 1 1. Introduction mechanisms involved in such variables as dormancy, root growth, carbohydrate reserves, and photosynthesis. It is becoming more and more apparent that in order to further understand the effects of storage, and conifer physiology in general, the interactions between single variables must be considered along with the effects of various planting site conditions. The ability of a seedling to become successfully established once planted is dependent on acclimation to its new environment, the production of new roots, and the resumption of photosynthesis. In the absence of light, stored seedlings are no longer able to photosynthesize and rely on stored carbohydrate reserves to maintain respiration. After storage and planting, if carbohydrate levels are low seedlings may suffer from lack of energy necessary to overcome planting shock and produce vigorous growth. The depletion of carbohydrate reserves has been noted in Pseudotsuga menziesii by Ritchie (1982), in Pinus ponderosa (Hellmers 1962), and in Picea engelmannii (Ronco 1973). Poor seedling performance has been attributed to depleted carbohydrate levels (Ritchie 1982, Ronco 1973). Pseudotsuga menziesii seedlings with low carbohydrate levels may suffer from reduced drought tolerance due to their inability to adjust osmotically and retain turgor (Ritchie 1982). It has also been suggested that seedlings unable to develop sufficient carbohydrate status will suffer high mortality because they are unable to develop necessary cold hardiness. A decline in cold hardiness during storage has been shown in Pinus contorta and Picea glauca engelmannii complex (Ritchie et al. 1985), which may be due to the depletion of carbohydrate required in the hardening process (Ritchie 1982). The effect of storage carbohydrate depletion will ultimately depend on the severity of the planting site and the potential stresses incurred. Dormancy has also been shown to be affected by storage. The placement of seedlings in dark, frozen storage removes them from normal environmental influences such as 2 1. Introduction fluctuating light and temperature. Cold storage temperatures below the optimum for normal dormancy release, and the lack of winter warm periods and a daily photoperiod, may slow dormancy release (Ritchie et al. 1985). In Pseudotsuga menziesii loss of dormancy was found to be much slower after storage than when influenced by normal winter conditions (Ritchie 1984). It has been suggested that in interior spruce, root growth potential (RGP) is related to loss of dormancy. Root growth potential was found to be high at storage durations where dormancy release was full (Ritchie et al. 1985). Additional storage reduced RGP possibly due to increased bud metabolic activity. Root growth potential is considered a good indicator of seedling performance as it integrates several important physiological processes, it also is clearly affected by cold storage. In Pseudotsuga menzeisii, RGP remained relatively constant until an increase at 6 months and a significant reduction at longer durations (Ritchie 1982). Root growth capacity of stored Thuja plicata seedlings showed increased root activity at two months storage and a general decrease thereafter with increasing storage durations up to 6 months (Hale et al. 1989). It was also found that larger, initial stock size increased the percent survival with long term storage (5-6 months). The influence of storage duration on root growth may be related to the effects of storage on bud break and cold hardiness. The period of maximum root growth may be determined by the period between loss of cold hardiness and the onset of bud break, noted in Pseudotsuga menzeisii and Picea engelmannii (Burr et al. 1989). The availability of photosynthate during this period would also determine new root growth. It has been found that new root growth in Picea sitchensis is partly, and in Pseudotsuga menzeisii, entirely dependent on carbohydrate from the shoots (Philipson 1988). Van Den Driessche (1987) showed, through the use of labelled 14C02, that in Pseudotsuga menziesii photosynthate was the primary source of carbon for new roots. Thus, it can be 3 1. Introduction seen that effects of storage on the processes of photosynthesis, bud dormancy, cold hardiness and the level of carbohydrate reserves are interrelated making simple interpretation of storage effects confusing without consideration of all factors and their interactions. Photosynthesis does not occur in dark, freezer storage. The effects of long term dark periods on the photosynthetic apparatus are largely unknown, although it is known that light is required for chlorophyll synthesis and chloroplast organization (Salisbury and Ross 1985). Photosynthetic decline has been noted in Pinus ponderosa and coastal Pseudotsuga menziesii after subfreezing temperatures (Pharis et al. 1970). The rate of recovery and also the amount of depression was dependent on the rate of temperature ascent. Recovery of photosynthesis varied from several days to several weeks dependent on the subfreezing temperature. It was found Pinus ponderosa could withstand lower temperatures than coastal Pseudotsuga menziesii. The effect of subfreezing temperatures on photosynthesis may be similar to winter photosynthetic depression noted in many conifers. Decline in carbon fixation has also been noted after periods of cold storage. Mattsson and Troeng (1986) working with Pinus sylvestris noted no difference in the photosynthetic recovery period of seedlings stored for 81 and 170 days. They found recovery was faster with seedlings that had been stored at 2 degrees Celsius (oC) rather than at -4oC due to the increased recovery of stomatal conductance, suggesting a stomatal limitation to carbon dioxide (CO2) gas exchange. Also the recovery period of frozen seedlings was much greater than for adequately thawed stock. Grossnickle and Blake (1985) also noted a poor stomatal response after storage and suggested that cold storage of Pinus banksiana and Picea glauca disrupted the normal stomatal light response mechanism. An increased depression of photosynthesis with increasing periods of storage was found in seedlings of Pinus mugo and Pinus radiata (McCracken 1978). At storage of 18 weeks, carbon fixation was reduced to 4 1. Introduction 40% of unstored seedlings. Water potential measurements were generally normal suggesting stomatal limitation to CO2 exchange was minor. McCracken (1978) noted that the persistent darkness and low temperature of storage may result in disruption of the CO2 fixation mechanism leading to an extended readaptation period when the seedlings are reexposed to light. It is unclear as to the mechanism(s) behind storage related decline of photosynthesis. However, effects on the photosynthetic apparatus may be related to changes in light conditions leading to photosynthetic decline through photoinhibition. Photoinhibition is caused when light saturating levels for the photosynthetic apparatus are exceeded by ambient light levels. Energy absorption by the chlorophyll that exceeds levels that can normally be dissipated, either the result of high intensity light or long term moderate light levels, may result in damage to the photosystem components. Under favourable growing conditions, rate of damage repair exceeds that of damage, but when rate of damage exceeds rate of repair, photoinhibition occurs and photosynthetic decline results (Powles 1984). If photoinhibition is severe it may lead to permanent damage through the oxidation of the chlorophyll pigments (photooxidation). Since photosynthesis is the major pathway for energy dissipation, conditions which reduce photosynthesis directly or indirectly such as low air temperature, drought stress, or frost will increase the probability of photoinhibition. In terms of the developing conifer seedling, the rapid establishment of photosynthetic capacity after storage is necessary to avoid photoinhibitory decline and produce the carbohydrate required for root growth and general maintenance of seedling vigor. Changes to the photosynthetic apparatus have been shown to occur in conifers exposed to winter temperatures. In Pinus sylvestris seedlings subjected to winter freezing conditions, inhibition of photosynthetic electron transport between photosystem II (PSII) and 5 1. Introduction photosystem I (PSI) possibly at the plastoquinone site was found (Oquist and Martin 1980). No inhibition was found of electron transport between cytochrome / and P700 (PSI reaction center chlorophyll). Lundmark et al. (1988) working with Pinus sylvestris, Pinus contorta, and Picea abies seedlings found photoinhibition damage during winter conditions (subfreezing temperatures and frozen soils) and evidence of recovery during spring growth. Recovery was temperature dependent and took up to several months before PSII activity was completely restored. Lundmark and Hallgren (1987) proposed high irradiances (clear days) following night frosts will induce photoinhibition and possibly photooxidation in planted seedlings. The decline in photosynthesis observed after cold storage (McCracken 1978) may have been due to disruption of the electron transport system similar to that noted after night frosts. Further research is required to develop a better understanding of the processes involved in photosynthetic decline noted after subfreezing temperatures. Not only would this provide information on the potential problems associated with cold storage, but it may also provide insights into long term seedling development and shade tolerance. 1.2. SOIL TEMPERATURE Soil temperature can also affect root development and ultimately seedling establishment. Scott et al. (1987) noted that annual height increment in tundra white spruce was related to the root zone soil temperature regime during the growing season. Growth form was largely due to ground temperatures limiting height increment and the length of the growing season. Husted and Lavender (1989) found cold soil temperatures (3°C) severely reduced new root growth in comparison to warm soil growth (17°C) in white spruce. Planting during the spring, at high elevations, and in soils covered with a thick 6 1. Introduction organic layer would certainly see cold soil temperatures for most of the early growing season and as such seedling establishment may be restricted by insufficient root growth. A relationship between root permeability and soil temperature has been suggested for some time based on temperature changes in water viscosity and species variation in soil temperature adaptation (Kramer 1942). In conifers, reduction in photosynthesis, stomatal conductance, and transpiration have been noted at low root temperatures (Babalola et al. (1968), Kaufmann (1975), Turner and Jarvis (1975), Running and Reid (1980), DeLucia (1986)). Differences in nonconiferous temperate species root and shoot temperature response have been noted which support a hypothesis of species cold soil tolerance based on optimal ecophysiological conditions (Anderson and McNaughton (1973), Lawrence and Oechel (1983)). 1.3. RESEARCH PROPOSAL Reforestation success depends on vigorous, healthy seedlings which are able to tolerate and grow under the stresses and limitations of their new environment. Site conditions ultimately determine plantation performance, therefore it is important to plant seedlings that are well suited to those conditions. The present system of reforestation in B.C. requires planting of later successional species, such as white spruce, in open logged areas which are subject to extremes in temperature and light. These species naturally regenerate in the understory and in small openings where the effects of shelter temper microclimate variation and provide a more tolerable light exposure. Planting seedlings in an environment more hostile than that experienced during their initial and determinant year of growth may predispose them to possible photoinhibitory damage. From the literature, it is evident physiological changes are occurring during storage which may affect seedling root 7 1. Introduction growth and photosynthetic capacity. These imposed stresses may exacerbate seedling efforts to successfully establish in its new environment. In December of 1988, a grant was obtained from the B.C. Science Council to study the effects of storage duration and outplanting soil temperature on white spruce seedlings. For the purpose of this thesis, the process of carbon fixation was of main interest, although root growth was also of concern. Through the interpretation of gas exchange measurements and chlorophyll fluorescence characteristics the effect of storage on photosynthesis was determined. Other morphological and physiological characteristics were measured in an attempt to develop a fuller understanding of the processes involved. The following set of hypotheses were developed to be either accepted or rejected. Hypothesis. The increasing duration of storage will produce a decline in net photosynthesis which will be similarly observed in photosynthetic efficiency (fluorescence characteristics). This effect will be compounded by cold soil conditions. Null Hypothesis. 1). The length of storage will have no effect on a) net photosynthesis and b) photosynthetic efficiency. 2). Root zone soil temperature will have no effect on a) net photosynthesis and b) photosynthetic efficiency. 8 2. MATERIAL AND METHODS 2.1. PLANT MATERIAL AND GROWTH CONDITIONS White spruce (Picea glauca (Moench.) Voss.) seedlings, seedlot number 8977 (Prince George, Ft. St. John wild stand collection), one year old were grown in polystyrofoam blocks (1+0, PSB 313A) by the B.C. Forest Service at their nursery located in Surrey, B.C. Ten blocks containing approximately 1980 seedlings were randomly selected from the seedlot for this project. Cold hardiness of the seedlot was monitored by nursery personnel to determine the appropriate lifting period. On December 14, 1989 the seedlings were lifted, and culled by the regular lifting crew at the nursery following ministry lifting specifications for white spruce (see Table 2.1 and Figure 2.1). After culling, seedlings were root wrapped in plastic-wrap, in bundles of 20, and placed upright in paper bag lined, wax cardboard boxes. From the initial number of seedlings approximately 43% were culled most of which were under height and some below caliper. The final number was placed randomly into 7 boxes for storage with 8 bundles per box including a cardboard retainer to secure the bundles in an upright position. One box was then selected for the zero storage trial and the rest were placed in freezer storage at the nursery. Storage temperature was maintained at -5 oC with seedling temperatures inside the boxes at -20C. Individual boxes were then removed at approximately one month intervals starting at 9.6 weeks. In keeping with the original objective to replicate operational production, lifting, and storage conditions, the boxes of seedlings were thawed for 7 days in a cool shaded area prior to shipment and planting. 9 2 . Material and Methods Figure 2.1 White spruce seedlings (1+0) PSB 313A after removal from the styroblocks and prior to packaging for freezer storage. 10 2. Material and Methods Table 2.1 Ministry Lifting Standards (1+0, PSB 313A) 1989 Cull Target Maximum Height (cm) Root Collar Diameter (mm) Root Dry Weight (g) 12 2.4 0.5 17 3.0 0.7 25 At 0, 9.6, 13.7, 17.9, 22, 26.1, and 30.6 weeks after storage and thawing, seedlings were transported to the University of British Columbia Forestry Nursery for growth chamber tests. For each storage trial, seedlings were randomly selected and planted 4 per pot in a 2:1 mixture of peat:perlite with 200 grams of dolomite to adjust mix to neutral pH (approximately pH of 4.5 prior to dolomite addition). The pots consisted of large plastic 4 liter wide mouth commercial food processing jars in which the tops had been removed leaving a water tight pot of approximately 14.5 cm diameter, 17 cm in height. Each was filled with 1.3 kg. of Forestry sand (Target Products Ltd., Burnaby, B.C.) covering the bottom 3.5 cm providing drainage and weight to maintain slightly negative buoyancy when the potting mixture was at field capacity and placed in the water bath temperature treatments. Peat:perlite mixture was wetted prior to potting to ensure even moisture content of the peat. Immediately after potting the pots were randomly placed in one of the three water baths located in the walk-in growth chamber. Ten pots were placed in each bath, evenly spaced and surrounded by a 3/4 inch styrofoam cover to minimize heat transfer to and from air above the pots (see Figure 2.2). The temperature controls of the water baths had previously been set to either 3, 7, or 11 degrees and the water temperature was continuously 11 2. Material and Methods Figure 2.2 A view of the pot arrangement in one of the water bath treatments. Thermometers were for monitoring soil temperature. 12 2. Material and Methods monitored over the entire project. Individual water bath settings were randomly selected prior to each storage trial. The growth chamber environment was as follows; day and night temperature of 11 degrees, relative humidity 80%, photoperiod 16 hours, and photosynthetic photon flux density of approximately 480 pmol m - 2 s-l from cool white fluorescent lights and 40 watt incandescent bulbs measured with a LI-COR Li-190S-l quantum sensor at seedling terminal bud height. Lights were ramped to provide a half hour of dawn and dusk conditions (only the incandescent source). Seedlings were grown for 28 days during which the pots were watered to maintain soil moisture levels at or near field capacity. 2.2. MEASUREMENTS For each storage trial seedlings were grown a total of 28 days during which gas exchange variables were followed every 3 days. Days to bud break, and height growth were also recorded. At day 28 seedlings were removed from their pots and new root growth measured. Seedlings were then stored in the freezer for later analysis of shoot and root dry weights, and individual seedling first and second year needle areas. 2.2.1. Gas Exchange Net photosynthesis, stomatal conductance, transpiration, internal stomatal and external CO2 concentrations were determined with the use of a LI-COR 6200 CO2 gas analyzer (see Figure 2.3). The LI-COR 6200 is composed of an infra-red CO2 analyzer, a microcomputer console, and leaf chamber housing containing sensors and fans. The system's microcomputer provides a rapid and accurate determination of gas exchange parameters, 13 2. Material and Methods and continuous monitoring of chamber conditions. A one liter chamber (LI-COR 6000-12) was used throughout the project. For each seedling, the top 3.7 cm of stem, buds and needles, approximately 2.0 cm below the terminal bud, was enclosed in the chamber for gas exchange measurements. A closed pore foam gasket sealed the chamber, Internal fans mixed the air and maintained a low boundary layer resistance around the needles. Leaf or needle temperature was determined with a chromel-constantan thermocouple, light intensity with a LI-COR Li-190S-l quantum sensor, and relative humidity with a Vaisala HUMICAP sensor. Calculations of net photosynthesis, stomatal conductance, internal CO2 concentration and transpiration follow those of Caemmerer and Farquhar (1981) with corrections for closed system design (Leuning and Sands 1987) (see Appendix 2). Over the entire period of the project, some 9000 individual gas exchange measurements were made. For any particular measurement day an efficient routine was necessary to allow all seedlings to be sampled and to maintain consistent measurement conditions within a minimum time span. Measurements were made in the growth chamber between 3 and 8 hours after dawn. Each pot was removed from its water bath and each seedling was subject to a saturating light intensity of 680 +/- 20 umol nr 2 s-l from a projector light source. The LI-COR chamber was placed around the upper stem of the seedling, and CO2 level monitored for 10 to 15 seconds before logging of gas exchange parameters commenced. Depending on the rate of carbon dioxide reduction, individual readings continued for another 25 to 45 seconds. Concern over maintaining relatively consistent ambient CO2 levels within the growth chamber while measuring gas exchange (operator respiration elevated ambient CO2 levels) led to maintaining the chamber door open approximately half a meter to enhance circulation. Care was also taken not to exhale near the LI-COR chamber. 14 2. Material and Methods Figure 2.3 LI-COR 6200 gas analyzer system used during gas exchange measurements. 15 2. Material and Methods Individual seedlings were identified by means of pot location within the water baths and by way of markings on each pot. In this way, individual performance over the growth period could be followed and leaf area correction could be made at a later date. To maintain this identification process and minimize confusion each individual (within a water bath) was sampled consecutively and water bath sampling was randomized every measurement day. Data were stored in the internal memory of the LI-COR 6200 microcomputer and transferred directly into ASCII file format for storage and analysis. 2.2.1.1. Leaf Area Initial leaf area (for use in gas exchange calculations) was estimated from a small sample of 15 randomly selected individuals. A 5 cm section of stem corresponding to the measurement region was removed and the needle projected area determined with the use of a LI-COR 3000 leaf area meter. Projected leaf area was then multiplied by a leaf factor of 2.31 to account for general spruce needle morphology. Leaf factor determination was based on needle crossectional shape approximating a four sided diamond pattern with angles of 60 and 120 degrees. Total needle area can then be approximated assuming needles are homogenous in shape and they are measured while lying flat and not on edge. This was not always the case and as a result error exists in obtaining an accurate needle area determination. After the 28 day growth period, projected needle area (first year needles) for each individual was determined (with the LI-COR 3000 leaf area meter) and gas exchange 16 2. Material and Methods parameters corrected accordingly. All results presented here, unless otherwise stated, reflect this correction for individual leaf area. The development of new foliage during the growth period should also be taken into consideration if gas exchange variables are to be considered on a total leaf area basis. However, as stated, measurements reflect the old (first year) leaf area only. Buds were not removed to avoid this inaccuracy in leaf area computation. It was felt that the potential stress and loss of hormones from removal of the developing buds and the loss of nutrient sinks, would not be representative of commercially planted seedlings and that interactions between soil and storage treatments and bud removal could not be ruled out. The contribution of the buds and subsequently the new foliage at the specific measurement dates should be determined if total leaf area results are required. This was not done as it was felt to be outside the scope of this project, and since consistency was maintained over the different treatments, comparisons can be made. As a result, after approximately day 19, increases in transpiration rate, and carbon fixation were noted at each storage duration with increasing days after outplanting. During the first week after bud break at some of the earlier storage durations, modelling putty was used to encase the buds to determine their contribution to gas exchange. This method was discontinued as it was found to be damaging to the developing foliage, time consuming, and blocked the infiltration of light. Other methods were tried with little success. 2.2.2. Fluorescence Induction Kinetics Measurements of fluorescence induction kinetics of the first year needles were made with a pulse modulated fluorometer, (PAM 101,102,103; H. Walz FRG) as described by Schrieber et al. (1986). Seedlings were dark adapted 15-20 minutes prior to fluorescence 17 2. Material and Methods induction. All measurements were taken from mature one year old needles. Initial fluorescence level was obtained by using a weak light intensity low enough not to induce induction transients. A continuous actinic light intensity of 65 umol nr 2 s-l was used to incite induction transients. Calculation of fluorescence parameters followed that of Schreiber et al. (1986). 2.2.3. Root Growth At the end of each growth trial period, seedlings were removed from their pots and the roots washed. The number of new roots in three size classes were counted; 1) roots less than 5 mm, 2) roots 5-10 mm, 3) roots greater than 10 mm Shoots were severed from the roots at the root collar and dry weights were measured after approximately 72 hours at 71°C. 2.3. STATISTICS 2.3.1. Experimental Design The design is completely randomized with a 3 by 7 factorial arrangement (3 soil temperatures by 7 storage durations). Soil temperatures consisted of three water bath treatments of 3, 7 and 11 degrees and storage durations were 0, 9.6, 13.7, 17.9, 22, 26.1, and 30.6 weeks. Seedlings were potted, 4 per pot, and 10 pots per water bath with the soil temperature treatments being randomly selected for each water bath at the start of each new storage growth chamber trial. The same three water baths were used for the entire project which may be viewed as pseudoreplication (Hurlbert 1984). Pseudoreplication in the context of this study refers to the testing for soil treatment effects without true replication of the 18 2. Material and Methods treatments. That is, since each soil temperature treatment was conducted with only one water bath for the entire growth period, there was no real replication of the soil temperature treatments. Replication of soil treatments would have required each replicate to be a separate water bath. It was felt that this situation although an example of pseudoreplication would not significantly influence the results. 2.3.2. Analysis Statistical analysis involved analysis of variance and Tukey-Kramer means test with significance level of p<0.05. Originally, analysis was to consider all treatments for each day of measurement (day 1 to day 28). In the final analysis, the zero storage treatment was dropped from gas exchange data analysis. Inconsistent measurement techniques during the zero storage duration growth period led to an overall poor confidence in that period's data. As a result, the analysis was concerned with a 3 by 6 factorial arrangement and in some cases, notably day 4, day 13, and day 25 with a 3 by 5 factorial arrangement due to missing data at 26.1, 22, and 26.1 weeks storage respectively. Two major concerns were taken into account during the final analysis. The first deals with one of the assumptions of analysis of variance, in particular the assumption of independent observations. The concern involves testing for differences between means of different days within a storage duration. Not only would the assumption of independence be violated if time series data were to be used, but the additional factor (3 by 6 by 10 factorial arrangement) would unnecessarily complicate analysis and interpretation of results. To maintain strict adherence to the assumptions of analysis of variance, differences between days were not tested. 19 2. Material and Methods The second point of concern involves the discussion of experimental and sampling error. Initially, the experiment had been designed with the objective to test whether sampling error (estimate of variation within and between groups or pots) was significantly different than experimental error (estimate of random seedling variation). A significant F-test (ratio of mean-square experimental error to mean-square sampling error) would suggest the variation between individuals was different than that associated within and between pots. If not significant then a new error term, mean-square error, a combination of the two would be used increasing degrees of freedom and precision. However, strong statistical argument continues to rage over the use of preliminary tests of significance in the analysis of incompletely specified models such as presented here (Bancroft 1964). Concern exists over combining or pooling mean squares and artificially inflating degrees of freedom affecting the critical F value. It appears a concise agreement on pooling of mean squares is not forthcoming although this technique continues to be used and is acceptable and commonplace in some fields of study. Sokal and Rohlf (1981) discuss pooling of mean squares based on work by Bancroft (1964) in which a further discussion of preliminary tests of significance can be found and a conservative method of pooling mean squares is given. In this experiment, it can be argued that changes in error degrees of freedom (DF) will have little affect on F critical. In the case of an increase in experimental error DF when the DF is already large, the change in F critical for a given a will be small. A similar increase from small experimental error DF will noticeably reduce the F critical value. Experimental error DF in this experiment is large, normally n>130. Pooling error terms and DF (n>400) has little affect on the interpretation of the analysis of variance results. In consideration of the controversy over when and when not to combine variation estimates, the conservative approach was taken. Statistical analysis proceeded without 20 2. Material and Methods pooling experimental and sampling error mean squares or their DF. Tukey-Kramer means test was done on a per pot, experimental unit basis, with n=10. Standard deviation as represented in the Appendix 1 are calculated on a per individual basis with a n=40. In proceeding with a 2 way repeated measures analysis of variance test for each of the 10 measurement days, several general steps were followed using the computer statistics program Systat. 1. Bartlett's test for homogeneous group variances. If variances were heterogeneous then, if possible, outlyers greater than 3 standard deviations from the mean were removed (as determined by Systat). In most cases this was enough to create homogeneity. Several days (day 7,10,13,25,28 for net photosynthesis and day 4, and 7 for stomatal conductance) required transformations to obtain homogeneous variances. Net photosynthesis data could only be successfully transformed with the cosine function, stomatal conductance with the sine function. All the usual transformations were tried before using these unlikely candidates. Any outlyers that may have been removed previous were replaced prior to transformations. 2. Once homogeneity had been established between groups, analysis of variance was conducted (for results see appendix). 21 3. STOMATAL CONDUCTANCE 3.1. RESULTS Conifer seedlings, like all other plants, are directly affected by changes in their environment. The stomata provide a means for the plant to control transpirational water loss and leaf temperature. Carbon fixation may be indirectly affected by changes in internal mesophyll CO2 levels at levels of stomatal conductance which restrict water vapour and CO2 movement into and out of the leaf. Stomata allow the plant to respond to changes in the environment to optimize photosynthesis and avoid or reduce drought stress. Observations of stomatal response can provide basic physiological information as well as a means to study the effects of environmental change. In this study, stomatal conductance along with net photosynthesis and internal mesophyll CO2 were followed in white spruce seedlings with the LI-COR 6200. Calculation of these individual gas exchange variables follow those of Caemmerer and Farquhar (1981) (see Appendix 2). The following results were obtained and are discussed in terms of the two treatment factors and interactions. 3.1.1. Soil Temperature In general, soil temperature (3-lloC) had little or no effect on seedling stomatal conductance. Very few significant soil temperature differences (probability (p)<0.05) were found. Even at p<0.10 the number of significant differences between soil treatments did not drastically change. However, considering specific storage durations, there was a major significant stomatal conductance increase with increasing soil temperature at 13.7 weeks on day 1, 4 (p=0.0611), 10, 13 (p=0.0739), and day 28 (see Figure 3.1). As a result, in terms of 22 3 Stomatal Conductance Figure 3.1 Stomatal conductance (cm s-i) changes with increasing storage duration (9.6 to 30.6 weeks) over 28 days growth at 3,7, and HOC soil temperature, n=40. 23 3 Stomatal Conductance d a y s a f t e r o u t p l a n t i n g 24 3 Stomatal Conductance stomatal conductance, it appears the 13.7 weeks storage duration seedlings were the most sensitive of all the storage periods to the soil temperature treatments. Other storage durations exhibited an apparent soil temperature difference in stomatal conductance, usually after day 7 (9.6, 17.9, 22, and 30.6 weeks), but this apparent effect was not significant when statistically tested. At a larger replication size (n=40), undoubtedly more and larger significant soil treatment conductance differences would have occurred, although it is unlikely that the overall suggestion of seedling soil temperature insensitivity (from 3-11°C) would have changed (degrees of freedom, n=10, was used). One final significant stomatal conductance increase due to the soil treatments occurred at 22 weeks on day 25 and 28. 3.1.2. Storage Duration Storage duration had a large significant effect on seedling stomatal conductance over almost all measurement days (see Figure 3.21). This effect involved a significant increase in seedling stomatal conductance after 22 weeks storage. The increase did not occur until day 4 and remained significant for the entire 28 day growth period. In general, seedlings outplanted after 26.1 and 30.6 weeks storage had significantly larger stomatal conductance than previous storage durations. Also, after 13.7 weeks storage, day 1 for the 7<>C and HOC soil temperature, seedling stomatal conductance was significantly larger than day 1 after any other storage duration. On day 4 measurements, after 9.6 and 22 weeks storage, seedling conductance values were significantly smaller than after 30.6 weeks (26.1 weeks data missing). In addition iNote: Figure 3.2 is a replot of data found in Figure 3.1 Similar replotting of net photosynthesis and internal CO2 / external CO2 ratio (Ci/Ca) data occurs in chapters 4 and 5. 25 3 Stomatal Conductance Figure 3.2 Stomatal conductance (cm s-l) changes over 28 days growth at 3,7, and 11<>C soil temperature after varying storage durations (weeks), n=40. 26 3 Stomatal Conductance A. K / ^ ^ 7C soli temp. 15 25 35 * A - -3C Boil temp. •er 15 25 storage duration (weeks) x DAY28 • DAY22 v DAY16 A DAY 10 O DAY 4 • DAY1 35 27 3 Stomatal Conductance to the increase in seedling conductance after 22 weeks storage, the conductance levels after 13.7 and 17.9 weeks were larger than seedling conductance levels after 9.6 weeks at 3<>C soil temperature and not significantly different than levels after 30.6 weeks storage. For 7°C soil temperature stomatal conductance after 9.6 weeks storage was less than after 13.7, 17.9, and 30.6 weeks storage at day 4. After 13.7 weeks storage, day 4 conductance levels at the 7°C soil temperature were significantly larger than day 4 after all storage durations except 30.6 weeks. The 11°C soil temperature treatment at day 4 saw stomatal conductance after 9.6 weeks significantly smaller than after 13.7 and 30.6 weeks. For measurement days 7 through 28, the only significant stomatal conductance storage effect was that dealing with the conductance increase after 22 weeks. Except for day 28 at the soil temperature 70C, stomatal conductance after 26.1 weeks or 30.6 weeks storage was significantly higher than conductance levels after 9.6 weeks storage. However, after 17.9 weeks storage, stomatal conductance was not different from other durations over the majority of measurement days at 7<>C and HOC soil temperatures (at 3°C there is a significant conductance difference). In Figure 3.2, specifically at the HOC soil temperature treatment, an apparent secondary increase in stomatal conductance occurs at the 13.7 and 17.9 weeks storage treatments from day 4 to day 16. At 3oC and 70C soil temperature treatments this variation in stomatal conductance was not as apparent. 3.1.3. Interactions On measurement days 1, 4, 10, and 28 there was a significant soil temperature, storage duration treatment interaction in the stomatal conductance measurements. These interactions can be explained by the changes in stomatal conductance over storage durations and the small differences in soil temperature which occur only at specific storage durations. 28 3 Stomatal Conductance For example, the rise in conductance with soil temperature from 9.6 to 13.7 weeks at HOC becomes a significant decrease from 13.7 to 17.9 weeks at HOC. 3.2. DISCUSSION The discussion has been divided into three sections; two on the main treatment factors, soil temperature and storage duration and a third on the changes occurring during the 28 day growth period after outplanting. 3.2.1. Days Changes in stomatal conductance over the 28 day measurement period suggested a stomatal recovery period of approximately 4-7 days after outplanting (see Figure 3.1). Except after 13.7 weeks storage, at the 11<>C and somewhat at 7©C soil temperature this period of rapid stomatal conductance change was present. Recovery of stomatal conductance has been noted in other conifers. Grossnickle and Blake (1985) showed an increase of 148% by day 6 in Picea glauca seedlings at 10oC soil temperature after removal from storage (-20C storage temperature). They also noticed an increase in conductance in Pinus banksiana which was soil temperature dependent, leveling off at 10oC after 6 days, but continuing to increase past 20 days (after outplanting) at 22oC soil temperature. Grossnickle and Blake (1985) suggested that dark freezer storage may disrupt the normally rapid, stomatal conductance light response mechanisms. Mattsson and Troeng (1986) suggested the decrease in photosynthesis they found after subfreezing storage (in Pinus sylvestris) was due to stomatal conductance inhibition of gas exchange (caused by water stress) which recovered after approximately 5 days. Seedlings stored at 2°C (versus - 4 0 Q showed a rapid photosynthetic recovery within 24 hours. Orlander (1986) working with bareroot and 29 3 Stomatal Conductance container grown Pinus sylvestris seedlings and a variety of planting and scarification techniques found recovery periods could last more than two years after planting in comparison to natural regeneration controls. Comparisons of the water relations of planted versus naturally established conifer seedlings have suggested recovery periods of several months to several years duration (Orlander 1986, Tranquillini 1973, Havranek 1975, Baldwin and Barney 1976). The results of these studies suggest the time period necessary for planted seedlings to reach an "established state" may be considerable and that the apparent steady level of conductance after 4-7 days may not represent similar levels in naturally established seedlings. The results presented here, even though they suggest a relatively short recovery period, are indicative of changes in seedling water relations upon removal from storage at the range of soil and air temperatures used. Evidence suggests the presence and duration of a recovery period is dependent upon species differences as well as a complexity of environmental and physiological variables such as storage conditions, and outplanting soil and air temperature. 3.2.2. Soil Temperature The effects of soil temperature on outplanted conifer seedling stomatal conductance have been studied in considerable depth. The results suggest a range of responses related to species and provenance adaptation to cold soil temperature. Soil temperature effects on seedling stomatal conductance are thought to be due to soil temperature influence on root resistance to water and nutrient uptake. Kramer (1942) suggested water uptake by plants is controlled by the viscosity of water (temperature dependent), root conductivity and species adaptation to the soil temperatures of their natural 30 3 Stomatal Conductance environment. Further work has suggested that species adaptation and plant physiological state (degree of cold hardiness) are the variables most responsible for the variation in plant water relations with soil temperature. Concrete evidence of stomatal response mechanisms is not available which is in part due to the complexity of stomatal responses and the present lack of knowledge on integrated functional plant physiology. Research involving plant water potential, conductance, and photosynthetic relationships have at times promoted plant water potential in a causative role. The correlation of tissue water potential with changes in conductance and photosynthesis should not be considered evidence of water potential's direct action on plant metabolism. The interpretation of a causal relationship between water potential and conductance is extremely dangerous (Schulze 1986). Passioura (1985) working with wheat demonstrated stomatal closure may occur under dry soil conditions even though the plants were not experiencing a water deficit. He further showed that leaf and soil water potential changed in parallel and changes in hydraulic conductivity from the soil through the plant affected stomatal size. A review by Schulze (1986) of the effects of drought on carbon dioxide and water vapour exchange, discusses in detail stomatal response mechanisms and the major theories of their control. Several points are worth mentioning here: 1) . Stomatal steady state response mechanisms are related to guard cell metabolism and their response to changes in humidity, root function, and mesophyll status. 2) . There appears to be a signal originating from the roots, related to root metabolic activity and independent of its hydraulic conductivity, to which stomata directly respond. This signal may be hormonal, possibly cytokinin. In this study, the effects of humidity on conductance can be omitted since vapour pressure deficit was relatively constant throughout the entire project. The internal CO2 31 3 Stomatal Conductance mesophyll status is discussed in chapter 5. The effect of soil temperature on root function is of direct concern here. Figure 3.3 shows the number of new roots >1 cm. after the 28 day growth period, and it was found that a critical root growth temperature exists which is greater than 7oC and at least 11°C soil temperature (Harper et al. 1989). If root activity alone were responsible for stomatal changes (directly or indirectly) one would surmise a soil temperature effect over all storage durations. As shown, this is not supported by the statistical results, although in most cases the trend exists. However, the apparent sensitivity of stomatal conductance at 13.7 weeks storage (see Figure 3.2) to soil temperature is interesting since this is also the storage duration at which the largest amount of roots >1 cm. (largest difference between treatments) was recorded. The results suggest that stomatal conductance response to soil temperature (3-ll°C) is related to root activity, and that the degree of stomatal soil temperature sensitivity is controlled by other unknown variables. Soil temperature effects in white spruce may be tempered by the physiological state such as degree of dormancy, carbohydrate status, and species cold soil adaptation. The presence of a critical soil temperature for stomatal response and photosynthesis has been shown in other conifer species (DeLucia 1986, Running and Reid 1980, Kaufmann 1976, Turner and Jarvis 1975). DeLucia (1986) working with Picea engelmannii suggested a critical soil temperature of 8°C was necessary to produce a sharp decline in stomatal 32 3 Stomatal Conductance soil temperature 50 r 8 B o - 40 a cd * 30 to a 3 degrees C. O 7 degrees C. A 11 degrees C O o o 4) a 20 10 i— —3E-0 5 10 15 20 25 storage duration (weeks) 30 35 Figure 3.3 New root growth >10 mm. after 28 days at 3,7, HOC soil temperature, and freezer storage to 30.6 weeks. Error bars are 1 SEM, n=40. (Harper et al. 1989) 33 3 Stomatal Conductance conductance and photosynthesis. Results from soil temperature studies on 2 year old Pinus contorta seedlings (Running and Reid 1980) showed an abrupt increase in plant resistance below 7oC due to water movement resistance from the root epidermis into the xylem. Kaufmann (1975) working with Picea engelmannii reported root permeability to water does not decrease drastically until below 7.5°C soil temperature. It was suggested that in temperate species, root permeability is less sensitive to cold soils than subtropical species (citrus root permeability decreased at 13.50©. Turner and Jarvis (1975) working with root temperatures of -8 to 20°C on Picea sitchensis, summarized gas exchange results by suggesting the decrease in photosynthesis and conductance in response to cold soil temperature was partially dependent on the physiological state of the seedling. Cold hardened seedlings showed a marked decrease at -loC and the unhardened seedlings at lo© Soil temperature above these levels had little affect on conductance and photosynthesis. The results were interpreted as due to changes in root permeability resulting from adaptation to cold temperatures. Studies on non-coniferous species also show evidence supporting species adaptation to natural soil temperature conditions. Several researchers have followed low soil temperature effects in temperate species. Lawrence and Oechel (1983) working on four taiga hardwood species (Populus tremuloides, Populus balsamifera, Betula papyrifera, and Alnus crispa) found varying degrees of response to soil temperature (5-250©. All species except A. crispa showed reductions in stomatal conductance with decreasing soil temperature. Results were explained in terms of evolutionary development. Alnus crispa, a later successional species, has adapted to low soil temperature conditions found in post disturbance plant communities. The other species, normally found in pioneer successional stages are less likely to have adaptive cold soil mechanisms. 34 3 Stomatal Conductance McNaughton et al. (1974) found that root chilling of low and high altitude Typha latifolia ecotypes showed reduced water and nutrient uptake (phosphorus) at the lower altitude ecotype. Cold soils may impose water and nutrient stress through decreased metabolic activity and root permeability. This may in turn promote ecological adaptation of plant water relations to tolerate or increase the plant functional efficiency in cold soil environments. Anderson and McNaughton (1973) surveyed 17 populations of 12 species over different elevations and soil temperatures (3-20OC). Relative water content results showed a significant decrease at 3<>C in comparison to 20°C, although there was little effect on transpiration and photosynthesis. An inverse relationship between elevation and decrease in relative water content was found, supporting the theory of adaptation of native populations through maintenance of turgor conditions necessary for normal function under cold soils. The relative insensitivity of white spruce stomatal conductance in this experiment to cold soil temperatures (except at 13.7 weeks) is in keeping with the hypothesis of species adaptation to native cold soil conditions. The white spruce used was from a provenance normally subject to cold soil conditions during the majority of the growing season and the temperature treatments (3,7,11°C) are not unlike those found in subalpine and boreal spruce forests. The results suggest white spruce are adapted to cold soils, possibly through changes in relative water content tolerance (Kaiser 1987, Anderson and McNaughton 1973). 3.2.3. Storage Duration The storage duration treatment effects on stomatal conductance are not understood. The factors affecting conductance and similarly transpiration remain unexplained. Very little literature exists on the actual mechanisms involved in conifer conductance responses. Most of the literature on plant stomatal behavior deals with angiosperms. A detailed 35 3 Stomatal Conductance discussion of stomatal behavior is beyond the scope of this study. Reviews by Schulze (1986), Kramer (1983), Schulze and Hall (1982) and others, cover the complexity of factors involved. The increase in stomatal conductance after 22 weeks storage, when considered along with other morphological and physiological data, suggests several hypotheses. It is known that long periods of storage reduce seedling carbohydrate levels (Marshall 1985). Although carbohydrate levels were not measured in this study, after storage for 26.1 weeks or longer, carbohydrate levels may have been reduced to a level below which normal stomatal function could not be maintained. Also, increased stomatal conductance may be a response to low rates of photosynthesis, an optimization of carbon fixation and water use response. Hormonally induced increase in conductance cannot be ruled out. Several authors have suggested root activity may affect stomatal conductance through growth regulators produced in the roots such as cytokinins (Anderson and McNaughton 1973, Livne and Vaadia 1972, Pallas and Box 1970). However, it is doubtful that higher levels of cytokinins or other hormones are responsible since root growth after 22 weeks is significantly diminished. It is also possible that an increase in the cuticular transpiration rate may occur after long storage durations due to freezer damage to the cuticle, epidermis or cell membranes. This is also unlikely since an increase in conductance did not register until day 4 after storage and no visual evidence of needle damage was observed. 36 4. NET PHOTOSYNTHESIS 4.1. RESULTS 4.1.1. Soil Temperature In general, statistical analysis at individual measurement days failed to show any significant difference (p<0.05) in seedling net photosynthesis due to the soil temperature treatments. There were four notable exceptions; 1) at day 4, after 9.6 weeks storage, 3<>C and HOC soil temperature treatments were significantly different (p=0.0673); 2) day 16, after 22 weeks, 3<>C and 7<>C ; 3) day 19, after 30.6 weeks, 3<>C and HOC (p=0.0656); and 4) at day 28, after 17.9 weeks, 3oC and HOC soil temperatures significantly different (see Figure 4.1). Variation in net photosynthesis between individual seedlings was found to be large, typically the coefficient of variation was around 25 to 30%. As such, 95% confidence levels increased to the extent that apparent treatment differences were also not statistically different. Figure 4.1 shows the progression of net photosynthesis values at each soil temperature over the 28 day growth period for each storage duration treatment. It can be seen that at the lloC soil temperature treatment higher values of net photosynthesis were maintained than at the 3<>C treatment. This is most apparent after 9.6, 26.1 and 30.6 weeks storage and less after 17.9 weeks. 37 4 Net Photosynthesis Figure 4.1 Net photosynthesis (umol nr2 s-i) changes over 28 days growth at 3,7,11°C soil temperature after various storage durations, n=40. 38 4 Net Photosynthesis d a y s a f t e r o u t p l a n t i n g 39 4 Net Photosynthesis 4.1.2. Storage Duration At all measurement days the storage duration treatments had a significant effect on seedling net photosynthesis (see Figure 4.2). The majority of differences were due to a drop in net photosynthesis values after storage durations greater than 22 weeks. This effect of long term storage on photosynthesis gradually diminished with days after outplanting until by approximately day 13 the significant effect had disappeared. The most dramatic decline in seedling photosynthesis occurred during the initial week after outplanting. At the 3°C soil temperature the photosynthetic decline after 22 to 26 weeks storage was abrupt and large (20% at day 1) with increasing decline at 30.6 weeks (36.6%). For the 7<>C and IPC soil treatments the seedling photosynthetic decline was not as abrupt (8.5% and 9.1% respectively after 22 to 26.1 weeks storage at day 1) and appeared to be a more gradual effect (31.2% and 17.7% respectively from 26.1 to 30.6 weeks storage at day 1). At the H O C soil treatment (at day 1), a significant difference in photosynthesis existed between storage treatments of 9.6 and 22 weeks suggesting an even earlier decline in net photosynthesis due to storage in interaction with the warmer soil temperature treatment. It appears the warmer lloC soil temperature has exacerbated, or the cold soil temperature has inhibited, the effects of the 22 weeks storage treatment on seedling carbon fixation. Another significant effect of the storage duration treatments was the increase in net photosynthesis after 9.6 weeks storage to after 13.7 to 17.9 weeks storage, again apparently tempered by the warmer lloC soil temperature treatment (see Figure 4.2). This effect was not manifested at measurement day 1. However, by day 4 at the 3oC and H O C soil temperatures, net photosynthesis after 9.6 weeks storage was significantly less than after 13.7, 17.9, and 22 weeks storage, but not after 30.6 weeks (26.1 weeks data missing). After 9.6 weeks at the 7<>C soil treatment, seedling photosynthesis at day 4 was not significantly 40 4 Net Photosynthesis Figure 4.2 Net photosynthesis (umol nr2 s-l) changes with increasing storage duration (9.6 to 30.6 weeks) over 28 days growth at 3,7, and HOC soil temperature, n=40. 41 4 Net Photosynthesis x DAY28 • DAY22 v DAY16 A DAY10 O DAY4 • DAY1 itorage duration (weeks) 42 4 Net Photosynthesis different than photosynthesis after 30.6 weeks storage although after 13.7, 17.9 and 22 weeks storage photosynthesis at day 4 was significantly different than after 9.6 weeks. This increase in net photosynthesis continued to be significant until measurement day 13 at the 3oC soil temperature and day 25 at the 7oC treatment. The lloC soil temperature photosynthesis values exhibit a similar trend through the entire growth period except at day 19, 25, and 28 where there was no significant increase with increasing storage duration treatments. After day 16, the highest seedling photosynthetic rate for each measurement day was reached after 17.9 weeks storage at the 7°C and lloC soil temperatures. This was most visible at the lloC soil treatment and not distinguishable at 3°C. For measurement days after day 16 at the two warmer temperature treatments, significant differences in net photosynthesis can all be related to this peak in activity, and as mentioned in section 4.1.1., the 17.9 weeks storage treatment has one of the few significant soil temperature effects. 4.1.3. Interactions Interactions are best portrayed by the three dimensional plots of Figure 4.3 where individual treatment means (n=40) have been joined together by a distance weighted least squares method (McLain 1974) to produce a response surface representing treatment relationships over the outplanting period. Changes in this surface between soil temperatures suggest how the two treatment factors are interacting. A significant interaction between storage duration and soil temperature treatments occurred on every measurement day except day 13 and 25. These interactions were due to storage duration differences in net photosynthesis being expressed disproportionately over the varying soil temperature treatments. For example, at measurement day 1 and the 3<>C 43 4 Net Photosynthesis Figure 4.3 Response surface relating changes in net photosynthesis to storage duration and outplanting period at the three soil temperature treatments (3,7, and HOC). 44 45 4 Net Photosynthesis soil temperature, there appeared to be an increase in photosynthesis after 9.6 to after 22 weeks storage but at the lloC soil temperature there was a significant decrease after 13.7 weeks to after 22 weeks storage. Similar interactions were associated with the rise in photosynthesis from the lower values after 9.6 weeks storage especially at the 3°C and 70C soil temperatures. After day 13 significant interactions occurred mostly due to the increased photosynthetic activity after 17.9 weeks storage at the 7°C and 11<>C soil temperatures which account for almost all interactions at measurement days 22, 25, and 28. Also a complex interaction existed between increases in photosynthesis after 22 and 26.1 weeks storage at day 16 (7<>C and HOC soil temperatures), day 19 (7oC soil temperature) and the significant soil temperature differences (day 16) and near significant (p<0.10) for measurement day 16 (after 22 weeks) and 19 (after 26.1 weeks). 4.1.4. Days As already stated in section 2.3.2., statistical analysis was not done on time series data or measurement days after outplanting. Figure 4.1 shows the treatment means over time for each storage duration treatment. Time is also represented in Figure 4.3. The only important trend over days is the general increase in photosynthesis after approximately day 19 which can be seen in Figure 4.3 as a rise in the response surface. This increase is due to the unaccounted-for contribution of the developing foliage after bud break (for discussion see section 2.2.1.1.). Correction for the new needle area at day 28 is shown in Figure 4.4. Little change was noted over the storage durations and between soil temperature treatments. The lower level of photosynthesis, in comparison to data prior to day 19, suggests a higher respiration rate may be occurring due to the high metabolic activity associated with the new 46 4 Net Photosynthesis needle area basis — l»t year — l«t year + new foliage soil temperature • 3 degree* C O 7 degree* C. A U degree* C. Figure 4.4 Net photosynthesis (pmol nr2 s-i) changes with increasing storage durations (9.6 to 30.6 weeks) at day 28 after outplanting and at 3,7, and HOC soil temperatures. Solid lines represent values based on first year needle area. Dashed lines represent values after addition of new foliage needle area, n=40. 47 4 Net Photosynthesis foliage development. Covering the developing buds with putty during the first week after bud break failed to show any change in photosynthesis. However, after the new foliage had developed further a reduction was noted. At this time, the putty technique became potentially damaging to the new foliage and was discontinued. It was felt that to fully understand the contribution of the new foliage to gas exchange parameters a detailed study would be required. Such a study is beyond the scope of this project, although such information would provide a more accurate estimation of photosynthesis after bud break. Prior to approximately day 19 there is little significant change in photosynthesis that can be associated with normal development after outplanting as was seen with stomatal conductance. 4.2. DISCUSSION 4.2.1. Soil Temperature The effects of soil temperature on net photosynthesis have been studied in conifer species (DeLucia 1986, Turner and Jarvis 1975, Linder 1973, Babalola et al. 1967, Rook and Hobbs 1976, for review see Kozlowski 1981). However, much of the present knowledge is based on extrapolation of root permeability, tissue water potential, and relative water content measurements and their correlation with transpiration and stomatal conductance. Little if any data exists that deals directly with physiological variables at the chloroplast level. As such, cold soil temperatures may cause a decline in carbon fixation through temperature effects on root permeability and water viscosity which limit plant root membrane water and nutrient uptake (Kramer 1982, McNaughton et al. 1974). Babalola (1968) found lower rates of photosynthesis and transpiration in Pinus radiata with 48 4 Net Photosynthesis decreasing soil temperature (26.7°C to 10oC) at a given soil water potential. DeLucia (1986) working with Picea engelmannii found a decrease in net photosynthesis and stomatal conductance only below 8<>C soil temperature and no effect between 10 and 20oC. The decrease in photosynthesis was concluded to be due to non-stomatal limitations. Work with non-coniferous temperate species has suggested cold soils affect photosynthesis only in certain species. Anderson and McNaughton (1973) followed the effects of cold soil temperatures on transpiration, photosynthesis and leaf relative water content in 12 species. They found that in species normally subject to cold soil conditions in their natural environment, root chilling failed to decrease photosynthesis or transpiration. However, leaf relative water content decreased significantly with decreasing soil temperature. These results led to their suggestion that species which have evolved under the presence of low soil temperatures have developed the ability to maintain leaf relative water content above critical values below which photosynthetic decline results, and that photosynthesis is not directly affected by cold soils but rather indirectly through plant water status. Lawrence and Oechel's (1983) work on four deciduous taiga hardwood species further suggests a species-ecosystem adaptation to cold soils. They found no significant change in photosynthesis of Populus tremuloides or Populus balsamifera seedlings when subjected to short term soil temperature cooling (20OC to 5oC). In Betula paprifera photosynthesis decreased at 5oC soil temperature and air temperature above 200C. On the other hand, response of Alnus crispa was to increase rates of photosynthesis at 5°C soil temperature. This is less confusing when you consider that P. tremuloides, P. balsamifera, and B. paprifera are pioneer hardwood species and A. crispa is a somewhat later successional species. The progression of disturbed sites from hardwood to conifer stands (in taiga forests) 49 4 Net Photosynthesis is also accompanied by a decline in soil temperature due to forest floor organic matter build-up and canopy closure. Lawrence and Oechel (1983) suggest long term decline in growing season soil temperature and "nonoptimal ecophysiological conditions" for the hardwood species, promote conifer establishment due to temporal and spatial changes in soil temperature characteristics. With this discussion in mind, the soil temperature results can be placed into perspective. Analysis of variance results failed to find a general significant soil treatment effect on seedling photosynthesis, although during certain storage durations and measurement days (see Figure 4.1), apparent soil temperature differences promote the hypothesis of an increase in photosynthesis with increasing soil temperature (3°C to l io©. If an effect is present, it is small in comparison to overall population variation. Of the four specific measurement days when a significant effect was found (see section 4.1.1.), day 19 and 28 are more likely to be due to true soil temperature effects since at day 4 and day 16 the lloC soil treatment photosynthesis value is between that of the 3»C and 7<>C treatments. The seeming insensitivity of white spruce photosynthesis in this study to soil temperatures in the range of 3<>C to lloC and lloC air temperature contradicts the results of others such as DeLucia (1986). This may be due to species and provenance differences. Seedlings in this study were grown from seed collected in northern British Columbia, an area subject to severe, long, cold winters and a short growing season. These restrictive growing conditions may favour the evolution of cold soil adaptive mechanisms. It is also possible the period of cold storage affected the seedling sensitivity to cold soil temperatures. Seedlings may decrease their sensitivity to cold soil temperature in response to periods of cold such as freezer storage or the normal onset of winter conditions. As such, comparisons involving soil 50 4 Net Photosynthesis temperature effects should consider plant material differences in soil temperature sensitivity related to species and plant adaptive behavior. 4.2.2. Storage Duration and Interactions Although many studies have looked at the effect of cold storage on height growth, bud dormancy, carbohydrate reserves, and survival, very little information is available on the process of carbon fixation. The information that is available gives conflicting results. Mattsson and Troeng (1986) working with Pinus sylvestris found the length of storage had no effect on the photosynthetic capacity of seedlings cold stored for three and six months.. Measurements were taken at light levels approximating saturation but carbon fixation results were based only on two seedlings per treatment. Differences were found in the rate of photosynthetic recovery after storage at different temperatures. Seedlings stored at 2oC recovered more quickly than those stored at -4oC during the first day outplanted. The slow increase in net photosynthesis of -4<>C seedlings was primarily caused by stomatal limitations to carbon dioxide exchange. Inhibition of net photosynthesis was also noted in planted seedlings inadequately thawed, also a result of stomatal limitations. McCracken (1978) working with Pinus mugo and Pinus radiata noted a greater depression in net photosynthesis with increasing periods of storage up to 18 weeks. In both species carbon fixation was clearly reduced by late lifting (spring) versus winter lifting. It was suggested that photosynthetic decline was due to disruption of the carbon fixation mechanism, since seedling water potential measurements were normal. The results of this study suggest an optimum period of storage of between 13.7 and 22 weeks, dependent upon outplanting soil temperature (see Figure 4.2 and 4.3). At the 3oC soil temperature, net photosynthesis significantly increased 37% by day 4 and 49% by day 10 51 4 Net Photosynthesis after 9.6 to after 13.7 weeks storage. At the 7»C soil temperature a significant increase in photosynthesis did not occur until after 17.9 weeks storage (17%) and at the lloC soil temperature there was a 20% increase between the 9.6 and 13.7 weeks storage treatments. The results also suggest that increasing periods of cold storage over 17.9 to 22 weeks decrease net photosynthesis upon outplanting and that this effect is also soil temperature dependent. The first day after outplanting appears to be the most sensitive period after storage to the storage effect. The increase in storage period sensitivity with warmer soil temperature is apparent in Figure 4.2 as a more gradual decline in net photosynthesis after 17.9 weeks storage with increasing storage duration than in the colder soil temperature (3oQ in which photosynthesis reduction is not significant and apparent until after 26.1 weeks storage. The interaction of storage duration and soil temperature treatment effects on photosynthesis suggests soil temperature is indirectly influencing the physiological changes induced by the storage treatments. In section 4.2.1. it was suggested seedlings are cold soil temperature acclimated due to storage conditioning and/or their evolutionary cold soil tolerance mechanisms. The storage, soil temperature interaction where photosynthesis reduction was noted earlier at the HOC soil temperature (after 22 weeks storage) in comparison to the 3oC soil temperature (after 26.1 weeks storage) at day 1 (see Figure 4.2 and 4.3), could be due to cold root temperature acclimation, induced by the storage period, influencing the storage treatment effect. The seedling roots may initially be more tolerant of 3oC than lloC soil temperature since they have been experiencing subfreezing temperatures (-20© in storage. Why the warmer soil temperature appears to have induced a significant storage duration effect in seedling photosynthesis sooner than the colder soil temperature is open to speculation but, it should be noted that this interaction occurs only at day 1. 52 4 Net Photosynthesis Interactions at later measurement days suggest a slower recovery from the decline in photosynthesis noted with the increasing storage duration treatments (26.1 and 30.6 weeks) at the 3°C soil temperature. This is most obvious after the 30.6 weeks storage period (see Figure 4.2). That is, by day 13 at the lloC soil temperature photosynthesis values after 30.6 weeks storage had equaled or surpassed those of seedlings subjected to shorter storage periods, while seedlings at the 3oC soil temperature (after 30.6 weeks) were still photosynthesizing at a considerably lower level than seedlings subjected to shorter storage durations. This type of soil temperature influence on photosynthetic changes over the storage duration treatments is also visible at the shorter storage duration treatments (9.6 to 17.9 weeks storage) where photosynthesis increase was moderated by the warmer soil temperature (see Figure 4.2). These results suggest that although soil temperature directly appears to have little significant effect on seedling photosynthesis in white spruce, soil temperature can influence the effects of other factors such as storage duration, through important interactions. The results also suggest that the interpretation of storage duration treatment effects on photosynthesis can be somewhat influenced by soil temperature. The mechanism(s) of these changes in photosynthesis are unknown, but comparison with stomatal conductance data suggests the processes are largely not due to stomatal limitations to CO2 exchange. It can therefore be speculated that the cold soil temperature, storage duration interaction may be affecting photosynthetic capacity through direct and interactive effects on plant root membrane water and nutrient uptake in combination with the physiological changes induced by the storage treatments. Since it appears that the storage treatment induced decline in seedling photosynthesis is related to nonstomatal limitations, seedling tolerance of cold soil conditions may be influenced through small changes in the 53 4 Net Photosynthesis tolerance of the photosynthetic apparatus to critical leaf relative water content (Anderson and McNaughton 1973). Until now the discussion has involved changes in photosynthesis over treatment effects at each measurement day. In Figure 4.5, the cumulative totals of net photosynthesis for the measurement days up to and including day 16 are shown for each treatment. The long term effect of cold soil temperature and storage duration on seedling photosynthesis is more easily discerned. After 9.6 and 30.6 weeks storage, apparent soil temperature effects are obvious. Surprisingly, the decrease in photosynthesis noted after 26.1 weeks storage (Figure 4.2) is not reflected at the 7«C or HOC soil temperature cumulative photosynthesis totals, although significant reductions during the early measurement days were noted. The results further support the hypothesis of a soil temperature, storage duration interaction effect on seedling photosynthesis. Maximum cumulative totals for all three soil treatments were attained after 17.9 weeks storage. Short and long term cold storage duration treatments appear to reduce the long term available photosynthate and this degree of reduction is dependent on soil temperature. However, after storage durations from 13.7 to 22 weeks long term seedling cumulative photosynthesis is unaffected by cold soil temperature (30C-110©. This suggests that in terms of maximizing the long term availability of photosynthate during the initial few weeks after outplanting an optimum storage period exists. 54 4 Net Photosynthesis soil temperature • 3 degrees C. 0 7 degree* C. | 11 degrees C. 10. 14. 18. 22. 26. 31. storage duration (weeks) Figure 4 .5 Cumulative net photosynthesis values, day 1 to day 19, for 3,7, and HOC soil temperatures after varying storage durations. 55 5. INTERNAL C02 / AMBIENT C02 RATIO 5.1. INTRODUCTION The calculation of intercellular CO2 (Ci) has been made available through the gas exchange modelling of mesophyll assmilation and stomatal conductance (Caemmerer and Farquhar 1981). A proper discussion of Ci cannot be made unless the assumptions and development of the model is understood. Numerous discussions have been written (for review see Farquhar and Sharkey 1982). In short, Ci has evolved from the Ohm's law analogy describing transpiration and net CO2 assmilation as the flow of current in an electrical circuit (Campbell 1986). From the formula: A = gc(Ca-Ci) therefore, Ci = Ca - A/gc where A is net photosynthesis, gc is the conductance associated with the diffusion of CO2 and calculated based on gs (stomatal conductance of water vapour)(ratio of gc/gs = 1.56). A correction must be made for transpirational mass flow of CO2, where E is transpiration rate, thus: A = gc (Ca - Ci) - (Ci + Ca) E/2 therefore, Ci = ((gc - E/2) Ca - A)/(gc + E/2) It can be seen that Ci is based on measurements of A and gs. In general, Ci can be regarded as an index which considers both mesophyll CO2 assimilation and stomatal conductance (Morison 1985). Since stomatal conductance is based on transpiration rate, Ci is a measure of plant water use relative to the rate of net CO2 fixation. Therefore, Ci/Ca is a means of determining seedling functional adaptation to the environment and response to stress. It is felt that plants tend to control transpiration 56 5 Internal C02 / Ambient C02 Ratio through stomatal conductance as a means of optimizing performance. Stomatal conductance may be a means of controlling transpirational loss, but rarely does CO2 stomatal resistance reduce photosynthesis. Stomata optimize conductance to minimize transpirational loss at a marginal cost to carbon assimilation (Farquhar and Sharkey 1982). 5.2. RESULTS Intercellular CO2 levels (Ci) were calculated using the LI-COR 6200 software following the model proposed by Caemmerer and Farquhar (1981). However, because ambient CO2 (Ca) was not controlled, Ci alone was not a good indicator of relative stomatal CO2 levels. Therefore the ratio Ci/Ca was used to discuss intercellular CO2 relative to background noise in Ca which averaged approximately 380 ppm, slightly above outdoor levels (330-340 ppm). Figure 5.1 shows the change in seedling Ci/Ca ratio over the growth period after each storage duration treatment. It is evident that a period of rapid increase occurs over the first 4-7 days leading to a somewhat constant Ci/Ca level dependent on the storage duration treatment. The rate of increase in seedling Ci/Ca was larger after 9.6 weeks storage than after 30.6 weeks and may be related to the effects of short and long term storage. The seedling Ci/Ca ratio showed a significant treatment interaction at every measurement day except day 10 (p=0.094). This interaction of soil temperature and storage duration is most easily discerned in Figures 5.1 and 5.3. At day 1, interactions center around a large increase in the Ci/Ca ratio at the 7<>C and lloC soil temperatures from after 9.6 weeks to after 13.7 weeks storage. The seedlings at the 3<>C and 7<>C soil temperatures after 13.7 weeks were significantly smaller than the 11°C at 13.7 weeks storage. After 17.9 weeks seedlings at the 3»C soil temperature had a Ci/Ca ratio increase from 9.6 weeks 57 5 Internal C02 / Ambient C02 Ratio Figure 5.1 Internal stomatal CO2 / external CO2 ratio (Ci/Ca) changes over 28 days growth at 3,7, and HOC soil temperature after varying storage durations, n=40. 58 5 Internal C02 / Ambient C02 Ratio 050 0.83 0.76 0.69 0.62 0.55 0.90 0.83 0.76 -0.69 -0.62 0.55 0.90 0.83 0.76 0.69 0.62 055 030 0.83 -0.76 0.69 0.62 0.55 0.90 0.83 0.76 0.69 0.62 0.55 050 0.83 -0.76 -0.69 0.62 -05S d a y s a f t e r o u t p l a n t i n g 59 5 Internal C02 / Ambient C02 Ratio although at the 7oC and HOC soil temperatures the Ci/Ca ratio decreased (significantly at lloC soil temperature). At all soil temperatures there was a significant increase in the seedling Ci/Ca ratio from after the 22 week storage treatment to the 30.6 week treatment. Similarly, seedling Ci/Ca values at all three soil temperature treatments after 9.6 weeks were smaller than at all soil treatments at 26.1 and 30.6 weeks storage. After the recovery period (day 1-7), the Ci/Ca ratio appeared to reach a constant level. Changes in this level were subdued in comparison to changes from day 1 to day 4, and interactive effects appeared to be concerned with soil temperature influence on storage treatment effects. In general, interactive effects can be associated with the decrease in seedling Ci/Ca ratio after 22 weeks storage at only the 7 0 C soil temperature. The 3 0 C soil temperature showed no significant decrease whereas the HOC soil temperature seedlings showed significant variation only after day 19. At day 16 all treatment effects were related to this decrease after 22 weeks storage at 7 o C soil temperature. After day 16, interactive effects are associated with the decrease in Ci/Ca ratio between the 13.7 and 17.9 week storage treatments which occurred at 7 o C and lloC but not the 3 o C soil temperature. The major treatment effect noted was that involving an increase in Ci/Ca ratio from after 9.6 weeks to after 26.1 and 30.6 weeks storage. This effect occurred from measurement day 1 to 16 at the 7 o C and lloC soil temperatures and over the entire 28 day period at the 3 o C soil temperature. After day 16 at the 7 0 C and the lloC treatments, seedling Ci/Ca ratio did not consistently show this effect. Storage duration treatment effects after day 16 were evident as a decrease in seedling Ci/Ca ratio from 9.6 and 13.7 to 17.9 and 22 weeks storage (see Figure 5.2 and 5.3). At the lloC soil temperature there was a significant increase from 9.6 to 13.7 weeks. 60 5 Internal C02 / Ambient C02 Ratio Figure 5.2 Internal stomatal CO2 / external CO2 ratio (Ci/Ca) changes with increasing storage duration (9.6 to 30.6 weeks) over 28 days at 3,7, and HOC soil temperature, n=40. 61 5 Internal C02 / Ambient C02 Ratio 62 5 Internal C02 / Ambient C02 Ratio Figure 5.3 Response surface relating changes in internal stomatal C02 / external C02 ratio (Ci/Ca) to storage duration and outplanting period at the three soil temperature treatments (3,7, and 11°C). 63 64 5 Internal C02 / Ambient C02 Ratio Soil treatment effects in seedling Ci/Ca were found only at the 13.7 and 22 week storage treatments. Day 1, 4, 10, 25 and 28 showed large differences in Ci/Ca ratio between soil temperatures after the 13.7 week storage treatment. Except for day 4, the seedling Ci/Ca ratio at the 3<>C soil temperature was smaller than at the 11°C temperature. After 22 weeks storage at day 16, and 19 the ratio at the 7<>C soil temperature was significantly less than at either the 3oC or the lloC soil temperature. Also, at day 25 and 28 after 22 weeks storage, the seedling Ci/Ca ratio at the 3<>C soil treatment was smaller than at the HOC temperature treatment. 5.3. DISCUSSION As already discussed, Ci was derived by modelling gas exchange through the stomata of a leaf or needle (see section 5.1.). Regarding the Ci/Ca ratio as an index of seedling response to the environment and stress is based on the calculation of Ci from A and gs. In many species it has been found that changes in the environment will cause A and gs to change proportionally in a linear relationship such that Ci remains relatively constant. However, work with water stressed plants suggests that Ci may be reduced in response to drought conditions. Also, Ci may increase when photosynthesis is reduced by factors affecting leaf metabolism. The Ci/Ca ratio, which is related to water use efficiency (the ratio of A over E), has been related to the discrimination against naturally occurring 13C during photosynthesis (Farquhar et al. 1982). In the strict sense, Ci represents the CO2 concentration found at the evaporation sites within the leaf (gs is based on transpiration rate). Since most transpiration is considered to occur through the stomata, this model is valid. However, concern over the assumption of uniform transpiration and photosynthesis across the leaf (or needle) has 65 5 Internal C02/Ambient C02 Ratio developed over evidence suggesting nonuniform stomatal size and response over the entire leaf surface (Laisk et al. 1980, Terashima et al. 1988). The effects of a patchy distribution of nonuniform photosynthesis and transpiration may be due to variation in stomatal conductance (Terashima et al. 1988). Thus, the method of conductance calculation may represent an average over the entire leaf, and because it fails to take into account the true stomatal size distribution, it may be a significant source of error (Laisk 1983). The results of conventional Ci calculations are further placed in doubt by evidence suggesting compartmentalization of leaf mesophyll cavities restricting diffusion and affecting Ci homogeneity over the leaf mesophyll (Terashima et al. 1988). This increased resistance to lateral diffusion of mesophyll CO2 has been seen in Helianthus annuus but not in Vicia faba (Terashima et al. 1988) and it remains to be seen whether Picea glauca or conifers in general display this trait. Cuticular transpiration, which is normally neglected since it is considered small in comparison to stomatal transpiration, may also cause error in Ci calculation. Changes in cuticle permeability due to changes in relative humidity, epidermis turgor or cuticle development (Schulze 1986) may affect transpiration such that at low stomatal conductance, Ci may be over-estimated. Changes in Ci have been related to changes in Ca, relative humidity, and light intensity. Since in this study, these variables were relatively uniform between treatments, and the small variation in Ca has been accounted for in the Ci/Ca ratio, comparisons amongst temperature and storage treatments are valid. In Figure 5.1 the period of increase in Ci/Ca, noted after outplanting (day 4 to 7), reflects the recovery of stomatal conductance and changes in net photosynthesis and suggests a decrease in the marginal cost of carbon assimilation. After this period the 66 5 Internal C02 / Ambient C02 Ratio seedlings appear to have reached an optimum ratio dependent somewhat on storage duration. This constant ratio reflects the proportionality between A and gg. Comparison of changes in seedling Ci/Ca ratio with changes in net photosynthesis (see Figure 4.1) show the period of rapid increase in Ci/Ca is not complemented by parallel changes in net photosynthesis. This suggests that stomatal limitations to CO2 exchange are not a major factor limiting photosynthesis. After 9.6 weeks and 22 weeks storage the comparison suggests photosynthesis is not under stomatal limitation to CO2 exchange. Because the relationship between photosynthesis and Ci/Ca does not appear to be consistent it is doubtful whether stomatal limitations play a role in regulating the level of carbon fixation even when an apparent correlation exists (after 17.9 and 30.6 weeks storage). However, stomatal limitations to photosynthesis may be important during the initially high stomatal resistance period found just after outplanting at day 1 (see Figures 3.1 and 4.1). The significant soil temperature, storage duration interactions, and the large differences between soil temperature treatments visible at day 1, 4, 10, 25 and 28 after 13.7 and 22 weeks (see Figure 5.2), suggest that soil temperature sensitivity, in terms of seedling Ci/Ca ratio, is variable. Although soil temperature appears to directly affect seedling Ci/Ca after 13.7 and somewhat after 22 weeks the lack of differences after other storage periods does suggest the seedlings were cold soil insensitive (3-lloC). However, the presence of interactions with storage treatments further suggests the seedlings were influenced by soil temperature indirectly. In general, the storage duration treatment effects in seedling Ci/Ca ratio were related to the significant increase in Ci/Ca after 22 weeks storage. This increase in the seedling Ci/Ca ratio suggests a decrease in the marginal cost of carbon fixation and may be due to storage induced physiological changes. The mechanism of these changes are unknown 67 5 Internal C02 / Ambient C02 Ratio however, Farquhar (1980) suggested increases in Ci due to reduction in carbon fixation are a result of changes in leaf metabolism. The increase in Ci/Ca noted here may be due to long term storage duration affects on photosynthesis at the metabolic level. If the decrease in carbon fixation noted after long term storage was due to stomatal limitations then the Ci/Ca ratio would have decreased. Since the opposite was found (stomatal conductance increased after long term storage), increased Ci/Ca ratio is evidence of nonstomatal limitations to seedling photosynthesis. The concomitant increase in stomatal conductance with decreased photosynthesis at the longer storage treatments may be evidence of a coupling mechanism between the two processes. The hypothesis of a relationship between photosynthesis and stomatal conductance has been suggested by other authors and may help explain the parallel increase in conductance with a decrease in photosynthesis over the increasing storage treatments. As a consequence of changes induced by the longer storage treatments, the seedlings may simply be trying to maximize mesophyll CO2 by increasing stomatal conductance in response to low levels of seedling photosynthesis. This suggests photosynthesis and stomatal conductance are mechanistically linked, though not directly. The maintenance of Ci/Ca ratio at a relatively steady level after all storage treatments does suggest the seedlings have a A and gs proportionality relationship. The significant decrease in the steady state Ci/Ca ratio noted after storage treatments 17.9 and 22 weeks, only at the 70C soil temperature suggests seedlings may be under water stress leading to stomatal closure and possible reduction of photosynthesis. Stomatal conductance (see Figure 3.2) does indeed show a decrease after 22 weeks storage and net photosynthesis (see Figure 4.2) shows an increase after 9.6 weeks but not after 17.9 or 22 weeks. This response (in relation to changes in storage duration treatments) is related 68 5 Internal C02 / Ambient C02 Ratio to a change in the proportionality relationship of A and g§. The decrease in Ci at the 7<>C soil temperature (seen as a decrease in Ci/Ca) after 17.9 weeks is due to the increase in A without a concomitant change in gs. At 22 weeks the seedling g s level drops without a proportional decline in A (with storage treatments). Both these scenarios result in the significant decline in seedling Ci/Ca ratio. At the other soil temperatures A and gg respond proportionally in similar directions maintaining Ci at a relatively constant level (Ci/Ca), relative to previous storage treatment trials. In summary, the changes in Ci/Ca steady level over storage duration treatments are related to changes in the seedling net photosynthesis, stomatal conductance relationship. Proportional changes in photosynthesis and conductance result in a constant Ci/Ca level. Changes in Ci/Ca result from nonproportional changes in photosynthesis and stomatal conductance over storage treatments influenced by the soil temperature treatments. The significant changes in seedling Ci/Ca suggest: 1) The reduction of photosynthesis after 26.1 and 30.6 weeks storage was due to a nonstomatal limitation to carbon fixation. 2) Soil temperature effects on photosynthesis are indirect and are important in the consideration of storage effects. 3) The decline of seedling Ci/Ca ratio after 17.9 and 22 weeks storage at 7<>C is similar to the response noted in water stressed plants (Farquhar 1980). However, seedling photosynthesis did not appear to be affected and the reason why only the 7°C treatment was affected is unclear. Observations of seedling Ci/Ca changes over time (the 28 day period) after each storage treatment does suggest seedlings are optimizing Ci through changes in net photosynthesis and stomatal conductance. 69 6. PHOTOSYNTHETIC EFFICIENCY 6.1. INTRODUCTION The use of chlorophyll fluorescence emission in plant physiology is not new. The first quantitative studies on fluorescence date back to 1931 (Kautsky and Hirsch 1931) and the initial description of the phenomena to Muller, 1874. In the last decade, advances in electronics have allowed the development of fluorescence as a measurement tool. The use of fluorometers in forestry has gained considerable interest in both research and field operations. By definition, fluorescence is a form of luminescence in which light energy is emitted from an irradiated object. In terms of conifer seedlings and plants in general, fluorescence is the emission of light by the chlorophyll involved in photosynthetic activity. Usually fluorescence from chlorophyll a is measured as an indicator of photosystem II (PSII) activity. About 10% of fluorescence quenching can be attributed to photosystem I (PSI) due to the presence a chlorophyll a (Schreiber et al. 1989). Fluorescence measurements are made after a period of dark adaptation. This dark period should be long enough to return all photoreduced plastoquinone (QA) to the oxidized state and eliminate the trans-thylakoid membrane proton gradient. The characteristic fluorescence induction curve observed upon excitation, also known as the Kautsky effect or curve follows OIDPSMT transients as proposed by Papageorgiou (1975) and shown in Figure 6.1. From Fo, the minimum fluorescence level when QA is fully oxidized, fluorescence increases to a maximum at the P-peak (Fp) where all QA is reduced. The ratio of variable fluorescence (Fy) to Fp where Fv = Fp - Fo, is considered a measure of the primary photochemical efficiency of PSII (Kitajima and Butler 1975) and is well correlated with 70 6 Photosynthetic Efficiency quantum yield of net photosynthesis (Demmig et al. 1987). Slow induction kinetics from S to T are due to fluorescence quenching dependent on changes in photochemistry within the chloroplasts. The introduction of modulated light and detection methods has allowed the partitioning of fluorescence quenching into photochemical and nonphotochemical processes (Schreiber 1983). The development of quenching component analysis relates changes in fluorescence kinetics to changes in PSII and PSI activity such as QA redox state, thylakoid membrane energization, and state changes in antennal pigments (Schreiber et al. 1986, Weis and Berry 1987). Figure 6.1 Fluorescence induction curve showing characteristic OIDPSMT transients. 71 6 Photosynthetic Efficiency For a more precise discussion of fluorescence induction kinetics and interpretation see Schreiber (1983), Sivak and Walker (1985), Hipkins and Baker (1986), Schreiber et al. (1986), Bilger and Schreiber (1986), Schreiber et al. (1989). In this study, interpretation of fluorescence results will be confined to discussion of Fy/Fp and M/Fo ratios. A discussion of modulated fluorescence results (26.1 and 30.6 weeks storage) can be found in Harper and Camm (1990)(in preparation). 6.2. RESULTS Figure 6.1 represents fluorescence kinetics similar to those found here. Induction curves for each individual seedling were measured for Fp, Fv, Fo, and M (maximum height between a line tangent to S and T transients and the secondary peak, M). Due to the delayed availability of the PAM fluorometer only measurements of storage duration treatments 17.9 (day 27), 22 (days 5,16,27), 26.1 (days 2,5,10,16,27), and 30.6 weeks (days 2,5,16,27) were made. The ratio Fy/Fp increased with time after outplanting and appeared to reach a maximum by approximately day 16 in the 7°C and lloC soil treatments (see Figure 6.2). The colder soil temperature had a significantly smaller ratio at day 16 over 22, 26.1, and 30.6 weeks storage and did not appear to reach the maximum steady state level of the warmer soil temperatures until day 27 at 22 and 30.6 weeks. These results suggest the primary photochemistry of PSII requires a recovery period the length of which is dependent on soil temperature and storage duration. After 26.1 and 30.6 weeks storage, seedling measurement day 2 Fy/Fp levels were equal at approximately 0.28. By day 5 a significant increase occurred at all soil temperatures except 3°C, after 30.6 weeks. Over the three storage durations 22, 26.1 and 72 6 Photosynthetic Efficiency Figure 6.2 Changes in Fv/Fp ratio over the 28 day growth period when planted at 3,7, and 11<>C soil temperature after 22,26.1 and 30.6 weeks storage, n=12. Error bars are 1 SEM. 73 6 Photosynthetic Efficiency 0.60 c, • 0.52 •S 0.44 h S 0.36 0.20 • 1 • 1 ' 1 • ' 1 • i • # / -* / - / / . / / •IT 30.6 weeks storage i . i i i 10 15 20 25 30 0.60 > 0.52 •§ 0.44 \-S 0.36 S 0.28 .O Q. 0.20 ~i ' 1 • 1 26.1 weeks storage 10 15 20 25 30 0.60 a > 0.52 [fa •g 0.44 p S 0.36 .a I 0.28 a. 0.20 ~i • 1 • 1 1 1 • r or 22 weeks storage soil temperature 3 degrees C O 7 degrees C A 11 degrees C 10 IS 20 days after outplanting 25 30 74 6 Photosynthetic Efficiency 30.6 weeks, at day 5, a significant decrease occurred with increasing durations at the 3oC and 7oC soil temperature (see Figure 6.3). A decrease also occurred at lloC from 22 to 26.1 weeks but not from 26.1 to 30.6 weeks. The change in seedling Fy/Fp with storage duration at day 5 suggests a drop in the maximum photosynthetic efficiency of PSII with increasing storage duration (22 to 30.6 week range). The initial recovery period may have been shorter at 22 weeks since Fy/Fp = 0.44 and this level is not attained until a few days later at 26.1 weeks. It remains to be seen whether the rate of recovery is dependent upon storage duration at shorter durations (<22 weeks). At day 16 there was no difference in seedling Fy/Fp between storage treatments for 70C and HOC soil temperatures (see Figure 6.2). However, seedlings at the 30C soil temperature had a larger Fy/Fp after 26.1 weeks than after 22 or 30.6 weeks storage. This was related to soil temperature differences where after 22 and 30.6 weeks the 3oC soil temperature treatment was significantly smaller than the 70C or lloC treatment, but not different after 22 weeks storage. For day 27 there appeared an increase after 26.1 to 30.6 weeks storage at the 30C soil temperature. In general there was a slight increase in seedling Fy/Fp after 22 to 26.1 weeks with little change from day 16 levels except at the colder soil temperature as already noted. An analysis of soil temperature effects was inconclusive. Figure 6.5 shows that after 17.9 weeks (day 27) there was an increase in Fy/Fp from the 3oC and 7oC soil temperature to the HOC temperature. After 22 weeks (see Figure 6.2) at day 16 and after 26 weeks at day 10, the 3oC soil temperature was less than the 70C and HOC temperatures. After 30.6 weeks at day 5 and day 16, the 3oC soil temperature was smaller than 7oC and HOC, although at day 27 70C was significantly smaller than 30C and lloC. It appears cold soils (3oC) may have an effect on Fy/Fp but the results suggest the effect is not always significant. 75 6 Photosynthetic Efficiency 24 26 28 30 storage duration (weeks) soil temperature 3 dcgrcei C. O 7 degrees C. A ]1 degrees C. 32 Figure 6.3 Day 5 changes in Fy/Fp ratio over increasing storage duration (22 to 30.6 weeks), n=12. Error bars are 1 SEM. 76 6 Photosynthetic Efficiency Fv/Fp results were compared with net photosynthesis measurements made the same day (see Figure 6.4). A poor correlation was found between Fy/Fp and net photosynthesis, although there was a significant increase in photosynthesis with increasing Fy/Fp (R2 = 0.156 for 26.1 weeks day 2, 5, and 16). This was probably due to differences in the time of measurement. Fluorometer readings were taken several hours after gas exchange measurements. For correlation studies, both gas exchange and fluorescence readings should be made concurrently. However in Figure 6.5, mean values of Fy/Fp and photosynthesis from similar measurement days reflect parallel changes and suggest a significant correlation. Figure 6.4 Relationship between net photosynthesis and photosynthetic efficiency in white spruce seedlings stored for 26.1 weeks and outplanted. Measurements were made from 1-16 days after outplanting. 77 6 Photosynthetic Efficiency storage duration (weeks) soil temperature 3 degrees C. O 7 degrees C. A l l degrees C. Figure 6.5 Changes in net photosynthesis and photosynthetic efficiency for similar days (day 4 and 5, day 27 and 28) and storage durations at 3,7, and HOC soil temperatures, net photosynthesis, n=40. Fy/Fp ratio, n=12. *(note at 26.1 weeks storage day 4 net photosynthesis data missing, therefore day 7 data is represented instead) 78 6 Photosynthetic Efficiency The ratio of M/Fo was also followed but it was found that changes in M were difficult to measure which led to high variation and inaccurate readings. M peaks diminished in size over the outplanting period. By day 16 the M peak was almost indistinguishable. Correlation with net photosynthesis was poor again (R2 = 0.317 at 22 weeks day 5 and 16) probably due to the difference in gas exchange and fluorometer measurement times. However, a significant increase in M/Fo occurred with increasing photosynthesis. 6.3. DISCUSSION As already stated, seedling Fy/Fp ratio is considered a measure of the photosynthetic efficiency of PSII photochemistry. That is, changes in this ratio reflect changes in the functional efficiency of the photosynthetic apparatus to use absorbed light energy. Any stress or condition which may affect normal photosynthetic function through decreased carbon fixation, light energy dissipation or damage to the photosynthetic apparatus will reduce Fy/Fp. Such things as low air temperature, freezing damage, water stress, and high light intensity may lead to reduced Fy/Fp either through direct damage to the photosynthetic apparatus or through reducing normal photosystem function leading to the build-up of excess energy which in turn results in photosystem damage. Photoinhibition is the term used to describe light induced photosystem damage and reduction in quantum yield. Greer et al. (1986) defined photoinhibition as the net difference between the rate of damage and the rate of damage repair. Thus, the degree of photoinhibition will be reflected in Fy/Fp and as such this ratio is considered an indicator of photoinhibitory damage. Studies on the winter depression of photosynthesis in conifers with fluorescence techniques have suggested photoinhibition may be partly responsible and that the susceptibility to photodamage is increased after subfreezing nights (Lundmark et al. 1988, 79 6 Photosynthetic Efficiency Lundmark and Hallgren 1987). The apparent Fy/Fp recovery period noted in this study upon outplanting after storage (see Figure 6.2) is similar to that noticed by Lundmark et al. (1988) where recovery from winter depression of photosynthesis was followed in Pinus contorta, Pinus sylvestris, and Picea abies. They found the rate and degree of Fy/Fp recovery to be air temperature dependent and they suggested PSII activity was affected by photoinhibition leading to chlorophyll degradation after subfreezing temperatures. Krause and Somersalo (1989) concluded photoinhibitory stress can be caused by even low levels of light and that inhibition of photosynthetic efficiency (Fy/Fp) can occur at low or subfreezing temperatures. Their work also suggested that the photoinhibitory response is dependent upon the degree of plant cold acclimation and the light intensity. It was found cold hardened and high light intensity acclimated plants were more tolerant of photoinhibition. In Figure 6.2, the increase in Fy/Fp after storage and outplanting suggests the seedlings were initially photoinhibited due to the effects of either long periods of darkness, or recovery from the subfreezing temperatures of storage, or both, upon return to the moderate light conditions. Cold soil temperature may increase the length of the photosynthetic efficiency recovery period and as a result prolong photoinhibition. This was evident after 30.6 weeks storage only. Since seedlings were subjected to lower light conditions (approximately 480 pmol nr 2 s-1) than would be normally found in plantations (approximately 1500+ pmol nr 2 s-1 on a clear day), it is felt that the potential for severe photoinhibitory stress would be greater in these open planting sites. Cold air and soil temperatures would also increase the length and extent of the recovery period. The effect of direct sunlight causing photoinhibition in temperate conifer and deciduous species is well documented (Strand and Lundmark 1987, Lundmark et al. 1988, Ogren 1988). 80 6 Photosynthetic Efficiency Soil temperature results in this study also suggest cold soils may decrease Fy/Fp. However, this response was not always significant and requires more research to determine interacting factors which may be involved. Long term storage for 30.6 weeks appears to increase the cold temperature effect. The mechanism of a possible soil temperature response is unknown. It may be related to the effects of soil temperature on seedling root permeability, root growth, or water viscosity. The influence of cold soil temperatures on the surrounding air temperature may also be an important factor. While the measurement of Fy/Fp is considered a useful indicator of photoinhibition, the loss in variable fluorescence associated with photoinhibition does not indicate the specific mechanism(s) of damage. Photoinhibition may act as a protective mechanism against excess light energy while the photosynthetic apparatus recovers and acclimates to changes in temperature and light intensity (Erause and Somersalo 1989). Under high light conditions the capacity of the photosynthetic system to function efficiently will limit the dissipation of excitation energy. The buildup of excess energy may cause damage to the Q B binding site of PSII (Kyle et al. 1985) inhibiting electron transport and photochemical quenching (qQ). Nonphotochemical quenching associated with the proton gradient (qE) may increase as part of a protective mechanism against severe photoinhibition through which thermal energy dissipation occurs in the antennal pigments (Demmig et al. 1987). Changes in the environment which induce photoinhibitory damage may require acclimation of the photosynthetic apparatus. These mechanisms of energy dissipation will minimize damage while cold temperature or high light intensity acclimation can occur. In terms of determining when cold stored white spruce seedlings are no longer under possible photosynthetic stress, an awareness of the maximum level of photosynthetic efficiency is required. In comparison with other work, the apparent steady level of Fy/Fp 81 6 Photosynthetic Efficiency attained after day 16 is indicative of further depression in the primary photochemistry of PSII. Bjorkman and Demmig (1987) followed the chlorophyll fluorescence in 44 species of plants and found the Fy/Fp ratio to be constant at approximately 0.832. The mean of several conifer species was 0.853. Results from Ogren (1988) show unstressed Salix species at approximately 0.85, and Lundmark et al. (1988) showed Pinus contorta, Pinus sylvestris and Picea abies at 0.80-0.85 after 4 months recovery from winter conditions. At day 27, white spruce seedlings in this study showed the Fy/Fp ratio at a maximum of 0.54. In comparison, it is clear that the seedlings have not fully recovered their photosynthetic system. Work by Lundmark et al. (1988) suggests 3 months may be required before an Fy/Fp ratio of approximately 0.8 is attained. It is possible that the first year needles measured may be unable to fully acclimate to the new environment and that a Fy/Fp ratio of over 0.8 can only be attained by new foliage developed in response to the new growing conditions. More work is required to follow the long term photosynthetic development of old and new needles. The depression of the Fy/Fp ratio has also been related to subfreezing temperature damage and is well correlated with the resulting reduction in the rate of carbon fixation (Krause and Somersalo 1989, Strand and Lundmark 1987, Klosson and Krause 1981). Both the reduction of Fy/Fp with increasing storage durations, at day 5, and its correlation with net photosynthesis (Figure 6.4) suggests increasing damage to the carbon reduction cycle with the increasing long term storage treatments. It appears the decrease in seedling net photosynthesis (as discussed in chapter 4) with storage treatments over 22 weeks may be due to freezing damage at the chloroplast level. No visible leaf discoloration was seen during the study indicating the freezing injury did not lead to photooxidation and severe chlorophyll degradation. Terminal bud break data also suggested that the seedlings planted after 26.1 and 30.6 weeks storage received freezing damage. Seedlings subjected to storage durations 82 6 Photosynthetic Efficiency shorter than 26.1 weeks showed no bud damage, but after 26.1 and 30.6 weeks, 6 and 11 seedlings respectively failed to break bud. Only terminal buds were affected and they appeared brown, necrotic and showed little sign of bud swell. Krause and Somersalo (1989) concluded freezing damage is more readily detected through analysis of quenching components qQ, and qE than Fy/Fp. They found in Spinacia oleracea only severe freezing damage caused a large drop in Fy/Fp. Analysis of qQ, qE, and Fo levels (Harper and Camm 1990, in preparation) showed seedlings after 26.1 and 30.6 weeks storage, during the initial 5-10 days, had low qQ and high qE, and Fo quenching (qo). However, qE levels after 30.6 weeks storage were significantly smaller than those after 26.1 weeks during the first 5-10 days after outplanting. Krause and Somersalo (1989) suggest freezing injury causes reduction in qQ which is correlated with CO2 fixation inhibition. Reduced CO2 fixation would lead to an increase in the proportion of reduced QA and therefore a decrease in qQ. They also suggest freezing damage results in the inhibition of qE through loss of thylakoid membrane protein gradient. This may be the case under severe membrane damage, but the results here suggest the qE, proton gradient mechanism of thermal de-excitation of PSII is still intact. Concomitant variation in qo also is evidence of an active energy dissipation mechanism and acclimation process. The reduction in seedling qE levels after 30.6 weeks storage (days 5-10) does however suggest freezing damage occurred, at least with seedlings stored for 30.6 weeks. Thus the decrease in Fy/Fp with increasing storage duration (Figure 6.2 and 6.3) may also indicate freezing injury to the seedling's photosynthetic apparatus. Up until now, gas exchange data has indicated that the reduction in net photosynthesis was due to nonstomatal limitations. The precise cause and mechanism of the reduction was unknown. Correlation of net photosynthesis results with photosynthetic efficiency (Figure 6.5) further 83 6 Photosynthetic Efficiency suggests the reduction in carbon fixation with the extended storage durations was due to disruption of photosystem electron transport activities. This disruption in electron transport may have been due to freezing injury during the storage period. This leads to the suggestion that the seedlings may have lost cold hardiness during storage after extended periods (possibly greater than 22 weeks). Once cold hardiness had been reduced, the freezing temperatures of storage could damage the photosynthetic apparatus which would lead to greater photoinhibition susceptibility upon returning the seedlings to the light. Normally, when conifer seedlings are placed in cold storage, high cold hardiness allows them to withstand extreme subfreezing temperatures. Hardiness provides a freezing avoidance mechanism protecting against damage to the photosynthetic apparatus as well as normal cell function. Cold acclimated or hardened plants are then less likely to receive photosystem damage at subfreezing temperatures. However, dehardening of seedlings during storage will increase the risk of photosystem damage. Ritchie et al. (1985) found a gradual loss of frost hardiness during storage of two year old Pinus contorta and Picea glauca engelmanii. Dehardening is controlled by the degree of chilling received and therefore longterm cold storage may cause a decline in hardiness in white spruce while in storage (Salim et al. 1990, personal communication). The susceptability of the photosynthetic apparatus to freezing damage may also be influenced by the long periods of darkness during freezer storage. Since the level of hardiness required to protect the photosynthetic system is unknown, and storage conditions are not directly comparable to natural conditions, the probability of damage is also unknown. However, the evidence suggests a loss of hardiness may be responsible for the apparent decline in photosynthesis and photosynthetic efficiency noted here through freezing damage to the photosynthetic system. 84 6 Photosynthetic Efficiency Seedlings grown under the influence of their natural environment will develop hardiness in response to photoperiod and temperature. The degree of hardiness may determine their ability to tolerate high light intensity during freezing conditions. Photoinhibition noted in conifers after frosts or subfreezing temperatures has been dependent on light conditions. The severity of photoinhibition may be a driving mechanism in seedling photosystem acclimation especially during the fall with the onset of winter conditions and also during the spring as growing temperatures become more favourable. Low temperatures inhibit photosynthesis and as such winter conditions represent a hostile environment to normal photosynthetic function. Changes may occur at the chloroplast level to reduce or channel the increased excess energy of light absorption in order to minimize photoinhibition damage during periods of seasonal climate change. Decreased chlorophyll content has been noted in seedlings of Pinus sylvestris and Picea abies after frosts (Lundmark and Hallgren 1987) and in Pseudotsuga menzeisii decreased total chlorophyll to carotenoid ratio during winter months (Hawkins and Lister 1985). Winter conditions may stimulate photosystem protective mechanisms such as increased thermal energy dissipation and reduce chlorophyll levels to avoid severe photoinhibitory damage. With the onset of spring conditions conifers lose their protective cold hardiness and warmer temperatures promote the return of normal levels of photosynthetic efficiency and photosynthesis. Acclimation of the photosynthetic apparatus will occur as temperature, light and growing conditions permit. It follows that spring and autumn frosts will be most damaging under high light intensity due to the greater freezing damage potential and the presence of low air and soil temperatures during these transitional seasonal periods. 85 7. SEEDLING MORPHOLOGY 7.1. SEEDLING GROWTH After the 28 day growth period, seedlings were harvested and shoot height, shoot dry weight and root dry weight were determined. Figure 7.1(a,b,c) show the changes in shoot and root dry weight and shootxoot ratio over storage duration at the three different soil temperatures. Total root weight is represented in Figure 7.1(c). Little real change in weight is noticeable with increasing storage duration, although an apparent decrease is visible after 26.1 weeks storage. Also, the 3oC soil temperature appears to consistently have a smaller root weight than either of the other soil treatments. At zero weeks storage, a significant increase exists with increasing soil temperature. This suggests that prior to any freezer storage, root weight development is sensitive to soil temperature. The effects of soil and storage treatments are not conclusive, mainly due to the overwhelming influence of the old root mass prior to new root development. That is, old root dry weight greatly exceeds new root dry weight and as a result any new root growth significant treatment differences are hidden. Shoot growth increased with storage duration, mainly due to the decrease in days to terminal bud break allowing a longer period of shoot development. Soil temperature appeared to have no effect on shoot growth. However, after 13.7 weeks storage shoot dry weight at the 7°C soil treatment was significantly smaller. The reason for this is unclear and may be related to growth chamber effects. Shoot:root ratio (S/R) reflects the changes in shoot dry weight and the zero weeks storage root weight differences. In general S/R increases with storage duration due to the 86 7 Seedling Figure 7.1 Dry weight values for various storage duration treatments after the 28 day growth period at 3,7, and HOC soil temperatures. (A) Shoot/root ratio, (B) shoot dry weight, (C) root dry weight. 87 7 Seedling Morphology 88 7 Seedling Morphology disproportionate increase in shoot growth in comparison to the seemingly insignificant root growth. The S/R ratios are large and suggest an unbalanced seedling development. The decrease in new root growth with increasing storage, as suggested by the number of new roots greater than 1 cm (Figure 3.3) and somewhat by root dry weight, along with increasing shoot growth suggests the shoots are the major sink of photosynthate. Since the seedlings were grown prior to this study under a commercial production regime (high density) where nutrients and water are normally in abundance and root growth is restricted, bud development may have been predetermined to "expect" similar conditions. The entire seedling resource allocation may be preconditioned to a favorable environment. This legacy may be partially responsible for the high S/R ratio noted here. 7.2. TERMINAL BUD DORMANCY Figure 7.2(a) shows the decrease in the number of days to terminal bud break (TBB) after outplanting with increasing storage duration. This decrease reflects the accumulation of chilling hours required to release dormancy. The zero weeks storage seedlings broke bud after approximately 20 days suggesting their chilling requirement had already been partially met prior to freezer storage. Soil temperature had no apparent affect on TBB. As already stated (section 6.3), terminal bud necrosis occurred at the two longest storage durations (6% at 26.1 weeks, 9.2% at 30.6 weeks) which may have been due to loss of tolerance to freezer 89 7 Seedling Morphology Figure 7.2 Change in, (A) days to terminal bud break (TBB) and, (B) height increment (cms.), for the various storage duration treatments after the 28 day growth period at 8,7, and HOC soil temperatures, n=40. 90 7 Seedling Morphology induced dehydration (Sakai 1983). This may be related to a loss of seedling cold hardiness while in storage causing terminal bud damage. 7.3. DISCUSSION In general, seedling morphological parameters such as height and dry weight measurements do not reflect the changes occurring in gas exchange variables. S/R ratio does suggest a disproportionate allocation of resources into shoot development which may be due to basic developmental patterns influenced by the storage treatments. The effect of storage duration on dormancy allows seedlings to meet their general chilling requirement thereby influencing the period to TBB. This effect on TBB period offers an opportunity to manipulate growth after planting. Early bud break may be advantageous for locations with short growing seasons but care must be taken to ensure the storage period has not reduced root growth potential and the ability to maintain adequate levels of photosynthesis. 91 8. CONCLUSION 8.1. HYPOTHESIS TEST RESULTS Prior to the commencement of this study several hypotheses were developed (as stated in section 1.3.). The results of analysis of variance tests on those hypothesis will now be reviewed. The null hypothesis 1-a), that the length of storage will have no effect on photosynthesis, can safely be rejected. That is, the alternative hypothesis, that increasing storage duration will produce a decline in seedling photosynthesis is accepted with 95% confidence. Similarly, null hypothesis 1-b) can be rejected also in favor of accepting the alternate, that photosynthetic efficiency (Fy/Fp) declines with storage length (in the range of 22 to 30.6 weeks storage). However, the data suggests this was only true early in the Fy/Fp recovery period (day 5 after storage) and that if only day 16 and 28 results were considered, rejection of the null hypothesis would not be possible. Soil temperature affects are not so clearly interpreted. Analysis of variance results of net photosynthesis and Ci/Ca ratio indicated there was a significant interaction effect between soil temperature and storage duration. Detailed analysis showed that soil temperature effects were significant only at specific measurement dates (null hypothesis 2-a) was rejected) and that in general, no significant soil treatment effect was realized although apparent differences were visible. Fluorescence results do not conclusively support a soil temperature effect. Significant soil treatment differences exist and as such the null hypothesis can be rejected. However, the inconsistency of the results suggests soil temperature effects are small (for the gas exchange and fluorescence variables). 92 8 Conclusion 8.2. VARIABLE RESULTS AND INTERACTIONS In response to the return of favourable growing conditions upon removal from storage and subsequent outplanting, all the seedlings appeared to follow a common recovery period (4-7 days duration), dependent on the length of storage. Stomatal resistance was initially high and decreased over the 4 to 7 days to an apparent optimum steady state level. The relatively rapid stomatal changes may be in response to the return of light (and photosynthesis), although normal stomatal light response is considerably more rapid. The fluorescence variable Fy/Fp also recovered significantly during this period from very low levels indicating poor photosynthetic efficiency. The increase in Fy/Fp ratio may be similar to photosynthetic decline and recovery noted in other conifers after subfreezing winter conditions. After the recovery period, Ci/Ca ratio suggests seedlings maintain an optimum level of Ci through proportional changes in stomatal conductance and photosynthesis and that this level is dependent on the storage treatments and soil temperature. Net photosynthesis results show a decline after durations of 22 or 26.1 weeks or longer dependent on outplanting soil temperature and length of storage. The warmer soil temperature ( l io© appears to induce the decline at earlier durations. An optimum period of storage for net photosynthesis is suggested, 13.7 to 22 weeks, with 17.9 weeks an apparent maximum (see Figure 4.2 and 4.5). The increase in stomatal conductance with a decrease in net photosynthesis after long term storage is evidence the decline in photosynthesis was due to inhibition of the carbon fixation process and not due to a stomatal limitation to CO2 exchange. This hypothesis is further supported by fluorometer readings (Fy/Fp) which show a decrease in photosynthetic efficiency with increasing storage over 22 weeks (at day 5). Changes in seedling photosynthetic efficiency (Fy/Fp) were shown to parallel changes in photosynthesis (see Figure 6.5). This also supports the view that the two variables are 93 8 Conclusion directly related. Long term storage appears to have reduced photosynthesis through photoinhibition induced by freezing injury to the photosynthetic apparatus possibly due to loss of cold hardiness while in freezer storage. The resulting inhibition of electron flow from PSII will result in lower levels of ATP and NADPH which in turn will reduce the rate of carbon fixation and RUDP regeneration. A low level of photosynthesis after 9.6 weeks storage was also noted which also may be due to nonstomatal limitations to carbon fixation. However, the reasons for this seedling photosynthetic decline are unknown. The increase in seedling stomatal conductance at long term storage (26.1 and 30.6 weeks), may be in response to the concomitant decline in net photosynthesis. Inhibition of electron flow leading to reduced photosynthesis may be mechanistically linked to stomatal response such that the stomatal conductance increase is in response to the low photosynthetic capacity. However, if stomatal conductance was linked directly to changes in photosynthetic capacity at the chloroplast level then one would expect a similar high level of conductance after 9.6 weeks storage since it appeared that these seedlings were also under nonstomatal limitations to carbon fixation. It seems apparent that if such a mechanistic link between photosynthesis and stomatal conductance exists, it is complex and involves other physiological variables. Cowan (1982) suggested such a link may be hormonal in nature. The effects of the soil temperature treatments were at a maximum after 13.7 and 17.9 weeks storage for stomatal conductance and photosynthesis respectively. In general, soil temperature effects were small and storage duration dependent. White spruce seedling cold soil temperature stomatal conductance sensitivity was low which may have been due to cold storage temperature acclimation and/or species cold soil adaptation. Net photosynthesis cold storage effects were dependent on soil temperature suggesting that although direct soil temperature influence was not significant, interactive effects are important. Long term 94 8 Conclusion cumulative effects of small soil temperature differences in seedling photosynthesis suggest the relative availability of photosynthate for growth and development. These cumulative effects suggest soil temperature may become an important determinant of photosynthate availability at nonoptimal storage durations. The importance of this long term cumulative photosynthetic effect in the field and over longer periods of growth is unknown, but undoubtedly any reduction in photosynthate will decrease growth potential. Photosynthate availability has been directly related to root growth in Pseudotsuga menziessii and Picea sitchensis seedlings (Van Den Driessche 1987). Root growth has also been related to levels of cold hardiness and bud dormancy (Burr et al. 1989). The period before bud break has been shown in Pseudotsuga menziesii and Picea engelmannii as a period of rapid root growth and is related to competition with shoot growth for photosynthate. It is unlikely, in this study, that the reduction in root growth was related to bud dormancy (TBB) after storage duration treatments where photosynthesis is optimal (13.7 to 22 weeks). However, after durations where the seedlings have been stressed or injured resulting in a low level of photosynthesis upon outplanting, then the decrease in the days to TBB and therefore earlier onset of shoot development will increase root to shoot competition and affect root development. In this study root growth potential was soil temperature and storage duration dependent. Maximum new root growth was after 17.9 weeks storage, at the lloC soil temperature. The colder soil temperature results (3°C and 70© indicate maximum root growth occurs with no storage treatment and suggests root growth potential at high soil temperatures does not represent the root growth potential of cold soil conditions (Harper et al. 1990). It is surprising that with this large difference in root activity between the soil temperatures there was not also a similar difference in stomatal conductance. This further 95 8 Conclusion supports the conclusion that in white spruce, the processes of photosynthesis and conductance have a low sensitivity to the soil temperature used here (3 to HOC). Apparent stomatal conductance soil temperature differences may only be due to temperature related water viscosity changes and not due to root activity. 8.3. CONCLUSIONS The results of this growth chamber study have shown white spruce seedlings are sensitive to varying lengths of commercial freezer storage dependent on the outplanting soil temperature. Further trials are necessary to determine if these effects are replicated under field conditions. The results of this study suggest the following points: 1) The range of storage of white spruce which appears to minimize deleterious effects is from 13.7 to 22 weeks with an optimum of 17.9 weeks. 2) Storage over 22 weeks can seriously affect seedling vigor through loss of photosynthetic capacity and root growth. 3) Short term storage (9.6 weeks) can also result in low rates of photosynthesis upon outplanting. 4) Soil temperature effects on gas exchange variables are in general small, although after nonoptimal storage durations long term cumulative effects of cold soils on photosynthesis may be important. The interaction of soil temperature with storage duration observed (seedling photosynthesis and root growth) suggests care must be taken in interpreting soil temperature results. It is possible that white spruce soil temperature sensitivity, in terms of photosynthesis, may change throughout the year in response to physiological and environmental developments. 96 8 Conclusion The importance of healthy, vigorous planting stock cannot be overemphasized. Planting white spruce seedlings which have been stored for more than 5.5 months (22 weeks) may reduce the plantation performance through decreased photosynthetic capacity, root growth and general vigor. Foresters should strive to avoid lengthy storage periods as well as to maximize soil temperature. The use of site preparation techniques which elevate root zone temperatures to at least HOC will greatly increase root growth at any storage duration. The use of long periods of storage may be unavoidable if seedlings are to be planted in northerly areas where late snow cover and frozen soils may be a problem. 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(C) 9 . 6 1 3 . 7 1 7 . 9 22 2 6 . 1 3 0 . 6 O v e r a l l Mean 1 3 3. .999 4.087 4 .173 4.443 3.556 2 .292 4.597 7 3 .986 4.192 4 .259 4.069 3.725 2 .562 4.64 11 4 .362 4.577 4 .287 3.765 3.421 2 .816 4.467 4 3 3 .522 4.842 5 .043 4.662 3 .234 4.727 7 4 .374 4. 615 5 .118 5.099 3 .396 4.774 11 4 .104 4. 911 4 .996 4.811 3 .732 4.594 7 3 3 .055 4.544 5.07 4.655 3.707 3 .596 4.575 7 3 .233 4.594 4 .986 4.241 4.298 3 . 633 4.598 11 3 .706 4. 993 4 .777 4.278 4.252 4 .021 4.596 10 3 3 .801 4.307 4 .924 4.268 4.381 3 .861 4.768 7 4 .384 4.399 4 .926 4.509 4.617 3 .563 4.847 11 4 .24 4.68 4 .798 4.784 4.834 3 .841 4.858 13 3 3 .786 4.338 4 .193 4.413 3 .962 4.604 7 3 .758 4.232 4 . 614 4.502 4 .162 4.7 11 4 .2 4.322 4 .73 4. 685 4 .793 4.89 16 3 3 . 692 4.495 4 .233 3.776 4.085 4 .202 4.312 7 3 . 95 4.083 4 .357 4.513 5.081 4.36 4.491 11 3 .972 4.398 4 .413 4.034 4.876 4 . 932 4.474 19 3 4 .095 4.601 4 .456 4. 667 4.265 4 .003 4.47 7 4 .117 3.985 5 .045 4.835 5.166 4 .488 4.649 11 4 .527 4.484 5 .095 4.444 4. 689 4 .776 4.666 22 3 4 .537 4.565 4 .701 4.867 4.502 4 .321 4.518 7 4 .249 4.384 1 5.41 4.772 4. 904 4 . 944 4.738 11 4 .66 4.889 6 .078 4.753 5.289 5 .368 5.034 25 3 4 .74 4 . 985 4 . 941 5.068 5 . 651 4.955 7 4 .657 4.566 6 .458 5. 64 6 .186 5.434 11 5 .643 5.04 6 .539 5.22 6 .406 5.633 28 3 5 .153 6.105 5 .674 5.94 5.558 5.89 5.574 7 5 .442 5.304 6 .392 5.994 5.802 6 .174 5.723 11 5 . 991 5.915 6 .726 5.627 5.944 6 .648 6.005 Overall Mean 4.265 4.648 5.047 4.731 4.606 4.394 4.826 105 Appendix 1. Stomatal Conductance (cm s-1) treatment means n=40 Storage Duration (weeks) Day Soil 0 9.6 13.7 17.9 22 26.1 30.6 O v e r a l l Temp. (C) Mean 1 3 0. .136 0.143 0. 164 0.125 0.159 0 .139 0.136 7 0. .115 0.187 0. 148 0.114 0.148 0.13 0.135 11 0. .147 0.284 0. 146 0.118 0.135 0 .157 0.159 4 3 0. .187 0.247 C 1.24 0.203 0 .269 0.216 7 0. .185 0.328 0. 241 0.196 0 .266 0.224 11 0. .281 0.274 0.24 0.224 0 .295 0.242 7 3 0. .214 0.21 0. 265 0.248 0.329 0 .314 0.255 7 0. .251 0.25 0. 284 0.246 0.346 0 .318 0.272 11 0. .236 0.251 0. 298 0.255 0.343 0 .347 0.285 10 3 0. .236 0.212 0. 257 0.225 0.31 0 .307 0.257 7 0. .266 0.242 0. 294 0.233 0.37 0 .328 0.297 11 0. .258 0.302 0. 287 0.265 0.316 0 .331 0.316 13 3 0. .215 0.245 0. 268 0.307 0 .322 0.257 7 0. .243 0.285 0. 277 0.32 0 .301 0.277 11 0. .235 0.298 0. 272 0.307 1 D.34 0.284 16 3 0. ,234 0.293 0. 267 0.252 0.288 0 .299 0.259 7 0. .26 0.302 0. 289 0.248 0.331 0 .321 0.277 11 0. .275 0.314 0. 314 0.264 0.322 0 .339 0.3 19 3 0, .238 0.269 o. 292 0.269 0.352 0 .349 0.286 7 0. .287 0.247 0. 336 0.282 0.358 0 .343 0.309 11 0. .286 0.266 0. 332 0.284 0.345 1 D.38 0.322 22 3 0, .286 0.277 0. 262 0.285 0.329 0 .348 0.311 7 0. .286 0.289 0. 287 0.322 0.33 0 .351 0.335 11 0. .293 0.328 0. 296 0.312 0.334 0 .378 0.35 25 3 0 .276 0.27 0. ,289 0.273 0 .396 0.326 7 0 .298 0.326 0. ,303 0.316 0.4 0.372 11 0 .303 0.322 0. ,304 0.355 0 .426 0.374 28 3 0 .302 0.267 0. ,308 0.293 0.372 0 .355 0.307 7 0 .357 0.323 0. ,341 0.31 0.361 0 .371 0.344 11 0 .316 0.341 0. ,358 0.368 0.424 0 .358 0.352 Overall Mean 0. 25 0.273 0. 275 0.255 0.314 0. .319 0.281 106 Appendix 1. Standard Deviations for Net Photosynthesis (umol nr2 s-i),treatments, n=40 Storage Duration (weeks) D a y S o i l 0 T e m p . (C) 9 . 6 1 3 . 7 1 7 . 9 22 2 6 . 1 3 0 . 6 1 3 0.734 1.049 1.016 1.007 1.004 0.861 7 0. 992 1.268 0.818 1.197 0.877 0.87 11 0. 955 1.098 0.888 1.016 1.071 0. 935 4 3 0. 938 0.753 0.703 1.204 0.721 7 0.792 0.758 0. 91 1.082 1.106 11 1.205 0.853 0.828 0. 979 1.027 7 3 1.197 1.01 0.726 0.925 0.959 1.15 7 1.665 0.82 0. 972 0. 96 0.875 1.092 11 1.397 0.673 1.026 0.783 1.055 1.236 10 3 0.827 0.752 0.688 1.157 1.406 0.943 7 1.191 0.729 0.916 0. 995 1.128 0. 932 11 1.178 0.926 0.964 0.835 1.109 0.798 13 3 1.021 0.837 0.73 1.22 1. 617 7 1.242 0.741 0.864 0.836 0.874 11 1.138 0.584 0.901 1.043 0. 92 16 3 0.7513 0.9996 0. 6232 1.0136 1.0449 0.832 7 0.9226 0.9877 0.8684 0.9193 1.1627 0.7622 11 . 1.2188 1.0144 0.9481 1.017 1.1039 1.0302 19 3 0.7999 0.9733 0.7853 1.2422 1.0003 0.9502 7 0.9041 0.6746 1.2443 1.4364 1.2068 1.3792 11 1.23 0.79 1.0052 0.9456 1.3266 1.1851 22 3 0.822 1.054 0.984 1.408 0.97 1.003 7 1.018 0.789 1.18 1.505 1.205 1.363 11 1.164 1.145 1.342 1.022 1.341 1.378 25 3 0.8014 1.0108 1.1175 1.6833 1.3026 7 1.0027 0.7653 1.9284 1.8627 1.1841 11 1.303 0.9741 1.6347 1.2078 1.9777 28 3 0.8857 1.0656 1.338 1.6656 1.396 1.6785 7 1.2151 1.0646 1.7389 1.6913 1.1124 1.4956 11 1.4357 1.6667 1.3294 1.4691 1. 97 2.4923 107 Appendix 1. Standard Deviations for Stomatal Conductance (cm s-1) treatments,n=40 Storage Duration (weeks) D a y S o i l 0 T e m p . (C) 9.6 13.7 17.9 22 26.1 30.6 1 3 0.12 0.041 0.041 0.043 0.036 0.038 7 0.035 0.043 0.034 0.046 0.034 0.033 11 0.097 0.063 0.034 0.04 0.034 0.047 4 3 0.114 0.075 0.037 0.05 0.063 7 0.071 0.071 0.05 0.063 0.049 11 0.302 0.053 0.06 0.052 0.059 7 3 0.051 0.047 0.036 0.067 0.056 0.068 7 0.039 0.041 0.052 0.076 0.054 0.056 11 0.07 0.049 0.075 0.05 0.072 0.063 10 3 0.053 0.045 0.035 0.058 0.055 0.064 7 0.042 0.04 0.071 0.066 0.057 0.06 11 0.064 0.07 0.068 0.051 0.062 0.062 13. 3 0.053 0.046 0.039 0.069 0.066 7 0.034 0.063 0.075 0.06 0.055 11 0.06 0.059 0.066 0.074 0.061 16 3 0.0522 0.0535 0.0397 0.0695 0.0651 0.0622 7 0.0359 0.0473 0.0658 0.0725 0.0612 0.0552 11 0.1111 0.0566 0.0747 0.0551 0.0776 0.0665 19 3 0.058 0.0597 0.0492 0.0731 0.0768 0.069 7 0.054 0.046 0.0681 0.0781 0.0636 0.0657 11 0.075 0.0501 0.0745 0.0633 0.0855 0.0745 22 3 0.063 0.059 0.057 0.074 0.053 0.074 7 0.05 0.049 0.059 0.085 0.061 0.078 11 0.066 0.057 0.055 0.067 0.071 0.091 25 3 0.0667 0.0634 0.0538 0.0763 0.085 7 0.0572 0.0636 0.0686 0.0837 0.0889 11 0.0792 0.0657 0.0702 0.075 0.1083 28 3 0.0583 0.055 0.0592 0.076 0.0873 0.0851 7 0.0758 0.045 0.0746 0.0798 0.0591 0.0836 11 0.0822 0.053 0.0867 0.0772 0.108 0.114 108 Appendix 1. Analysis of Variance - Net Photosynthesis Day Source Transformation Sum-of-Squares DF Mean-Square F-Ratio P Soil Temperature 1.371 Storage Duration 264.015 2 0.6855 0.5862 0.5577 5 52.803 45.153 0 Interaction 23.393 10 2.3393 2.0004 0.0372 Experimental Error 170.736 146 1.1694 Sampling Error 425.448 492 0.8647 Soil Temperature 9.552 Storage Duration 195.369 2 4.776 3.905 0.023 4 48.842 39.932 0 Interaction 19.402 2.425 1. 983 0.054 Experimental Error 149.223 122 1.223 Sampling Error 286.806 411 0.698 7 Soil Temperature 1.693 cosine(1.25) Storage Duration 44.931 2 0.8464 2.2468 0.1092 5 8.9862 23.8544 0 Interaction 8.93 10 0.893 2.3706 0.0122 Experimental Error 58.767 156 0.3767 Sampling Error 173.134 522 0.3317 10 Soil Temperature 2.653 2 cosine(1.15) Storage Duration 47.471 5 1.3262 3.1224 0.0469 9.4942 22.352 0 Interaction 8.033 10 0.8033 1.8913 0.0501 Experimental Error 66.262 156 0.4248 Sampling Error 170.482 522 0.3266 13 Soil Temperature 3.861 cosine(1.17) Storage Duration 19.523 2 1.9304 4.0703 0.0192 4 4.8806 10.2907 0 Interaction 3.785 8 0.4731 0.9975 0.4411 Experimental Error 62.604 132 0.4743 Sampling Error 150.132 441 0.3404 109 Appendix 1. Analysis of Variance - Net Photosynthesis Day Source Transformation Sum-of-Squares OF Mean-Square F-Ratio P 16 Soil Temperature 17.167 Storage Duration 50.09 2 8.584 9.21 0 5 10.018 10.749 0 Interaction 25.612 10 2.561 2.748 0.004 Experimental Error 137 Sampling Error 351.556 147 0.932 495 0.7102 19 Soil Temperature 11.413 2 5.707 6.72 0.002 Storage Duration 29.764 .5 5.953 7.01 0 Interaction 27.095 10 2.709 3.191 0.001 Experimental Error 120.586 142 0.849 Sampling Error 408.461 480 0.851 22 Soil Temperature 34.398 2 17.199 10.436 0 Storage Duration 56.475 5 11.295 6.853 0 Interaction 32.904 10 3.29 1.997 0.037 Experimental Error 243.917 148 1.648 Sampling Error 511.476 498 1.027 25 Soil Temperature 1.807 cosine(1.27) Storage Duration 12.887 Interaction 5.672 2 0.9035 2.0499 0.1329 4 3.2217 7.3093 0 8 0.709 1.6085 0.1284 Experimental Error 56.858 129 0.4408 Sampling Error 169.869 432 0.3932 28 Soil Temperature 4.259 cosine(0.725) Storage Duration 8.641 2 2.1295 5.2323 0.0063 5 1.7282 4.2462 0.0012 Interaction 10.051 10 1.0051 2.4695 0.009 Experimental Error 63.4 9 Sampling Error . 187.198 156 0.407 522 0.3586 110 Appendix 1. Analysis of Variance - Stomatal Conductance Day S o u r c e T r a n s f o r m a t i o n S u m - o f - S q u a r e s DF M e a n - S q u a r e F - R a t i o P Soil Temperature 0.058 2 Storage Duration 0.551 5 0.029 11.999 0 0.11 45.271 0 Interaction 0.388 10 0.039 15.959 Experimental Error 0.372 Sampling Error 0.772 153 0.0024 513 0.0015 sine(2.65) Soil Temperature 0.1375 Storage Duration 3.5523 2 0.0687 2.8567 0.0611 4 0.8881 36.9138 0 Interaction 0.514 8 0.0643 2.6708 0.0095 Experimental Error 3.1035 129 0.0241 Sampling Error 7.2961 432 0.0169 sine(2.0) Soil Temperature 0.198 Storage Duration 3.3556 2 0.099 7.9593 0.0005 5 0.6711 53.9517 0 Interaction 0.1554 10 0.0155 1.2489 0.2643 Experimental Error 1.9406 156 0.0124 Sampling Error 4.5342 522 0.0087 10 Soil Temperature 0.134 Storage Duration 0.826 2 0.067 17.597 0 5 0.165 43.407 0 Interaction 0.196 10 0.02 5.147 Experimental Error 0.533 140 0.0038 Sampling Error 1.039 474 0.0022 13 Soil Temperature 0.036 2 0.018 3.561 0.031 Storage Duration 0.594 4 0.148 29.131 0 Interaction 0.076 0.01 1.875 0.069 Experimental Error 0.673 132 0.0051 Sampling Error 1.398 441 0.0032 111 Appendix 1. Analysis of Variance - Stomatal Conductance Day Source Transformation Sum-of-Squares DF Mean-Square F-Ratio P 16 Soil Temperature 0.118 Storage Duration 0.436 2 0.059 10.727 0 5 0.087 15.864 0 Interaction 0.041 10 0.004 0.746 0.68 Experimental Error 0.857 156 0.0055 Sampling Error 1.993 522 0.0038 19 Soil Temperature 0.045 2 Storage Duration 1.055 5 0.023 3.545 0.031 0.211 33.097 0 Interaction 0.104 10 0.01 1.627 0.103 Experimental Error 0.994 156 0.0064 Sampling Error 2.061 522 0.004 22 Soil Temperature 0.073 2 Storage Duration 0.515 5 0.036 . 6.163 0.003 0.103 17.423 0 Interaction 0.052 10 0.005 0.873 0.56 Experimental Error 0.923 156 0.0059 Sampling Error 2.073 522 0.004 25 Soil Temperature 0.157 2 Storage Duration 1.073 4 0.079 10.262 0.268 35.052 Interaction 0.068 8 0.009 1.114 0.358 Experimental Error 0.988 129 0.0077 Sampling Error 2.222 432 0.0051 28 Soil Temperature 0.228 2 Storage Duration 0.473 5 0.114 13.717 0.095 11.36 Interaction 0.186 10 0.019 2.236 0.018 Experimental Error 1.299 156 0.0083 Sampling Error 2.797 522 0.0054 112 Appendix 2. LI-COR 6200 System Equations from LI-6200 Technical Reference Manual, September 1987 Parameter Units Description Ta C Chamber air temperature T[ C Needle temperature Fd umol s'1 Flow through desiccant e mb Vapour pressure of air E mol m-2 s-l Transpiration rate de/dt mbs-l Rate of change in e es() mb Saturation vapour pressure function gbwo m ° l m " 2 s _ 1 One-sided boundary layer conductance K Stomatal ratio Kabs Absorption coefficient C ul 1_1 CO2 concentration dC/dt 1^ l-l s-l Rate of CO2 change P mb Atmospheric pressure S cm2 Leaf area Vt cm3 Total volume V g cm3 IRGA volume gtc mol m-2 s-l Total conductance to CO2 gs mol m-2 s-l Stomatal conductance to water A umol rn-2 s-l Net photosynthetic rate G Correction factor relating chamber ac/at with I R G A ac/at Transpiration (E) Fde Kabs Fd 3e + ( ) (V t - — V g ) -100 P 8.314 (Ta + 273) F x dt E - : :  e S( 1- -) P 113 Appendix 2. Stomatal Conductance (gs) gs = es (T/) - e (K2 + 1) E ( P - ) Photosynthesis (A) FdeC PGVt 3C A = X — - C E G 100 S P 8.314 (Ta + 273) S 3t Intercellular C 0 2 (Ci) E ( etc - — ) Ca - A 2 Ci = E ( gtc + ) 2 Unit Conversion Conductance from mol m-2 s-1 to cm s-1 multiply by 8.314 (Ta + 273) 114 

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