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Towards locating and quantifying respiration in the soil and in the plant using a novel 18-oxygen labelling… Chillakuru, Dheeraj R. 2009

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   TOWARDS LOCATING AND QUANTIFYING RESPIRATION IN THE SOIL  AND IN THE PLANT USING A NOVEL 18-OXYGEN LABELLING  TECHNIQUE   by  DHEERAJ R. CHILLAKURU  M.Phil., Madras Christian College, India, 2004       A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR  THE DEGREE OF  MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES  (Forestry)    THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)    APRIL 2009  © Dheeraj R. Chillakuru, 2009  Abstract Respiration is typically measured by monitoring either carbon dioxide release or oxygen consumption. An alternative approach, used here, is to monitor the product of oxygen consumption, namely water. Although the metabolic water produced will, like carbon dioxide, ultimately diffuse away from mitochondria or be carried off by other processes, it will do so at a much slower rate and may therefore be captured at or near the site of respiration. In this way, the amount and within limits the location of the label provided can be known. Plant species and tissues under investigation were vacuum infiltrated with 18C>2 (99.97%) and kept devoid of light for a 30 minute period to prevent photosynthesis and allow sufficient time for the label to be incorporated. Labelled metabolic water was easily detected and recovered against a large background of normal tissue water, in Medicago sativa L. sprouts. We investigated four other species, representing different functional groups: a C3 plant {Helianthus giganteus L.), a CAM plant (Crassula ovata (Miller) Druce), a desert plant {Disocactus flagelliformis (L.) Barthlott), and a tree (Populus trichocarpa Torr & Gray). The <5180 values of tissue water ranged from +7.93%o to +216%o. Higher values were associated with tissues with high respiration rates per unit water content. In some experiments, labelling was followed by immersing the tissues in unlabelled water to monitor exchange (i.e. leakage) over periods of 5 to 60 minutes. Exchange of labelled water was more rapid during the first 5 minutes (X = 63.4% complete over all species, tissues and treatments) than over the 5-10 min interval, suggesting the existence of two pools of water available for exchange; namely apoplastic and symplastic water. Attempts to modify aquaporin activity failed to influence exchange. A model system was constructed to test whether high rates of exchange could also be expected in a soil environment, using Helianthus. In conclusion, the movement of metabolic water across cellular membranes was very rapid, severely limiting the utility of 1  O labelling for pin-pointing sites of respiratory activity. ii Table of Contents Abstract u Table of Contents Hi List of Tables v List of Figures vi List of Illustrations vii List of Abbreviations viii Acknowledgements ix Dedication x 1 INTRODUCTION AND OBJECTIVES 1 1.1 Soil respiration 1 1.1,1 Autotrophic vs. heterotrophic respiration 2 1.2 The oxygen isotope system 4 1.3 Natural variation in isotopic composition of water 5 1.3.1 Source 6 1.3.2 Transpiration 7 1.3.3 Mixing 7 1.3.3.1 Back diffusion of water 7 1.3.3.2 Phloem transport 8 1.3.3.3 Fidelity of stem water 8 1.4 Metabolic water 9 1.4.1 Role of aquaporins in water movement across membranes 10 1.5 OBJECTIVES 12 2 MATERIALS AND METHODS 13 2.1 Plant material 13 2.2 Source of plant material 13 2.3 Prior to label incorporation 13 2.4 Labelling of plant parts 14 2.5 Exchange of labelled water by plant parts 15 2.6 pH and inhibitor experiments 16 2.7 Diurnal study and transpiration experiment 16 2.8 Closed circulating system 17 2.8.1 Experimental design 17 2.8.2 Mechanism 18 2.8.3 Calculation of root respiration rate in the model system 20 2.9 Cryogenic extraction from a bulk sample 21 2.9.1 Mechanism 22 2.9.2 Extraction of water 22 2.10 Isotope analysis 24 2.11 Calculation of respiration rate by * 8 0 labelling of metabolic water 24 2.12 Calculation of % exchange 26 2.13 Statistical analysis 27 3 RESULTS 28 3.1 1802 uptake (respiration rate) 28 3.2 Exchange of labelled water by tissues and species 31 iii 3.3 Role of aquaporins 38 3.3.1 Effect of mercuric chloride 38 3.3.2 Effect of pH 39 3.3.3 Diurnal studies 41 3.3.4 Transpiration experiment 41 3.4 Closed circulating system 43 4 DISCUSSION 53 4.1 "Voila", clear detection of metabolic water 54 4.1.1 Factors accountable for the variations in respiration rates 57 4.2 Exchange of metabolic water in "untreated" tissues 57 4.3 Exchange of metabolic water 59 4.4 Role of aquaporins remains elusive 59 4.4.1 Issues related to exchange rates 61 5 CONCLUSION 64 5.1 Future applications for studying water relations and the role of metabolic water 64 6 LITERATURE CITED 65 7 APPENDICES 77 Appendix A 77 7.1 Calculation of percent exchange 77 Appendix B 79 7.2 Application of O labelling under field conditions 79 7.2.1 Global scale 79 7.2.2 Site specific: relevant to any field study 80 7.3 Field study of Pseudotsuga menziesii 80 iv List of Tables Table 1. Initial and final SO values of water extracted from intact Medicago sprouts.. 28 Table 2. Respiration rates of study species 29 Table 3. Respiration rates of Helianthus tissues 30 Table 4. Respiration rates of Populus roots 31 Table 5. <5180 values and calculated % exchange of water in "untreated" Helianthus tissues 46 Table 6. <S180 values and calculated % exchange of water in roots of Populus and Disocactus and leaves of Crassula 48 Table 7. dnO values and calculated % exchange of water in "treated" Helianthus tissues 49 Table 8. ^180 values and calculated % exchange of water in Populus roots 50 Table 9. Label distribution in water from isolated parts of Helianthus across different media in a model system 52 v List of Figures Figure 1. Example of annotated computer trace of output from the SI 02 O2 sensor 20 Figure 2. Schematic diagram of a single detatchable cryogenic distillation unit on the vacuum-preparation line 21 Figure 3. Decay in 8 values of water from intact Medicago sprouts during exchange with DDW 32 Figure 4. Total % exchange of metabolic water in "untreated" tissues of Helianthus 35 Figure 5. Total % exchange in roots of Populus and Disocactus and leaves of Crassula 37 Figure 6. Total % exchange in "treated" tissues of Helianthus 40 Figure 7. Total % exchange in "untreated" and "treated" Populus roots 42 1 o Figure 8. 8 Ofmai values of transpiring Helianthus shoots and roots in a model system. 45 Figure 9. Natural variation in the isotopic composition of water from Pseudotsuga menziesii 81 vi List of Illustrations Illustration 1. Components of labelling apparatus 14 Illustration 2. Steps involved in exchange of labelled metabolic water against background water 15 Illustration 3. A model system to assess the utility of O2 in exploring spatial patterns of belowground respiration 19 vii List of Abbreviations Carbon C Carbon dioxide CO2 Deionized Distilled Water DDW Global Meteoric Water Line GMWL Isotope Ratio Mass Spectrometer IRMS Isotopic composition (%o) where subscript indicates the source of water analyzed Subscript Labelled metabolic water 180-H20 Liquid Nitrogen LN2 Local Meteoric Water Line LMWL Mercuric chloride HgCL; Oxygen 0 2 Stable oxygen isotope composition (%o) <5180 Temperature sensitivity of respiration Q10 Vienna-Standard Mean Ocean Water V-SMOW Vienna-Standard Light Antarctic Precipitation V-SLAP 180/160 isotope ratio R viii Acknowledgements I profusely express my gratitude to my academic supervisor Dr. Robert Guy, for his panache guidance and encouragement throughout; Dr. Andrew Black and Dr. Christopher Chanway, for serving on my committee; Dr. Mahesh Upadhyaya for serving as my external examiner; My friends and colleagues: Amer Khan, Denise Brooks, Limin Liao, Raju Soolanayakanahally, Shofiul Azam, Tyler Abbey, and Virginie Pointeau. And to David Kaplan, Manager of UBC Greenhouse: Thank you for all your help and support. Funding was provided by an NSERC Discovery Grant to RDG. ix To My Professor: Dr. Robert Dean Guy & My Wife: Thangam Moments are born out of Opportunities. Thank you for such a fantastic opportunity. x 1 INTRODUCTION AND OBJECTIVES 1.1 Soil respiration Soil respiration (or belowground respiration) is the process by which carbon dioxide (CO2) produced by plant roots and soil microorganisms is released at the soil surface (Witkamp and Frank 1969; Rochette et al. 1991; Akinmeri et al. 1999). Soil respiration is influenced by several factors such as soil temperature, soil water, fine roots, microbial activity and soil physical and chemical properties (Tang et al. 2005). Temperature is often the most influential factor but the exact relationship between respiration and temperature is still unclear. Soil respiration is often modeled as either a simple Qio function or as a step relationship based on temperature response curves. Several studies (Raich and Potter 1995; Akinremi et al. 1999; Rustad et al. 2000) report that soil moisture is also an important variable influencing respiration, second in importance to temperature. Soil respiration consists of both autotrophic (plant-based respiration supported by recent photosynthate or mobilized reserves) and heterotrophic (respiration by organisms consuming or decaying soil organic matter) components. Autotrophic respiration is variously defined as either respiration by live roots alone, or by roots plus their associated microorganisms, including rhizosphere bacteria and mycorrhizal fungi (Hanson et al. 2000; Scott-Denton et al. 2006). Regardless of the definition used, the distinction between autotrophic and heterotrophic respiration is not always clear as they may not be independent. Interactions of the root with soil biota are an important unknown (Eissenstat and Yanai 1997). Dead roots (supporting heterotrophic respiration) can be an important carbon pool accounting for up to one-third of total belowground respiration in temperate soils (Bowden et al. 1993). 1 1.1.1 Autotrophic vs. heterotrophic respiration Most studies have assigned roughly equal contributions to autotrophic and heterotrophic components of soil respiration (Ryan 1991; Trumbore and Torn 2003; Scott-Denton et al. 2006). For example, in boreal pine forest, Hogberg et al. (2001) reported a value of 56% whereas Scott-Denton et al. (2006) revealed a value of 44% for the rhizospheric component. In Scot's pine, Bhupinderpal-Singh et al. (2003) reported that heterotrophic respiration increased from 56 to 67% during the first year after tree girdling. An understanding of belowground respiration and its component processes is hampered by a lack of measurement techniques (Eissenstat and Yanai 1997). Total respiration can be measured using the simple static chamber method (Edward 1975), open/closed chamber methods using gas chromatography (Rochette et al. 1991) or infrared gas analysis (IRGA), isotopic tracers (Andrews et al. 1999), and techniques based on concentration gradients and eddy correlation (Chahuneau et al. 1989). As many have pointed out (Philipson et al. 1975; Rouhier et al. 1996; Thierron and Landelont 1996), the quantification of the contribution of roots to total respiration is challenging in most circumstances. Component integration, root exclusion and isotopic methods are the three categories recognized by Hanson et al. (2000) to separate autotrophic and heterotrophic respiration. Component integration is a relatively simple technique wherein roots and litter are typically removed from soil samples and respiration rates of each can be measured. If in situ measurements are not possible to estimate or model respiration, measuring soil bulk density and root mass by species (if species and site-specific respiration values are known) is practical. Root exclusion experiments compare respiration measurements taken from soil with and without living roots, involving 2 trenching (measuring soil respiration without roots on relatively undisturbed soil) and physically excluding new root growth into sample plots. Both techniques cause disturbance to the natural matrix. The isotopic method involves the use of stable isotopes. For example, stable isotopes of carbon can be used to partition soil respiration components in situ, thus avoiding disturbance of the root-soil system. One can distinguish between root-derived carbon (root respiration) and soil-derived carbon (soil organic matter) by examining the differences in isotopic composition of evolved CO2. Estimation of root respiration can be made using isotopic tracing techniques by pulse labelling (e.g. Howarth et al. 1994; Liljeroth et al. 1994). When restricted to the lab or greenhouse, these studies do not reflect conditions found in the natural environment (Andrews et al. 1999). Under field conditions, Rochette et al. (1999) used differences in 13C natural abundance to separate belowground respiration into plant and soil components. Since different methods are used at different sites, it is difficult to compare data. A comprehensive study contrasting all these techniques has not been reported. To date, all approaches using tracers have relied on isotopes of carbon to estimate respiration rates or to separate heterotrophic and autotrophic contributions. Necessarily, the label of interest is detected in CO2 released into the air. Although the label can be applied to known ecosystem components (typically the aerial portions of plants) it is not necessarily released from these components, and the point of release, in time or space, is not easily determined. If the label is released soon after provision, it is assumed to originate from autotrophic respiration; if late, then perhaps heterotrophic. The "specific activity" of the carbon substrate used in respiration is not well-constrained and the overall information content is low. An alternative approach, proposed here, is to instead label the oxygen consumed in respiration, yielding metabolic water. In this way, the amount and 3 potentially the location of the label provided (as 1802) can be precisely known. The exact stoichiometry of O2 uptake relative to CO2 release depends on the substrate utilized, but must approach unity overall. Although the 180-H20 produced will, like C02, ultimately diffuse away from mitochondria or be carried off by other processes, it will do so at a much slower rate than C02 and may therefore be captured at or near the site of respiration. 1.2 The oxygen isotope system The element oxygen comprises about 21% of the atmosphere, 89% of water and approximately 51% of the inorganic crust (Urey 1948). 1 60, 170 and 1 80, are the three 1 ft • 1R 17 stable isotopes of oxygen. O is by far the most abundant, followed by O and O, 1 R respectively. The natural abundance of O is 0.20%. Atmospheric oxygen is not in isotopic equilibrium with either ocean water or CO2. The value of an isotope ratio in a sample relative to a reference is expressed using delta {§) notation. For reporting purposes, the '<?' value is commonly multiplied by 1000 and reported in units per mil (%0). The <5180 of atmospheric oxygen is determined by isotope fractionation associated with oxygen production and consumption by terrestrial and marine photosynthesis, aerobic respiration and photorespiration and is a relatively constant +23.5%o relative to standard mean ocean water (Dole 1935; Kroopnick and Craig 1972; Guy et al. 1993). Insight into chemical, kinetic, thermodynamic, geologic, climatic and physical processes in the earth's surface is possible with diagnostic tools such as the isotopic ratios of oxygen. For example, recently it was discovered that <S180/<5170 values of dissolved oxygen can be used as a process indicator to estimate global marine oxygen production 4 (Luz and Barkan 2000) and global paleoproductivity. Determination of the stable isotope composition of water (both 2H and 180) provides a significant range of applications in plant physiology and plant ecology. For example, in understanding the isotopic signatures of H, O or both, evaluation of the proportion of recent precipitation to ground water used by the plant (White et al. 1985; Flanagan et al. 1992), and plant sap flow measurements (transpiration, water-use, dynamics within a plant, etc.) (Leaney et al. 1985; Yakir et al. 1990; Flanagan and Ehleringer 1991) are possible and could be further analyzed. In future, several stable isotopes will be used as markers in the integration of ecological processes (Griffiths 1991). 1.3 Natural variation in isotopic composition of water An indication of a range of hydrological processes and plant functions is given by the distribution of stable isotopes of water within the soil-plant-atmosphere continuum (Burgess et al. 2000). Measurement of <S180 is one of the precise approaches for studying the origin of water (Hardegree et al. 1995; Brunei et al. 1997). This type of information can be helpful in the identification of different water resources and investigation of their availability to plants (Smith et al. 1997). Variation in the oxygen isotope ratios in plant water results from the following: (1) variation in <5180 of source water taken up by the plant, (2) variation in the enrichment of leaf water during transpiration, and (3) mixing of enriched leaf water and unenriched source water within the plant (Roden et al. 2000; Barbour et al. 2000). 5 1.3.1 Source Soil depth profiles (Mathieu and Baraic 1996a), enrichment in stems (Dawson and Ehleringer 1993) and seasonal effects (Dawson and Pate 1996) contribute to variation in the isotopic composition of source water. The ultimate source of water for plant roots is precipitation which includes rainfall (recent precipitation) during summer, soil moisture and deep ground-water, which may have different isotopic signatures (White et al. 1985). The 180 content of precipitation decreases as a function of temperature relative to the distance from its source (e.g., from the ocean towards land, or from the tropics to the poles). The gradual depletion of heavy isotopes in precipitation influences the soil and leaf water in the plant system. A long term average of the isotopic composition of precipitation that falls in an area is represented by the isotopic composition of ground water (Flanagan and Ehleringer 1991). Precipitation shows a seasonal cycle with high S2R or SnO values in summer and low <52H or <5180 values in winter (Dansgaard 1964). As suggested by Zimmerman et al. (1967) the downward movement of water in soil is thought to be a layer by layer process; traces of the yearly cycle of S2H values in precipitation have been noted in soil moisture profiles. Lin et al. (1999) report that the isotopic composition of soil water can vary significantly in both space and time. Though observed variation depends on the mixing of different precipitation sources (i.e. rain, fog, and snowmelt) with existing ground water, within the rhizosphere the <52H and <5180 composition of soil water will be influenced by soil characteristics as well as environmental factors which influence water movement into, through, and from the soil profile (Barnes and Allison 1988). 6 1.3.2 Transpiration Water absorbed by roots is transported with little isotopic modification to the leaves where it is lost to the atmosphere. Due to evaporation, water molecules are enriched in heavy isotopes (2H and 180) since the lighter isotopes (!H and 160) diffuse faster away from the liquid-vapor interface (Craig and Gordon 1965) until an isotopic steady-state condition is reached. All leaves have a distinct boundary layer which separates the evaporative leaf surface from the chaotic atmosphere around it. Such inherent boundary 1 o layers also influence leaf water enrichment and d O value (Cooper and De Niro 1989; Buhay et al. 1996). In a fully stagnant layer (e.g., within the leaf sub-stomatal air spaces) where water vapor transport is dominated by molecular diffusion, a maximum diffusional fractionation of about 28%o for 180-H20 (Merlivat 1978) would be expected. Though no discrimination is expected in the bulk atmosphere, an intermediate effect of about 19%o (Yakir et al. 1993) can be expected near the leaf surface where laminar flow is more likely. As suggested by Farquhar and Lloyd (1993) the effects of different boundary layers should be represented by the overall fractionation factor in the vapor pathway (i.e. from the evaporating sites in leaves to the mixed bulk atmosphere). 1.3.3 Mixing 1.3.3.1 Back diffusion of water Modeling transpiration in terms of diffusion path lengths has made use of the processes where 180 is enriched (Leaney et al. 1985; Walker et al. 1989). The principal mechanism of mixing operating in a leaf is the back diffusion of water (a relatively slow process) opposed by a flood of liquid water to the evaporating sites. Isotopic enrichment of the bulk water extracted from transpiring leaves is generally less than the predicted (Flanagan 7 et al. 1991; Farquhar et al. 1982) because the mixing of source water entering the leaf with evaporating water may be limited when transpiration is rapid. 1.3.3.2 Phloem transport A common assumption is that there is no modification to the isotopic composition of water within the plant conductive system before it reaches the leaf. However, several exceptions have been reported. For example, in Tamarix trees, between the main branch and the twigs, an enrichment of more than 2%o in <5180 values was observed in the summer but not in winter (Adar et al. 1995). This may indicate progressive transpirational enrichment of stem water, or could also be due to exchange with highly enriched leaf water flowing down in the phloem system. For example, in Tamarix trees, a 1 Q gradient of 10%o across the stem to deeper xylem sap layers that maintained the d O value of source water was observed. The isotopic composition of phloem water from photosynthesizing leaves is less well known though it might play an important role in quantitatively assessing the processes contributing to the oxygen isotope composition of plant organic material and plant cellulose (Cernusak et al. 2003). 1.3.3.3 Fidelity of stem water The isotopic composition of water in roots and stems reflects the isotope composition of water available to the plant. In one study, it was noted that there was no change with time or height for stem water <5180 values (reflecting soil water) in coniferous species, implying a constant water source (Fessenden et al. 2002). Observations of plants from pot experiments (Walker and Richardson 1991) and alfalfa in controlled glasshouse environment (Thorburn and Mensforth 1993) have showed that stem water has a 5nO 8 similar to the surrounding soil water, thereby proving that stable isotopes of water are potentially useful in the determination of the source of water to plants. As water is obtained from various depths in the soil by plant roots, inference could be made from the isotopic signature of plant water that reflects the soil-water sources (Takahashi 1998). For example, comparison of stem water isotope signatures with those of soil water at different depths was made to assess the water source of plants in tropical forests by making use of the vertical change in the isotopic ratio in water along the soil profile (Jackson et al. 1995). Except in actively transpiring leaves (Walker and Richardson 1991; Thorburn et al. 1993) it is now clear that the isotopic composition of xylem sap is typically identical to the water drawn from the soil by the roots. Gaps still exist in our knowledge on linking stable isotope ratios of plant xylem sap with plant function (Snyder and Williams 2003). 1.4 Metabolic water Natural variation in the <S180 of water in plants and other ecosystem components is clearly of great interest and may have numerous applications. The present challenge, however, is to provide sufficient 18Oto be able to detect metabolic water over-and-above this natural variation with sufficient confidence to estimate rates of respiration. Another concern is the path of water movement through plants and the rate at which recent metabolic water is swept away by this flux. Apoplastic, symplastic, and transcellular are three pathways that co-exist for water transport across living root tissue (Javot and Maurel 2002). Water has to flow radially across a chain of concentric cell layers (epidermis, cortex, endodermis, pericycle, and finally xylem) from the soil solution into the root vascular tissues (Steudle and Peterson 1998). Once water reaches the vascular tissues, it moves axially towards the aerial parts. 9 The isotopic composition of the xylem water remains unaltered from that in the soil during water transport between the root and the shoot, until it reaches tissues undergoing water loss where evaporative enrichment in the heavier isotopes of hydrogen and oxygen takes place (Lambs and Berthelot 2002). Yakir et al. (1993) considered the SnO value of water in the metabolic compartment of leaf cells in light of the reports of Yoshida and Mitzutani (1986) [where carbon dioxide was used to "probe" leaf water] and Guy et al. (1987; 1993) [who reported the dnO of the O2 from the photosynthetic splitting of water]. Yakir et al. (1993) concluded that leaf metabolism in transpiring sunflower leaves uses a water fraction the oxygen isotopic composition of which is clearly less positive than that predicted for water at the sites of evaporation. They suggested that metabolic water is closer in isotopic composition to source water than to water at evaporating sites and is about 6%o less positive than the bulk leaf water. Thus, there is evidence that chloroplast water is at least in some situations not in isotopic equilibrium with the apoplastic water of closely adjacent evaporating sites within leaves (Yakir et al. 1993). This suggests that the water in these two pools is not well mixed and that there may be a significant limitation to the rapid diffusion of water across membranes. 1.4.1 Role of aquaporins in water movement across membranes During day time, in most plants, the uptake of water from the soil ends with the loss of water vapor from the leaves into the atmosphere. The apoplastic pathway (movement of water outside of living cells, through cell walls or in xylem vessels and tracheids) and the symplastic/transcellular pathway (movement of water through living cells, including plasmodesmata) are the only means of water transport through the tissues. Water, the 10 major component of all living cells, moves via both pathways all the time. The symplast is bounded by the plasma membrane, which is an effective barrier to mass flow of water; therefore salts, sugars, proteins, and other components move across this membrane through protein channels and transporters. The general assumption until recently was that water transport into and out of the cells was by passive diffusion across cell membranes (Booth and Louis 1999). However, it is now clear that membrane channel proteins, namely aquaporins, assist water transport (Calamita et al. 1998; Stroud et al. 2003). Water can diffuse passively across the plasma membrane, but aquaporins facilitate much more transport of water. Although first discovered in animals (Gorin et al. 1984; Macey 1984; Agre et al. 1987), the existence of passageways for water across plant membranes was discussed as early as the 1960's by Ray and Dainty (Maurel et al. 1997). The first plant aquaporins, namely tonoplast intrinsic proteins (TIPs) were identified due to their location in the membrane that surrounds the protein storage vacuoles of storage parenchyma cells of bean cells (Schaeener 1998). Aquaporins are controlled (gated) by phosphate ions (Maurel et al. 1997) and pH (Townaire-Roux et al. 2003). Their sensitivity to mercury is such that it inhibits their functioning. Heymann et al. (1998) and Maggio and Joly (1995) further report that the shape of aquaporins changes, impeding water movement when exposed to mercury. Aquaporin involvement in controlling cell-to-cell water flow (especially transcellular) within tissues and the overall hydraulic conductivity of plants is a very active area of research. 11 1.5 OBJECTIVES The aim of this thesis was to determine (and to quantify) the location of respiration in the • • IS • soil and in the plant, under laboratory conditions, using a novel O labelling technique. I examined: 1. Whether sufficient 100 can be incorporated into cell water to be detected against background variation, with precision and speed. 2. The distribution of labelled metabolic water in different plant parts (reflecting location, specific respiration rates and/or water movement). 3. The rate at which metabolic water is exchanged with extracellular water under a) normal conditions, and b) conditions expected to influence aquaporin activity. 4. The prospects for separating autotrophic and heterotrophic respiration in a simple system containing transpiring plants. 12 2 MATERIALS AND METHODS 2.1 Plant material Bearing in mind that this research is more of a 'proof of concept' approach, the choice of study species and their respective parts experimented upon in vitro varied widely and consisted of intact Medicago sativa L. sprouts, Helianthus giganteus L., Populus trichocarpa Torr. & Gray, Crassula ovata (Miller) Druce, and Disocactus flagelliformis (L.) Barthlott; henceforth referred to as Medicago, Helianthus, Populus, Crassula, and Disocactus. 2.2 Source of plant material Medicago sprouts market-purchased on 28 July, 2006 and Helianthus achemes purchased on 09 April, 2006 (West Coast Seeds, Canada) were stored at 5°C in the Tree Physiology Laboratory, University of British Columbia (UBC) and used as and when required. Populus roots of local provenance grown hydroponically from 26 October, 2006 to 09 January, 2007 at UBC greenhouse, were selected. Crassula leaves were obtained from a potted plant at UBC greenhouse on 01 August, 2007. Two potted Disocactus plants were obtained on 17 October, 2007 from the UBC greenhouse. 2.3 Prior to label incorporation Helianthus seeds were stripped of their fruit coat and pre-treated with 1 % hydrogen- peroxide (H2O2) for 5 minutes. Seeds were grown in vitro on 35 x 25 cm germination paper (Anchor Seed Company, Minnesota, USA). Germination paper was always submerged overnight in tap water prior to sowing of seeds ensuring moist conditions for ideal growth. The relative humidity was 100% and the temperature ranged from 20°C- 13 24°C. Populus roots were cut under water and immersed in deionized distilled water (DDW) and processed in the laboratory. The selection of Crassula leaves was pre- determined; i.e., small intact leaves that could fit into 1 mL reacti-vials were cut at the base of the petiole to avoid minimum water loss, immersed in a beaker containing distilled water and quickly processed in the laboratory. Water loss from the leaves was minimal during processing. Disocactus shoot system was discarded and the roots were separated from the potting mix in which they were growing to the maximum extent possible and used. 2.4 Labelling of plant parts. i n i >8>wTmiiif>MMMII'W>lWIBIWMMMMBBMBMW Illustration 1. Components of labelling apparatus. 1). Reacti-vials; 2). Teflon discs; 3). Syringe and, 4). Gas sampling bag containing 18C>2. After selection, about 200 mgs of individual plant parts, irrespective of species as well as experiment, were weighed, introduced into 1 mL reacti-vials (Pierce Chemical Co., Illinois, USA) and then sealed with serum caps lined with 12 mm laminated Teflon discs (Pierce Chemical Co., Illinois, USA). Most of the air in the reacti-vials was removed with 14 a 5 cc syringe (Becton and Dickinson Co., New Jersey, USA) followed by vacuum infiltration in vitro (ranging from 20°C to 24°C) with 1 mL 180 (99.97%) (Isotech, Sigma-Aldrich, USA) (Illustration: 1). During labelling the reacti-vial, containing tissue, was kept in darkness to prevent photosynthesis. An incubation time of 30 min was sufficient for the label to be incorporated into plant tissue. 2.5 Exchange of labelled water by plant parts Illustration 2. Steps involved in exchange of labelled metabolic water against background water. 1). Intact Helianthus seedlings (2 weeks old); 2). Segregating Helianthus seedling into cotyledons, hypocotyl (1); hypocotyl (2); and roots; and 3). Exchange of metabolic water of individual tissues each in a different exchange period with background water (containing 10 mL DDW). 15 'Exchange' refers to the process whereby labelled water was lost to the media and replaced with unlabelled water when the isolated plant tissues were immersed in their respective solutions after label incorporation, be it DDW, mercuric chloride solution (to block aquaporins) or solutions with different pH (to reduce water channel activity) (Illustration 2). Exchange periods for Medicago were 5, 30, and 60 minutes; Helianthus: 5, 10, 15, 20, and 30 minutes; Populus: 5, 10, 15, 20, and 30 minutes, Crassula: 5, 10, and 30 minutes, and Disocactus: 5, 10, and 30 minutes. Additional water gained by the plant parts prior to label incorporation was kept to a minimum by sandwiching the plant tissue between two filter papers (Whatman). Exchange was always followed by plant parts being sandwiched with filter paper once again and immediately freezing the sample in liquid nitrogen (LN2) for 10 min before saving at -25 °C for cryogenic water extraction. 2.6 pH and inhibitor experiments Experiments related to pH were limited to roots of Helianthus (10 September, 2007) and Disocactus (17 December, 2007) only. The exchange period (5 minutes) was constant with varying pH's of 5, 6, 7, and 8 with respect to Helianthus whereas a pH of 6 was constant with exchange periods of 5, 10, and 30 minutes with respect to Disocactus. HgCl2 experiments were limited to Helianthus roots and radicle (4 days after germination, measuring 1-1.5 cm in length) and Populus roots. 2.7 Diurnal study and transpiration experiment Roots grown hydroponically at UBC greenhouse were harvested from individual Populus plants under water at 12 noon and 12 am on 21, 22, and 23 November, 2007. They were then immersed in DDW and processed in the laboratory with exchange periods of 5 and 10 minutes. Four Populus plants (including shoot and root system) were obtained from 16 the UBC greenhouse. Two plants were suspended in the air by threads with their respective root system floating in a 500 mL beaker of DDW (Control) and the other two plants in a similar volume of 50 uM HgCl2 (Treated). A photosynthetic photon flux density (PPFD) of-250 umol quanta m"2 sec"1 from 10 tube lights (34 W, Sylvania cool white, Canada) was provided for 1 hr duration with fan. This was followed by discarding the shoot system and quickly processing the roots for 5 and 10 minute exchange periods. 2.8 Closed circulating system The purpose of constructing the closed circulating system was to locate the distribution of labelled metabolic water in different plant parts (reflecting location, specific respiration rates and/or water movement) and also to check feasibility of separating autotrophic/heterotrophic respiration in a simple system. This system was used to expose roots and the simple rooting media to 21% 1802 in N2. Based on results from labelling and exchange experiments, I sampled various portions of the roots and the freely transpiring shoot to detect metabolic water in these parts. 180-H20 detected in leaves in these experiments or in subsequent chase studies gave some indication of mixing of symplastic (root) water into apoplastic (xylem) water. The same experiment was repeated using non-sterile media. In the absence of significant exchange of labelled metabolic water from roots with soil water, 180-H20 detected in the media should in this case reflect heterotrophic respiration. 2.8.1 Experimental design Pots constructed from capped PVC (polyvinyl chloride) piping (4.5 cm diameter, 0.2 mm wall thickness, 12 cm in height, 1 cm diameter tubing connectors at the bottom) were used for growing plants. The top end of each "root-chamber" was covered with another 17 PVC cap with a slit to accommodate the growing shoot system. Helianthus seeds were germinated (1 seed per pot) in four different media (gravel, silicon sand, potting-mix, and soil,). Gravel, potting-mix and silicon sand were commercially obtained from Delta Aggregates LTD, Canada and David Hunter Garden Centers, Canada. Soil was obtained from Totem Field, UBC campus. Commercial tank 0 2 (99.76% 160) was taken as control with gravel as the growth medium. Planting was conducted on 13 May, 2008. Plants were grown under natural light (next to a window). The ambient air temperature in the laboratory was 20 °C. Soil moisture content in each pot was maintained by weighing and watering. Sampling was conducted between 27 May and 03 June, 2008. 2.8.2 Mechanism The closed circulating system (Illustration: 3) consisted of: (1) Plant-media system, designed to contain the living roots and respective media in the root-chamber, (2) Air- flushing valves with lA inch tubing, connected by 'T' joints, and (3) tubing, connected to an E-series peristaltic pump (Model No. 72-410-108, Manostat Corporation, Illinois, USA) and SI02 oxygen sensor (Qubit Systems Inc., Kingston, ON, Canada) which in turn was connected to a data-collection device, Vernier Lab Pro® (Qubit Systems Inc.). The sensor measures the oxygen concentration in the range of 0-27% using an electrochemical cell. A needle valve served as pressure release as well as an inlet for air, N2 or O2 gas. Pots, etc, were tested for possible air leaks because of the many connecting joints and valves, by pumping air into the system through the air inlet and submerging the air outlet tubing into water. At the time of sampling the respective pot was placed in position and sealed tight with sealant (Qubit Systems Inc., Kingston, ON, Canada) where necessary. 18 "*1 H i i ^ ^ JP'MJ HP —*"—JSS ^•••^s. . . \ MR f 1 *S$il\trnm if S8» BH^yS^Pl • I f —' P U ^ _ f 1 MM \ I*i35^-'": • ."v Mm - ~^M\ mi V*^. -̂̂ .4 1 R Illustration 3. A model system to assess the utility of O2 in exploring spatial patterns of belowground respiration An example of the output from the oxygen sensor is presented in Fig 1. Prior to the actual 'experimental run', the air within the system was displaced with a known quantity of nitrogen. The total gas volume of the system was calculated as follows: Total gas volume = ("Volume of N2 gas injected) New 0 2 concentration ^ Initial O  2 concentration 1 [1] The system was purged with N2 gas to remove most of the O within the chamber. Based on the total gas volume of the closed system, the volume of the oxygen isotopic label was introduced into the closed circulating system. A 30 min period was allocated for the label 19 to be incorporated by the transpiring plant, which was provided with a PPFD ranging 0 1000 2000 3000 Figure 1. Example of annotated computer trace of output from the SI02 O2 sensor. from 1000-1150 umol quanta m"2 sec"1. Care was taken to separate the roots, shoots and soil media with minimum stress as quickly as possible. Isolating the intact root system from the growth media was a challenge. During isolation, some of the 'fine roots' branching off of the main roots were difficult to collect and were left behind in their respective growth media. The shoots, isolated roots and growth media were weighed and frozen in LN2 and saved for later extraction. 2.8.3 Calculation of root respiration rate in the model system Given the air volume of the model system, one can calculate the total "belowground" respiration rate based on the mmols of O2 used, time and tissue mass relevant to each pot. Root respiration rates were calculated from the linear portion of the oxygen depletion graph: 20 Rate of 0 2 consumption = (Volume x Slope) (RxlOO) ~ FW umol O2 min" 1 where, Slope = change in O2 concentration over time (% min"1), Volume = volume of the air (mL) within the closed system, and R = molar volume of air at 20°C and 1 atmosphere pressure (20405 mL mol"1) 2.9 Cryogenic extraction from a bulk sample ©• > < * > < 7 ^ LN, u - h o t H20 . v. J >< X Roughing Main Vacuum Pump Pump Figure 2. Schematic diagram of a single detatchable cryogenic distillation unit on the vacuum-preparation line. 1) Cajon Ultra-Torr fitting; 2) 2-way vacuum stopcock; 3) V2 inch Pyrex® collection test-tube; 4) 1 inch Pyrex extraction tube containing 1 mL reacti-vial with plant tissue; 5) Rotary pumps; 6) Liquid nitrogen trap; 7) Detachable unit; 8) PG: Pirani vacuum gauge; 9) TC: Thermocouple vacuum gauge. Water was extracted from all materials by cryogenic vacuum distillation (Ehleringer and Osmond, 1989). A vacuum line and detachable distillation units (Fig 2) were constructed for this purpose in the Tree Physiology Laboratory, Department of Forest Sciences, UBC. 2.9.1 Mechanism The cryogenic distillation apparatus consisted of four independent detachable glass units that could be coupled to a 1-inch glass vacuum manifold (Fig 2). Each unit consisted of % inch glass "T" arm connected to the manifold via an Ultra-Torr fitting and could be isolated from the manifold by a vacuum stop-cock. Attached via Ultra-Torr fittings to either side of the "T" arm were a collection tube QA inch Pyrex®) and an extraction tube (1-inch Pyrex ). The entire vacuum line was connected to two vacuum pumps (Edwards, Model E2M2); one for 'rough-pumping' (to remove moist air) and another, cleaner pump to maintain a good vacuum. Pressure was monitored with a thermocouple vacuum gauge, connected to a Granville Phillips 307 Vacuum gauge controller, and a 501 Pirani gauge (Edwards). 2.9.2 Extraction of water To extract water from a sample, a previously evacuated unit was isolated from the vacuum manifold. The extraction tube was removed and a sample vial, containing the material of interest, was placed directly inside. The extraction tube was then reconnected to the unit and immersed in liquid nitrogen, freezing the sample and any water vapor in the unit. Soil media was an exception; it was frozen in a 500 mL round-bottom flask in a mixture of dry ice and 95% ethanol for 30-45 minutes prior to extraction and then connected to the roughing pump. Care was required to ensure that the vacuum seal of the 22 extraction tube was not compromised. Once the sample was frozen, the entire line was pumped down to a pressure of approximately 60 mTorr. At this point the valve isolating the unit from the vacuum manifold was closed and the pressure in the isolated unit was monitored. If the vacuum (6><10"2) was maintained, the dewar containing liquid nitrogen was removed from the extraction tube and was replaced with a beaker filled with water and containing a heating element. This water was maintained at boiling point throughout the duration of the extraction. Periodic additions of water were required to keep the water level constant. The dewar of liquid nitrogen was placed on the collection tube in order to freeze out the water vapor emanating from the sample. At the completion of the distillation, the boiling water and liquid nitrogen were removed from the collection tube and the extraction tube, respectively. The collection tube was removed, plugged with a serum cap, sealed with Parafilm®, allowed to thaw and then centrifuged at 1000 rpm. The water was then pipetted (Redi-TipT , Fisher, Canada) into 200uL conical inserts, sealed with Parafilm® and inserted into clear glass vials (Model C223682C, Chromspec Inc). An aluminum with rubber septa (teflon/orange) (Model 221150) was placed on the clear glass vial and sealed with a crimper (Model 21170, Restek, USA). The sample was retained for isotopic analysis. For the purposes of this study, extractions were timed (2 hrs of boiling time for plant tissues and 5 hrs for soil water) from the moment the extraction tube was placed in the boiling water until the collection tube was removed from the line. These extraction times were based on preliminary tests to determine the time required to get full recovery based on measurements of tissue FW and DW, and mass of water recovered. Yields were routinely 100 ±1%. 23 2.10 Isotope analysis Water distilled from samples was sealed into ampoules as described above for injection onto a stable isotope ratio mass spectrometer (TRMS). The samples were analyzed at the Pacific Centre of Isotope and Geochemical Research (PCIGR), Department of Earth and Ocean Sciences, UBC in continuous flow mode by pyrolysis in a Thermal Combustion Elemental Analyzer attached to a mass spectrometer (Thermo-Finnigan Delta plus XL). Some samples sent to UC Davis, California were analyzed by Laser-Absorption Spectroscopy (Las Gatos Research DLT-100). The analyses were corrected for isotope fractionation using three internal laboratory standards, calibrated against international water standards V-SMOW and V-SLAP. The precision was <0.3%o. Isotopic composition of oxygen is expressed in delta notation (<5) as shown: ( R. 8 = . ^sample n standard J xl000%o [3] Where, i?sampie and i?standard are the molar ratios of 180/160 of the sample and standard respectively. The standard, V-SMOW, has 8180 of 0%o. 2.11 Calculation of respiration rate by l sO labelling of metabolic water Respiration rate was calculated from the differences between the initial and final 180 content of tissue water. The absolute ratio of V-SMOW used in these calculations (Standard)was 0.0020052 (Ehleringer and Osmond 1989). The i?initiai was calculated as: ~( 8X%0„ ^ -"initial 1 4- ~ — 'n 't'a* 1000 X  ""standard [4] 24 and the i?finai was calculated as: -^final — ( x18r> A £ l sO f 1 , ~ ~ final 1000 xR standard The % Oinitiai was given by: %1 80 initial R, initial V + "^initial J xlOO 18 and % Ofmai was given by: %18n /o ufi„„, — final -"•final V* + "final J XlOO The mean final molecular weight (MW) of tissue water was then obtained from: MW = ((l00 - %18 Ofmal )x 18)+ (%18 Ofmal x 20)' 100 [5] [6] [V] [8] The umol of H2180 generated was calculated as: Metabolic water generated (%,,OM-%'«OUM)xf^^lxl«'0 V MW J where water yield is the FW-DW in mg. Finally, respiration rate was obtained from: Metabolic water generated Respiration rate = ( FWtissue x (Time) i_ - i i [9] umol 0 2 m i n V F W [10] ^ 1000 where the factor 2 accounts for the stoichiometry of the 0 2 to H20 conversion at the end of the mitochondrial electron transport chain (note: this is not equivalent to the overall stoichiometry of respiration). 25 2.12 Calculation of % exchange Exchange of labelled tissue water with bulk water upon immersion was expressed in terms of percent conversion on a theoretical end-point where isotopic equilibrium is achieved. The expected equilibrium <5180 will be the weighted average of the tissue water and exchange solution combined: IpLsoln X 8 Osoln j + \°°^tissue water X $ O tissue (0 min) j S"0 end-point l m L S 0 l n + m L tissue water) [11] Where <518Osoin is the isotopic composition of the exchange solution (DDW or other) and <5180tissue (o mm) is the isotopic composition of tissue water at the beginning of the exchange period. Percent conversion on the end point was then given by: % exchange (tmin) ° *-* tissue (Omin) tissue (tmin) V ^tissue(Omin) ° ^end-point , xlOO [12] Where (518OtiSsue (tmin) is the isotopic composition of the tissue water after an exchange period oft minutes. For component exchange periods, percent exchange of residual 180- labelled tissue water (e.g. exchange occurring between 5 and 10 minutes) was given by: % exchange of residual label over tj to 15 f  % exchange^  min) - % exchange(ti m ^ 100-%exchange(timin) xlOO [13] An example step-by-step implementation of these calculations is presented in the appendix. 26 2.13 Statistical analysis T-test and one-way ANOVA were used to test for differences between system components (media and tissues). Statistical differences were calculated using MS Excel spread sheet and considered significant at 'p ' < 0.05. 27 3 RESULTS Because the labelling of metabolic water is essentially unprecedented as a tool to study plant respiration and water relations, many of the experiments conducted were exploratory in nature and meant to stand on their own. Species and /or tissue comparisons were made where possible. 3.1 * 8 0 2 uptake (respiration rate) I was easily able to detect and recover metabolic water labelled with O2 against a large background of normal tissue water (Table 1). Respiration rates were calculated from the rate of label incorporation. The respiration rates of the study species and their respective parts were expressed in |imol C^min"1 g"1 FW. Table 1. Initial and final <5180 values of water extracted from intact Medicago sprouts O Oinitial (%o) -12.49 -13.75 - - <> Ofinal (%o) 120.57 152.15 95.91 125.79 Respiration rate (nmol02niin"1g1FW) 24.2 28.5 16.4 16.9 The <5180jnitiai value of-13.12%o (n = 2) of intact Medicago sprouts was slightly negative (-11.0%o) relative to DDW. In contrast, water from germinants exposed to 1802 was highly enriched. One batch showed a relatively low enrichment value of 95.91%o, compared to the other three, indicating that 180 label was not incorporated into the intact germinants as much when compared to the others (Table 1). Of the four batches of intact Medicago sprouts, two batches analyzed on 13 November, 2006 showed respiration rates of 24.2 and 28.5 umol 0 2 min"l g"1 FW. The other two analyzed on 04 December, 2006 28 showed respiration rates of 16.4 and 16.9 umol 0 2 min"l g"1 FW. Overall, the respiration rates of intact Medicago sprouts ranged between 16.4 and 28.5 umol O2 min"1 g"1 FW (Table 1). Table 2. Respiration rates of study species. All isolated tissues were immersed in 10 mL DDW followed by label incorporation. [In Expt. 1, the hypocotyl was separated as one unit with a cut just below the petiole of the growing cotyledons and a cut just above the root system, and then further divided into two sections approximately in the middle. The upper section was named Hypocotyl (1) and the lower section was named Hypocotyl (2)]. Plant name Helianthus (Expt. 1) Helianthus (Expt. 2) Helianthus (Expt. 3) Helianthus (Expt. 4) Crassula Disocactus Plant part analyzed Cotyledons Hypocotyl (1) Hypocotyl (2) Roots Cotyledons Roots Cotyledons Leaves Roots Leaves Roots Respiration rate (umol 0 2 min"1 g_1FW) 14.0 8.1 7.5 11.2 32.3 15.6 7.6 37.7 10.4 12.4 2.9 When cotyledons, two sections of hypocotyl and roots of the pooled one-week old Helianthus seedlings were analyzed, the individual parts showed respiration rates of 14.0, 8.1, 7.5, and 11.2 umol O2 min"1 g"1 FW respectively (Table 2, Expt. 1). When Helianthus cotyledons and roots (one week old) were compared, cotyledons showed a respiration rate of 32.3 umol 0 2 min"1 g"1 FW whereas the respiration of roots appeared to be 15.6 umol 0 2 min"1 g"1 FW (Table 2, Expt. 2). In another experiment, the cotyledons showed a respiration rate of 7.6 umol O2 min"1 g"1 FW (Table 2, Expt. 3). When leaves and roots were compared, leaves showed a respiration rate of 37.7 umol O2 min"1 g"1 FW whereas the respiration rate of roots was 10.4 umol O2 min"1 g"1 FW (Table 2, Expt. 4). Overall, 29 the respiration rate of cotyledons and roots ranged from 7.7 to 32.3 umol O2 min" g"1 FW and 7.8 to 15.6 umol O2 min"1 g"1 FW, respectively (Table 2). Crassula leaves respired at a rate of 12.4 umol O2 min"1 g"1 FW, whereas Disocactus roots exhibited a respiration rate of 2.9 umol O2 min"1 g"1 FW, the lowest value obtained for any of the experiments conducted (Table 2). In all the above experiments, the labelled parts were immersed in 10 mL DDW without any additions; yet, the respiration rates observed were quite variable. Table 3. Respiration rates of Helianthus tissues. After label incorporation, all isolated parts immersed in 10 mL DDW (Control) and 10 mL 200 uM HgCl2 (Treated) respectively Plant name Helianthus (Expt. 1) Helianthus (Expt. 2) Helianthus (Expt. 3) Plant part analyzed Shoots Roots Radicle Treatment Control Treated Control Treated Control Treated Respiration rate (umol 0 2 min"1 g"1 FW) 25.7 20.6 7.8 10.0 39.6 17.0 There seemed to be no consistent effect of HgCi2 treatment on respiration rate. When young Helianthus shoots (cotyledons and hypocotyl) were analyzed, respiration rates of 25.7 umol Chmin"1 g"1 FW for Control (DDW) and 20.6 umol 02min"1 g"1 FW for Treated (200 uM HgCl2) were observed (Table 3, Expt. 1). Helianthus roots showed a respiration rate of 7.8 umol 0 2 min"1 g"1 FW for Control (DDW) and 10.0 for Treated (200 uM HgCy (Table 3, Expt. 2). A similar experiment with Helianthus radicles (four days after germination) showed a respiration rate of 39.6 umol O2 min"1 g"1 FW for Control (DDW) and 17.0 umol O2 min"1 g"1 FW for Treated (200 uM HgCb). Radicles appeared to have higher respiration rates than roots (Table 3, Expt. 3). 30 Table 4. Respiration rates of Populus roots in diurnal study analysis and transpiration effect experiments after label incorporation, all isolated parts immersed in 10 mL DDW (Control) and 10 mL 50 uM HgCl2 (Treated) respectively Experiment DDW Day time Night time 'Transpiration effect' Respiration rate (jimol Oz mm1 g1 FW) 24.4 19.8 24.0 20.6 20.1 17.1 25.1 13.4 24.3 13.5 17.2 Mean respiration rate (Hmol 0 2 min"1 g"1 FW) n/a 21.4 (n = 3) 20.7 (n = 3) 18.8 (/i = 2) 15.3 (n = 2) Initial analysis of untreated Populus roots on 10 September, 2007 showed a respiration rate of 24.4 umol 0 2 min"1 g"1 FW (Table 4). Diurnal analysis of Populus roots on three consecutive days (21, 22 and 23 November 2007) showed that the respiration rate was 21.4 umol 02 mm"1 g"1 FW at 12 AM and 20.7 umol C^min"1 g"1 FW at 12 PM, indicating no significant difference in O2 uptake rates, day or night (Table 4). In the 'transpiration effect' experiment on Populus roots, the mean respiration rate was 18.8 umol Chmin"1 g"1 FW for Controls (DDW) and 15.3 umol 0 2 min"1 g"1 FW for Treated (50uM HgCl2) samples, again suggesting no great effect of HgCl2 on respiration (Table 4). 3.2 Exchange of labelled water by tissues and species 1 Q After O2 label was incorporated into plant tissues as metabolic water, exchange experiments were conducted to assess the rate of movement of that water out of the tissues, in exchange for unlabelled water. It was expected that after 30 minutes of 31 labelling 180-H20 would be well distributed throughout the cytoplasm of all living cells. Depending on the rate of exchange across membranes, the isotopic composition of vacuolar and apoplastic (cell wall and xylem) compartments may or may not have been at or near equilibrium with the cytoplasm. In any case, exchange would commence upon submersion of labelled tissues into the unlabelled bulk media water. Over time, the isotopic composition of the plant tissue was expected to approach that of the medium, and vice versa, but the much larger volume of water external to the plant tissue would have the proportionally larger influence over the final end-point, where all water in the system would be at isotopic equilibrium. Expected end-points were calculated based on water content and initial duO values. This information was used to determine the percent exchange over a given time interval to give an indication of the tissue "leakiness". The Percent exchange of residual 180 (e.g. label remaining in tissues at 5 min) was also calculated over the subsequent intervals (Appendix: 7.1). Time (Mini • DDW —•—Medicago Figure 3. Decay in 5 values of water from intact Medicago sprouts during exchange with DDW 32 The <S180 of water from Medicago sprouts after various periods of exchange, and the 5DDW in which the germinants were submerged, are represented in Fig 3. Most of the label incorporated into the tissue exchanged with DDW during the first 5 min period. After 30 minutes there was still some perceptible difference between tissue water and the bulk media, but near equilibrium was achieved over the full 60 minutes. It should be noted that as <S180 analysis of tissue water is destructive, it is not possible to obtain the initial isotopic composition (i.e. prior to exchange) for each sample. However, for this experiment the starting Sl80 was estimated as a mean (n = 4) of the enriched values in Table 1 (i.e. 123.60%o). The <5180 values over the subsequent 5, 30, and 60 min exchange periods were 10.2, -7.8, and -10.43%o (Fig 3). Due to the irregularity of the experiment (water yields of Medicago sprouts were not recorded) the total percent exchange of O label and the percent exchange of residual 180 could not be precisely calculated, but it was at least 84% by the end of 5 minutes. In an early experiment with one week old Helianthus seedlings (Table 5, Expt. 1), labelled parts [cotyledons (0 min and 5 min), hypocotyl (1) (10 min), hypocotyl (2) (15 min), and roots (20 min)] were immersed in DDW (<5180 value of -12.43%o); each part with varying exchange period as shown in parentheses. The dlsO of the immersion media was also analyzed at the end of each period [-13.85%o(« = 4)]. Except for cotyledons, the pre-exchange <5180 of tissue water (<S18Ojnjtiai) in these parts was not estimated. Due to the irregularity of the experiment (not having «5180initiai and consistent exchange periods), the total percent exchange of 180 label and the percent exchange of residual 180 could not be calculated. Overall, I am left to believe that cotyledons are less leaky when compared to the other organs; it appears that most of the label was likely exchanged during the first 5 minutes in the other parts. 33 A second similar experiment (Table 5, Expt. 2) was conducted with one week old Helianthus seedlings where the labelled parts consisted of cotyledons, hypocotyl (1), hypocotyl (2), and roots. Each of the isolated parts was this time represented by a 'native sample' (represents the beginning <5 value of plant water prior to labelling), <5180injtiai and exchange periods of 5, 10, 15, and 20 minutes. The JDDW was -12.21%o. All parts exhibited high enrichment upon labelling, as expected. Total % exchange of labelled water in these tissues is shown in Fig 4A. Cotyledons showed a relatively slow but progressive depletion of labelled water during the subsequent exchange periods. The performance of hypocotyl (1) and hypocotyl (2) during the subsequent exchange periods seemed to 'bounce' around indicating sampling error and lower rates of exchange in general. Roots were more permeable to water. Two individual experiments were conducted in which pools (« = 10) of one-week old Helianthus seedlings were segregated into leaves or cotyledons and roots. The performance of leaves vs. roots (Table 5, Expt. 3) and cotyledons vs. roots (Table 5, Expt. 4) was the main objective. Each of the isolated parts was represented by <5180jnjtjai and exchange periods of 5, 10, and 30 minutes. Overall, leaves were highly enriched relative to cotyledons, and the roots were least enriched. Mean (n = 6) <5DDW of the two experiments was -13.45%o. Labelled leaves, cotyledons, and roots exhibited high enrichment, as usual. Over the subsequent 5, 10, and 30 minute exchange periods, the calculated total percent exchange of 180 label in leaves was 79.7%, 77.1%, and 90% respectively (Fig 4B.) The respective values for roots were 71.3%, 89.2% and 91.3%. Similarly, the total percent exchange of 180 label in cotyledons was 62.5%, 58.8%, and 79.3% (Fig 4C). The respective values for roots in this case were 72.5%, 79.4% and 91.6%. 34 100 u 80 20 25 5 10 15 Time (M in) -Cotyledons —•— Hypocotyl(l) —A—Hypocotyl(2) X Roots Time (Min) •Leaves • Roots 100 x n o H 15 20 Time (Min) -•—Cotyledons — • 30 35 • Roots Figure 4. Total % exchange of metabolic water in "untreated" tissues of Helianthus. A) Performance of each segregated part in subsequent exchange periods. B) Comparison between leaves versus roots and C) Cotyledons versus roots in initial and subsequent exchange periods. 35 The performance of Populus roots during initial and subsequent exchange periods was tested in DDW (Table 6 and Fig 5A). The (5DDW was -12.88%o and <518Oinitiai was 122.34%o. The tissue water <S180 values at the ends of each subsequent exchange period were 5roots(5min) = -0.09%o, <Ws (io min) = -6.68%o and t>roots(30 min) = -9.42%o. The calculated total percent exchange of 180 label of roots was 89.8, 95.5, and 97.5% respectively. The percent exchange of residual 180 was 54.6% between 5-10 min and 45.2% between 10-30 min. In the experiment related to Disocactus roots, an estimate of 6 Oimtiai was obtained and exchange periods of 5, 10, and 30 minutes in pH 6 buffer solution were used (Table 6 and Fig 5B). The <?DDW was -12.1%o and <518Ommai was 7.9%o.The enrichment values following the subsequent exchange periods were <Sroots(5 min) = 3.2%o, <5roots(io min) = -4.9%o and <Sroots(30 min) = -11.7%o. Over the subsequent 5, 10, and 30 min exchange periods, the calculated total percent exchange of 180 label of roots was 23.9, 64.6, and 98.7% respectively. The percent exchange of residual 180 label was 53.48% between 5-10 min and 96.53% between 10-30 min. In the experiment related to Crassula leaves, there were data for <518Omraai and exchange periods of 5, 10, and 30 minutes respectively (Table 6 and Fig 5C). The <5DDW was - 13.5%0 and S Omitiai was 66.7%o. The tissue enrichment values at the end of each subsequent exchange period were <Sieaves(5 min) = 44.7%o, <Sieaves(io min) = 52.4%0 and <SieaVes(30 min) = 47.7%o. Over the subsequent 5, 10, and 30 min exchange periods, the calculated total percent exchange of 180 label of leaves was 26.5, 17.2, and 22.8% respectively. The percent exchange of residual 180 label was -12.75% between 5-10 min and 6.80% between 10-30 min. 36 4> CO a 1 & •a E2 80 - 60 - 40 - 20 - 0 t A 4 * f * | •- - • * f * i 10 20 Time (M in) 30 40 15 20 Time (Min) 15 20 Time (Min) Figure 5. Total % exchange in roots of Populus and Disocactus and leaves of Crassula . A). Exchange of Populus roots in DDW; B) Exchange of Disocactus roots in a constant pH 6 solution in initial and subsequent time periods; C) Performance of Crassula leaves. 3.3 Role of aquaporins 3.3.1 Effect of mercuric chloride An experiment was conducted using one-week old Helianthus shoots (cotyledon and hypocotyl) to compare isotope labelling and exchange in DDW (Control) and 200(4,M HgCl2 (Treated) (Table 7, Expt. 1 and Fig 6A). This was the only experiment in which Helianthus was treated with 200uM HgCb for an hour prior to vacuum infiltration. The <S18Ojnitiai values were 116.4%o (Control) and 78.7%o (Treated). The enrichment values following the subsequent exchange periods (5 and 10 min) were <5Shoots(5 min) = 82.7%o and <5shoots(io min) = 12%o for Control and <5sh0ots(5 min) = 28.5%o and <5sh0ots(io min) = 41.1%o for Treated. The total percent exchange of 180 label was 26.5% and 82.5% for Control shoots, and 55.7% and 41.7% for Treated shoots, over 5 and 10 min, respectively. The percent exchange of residual 180 label over the 5-10 min interval for Control was 75.8% and for Treated was -31.5%. An exact replica of the above experiment was this time conducted without HgCk treatment prior to vacuum infiltration using one-week old Helianthus roots (Table 7, Expt. 2 and Fig 6B). The <S18Oinitiai values were 10.9%o (Control) and 19.6%. (Treated). The enrichment values following the subsequent exchange periods were <5roots(5 min) = 0.8%o and <5roots(io min) = 5.2%o for Control and <5roots(5 min) = -0.3%o and <5roots(io min) = -4.1%o for Treated. Over the subsequent 5 and 10 min exchange periods, the calculated total percent exchange of 180 label of roots for Control was 44.5 and 25.2% and that of Treated roots was 64.3 and 77.7% respectively. The percent exchange of residual 180 label over the 5-10 min period was -34.83% for Control and 37.59% for Treated. Another replica of the above experiment (without HgCfe pretreatment) was conducted with Helianthus radicle only (Table 7, Expt. 3 and Fig 6C). The 518Omitiai values of 38 radicle were 167.98%o (Control) and 73.32%o (Treated). The enrichment values following the subsequent exchange periods were <5radicie(5 min)= 34.19%o and <5radiCie(iomin)= 1.14%ofor Control and <5radicie(5 min) = 6.24%o and <5radicie(i0min) - -1.14%o for Treated. Over the subsequent 5 and 10 min exchange periods, the calculated total percent exchange of 180 label was 76.2 and 94.7% for Control and 80.2 and 88.9% for Treated radicles, respectively. The percent exchange of residual 180 label over the 5-10 min exchange period was 77.8% for Control and 44.1% for Treated. 3.3.2 Effect of pH In an experiment related to the effect of pH on Helianthus roots, the exchange period of 5 minutes was kept constant with varying pH (5, 6, 7, and 8) (Table 7, Expt. 4). The <SDDW was -12.5%o. The enrichment values after exchange were 8.2%o, 1.4%o, -0.3%o, and 2.1%0 in pH's 5, 6, 7, and 8 respectively. The <S18Oimtiai was not estimated for this experiment; hence percent exchange of total 180 label could not be calculated. 39 00 § o X W f2 4 6 8 Time (Min) -•- -Control —•—Treated \ 6 8 Time (Min) -•- - Control — • — Treated 10 12 4 6 8 Time (Min) -•- - Control — • — Treated 10 12 Figure 6. Total % exchange in "treated" tissues ofHelianthus. A). Performance of shoots in the absence and presence of 200 uM HgCb; B) Performance of roots in the absence and presence of 200 uM HgCk; C) Performance of radicle in the absence and presence of 200 uM HgCl2. 40 3.3.3 Diurnal studies In the experiment related to day vs. night time study of Populus roots (Table 8, Expt. 1 and Fig 7A), the &ydroponic water was found to be -ll.l%o. The <5180inraai across day-time sampled roots ranged from 99.5 to 124.4%o and that of night-time sampled roots ranged from 84.9 to 132.7%o. The range of enrichment values for the subsequent exchange periods of day-time samples were 7.0 to 30.1%o for <5roots (5 mm) and -0.4 to 23.7%o for <5roots (io min) and that of night-time samples were <5roots (5 min) 3.6 to 23.3%o and <5roots (10 min) 1.4 to 15.5%o. Over the subsequent 5 and 10 min exchange periods, the calculated total percent exchange of 180 label of roots ranged from 71.2 to 85.4%> and 75.6 to 91.6%> respectively for day-time samples. Similarly, the total percent exchange of 180 label of night-time roots ranged from 77.4 to 86% and 77.9 to 91.6%, respectively. The percent exchange of residual 180 label for day-time samples ranged from 0.74 to 42.5% for the 5-10 min exchange period and that of night-time samples ranged from 2.4 to 62.2% for the 5-10 min period. 3.3.4 Transpiration experiment In the experiment related to the transpiration effect of Populus roots (Table 8, Expt. 2 and Fig 7B), Control and Treated tissues were replicated twice. The ^DDW was -12.10%o and the <S18Ojnitjai for 'Control 1' was 64.07%o and that of 'Control 2' was 132.51%o. The enrichment values after exchange for 5 and 10 minutes were 2.3%o and 2.5%o for Control 1 and 56.5%o and 27.9%o for Control 2, respectively. Over the subsequent 5 and 10 min exchange periods the total percent exchange of 180 label for roots was 82.4 and 82.3% and 53.7 and 74.1% for Control 1 and 2, respectively. The percent exchange of residual 180 label between 5 and 10 minutes was -0.20% and 44.06% for Control 1 and Control 2, 41 respectively. The J18Oinitiai for 'Treated 1' was 64.33%o and that of 'Treated 2' was 90.29%o. The enrichment values after exchange for 5 and 10 minutes were 9.0%o and 7.9%o, and 8%o and 2.1%o, for Treated 1 and Treated 2. Over the subsequent 5 and 10 min exchange periods the total percent exchange of 180 label for roots was 74 and 75.5% and 82.2 and 88% for Treated 1 and 2, respectively. The percent exchange of residual 180 label between 5 and 10 minutes was 5.0% and 32.4% for Treated 1 and Treated 2, respectively. -•- -Day 1 — • Night 1 - -A- -Day2 —*—Night 2 - -m- -Day 3 -Night 3 100 4 6 8 Time (Min) 10 12 • Control 1 — • Treated 1 - -A- -Control2 —A—Treated2 Figure 7. Total % exchange in "untreated" and "treated" Populus roots. A) Diurnal variation of untreated roots; B) Effect of transpiration on roots in the absence and presence of 50 uM HgCk- 42 3.4 Closed circulating system As a rule, to calculate respiration rate, one should use the dlsO value of the background water (DDW) in which the plants were grown (i.e., water distilled from respective media), and the dnO value of native samples of the respective plant as the starting points. With reference to the model system (Illustration 3, Table 9 and Fig 8), however, a 'Control' plant was "labelled" with commercial tank 0 2 (99.76% 160). The <S180 values of water from shoots (-2.2%o), roots (-14.0%o), and soil water (-12.2%o) (i.e. water distilled from gravel, in this case) were used as the <5180initiai 'starting points' for comparison with <5180finai values across all media labelled with180. The values of «5roots tended to track values of b̂ackground water but were slightly more enriched, indicating a rapid exchange, which is consistent. Compared to the other media, loss of label from roots in silicon sand was far less. This could be interpreted to indicate that diffusion of labelled water from roots to soil was at a faster pace in gravel rather than in silicon sand. The value of <SShoots increases with the value of <5roots, indicating that some labelled water was presumably making it into the shoots in the course of the experiment (Fig 8). The shoot is also enriched from transpiration (to -2.17%o in the Control). <5roots and background water values are on the same page more or less, except silicon sand, where there may have been relatively more transpirational movement of labelled metabolic water to the shoot. There may also have been progressive enrichment of media water due to daily watering and the evaporation of water from the soil surface during the two week old growth period. All six plants were harvested within a week, and however small the time interval was between each harvest, this window may have provided an opportunity for O-H2O to accumulate. The effect of this situation would be on apparent uptake of 43 the isotopic tracer, which in turn would affect the calculated respiration rate of each analyzed plant as a whole (Table 9). Although rate of exchange is high for excised roots immersed in water, it may not be for intact plants rooted in soil (i.e. because of reduced aqueous media, and transpiration pull). Based on the above observations, the shoots were mostly more enriched than roots and the rate at which metabolic water is lost from roots in intact plants was pretty fast (Table 1 8 9). The overall belowground respiration rate (from O-H2O (ug) found in individual shoot and root systems as well as the water, corrected for Controls) was highest in silicon sand (43.04 umol O2 min"1 g"1 FW) and least in potting mix (5.61 umol O2 min"1 g"1 FW). Rapid diffusion accounts for most of the label lost to the soil in transpiring plants. Compared to the three growth media, the maximum percent of the label lost to growth medium (94.06%), the minimum percent of the label retained by harvested roots (2.69%), and the minimum percent exported to shoots (3.24%) was in gravel (Table 9). The percent of the label exported to shoots is far less compared to the maximum percent of the label lost to the media (Table 9) indicating that labelled water from roots is mostly lost to soil water rather than entering the transpiration stream. Respiration rates based on the slope from the computer were 74 and 109 umol C^min"l g 1  FW for plants grown in silicon sand and gravel respectively. These rates are three times higher than expected for intact Medicago sprouts, suggesting that either some of the measured O2 uptake may have been by "soil" microorganisms, or that root mass was underestimated (some finer roots could not be efficiently recovered). 44 Control (Gravel) Gravel 1 Gravel 2 Silicon-sand Potting-mix Soil • Shoots A Roots • Water Figure 8. <5180finai values of transpiring Helianthus shoots and roots in a model system. 45 Table 5. Sxs0 values and calculated % exchange of water in "untreated" Helianthus tissues Plant name Helianthus (Expt. 1) Helianthus (Expt. 2) Plant part analyzed Cotyledons Hypocotyl (1) Hypocotyl (2) Roots Cotyledons Hypocotyl (1) Hypocotyl (2) Exchange period (min) 0 5 10 15 20 N.S* 0 5 10 15 20 N.S* 0 5 10 15 20 N.S* 0 8 Ofinal (%o) 61.55 46.03 3.17 5.50 -4.46 N/A** 66.58 31.67 31.80 29.92 26.60 -3.72 32.78 18.76 22.74 23.43 14.61 -7.15 29.46 Total percent exchange (%) Exchange period (Min) 5 - 21.48 - - - - - 45.29 - - - - - 31.82 - - - - - 10 - - 80.87 - - - - - 45.10 - - - - - 22.75 - - - - 15 - - - 77.46 - - - - - 47.48 - - - - - 21.23 - - - 20 - - - - 91.88 - - - - - 52.10 - - - - - 41.40 - - 30 - - - - - - - - - - - - - - - - - - - Percent exchange of residual l sO label Subsequent time intervals (Min) 5-10 - - - - - - - - -0.29 - - - - - -13.30 - - - - 10-15 - - - - - - - - - 4.27 - - - - - -1.97 - - - 15-20 - - - - - - - - - 8.80 - - - - - 25.60 - - 10-30 - - - - - - - - - - - - - - - - - - - *N.S - Native sample; **N/A - Not applicable contd.... Plant name Helianthus (Expt. 3) Helianthus (Expt. 4) Plant part analyzed Hypocotyl (2) Roots Leaves Roots Cotyledons Roots Exchange period (min) 5 10 15 20 N.S* 0 5 10 15 20 0 5 10 30 0 5 10 30 0 5 10 30 0 5 10 30 <5 Ofmai 17.43 10.56 28.05 24.34 -6.51 52.50 10.61 3.79 -0.66 1.46 216.38 34.10 41.07 11.50 44.77 2.63 -7.40 -9.04 166.86 55.27 63.15 26.71 48.16 3.07 -1.15 -8.57 Total percent exchange (%) Exchange period (Min) 5 29.60 - - - - - 66.20 - - - - 79.68 - - - 71.34 - - - 62.51 - - - 72.51 - - 10 - 46.42 - - - - - 76.86 - - - 77.13 - - - 89.19 - - - 58.78 - - - 79.38 - 15 - - 3.45 - - - - - 83.83 - - - - - - - - - - - - - - - - - 20 - - - 12.5 - - - - - 80.6 - - - - - - - - - - - - - - - - 30 - - - - - - - - - - - - - 90.0 - - - 91.3 - - - 79.3 - - - 91.6 Percent exchange of residual lsO label Subsequent time intervals (Min) 5-10 23.89 - - - - - 31.55 - - - -12.57 - - - 62.29 - - - -9.96 - - - 24.98 - 10-15 - -80.20 - - - - - 30.10 - - - - - - - - - - - - - - - - - 15-20 - - - 9.46 - - - - - - - - - - - - - - - - - - - - - - 10-30 - - - - - - - - - - - - - 56.37 - - - 20.32 - - - 49.93 - - - 59.46 *N.S - Native sample Table 6.3 0 values and calculated % exchange of water in roots of Populus and Disocactus and leaves of Crassula Plant name and part analyzed Populus roots Disocactus roots Crassula leaves Treatment Control Untreated - Bathing solution of pH6 - Untreated Exchange period (min) DDW 0 5 10 30 Control (DDW) 0 5 10 30 Control (DDW) 0 5 10 30 3 Of,nal (%.) -12.88 122.34 -0.09 -6.68 -9.42 -12.08 7.93 3.17 -4.90 -11.67 -13.54 66.76 44.70 52.49 47.79 Total percent exchange (%) Exchange period (Min) 5 - - 89.83 - - - - 23.98 - - - - 26.54 - - 10 - - - 95.38 - - - - 64.64 - - - - 17.17 - 30 - - - - 97.47 - - - - 98.77 - - - - 22.80 Percent exchange of residual lsO label Subsequent time intervals (Min) 5-10 - - - 54.64 - - - - 53.48 - -12.75 - 10-30 - - - - 45.19 - - - - 96.53 - - - - 6.80 48 Table 7. <S180 values and calculated % exchange of water in "treated" Helianthus tissues Plant name and part analyzed Helianthus shoots (Expt. 1) Helianthus roots (Expt. 2) Helianthus radicle (Expt. 3) Helianthus roots (Expt. 4) Treatment Control (DDW) 200 pM HgCl2 N.S* Control (DDW) 200 nM HgCl2 N.S* Control (DDW) 200 uM HgCl2 Control pH5 pH6 pH7 P H 8 Exchange period (min) 0 5 10 0 5 10 N/A** 0 5 10 0 5 10 N/A** 0 5 10 0 5 10 DDW 5 5 5 5 8 Of,„al (%.) 116.40 82.71 11.99 78.78 28.51 41.09 -10.27 10.92 0.85 5.25 19.66 -0.38 -4.61 -5.02 167.98 34.19 1.14 73.32 6.24 -1.14 -12.51 8.15 1.44 -0.30 2.12 Total percent exchange (%) Exchange period (Min) 5 - 26.51 - - 55.73 - - - 44.55 - - 64.35 - - - 76.20 - - 80.28 - - - - - - 10 - - 82.25 - - 41.76 - - - 25.24 - - 77.76 - - - 94.72 - - 88.99 - - - - - Percent exchange of residual lsO label Subsequent time intervals (Min) 5-10 - - 75.84 - - -31.54 - - -34.83 - - 37.59 - - - 77.81 - - 44.19 - - - - - *N.S - Native sample; N/A - Not applicable 1 Q Table 8. S O values and calculated % exchange of water in Populus roots Plant name and part analyzed Diurnal study (Expt. 1) Treatment Control Nov 21, Noon Nov 22, Noon Nov 23, Noon Nov 21, Night Nov 22, Night Nov 23, Night Exchange period (min) DDW 0 5 10 0 5 10 0 5 10 0 5 10 0 5 10 0 5 10 <5 Ofinal (%o) -11.14 99.49 6.95 -0.39 124.37 30.12 23.70 105.32 18.68 18.74 100.74 16.01 15.47 89.49 3.58 1.41 132.67 23.33 3.65 Total percent exchange (%) Exchange period (Min) 5 - - 85.40 - - 71.25 - - 75.47 - - 77.43 - - 86.01 - - 77.93 - 10 - - - 91.60 - - 75.97 - - 75.65 - - 77.98 - - 88.12 - - 91.66 30 - - - - - - - - - - - - - - - - - - - Percent exchange of residual 180 label Subsequent time intervals (Min) 5-10 - - - 42.49 - - 16.42 - - 0.74 - - 2.41 - 15.14 - - - 62.20 10-30 - - - - - - - - - - - - - - - - - - - contd.... Plant name and part analyzed Transpiration effect (Expt. 2) Treatment Control N.S* DDW 50 uM HgCl2 DDW 50 uM HgCl2 Exchange period (min) DDW N/A** 0 5 10 0 5 10 0 5 10 0 5 10 <5 Ofinal (%.) -12.10 -10.28 64.07 2.27 2.52 64.33 9.02 7.92 132.51 56.51 27.93 90.27 7.96 2.11 Total percent exchange (%) Exchange period (Min) 5 - - - 82.38 - - 74.03 - - 53.78 - - 82.23 - 10 - - - 82.34 - - 75.34 - - 74.13 - - 87.98 30 - - - - - - - - - - - - - - Percent exchange of residual lsO label Subsequent time intervals (Min) 5-10 - - - - -0.20 - - 5.04 - - 44.06 - - 32.37 10-30 - - - - - - - - - - - - - - *N.S - Native sample; **N/A - Not applicable 51 Table 9. Label distribution in water from isolated parts of Helianthus across different media in a model system Growth medium Gravel Potting- mix Soil Silicon sand Gravel Treatment «Q 18o 18Q 1 8 0 18Q Plant part Shoots Soil water Roots Shoots Soil water Roots Shoots Soil water Roots Shoots Soil water Roots Shoots Soil water Roots S Ofinai Values (%.) -1.48 -11.21 -10.94 3.14 -9.83 -5.92 9.82 -8.74 -5.01 16.61 -6.72 18.22 8.7 -5.54 -2.45 Root respiration with respect to each pot. (nmolOzmin1 g"1 FW) Maximum percent of label lost to soil (%) Minimum percent retained by harvested roots (%) Minimum percent exported to shoots (%) N/A** 5.61 9.52 43.04 40.60 78.39 81.19 90.29 94.06 - - 9.93 - - 6.80 - - 5.89 - - 2.69 11.66 - - 11.99 - - 3.81 - - 3.24 - - Based on a O control experiment, initial unlabelled values were taken as -2.17%o (shoots), -12.22%o (soil water), and -14.00%o (roots). N/A** Water yield for roots was in this case not noted because of contamination and water across distillation tubes. One cannot calculate the respiration rate without water yield. 52 4 DISCUSSION The first known application of 1802 as a tracer to label metabolic water was initially conducted in animals (silk worm: Bombyx mori) and later on in intact whole plants (Cactaceae: Cereus hexagonus; Solanaceae: Lycopersicon esculentum) by Vartapetian (1973). Vartapetian was interested in the recycling of free oxygen generated in photosynthesis, and in contributions of metabolic water to total tissue water. His intent was not to identify sites of respiration or to trace the movement of metabolic waterier se. It seems to me that the 'true sense of labelling' was lost here. I am unaware of any other attempts to label metabolic water in plants. A little over a decade ago, the assumption was that the path of water into and out of the cells, or between cellular compartments, was primarily through simple diffusion directly across membranes. It is now evident that water can also be transported through aquaporins (water channels), which are crucial for rapid transport of water in large amounts during osmotic stress (Booth and Louis 1999; Calamati et al. 1998). However, in bacteria (Escherichia coli), Kreuzer-Martin et al. (2005) claimed on the basis of O2 labelling that during exponential growth, approximately 70% of the water within the cells was generated in metabolism and was not in equilibrium with the growth media. Furthermore, the metabolic state of the cells influenced the degree of exchange, which was much greater in other phases of growth. By measuring the isotopic composition of O2 generated from water in photosynthesis, Yakir (1992; 1993) concluded that chloroplast (and by extension, cytoplasmic) water was not in equilibrium with apoplastic (cell wall) water in transpiring leaves. On the other-hand, Farquhar et al. (1993) concluded that chloroplast water has an isotopic composition indistinguishable from cell wall water. 53 My quest began with this background knowledge; bearing this in mind as well as wanting to explore the untapped potential of 180 labelling. Almost all of the attention given to metabolic water in the literature has focused on the physiology or eco-physiology of animals, particularly desert animals (for example in kangaroo rats as reported by Schmidt-Nielsen and Schmidt-Nielsen 2005). Such animals depend directly on their carbohydrate supplies (from food) to generate water. On the other hand, we typically view plants as being indirectly dependent on water to generate carbohydrates. The absence of plant studies was not particularly encouraging at the outset of my studies. One • 1R concern was that it might be difficult to detect O labelled metabolic water against the large amount of unlabelled water typically present in plants. "Back-of-the-envelope" calculations, however, indicated that labelling should be possible within 10-30 minutes in a closed system. Entering into the virtually uncharted territory of the labelling process per se, the initial small step, literally (Objective 1) was to examine "Whether sufficient 180 can be incorporated into cell water to be detected against background variation, with precision and speed". If successful, the next step (Objective 2) was to examine "The distribution of labelled metabolic water in different plant parts" (reflecting location and specific respiration rates). 4.1 "Voila", clear detection of metabolic water Past experience (Guy et al. 1989; 1992) indicated that respiration rates are high in intact Medicago sprouts, which are easily handled and readily available from the local market at just the right stage. My initial trials with intact Medicago sprouts revealed that I was easily able to label and detect sufficient 180 incorporated into cell water as well as recover all 180-H20, against the bulk tissue water. By the end of a 30 minute labelling 54 period, intact sprouts labelled with 18C>2 were highly enriched (i.e. 123.60%o;(« = 4)). The 5180jnitiai value of-13.12%o (n = 1) of unlabelled intact spouts was slightly negative relative to DDW (-11.0%o), likely reflecting a slight difference in the source water used in their production or taken up during handling at the market. Labelling was within an air- tight reacti-vial with no additional water added, so the label recovered was a pure reflection of water generated in aerobic metabolism. Given the precision of isotope analysis (± 0.3%o), the very large enrichment obtained after 30 minutes was definitely more than sufficient to support further investigation. Consequently, each and every 1 o sample was always labelled with 1 mL O2 (99.9%) for half an hour. Given the amount of 1802 label introduced over time, it was straight forward to calculate respiration rates. Knowledge of the 518Obackground water value is required to calculate respiration rate from the d O of labelled samples. Some of the intact Medicago sprouts used in later exchange experiments had relatively low <S18Obackground water values, ascribable to their market origin. The respiration rates of intact Medicago sprouts ranged between 16.4 and 28.5 îrnol O2 min"1 g"1 FW. These rates are consistent with those of Guy et al. (1989; 1992). Their reported respiration rates for intact Medicago sprouts ranged from 17.5 to 27.7 |j.mol O2 min ! g"1 FW. Encouraged by the high enrichment of S values observed in intact Medicago sprouts, I chose to investigate four other species: a C3 plant {Helianthus), a CAM plant (Crassula), a desert plant (Disocactus), and last but not the least, a tree (Populus). Some tissues showed relatively low enrichment consistent with low rates of respiration per unit fresh weight. The enrichment values across all species analyzed ranged from +7.9%o to +216%o. At the high end were Helianthus leaves (216%o) and cotyledons (167%o). At the 55 low end were Crassula leaves (66.8%o) and Disocactus roots (7.9%o), tissues with low respiration rates per unit water content. Higher values are associated with tissues with high respiration rates per unit water content. Helianthus leaves in particular were quite enriched; they showed a respiration rate of 37.7 umol O2 min"1 g"1 FW. Respiration rates of cotyledons and roots ranged from 7.6 to 32.3 umol O2 min"1 g"1 FW and 10.4 to 15.6 umol O2 min"1 g"1 FW, respectively. The range of enrichment in Helianthus roots varied between 44.8%o to 52.5%o. Radicles, like leaves, were strongly enriched, consistent with a respiration rate (39.7 umol O2 min"1 g"1 FW; the highest value obtained for any of the experiments conducted) in support of rapid growth from the root tip. Goddard and Bonner (1960) showed that the respiration rate of the primary root varies with distance from the root tip; hence a decrease in respiration rate is obvious with an increase in distance from the root tip. It should be borne in mind that the data discussed here pertain to the primary (growing) root. There is still very little information on the partitioning of respiratory activity over the entire root system (Lambers et al. 1991). No data are available on respiratory patterns in lateral roots, which may be of particular interest because of their restricted growth period. Populus roots, enriched by 122.3%o, showed a respiration rate of 24.4 umol O2 min"1 g"1 FW. Crassula leaves respired at a rate of 12.4 umol O2 min"1 g"1 FW whereas Disocactus roots exhibited an enrichment value of 7.93%o and a respiration rate of 2.9 umol O2 min"1 g"1 FW; the lowest value obtained for any of the experiments conducted. This fits with the slow growth rates and high succulence of these tissues. 56 4.1.1 Factors accountable for the variations in respiration rates In all the above cases (Medicago, Helianthus, Populus, Crassula, and Disocactus), the respiration rates were influenced by the isolated plant part taken for analysis (the part analyzed was pooled from different plants within the same species). Within species or tissue types there is bound to be subtle sample-to-sample variation in the uptake rates. Another possibility that needs to be taken into account is the 'handling' of plant material at various stages of labelling and during extraction. Although care was taken to minimize stress, wounding and loss of water from the sample likely lead to some experimental error. Loss of water or wounding prior to labelling could affect respiration rates directly. Loss of water after labelling can alter <5180 values due to evaporative enrichment. After label incorporation into tissue water it was then possible to conduct exchange experiments on the four species representative of different functional groups. The purpose of these experiments was to meet Objective 3: "To examine the rate at which metabolic water is exchanged with extra-cellular water under a), normal conditions and, b). conditions expected to influence aquaporin activity. 4.2 Exchange of metabolic water in "untreated" tissues Although every sample taken in these experiments, regardless of exchange time, represents an independent labelling event, every sample after 5 minutes of exchange was always depleted in 180 relative to the sample at zero time. Hence, exchange appeared to be very rapid over this initial 5 minute period. Because exchange slowed over the next 5 minutes, some samples taken at 10 minutes were enriched relative to earlier samples taken at 5 minutes. This does not indicate that 180-H20 moved back into the tissue; rather 57 it simply reflects sample-to-sample variation in initial labelling and, possibly, membrane permeability. To overcome this variation, I calculated mean rates of exchange from 0-5 minutes versus 5-10 minutes. Exchange was clearly more rapid over the first interval (X = 63.4% complete over all species, tissues, and treatments). This suggests that a large fraction of water exchanged in this interval had more direct access to the bulk medium than did water that was subsequently exchanged. This would be consistent with the existence of two pools of labelled water available for exchange; namely apoplastic and symplastic water. One of the more interesting outcomes of this work was the observation that the general pattern of exchange across species and tissues analyzed was consistent with expected differences in water retention. When exchange was examined using different parts of Helianthus over identical periods, it was clear that roots were far more "leaky" than any other tissue, and true leaves appeared to be more leaky than either cotyledons or hypocotyls. Crassula leaves, which have a thick cuticle and low surface area to volume ratio, retained label particularly well. Less than 25% of the tissue water was exchanged after 30 minutes. Although there was only one experiment, Disocactus roots were also relatively impermeable. Though Disocactus is a desert plant, this was nonetheless surprising given that roots do not possess a cuticle. This may reflect a greater need to retain water in a desert environment, hi contrast, roots of hydroponically grown Poplar seemed particularly leaky, with ~ 75-85% of labelled water exchanged within the first 5 minutes, regardless of treatment. Populus trichocarpa is typically found in riverine habitats that are well supplied with water. 58 4.3 Exchange of metabolic water As noted above, on average approximately 63.4% of metabolic water was exchanged during the first 5 minutes of immersion, whereas only -21.9% of the remaining metabolic water exchanged over the 5-10 min interval. This difference was significant by T test at p < 0.005. Unfortunately, leakage was very severe. I anticipated the disequilibrium between the apoplastic and symplastic pools to last longer, hoping that metabolic water would diffuse at a much slower pace into the bulk media water. The movement was surprisingly fast. Previous work suggested that it might take a much longer period to reach isotopic equilibrium. For example, exchange was reportedly very slow, requiring many hours, in log phase E. coli cells (Kreuzer-Martin et al. 2005). Yakir (1991) observed a two hour period of uniform environmental conditions was required by Helianthus leaves to reach a state of isotopic equilibrium. In Phaseolus plants, the recorded time was one hour and almost an entire day in Helianthus leaves under natural conditions (Flanagan et al. 1991). The rate of exchange that I observed in roots was so high that it did not bode well for using the technique to separate autotrophic and heterotrophic respiration, this is because metabolic water diffuses from the symplast to the apoplast and into the bulk medium more rapidly than it can be labelled and recovered. At the same time however, the rate of exchange suggested an approach to study controls on movement of water across membranes or tissues. 4.4 Role of aquaporins remains elusive The movement of water across membranes is influenced by the cytoplasmic pH within the cells and aquaporin activity (Townaire-Roux et al. 2003). With this in mind, I went on 59 to look for evidence of the involvement of aquaporins in modulating water movement. This was done in three ways: 1. I attempted to block aquaporins with HgCk. The expectation was that HgCk treatment would increase the exchange time (Javot and Maurel, 2002). 2.1 used bathing solutions of varied pH, because low pH has been shown to reduce water channel activity in Arabidopsis (Townaire-Roux et al. 2003). 3. I looked for a diurnal pattern in exchange rate that would be consistent with reduced water channel activity at night. Cochard et al. (2007) have associated night-time reductions in leaf hydraulic conductance of walnut (Juglans regia) with down regulation of aquaporin transcripts. In a recent study (Vandeleur et al. 2009) the root water of grape (Vitis vinifera) channeled by aquaporins influenced the diurnal pattern in hydraulic conductivity. In neither Helianthus roots nor radicles, 200 uM HgCb did not influence the rate of exchange. Likewise, Populus roots at 50 uM HgCk and Controls were within ±10% of each other. Day vs. night time analysis of Populus roots revealed that over the 5 and 10 minute exchange intervals, the percent exchange of residual 180 label for day time sampled roots (n = 3) was 77.4% and 19.9%, and that of night time sampled roots was 80.5% and 26.7%. Clearly, there was no appreciable difference. In all three cases, sample sizes were low. Consequently, it's possible that one or more of these three experimental conditions might have a small effect on isotopic exchange. When Helianthus roots were treated in pH of 5, 6, 7, and 8 with a constant 5 min exchange period, there may have been greater retention of label at pH 5. It was not a large effect however and being a single unreplicated experiment no real conclusions are possible. This would be worthy of further investigations. 60 Regardless of the plant species experimented upon, mercuric chloride did not seem to have any effect. Susceptible aquaporins may have been unimportant in the tissue I used, or already closed. Alternatively, it's possible that mercuric chloride did not penetrate the tissue. I am left to conclude, however, that the exchange of metabolic water was not influenced by treatments believed to influence water flux across membranes. This aspect of my work is in dire need of further intense scrutiny; in future this may help in identifying the sites of respiration. 4.4.1 Issues related to exchange rates Because all sampling was destructive, the <5180i„itiai was always specific to the sample taken at time zero, and therefore may have been high or low relative to the "true" <5 Oinitiai of subsequent samples. This error was more severe when comparing samples taken at later time intervals, hi addition, some samples had a tendency to float more than others, thereby reducing the surface area for exchange, which would cause some error, (additional sources of error described in section 4.1.1) Up to this point in my studies, the rate of exchange observed in non transpiring plant parts was high with respect to bulk tissue water. Isotopic equilibrium was approached rapidly between the tissues, the cells within them and their surroundings. Although this was the case for completely submerged samples, metabolic water might be better retained by roots in soil (where there are air spaces) or where transpirational flow directs water into the plant. So, Objective 4 was to examine "The prospects for separating autotrophic and heterotrophic respiration in a simple system containing transpiring plants". The rate of exchange in "soil" was assessed in a model system, using transpiring plants (two week old Helianthus seedlings). The dx%0 values for shoot water were high 61 compared to dlsO of media water and most roots, whether labelled or not. There was also 1 o a tendency for media and plant S O values to be higher where pots sat longer before labelling, consistent with transpirational enrichment from shoots and evaporative enrichment from the soil surface. In silicon sand at least, where labelling of metabolic water was most successful, both evaporative/transpirational enrichment and root-to-shoot translocation of labelled water probably contributed to a higher shoot water S O value. The focal point here is the partitioning of label between soil medium, roots and shoots in the model system (Table 9). The maximum percent of labelled metabolic water lost to the medium in the transpiring Helianthus plants was 78.4% in potting-mix, 81.2% in soil, 90.3%) in silicon sand, and 94.1% in gravel. Similarly, the minimum percent of the label retained by harvested roots was 9.9%, 6.8%, 5.9%, and 2.7% respectively. The minimum percent of the label exported to shoots was 11.7% in potting-mix, 12% in soil, 3.8% in silicon sand, and 3.2% in gravel. On an average, 85%) of the label was lost to the growth media, 6% of the label was retained by the harvested roots, and 7% of the label was exported to the shoots. Clearly, then, it seems that also in soil media the roots were not able to retain metabolic water. Apart from this, respiration by microorganisms that may have been present in each media may have also contributed to the label found in the soil (i.e. the heterotrophic component). Furthermore, not all of the fine roots were successfully collected and this may account for some portion of the label "lost" to the medium. If the entire label recovered is ascribed to the root mass measured, then the estimated respiration rates are three times higher than expected. Therefore, it is likely that some large fraction of the label found in the medium does indeed originate heterotrophically. 62 I was obsessed by the movement of metabolic water via the symplastic and apoplastic components of tissues in various non-transpiring plants, as well as the rate of mixing of the symplastic root water with that of xylem apoplastic water in the transpiring Helianthus. To my dismay, metabolic water proved not much of a die-hard fan of movement (diffusion) in the presence of non-sterile rooting media. Rather than being retained, labelled water was rapidly lost to the soil medium. Had metabolic water been better retained in roots (tough luck), its movement in the cellular compartments would have been a whole new ball game of trying to prove its utility in detecting heterotrophic vs. autotrophic respiration. Perhaps, some day this sweet dream may come true. 63 5 CONCLUSION 5.1 Future applications for studying water relations and the role of metabolic water Metabolic water was easily labelled and detected. The successful application of 1802 labelling revealed its rapid movement from symplast to apoplast across cellular membranes, severely limiting its utility for pin-pointing the location of sites (cytochrome oxidase and alternative oxidase) of respiratory activity. However, as long as all metabolic water is experimentally retained and not lost through diffusion and transpiration, this labelling technique is equal or superior to any other available method for measuring respiration rate. The major drawbacks to this technique are that it is expensive and time consuming. Under special circumstances, this technique holds promise in investigating contributions of metabolic water in "dry" tissues or soils. 180 labelling of metabolic water under field conditions is clearly worth exploring. An attempt was made to assess this possibility in the field based on the natural abundance of 180 in air (Appendix: 7.3). If water goes through aquaporins, there has to be an effect on the rate of exchange. It is worth pursing what isotopically labelled metabolic water can tell us about aquaporin activity (functioning and control). This area is totally unexplored. If aquaporins are involved in water transport across membranes, then their modulation must certainly affect the approached isotopic equilibrium across those membranes. 64 6 LITERATURE CITED Adar, E.M., Gev, I., Lipp, J., Yakir, D., and Gat, J.R. 1995. 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A particular experiment (from Table 7, Expt. 1 and Fig 6A) was chosen as an example to address the above. Briefly, 1 S3 ^DDW value was -13.38%o. Control d Oimm value of shoots was 116.4%o; <5Sh00ts (5 mm) was 82.7%o; and Shoots (io mm) was 12.0%o. Based on <5DDW, <S18Omitiai values and water yield (mL) of the respective sample, the expected end-point was calculated using equation 11 in Chapter 2: <518Oend.point=(l0x£DDW)+ f  Water yield  C.1S/ 1000 xSl*0 initial 10 + f  Water yield 1000 [14] Where 10 (mL) is the amount of DDW in which the tissue was immersed, after label incorporation and "water yield" is the volume of water (uL) extracted from the tissue. Substituting the values, <?18Oend,oint=(l0x(-13.4))+ ' 2 1 1 - 3  , 1 , / x 116.4 1000 10 + 211.3 1000 = -10.96 [15] Based on the end-point derived (-10.96), the percent conversion on end-point was calculated using <S18Omjtiai and <5180finai of subsequent exchange periods (5 and 10 minutes) in the formula that follows: 77 %exchange(5mi in) ^A18n _>rI8n ° ^ ini t ia l ° V^shoots(5min) <S180initial-End point xlOO [16] Substituting the values, %exchange(5 ̂  = (116.4-82.7) 116.4 -(-10.96) > x 100 = 26.5% [17] So, 26.5% is the percent conversion on end point by the end of 5 minutes. Similarly, the calculated percent conversion on the end point was 82.25% by the end of 10 minutes. Given the end-point conversions (%), it is now possible to calculate the percent exchange 1 o of residual O (i.e. label remaining in tissues at 5 min) over the subsequent 5-10 min interval using the formula: Percent exchange of residual O = Substituting the values, f% exchange(10min) - % exchange(5min) N 100-% exchange (5 min) xlOO [18] Percent exchange of residual O (82.2-26.5) 100-26.5 x 100 = 75.5% [19] Put another way, exchange of residual labelled water remaining after 5 minutes was 1 o estimated by using the S O of tissue water at 5 min as the initial value. For example, in Control, the label exchange was 26.5% and 82.2% by the end of 5 and 10 min exchange periods respectively. The percent exchange of residual 180 label was 75.5% over the subsequent 5-10 min interval. Similarly, for Treated, the labelled metabolic water exchanged was 55.7% and 41.7% by the end of 5 and 10 minutes time period. The 1 o percent exchange of residual O label was -31.5% over the subsequent 5-10 min interval. 78 Appendix B 7.2 Application of 1 80 labelling under field conditions 7.2.1 Global scale Most water vapor in the atmosphere originates by evaporation from low latitude oceans. 9 i o Precipitation derived from this vapor is always enriched in deuterium ( H) and O relative to vapor as a function of condensation temperature. These two main factors control the isotopic character of precipitation at a given location. For water, the higher the mass number, the lower the vapor pressure. Thus, 160 and !H preferentially enter the vapor phase, whereas 180 and 2H preferentially concentrate in the liquid phase. Consequently, in evaporation, water vapor is enriched in 160 and !H, whereas the 1R 9 1 R remaining liquid water is enriched in O and H. More specifically, H2 O is enriched in liquid water by - 1 % relative to its concentration in water vapor at the same temperature. I Q For example, evaporation from the ocean with a S O of 0%o produces vapor of -12%o. Later, 50% condensation of rain from this vapor will result in water with a <5180 of -3%o and a residual vapor with a (5180 of -21%o. The <52H and <5180 values for precipitation worldwide behave predictably, falling along the Global Meteoric Water Line (GMWL) as defined by Craig (1961b), who expresses the relationship between 180 and 2H in meteoric waters as follows: <S2H = 8 <5180 +10%o [20] With increasing temperature, precipitation becomes enriched in the heavier isotopes, O and H, in a linear relationship. This relationship of the isotopes is primarily a reflection of differences in their equilibrium fractionation factors. The GMWL has an r2>0.95. This high correlation coefficient reflects the fact that the oxygen and hydrogen stable isotopes in water molecules are intimately associated (consequently the isotopic ratios and 79 fractionations of the two elements are usually discussed together). The slope of the GMWL expresses this ratio, which is eight times greater for oxygen than hydrogen. 7.2.2 Site specific: relevant to any field study At a given location, the slope and intercept of any Local Meteoric Water Line (LMWL), from a single site or set of 'local' sites can be significantly different from the GMWL. In general, most of these local lines have slopes of 8+/-0.05, but the line shifts toward increased <5180 because the phase change leans toward liquid precipitation, hi arid environments, LMWLs will exhibit the same slope, but plot higher in relation to d H because of increased evaporation. In general, rain in summer is isotopically heavier than rain in winter. Factors such as humidity, salinity, and temperature affect the kinetic fractionation during evaporation. The lower the relative humidity, the faster the evaporation rate and greater the kinetic fractionation. Humidity affects oxygen and hydrogen differently such that the slope of the evaporation line will vary due to changes in relative humidity. At very low relative humidities (<25%) the slope of the evaporation line will be close to 4; for moderate relative humidities (25% to 75%) the slope will be between 4 and 5; only for relative humidities above 95% does the slope approach 8, the slope of the meteoric water line (Clark and Fritz, 1997). 7.3 Field study of Pseudotsuga menziesii An attempt was made to detect the signature of metabolic water in the field based on the natural abundance of O in air. The field component of the study was conducted at UBC Farm on 07 August, 2007 (a cloudy day with a temperature of 16.4°C and a relative humidity of 75%). A very light drizzle of rainfall occurred mid day. The study species, P. 80 menziesii, was growing in a relatively undisturbed area adjacent to the main farm road. A cork borer was used to collect xylem and phloem from four trees; their placement similar to the four corners of a square. Roots, and root proximal parts were collected manually within this square. All samples were removed from the plants and placed in glass tubes sealed with a rubber stopper and wrapped with Parafilm®. The plant samples were then frozen until water was extracted from the tissue. <52HvS(S180 )0 X-- -90 -80 -70 -6" • • • * • # ; : * •A * • -50 •  l -40j -30 .. • - - A (1 - • phloem A xylem • root tip • soil water --->£-— GMWL Figure 9. Natural variation in the isotopic composition of water from Pseudotsuga menziesii The dnO value of the Earth's atmosphere is 23.5%o (Dole effect). In respiration, discrimination against O2 varies, but it's about 19 to 22%o (Guy et al. 1993). The expected O-H2O of metabolic water in nature therefore has a fairly constant isotopic composition of about 1.5 to 4.5%o. In contrast, the (52H of metabolic water will be equal to S2H of meteoric water or bulk tissue water, assuming that this is the ultimate source of all hydrogen atoms in plants (some might be derived from ammonium in the soil). In the field study (Fig 9), xylem water is consistent with GMWL whereas soil and phloem water 81 appear to be influenced by evapo-transpirational enrichment. Root tips are associated with higher d O and 5 H values, indicating that water is enriched due to evaporation. i n Although soil and root water appear to be preferentially enriched in 8 O, which would be expected if there was contribution from metabolic water, it is indistinguishable from the enrichment that would occur during evaporation. In the absence of applying artificial label (either 180-labelled O2 or 180/deuterium-labelled H2O) it is not possible to detect metabolic water against the large background variation. However, this may yet be possible in arctic or subarctic ecosystems where precipitation is lighter in isotopic composition. 18C>2 labelling of metabolic water under field conditions should also be explored. 82

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