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¹⁵N discrimination as an indicator of nitrogen dynamics in Populus trichocarpa Buschhaus, Hannah Ariel Elizabeth 2007

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'N Discrimination as an Indicator of Nitrogen Dynamics in Populus trichocarpa By Hannah Ariel Elizabeth Buschhaus B.Sc. Hon. (Biology) Trinity Western University, 2003 A.Sc. Tacoma Community College, 2000 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Forest Sciences) THE UNIVERSITY OF BRITISH C O L U M B I A April 2007 © Hannah Ariel Elizabeth Buschhaus, 2007 A B S T R A C T The current understanding of nitrogen stable isotope ratios in plant tissues increasingly emphasizes the relationship between plant growth and nitrogen nutrition in determining plant 8 1 5 N. This demand relative to supply is thought to influence the plant's ability to discriminate against the heavier 1 5 N . Discrimination (Dpiant) is then linked to the ratio of efflux to influx. Factors which influence either of these thus affect Dpiant- This thesis examines genotypic differences and physiological manipulations in Populus trichocarpa to further test the proposed efflux/influx model for Dpiant. Substrate depletion experiments used ramets grown in hydroponic media containing 200uM N H 4 + . Root pruning to reduce the plant's capacity to supply N correspondingly increased the rate of N H 4 + uptake and decreased Dpiml. Shoot pruning and genotypic variation did not appear to play a significant role in determining Dpiant as assessed by this method. Substrate depletion experiments also allowed us to calculate the root N H 4 + influx and efflux from the 8 1 5 N and the net uptake rate. This novel application of the efflux/influx model for discrimination generated efflux and influx values that corroborated existing radiolabelled 1 3 N studies. The ability to accurately calculate efflux and influx using stable isotope methods at natural abundance levels provides a new, non-radioactive approach for further nutrient-uptake efficiency studies. In steady-state experiments, ramets were grown at either ambient (400ppm) or elevated (800ppm) atmospheric CO2 concentrations in either 200uM or 400|j.M N H 4 + hydroponic media. Within the treatments, Dpiant corresponded to the relative growth rate responses, signifying its dependence on physiological growth factors. Genotypic differences in the discrimination values of P. trichocarpa provenances could be manipulated by changing the supply/demand regimes. Plant tissue 8 1 5 N revealed an unexpected but distinct foliar enrichment. These data prompted the development of a revised efflux/influx model that accounts for translocation and subsequent assimilation of N H 4 + in the leaves. This newest model now provides testable hypotheses for future N H 4 + translocation and assimilation studies. ii TABLE OF CONTENTS ABSTRACT " LIST OF TABLES v LIST OF FIGURES vi ACKNOWLEDGEMENTS viii DEDICATION ix 1 Introductory Chapter 1 1.1 Introduction 1 1.1.1 Stable isotopes of nitrogen 1 1.1.2 Fractionation: changing the isotopic composition 2 1.1.3 Plant discrimination: the efflux/influx ratio 4 1.1.4 Plant material: Populus trichocarpa 5 1.1.5 Nitrogen dynamics in Populus trichocarpa 5 1.2 Research obj ectives , 6 1.2.1 Research questions and purpose 6 1.2.2 Approach 7 1.3 References 8 2 5 1 5N Discrimination in Populus trichocarpa by the Substrate Depletion Method 11 2.1 Introduction 11 2.2 Methods 13 2.2.1 Plant material and growth 13 2.2.2 Substrate depletion and diffusion techniques 13 2.2.3 Provenance experiments 16 2.2A Pruning experiments 16 2.3 Results 17 2.3.1 Substrate depletion analysis 17 2.3.2 Efflux and influx 19 2.3.3 Provenance comparisons 20 2.3.4 Pruning effects 22 2.4 Discussion 25 2.4.1 8 1 5 N and discrimination 25 2.4.2 Estimating efflux and influx 27 2.4.3 Genetic variation in N isotope discrimination 28 2.4.4 Pruning as a means of altering N isotope discrimination 29 2.4.5 Conclusion 29 2.5 References 30 3 Tissue 6 1 5N of Populus trichocarpa Grown in Steady-State N H 4 + Nutrition.... 33 3.1 Introduction 33 3.2 Methods 36 3.2.1 Plant material and maintenance 36 3.2.2 Isotopic analysis 37 i n 3.2.3 Statistical analysis 37 3.3 Results 38 3.3.1 Relative growth rate 38 3.3.2 Nitrogen content 40 3.3.3 5 1 5 N analysis 41 3.4 Discussion , 46 3.4.1 Growth rate and biomass accumulation 46 3.4.2 Tissue % N 46 3.4.3 Whole plant 5 1 5N 46 3.4.4 Provenance 8 1 5 N 47 3.4.5 Plant tissue component S 1 5 N 47 3.4.6 Comparative analysis 49 3 A.l N H 4 + transport and assimilation model 50 3.4.8 Conclusion 53 3.5 References 54 4 Conclusion 58 4.1 Recommendations for future research 58 4.2 References 61 iv LIST OF T A B L E S Table 2-1. Quantification of 8 1 5 N for the N H 4 + salt and for the contamination associated with glassware cleaning and Teflon 15 Table 3-1. Total plant tissue averages of P. trichocarpa for 6 weeks of growth in either 200 or 400 uM N H 4 + at either ambient or elevated CO2. Averages represent three provenances: Bell Irving River, B C (IRVD); Quesnel Lake, BC (QLKE); and Jasper River, OR (JASP). RGR is the relative growth rate 38 Table 3-2. Model input data obtained from the P. trichocarpa steady-state growth experiment with the specific treatment conditions as noted. The treatment averages are inclusive of all three tested provenances 52 Table 3-3. Model outputs for the P. trichocarpa treatment data given in Table 3.2. Tj/T t equals the proportion of foliar N translocated to the leaves as inorganic NH4; / equals the fraction of total plant N assimilated in the roots; E/I equals the ratio of efflux to influx in the root-growth medium continuum; 8 1 5N c ytopiasm equals the isotopic composition of the N in the root cytoplasm 52 v LIST OF FIGURES Figure 2.1. Data from a typical substrate depletion experiment showing the changes in residual N H 4 + 8 1 5 N as the substrate was consumed (A), and the same data plotted to show how discrimination is estimated as the slope of the line between two sample points (B). The relationship between the estimated discrimination from Panel B and N H 4 + concentration is shown in Panel C 18 Figure 2.2. The E/I ratio (A) was calculated from Eq. 1 and the discrimination factors in Figure 2.1C. Estimations of the net N H 4 + influx and efflux (B) were also made using the net N H 4 + uptake rate and the corresponding Dpiant according to Eq. 5 20 Figure 2.3. The average provenance fresh weight biomasses (bars) and root-to-shoot fresh weight ratios (line) obtained at the time of the drawdown experiments. The three provenances were as follows: Jasper River (JASP), OR (44°00'), Quesnel River (QLKE), BC (52°58'), and Bell-Irving River (IRVD), BC (56°51'). Error bars represent the standard error of the mean 21 Figure 2.4. The discrimination factors of the three provenances as calculated from approximations of the curve generated from Eq. 4. The provenance labels are identical to Figure 2.3 22 Figure 2.5. P. trichocarpa 1 5 N discrimination obtained via the drawdown technique for three days prior to and post shoot-pruning 23 Figure 2.6. The net uptake rate of N H 4 + per root mass in the root-pruned and unpruned P. trichocarpa (p=0.09) 24 Figure 2.7. The root-pruned and unpruned P. trichocarpa discrimination against 1 5 N . The correlation lines highlight the difference between the pruned (r2=0.203, p=0.341) and unpruned (r2=0.50, p=0. 019) treatments 25 Figure 3.1. P. trichocarpa relative growth rate (RGR) values were for the first 6 weeks of ramet establishment on either 200 u M N H 4 + 400ppm CO2 (200 Ambient), 200 uM N H 4 + 800ppm C 0 2 (200 Elevated), 400 uM N H 4 + 400ppm C 0 2 (400 Ambient) or 400 uM N H 4 + 800ppm C 0 2 (400 Elevated) . RGR values are the averages of the three provenances: Bell Irving River, BC (IRVD); Quesnel Lake, BC (QLKE); and Jasper River, OR (JASP) (n=12). The letters (a, b) differentiate statistical significance of p<0.05 39 Figure 3.2. P. trichocarpa relative growth rate (RGR) values were for the first 6 weeks of ramet establishment. RGR values are the averages of the three clones representing each provenance. Treatment and provenance labels are identical to Figure 3.1. Statistical significances for the two N treatments were calculated separately and are indicated by the lower case letters (p<0.05) 40 vi Figure 3.3. P. trichocarpa root and leaf tissue %N were calculated as a percentage of the respective tissue's dry mass at the conclusion of the 6 week growing period. Treatment and provenance labels are identical to Figure 3.1. Statistical significances for the two N treatments were calculated separately and are indicated by the lower case letters (p<0.05) 41 Figure 3.4. P. trichocarpa 8 1 5N values measured for the first 6 weeks of ramet establishment. 8 1 5N values are the averages of the three clones representing each provenance. Treatment and provenance labels are identical to Figure 3.1. Statistical significances for the two N treatments were calculated separately and are indicated by the lower case letters (p<0.05) 42 Figure 3.5. P. trichocarpa root and leaf tissue 8 1 5N measured at the conclusion of the 6 week growing period. Treatment and provenance labels are identical to Figure 3.1. Statistical significances for the two N treatments were calculated separately and are indicated by the lower case letters (p<0.05) 43 Figure 3.6. Leaf and root 8 1 5N values are plotted as a function of tissue %N. The correlation is for the 200 uM N H 4 + elevated CO2 treatment (r2=0.150, p=0.035). Treatment and provenance labels are identical to Figure 3.1 44 Figure 3.7 Root to leaf 8 1 5N differences are plotted as a function of leaf %N. The correlation highlights the 200 uM N H 4 + elevated C 0 2 treatment (r2=0.372, p=0.0003). Treatment and provenance labels are identical to Figure 3.1 45 Figure 3.8. Modeled diagram of N fluxes during the uptake of N H 4 + from the source pool in the growth media and assimilation into the organic sink pools in both root and leaf tissues. The cytoplasmic intermediate pool is considered to be in a steady state with respect to both total mass and isotopic composition. Arrows refer to the total unidirectional fluxes of both isotopes with labeling according to the specific flux: E = efflux, I = influx, GS = glutamine synthetase, T 0 = organic N translocated to the shoot, Tj = inorganic N translocated to the shoot, and T t = total N translocated to the shoot 50 vii A C K N O W L E D G E M E N T S I gratefully acknowledge the many people who have facilitated the work contained in this thesis. First, I thank my parents. Guiding and assisting with 4-H projects on everything from cooking and sewing to animal breeding and showing as well as with science experiments ranging from simple lab homework to collecting biological samples in the sleet, they loved to learn and teach in a way that someday I hope to achieve. They taught me the perseverance needed to complete this graduate degree through the many projects and trips we went on as a family. Specifically, I thank my father for his love of nature and research that helped me to develop similar interests throughout high school and university. I also thank my mother for contributing her English grammar editing skills to this thesis. I would particularly like to thank those who worked with me as I discovered scientific research through completing numerous course research projects during my undergraduate degree at Trinity Western University. Prof. Karen Steensma's passion for ecological research carried through into zoology projects that I completed in her courses. My interest in research was further stimulated through working with Dr. David Clements on Scotch broom ecology in Garry oak meadows. Dr. Paul Brown introduced me to plant physiology through course projects on nutrient deficiencies. I would also like to thank the many fellow students I worked with at TWU. The work of this thesis would not have been possible without the wonderful help from the faculty, staff and fellow students in the Faculty of Forestry at the University of British Columbia. I am continually inspired by their work. I owe a particular thanks to Dr. Rob Guy, my M.Sc. supervisor, for his endless patience and valuable insights. I thank my lab mates Raju, Tyler, Dheeraj, Virginie, Azam, Ankit and Limin for helping me with lab work and with morale support as we toiled together. I specifically would like to thank Raju for sharing an office with me over the past several years and for introducing me to Indian music during the afternoons. I also wish to thank the amazing summer students Rhianna, Meghan, Yuriko, and Megumi for all the hands-on work that they contributed for this thesis. Finally I thank my husband, Christopher Buschhaus, for helping me through many undergraduate projects, seeing me through this whole thesis process, and encouraging me to think more critically. I love you. viii DEDICATION I dedicate this thesis to my grandfather, Fredrick Shelton, who for years has studied forest product chemical engineering and continues to delight in research. He took me to my first conference and challenged me to pursue education. ix 1. Introductory Chapter 1.1 Introduction Nitrogen, the fourth most common element in living systems, is a foundational element within the biosphere. It forms an essential component of proteins and is necessary to all living organisms for survival. Plants acquire the majority of their mineral nitrogen nutrition from soil solution as NO3" and N H 4 + . The nitrogen stable isotopes within these compounds are unequally incorporated into various plant metabolites, and consequently, plant parts. The unequal incorporation or "fractionation" of stable isotopes among these various pools results from discrimination at entry and/or exit into or out of the plant, and presumably, at metabolic branch points within the plant. Nitrogen isotope discrimination during assimilation is not yet thoroughly understood, but it seems to be influenced by changing nutrient dynamics and growth demands. As such, this study sought to examine physiological and genetic factors affecting the relationship of nutrition and growth in black cottonwood (Populus trichocarpa Torr. & Gray) with the aim of gaining further insight into the mechanisms controlling nitrogen isotope discrimination in woody plants. 1.1.1 Stable isotopes of nitrogen Nitrogen exists as two stable isotopes which are present in both inorganic (e.g. NO3", NO2 \ N H 4 , NH3, N2) and organic compounds. The majority of nitrogen contains seven neutrons and seven protons giving in an atomic mass of approximately 14 amu (hence 1 4 N). A small percentage (only 0.3663%) of global nitrogen contains an extra neutron which increases the mass to 15 amu ( 1 5N). Although the lighter of the isotopes ( 1 4N) is by far the most abundant, small shifts in the abundance of 1 5 N offer tremendous potential for new approaches in researching ecological processes. Stable isotope abundances are expressed on the basis of the isotopic ratio (R) of heavy to light isotopes, e.g. 1 5 N / 1 4 N . Comparing the ratio of a material sample (Rsamp!e) to the ratio of a well-characterized standard (Rstd), namely air, provides a measure of the absolute isotopic content of the sample (Ehleringer and Rundel, 1988). This technique of differential comparison between a sample and standard was first introduced for stable isotope analysis 1 almost sixty years ago by Nier (1950). In differential notation the basic equation is as follows: [ 1 ] 5 X s a m p l e = (Rsample/Rstd - 1) X 1000 where 8X s a m p i e is the isotope ratio in del (5) units relative to the standard. Multiplying by 1000 allows convenient expression in parts per thousand (%o) or "per mil ." Expression in this manner provides a precise focus on the differences between the samples. Thus, while the standard is N 2 in air (8 1 5 N = 0 %o), natural variations in 8 1 5 N generally range from as little as -20%o to as much as +20%o relative to this standard (Ehleringer and Rundel, 1988). Biologically significant differences are often less than l%o (Handley and Raven 1992). 1.1.2 Fractionation: changing the isotopic composition Differences in the sample 8 1 5 N values arise through processes of discrimination which produce a measured difference in the isotope ratio referred to as fractionation. Discrimination results from differences in equilibrium and kinetic constants of the stable isotopes in both physical and (bio)chemical reactions respectively (Broeker and Oversley, 1971). In chemical interactions, the lighter isotope tends to react more readily than the heavier one, resulting in 1 5 N enrichment of the source and matching depletion of the sink (Handley and Scrimgeour, 1997). Biologically speaking, kinetically-activated enzymatic reactions all have large fractionation potentials due to the energy requirements associated with breaking bonds with the heavier 1 5 N (Handley and Scrimgeour, 1997). Besides these processes which result in fractionation, there are numerous influences on the expression of isotopic effects such as source effects, diffusional constraints, enzyme selectivity, and/or interactions between compounds (Shearer and Kohl, 1989a). In principle, discrimination is readily calculated as the instantaneous difference between the source and product isotopic ratios. In practice, however, the composition of either the source, or its (instantaneous) product, may be difficult to obtain. Quantifying discrimination is further complicated by the multiple processes that could be contributing to the 8 1 5 N difference. Since all isotopic changes must conform to the law of conservation of matter, as discrimination occurs one pool of nitrogen will increase in its isotopic signature while another will decrease. For this to be measurable as net fractionation, each pool must be 2 measured separately or there must be a physical loss or gain from the N pools of interest (Handley and Scrimgeour, 1997). Thus, in closed systems, net fractionation is not realized when all of the substrate is converted to product because none is left to report a unique isotope ratio. Nature, however, does not generally support a closed system, although some systems do behave as such, due to slow substrate replenishment rates. In the context of plant N uptake, a lack of source to sink 8 1 5 N difference indicates limited N supplies (Evans et al. 1996). In contrast to the closed system, a fully open system boasts an infinite supply of substrate relative to the demands of the reaction and thus fractionation between substrate and product is realized and can be easily measured (Handley and Scrimgeour 1997). Plant 8 1 5 N studies quantify N isotope discrimination using these two general systems in various ways. In practice, there are three approaches. One attempts to establish an essentially open system whereby the plants are grown for extended periods without substantial depletion of the nutrient medium or changes in the nutrient demand. As a non-exhaustible source is a difficult predicating circumstance to meet, experiments conducted in this manner at low N concentrations tend to report lower than expected discrimination values, whereas experiments conducted at higher N concentrations result in increasing discrimination (Yoneyama et al. 1991; Evans et al. 1996). The two remaining approaches rely on a closed system where the media N is progressively consumed. The first of these measures the product's isotope enrichment as it accumulates in the plant. Because this requires repeated destructive sampling, it is not amenable to intact, actively growing higher plants; however, it has been extensively used in phytoplankton (Needoba et al. 2004). The second closed system approach analyzes the isotopic enrichment of the nutrient media. As the plant consumes the media N , the remaining N is progressively enriched. Although rarely used for N isotopes, this method has the advantage of providing short-term discrimination data over a range of concentrations (Kolb and Evans 2003; Pritchard and Guy 2005). In principle, plant-mediated fractionation of 1 4 N and l 5 N could occur during uptake of inorganic N or at metabolic branch-points in plant N utilization (Comstock 2001; Evans 2001; Robinson 2001). The first opportunity for isotope discrimination, the absorption of inorganic N across root cell membranes, does not result in measurable fractionation (Shearer and Kohl 1989b). The next opportunity for fractionation lies with the kinetic preferences of 3 the assimilatory enzymes. N H 4 + is assimilated into glutamine by glutamine synthetase (GS). Based on in vitro measurements, discrimination by GS has been estimated to be 16.5 ± 1.5%o (Yoneyama et al. 1993), whereas in vivo estimates are highly variable (Robinson 2001). There are additional opportunities for tissue level fractionation within the plant i f 1 5 N is disproportionately translocated or exuded at metabolic branch-points from specific N pools (Comstock 2001; Evans et al. 1996). 1.1.3 Plant discrimination: the efflux/influx ratio Because discrimination does not appear to occur during membrane transport, but instead occurs during and after assimilation, fractionation between the media and plant can only happen i f isotopically enriched nitrogen effluxes back into the bulk medium (Comstock 2001; Evans 2001; Robinson 2001). When plants absorb net quantities of N H 4 + or NO3", a substantial level of efflux also occurs (Handley and Raven 1992 and citations therein). Efflux must be considered in relative proportion to influx; thus, it is the efflux/influx ratio that ultimately varies in direct proportion to discrimination (Handley and Raven 1992). To this end, i f the external N concentrations were high or demand low, and efflux equaled influx, the resulting source-to-plant difference would approximate the discrimination by the enzyme. Since these conditions are impractical, most studies report much smaller source to sink differences in 8 1 5 N. In experiments where the substrate concentration varied, increasing the nitrogen concentration so that efflux/influx approached unity resulted in greater differences in 8 1 5 N values (Kolb and Evans 2003; Hoch et al. 1992; Hogberg et al. 1999; Yoneyama et al. 1991, 2001). Conversely, i f influx is substantially greater than efflux (i.e. efflux<influx), as in the case of a more limited nitrogen supply, source-to-plant differences in 8 1 5 N decrease. This trend was confirmed in diatoms where nitrogen demand was high following nitrogen starvation and less nitrogen was effluxed from cells, thereby reducing fractionation (Waser et al. 1999). The correlation between discrimination and the efflux/influx ratio, as controlled by nutrient supply and demand, provides a foundation for examining the growth and nutritional factors affecting plant discrimination. 4 1.1.4 Plant material: Populus trichocarpa Poplars provide a unique opportunity to study N isotope discrimination and N dynamics in trees as they are a fast-growing, easily propagated, temperate hardwood species. Their rapid growth requires large nutrient quantities, specifically N , to be quickly accumulated, making them an ideal study candidate. Numerous species selections, cultivars, and hybrids exist on which to base comparative studies. Natural variation can be explored through sampling wild populations. For the present study, a large collection of 880 P. trichocarpa clones, spanning a considerable latitudinal range, was made accessible courtesy of Dr. Cheng Ying (BCMoFR/Research Branch, Surrey, BC). In a recent review, Taylor (2002) outlines the many other advantages (and some drawbacks) of using poplar as a model tree. One compelling reason is the completed genome sequencing of Populus trichocarpa, the first tree species, and only the third plant, to be entirely sequenced (Tuskan et al. 2006). 1.1.5 Nitrogen dynamics in Populus trichocarpa The influences of plant supply and demand dynamics on 1 5 N discrimination can be used to further elucidate the role of the efflux/influx ratio in determining plant discrimination. Supply and demand are linked to plant growth and nutrition, and these are controlled by a number of factors. Genetic variation by latitude affects plant growth and morphology through such physiological adaptations as photosynthetic rate differences (Benowicz et al. 2000), nitrogen-use-efficiency (NUE) (Li et al. 1991), and many other factors (Pilon et al. 2002; L i et al. 1998). These differences serve to alter the plant's need for N , and thus potentially alter demand relative to supply, with subsequent effects on discrimination (Pritchard and Guy 2005; Kolb and Evans 2003; Yoneyama et al. 2001; Robinson et al. 2001; Handley et al. 1997). Elevated atmospheric CO2 affects plant growth and might, therefore, influence discrimination. In greenhouse experiments conducted by Tupker et al. (2003), CO2 enrichment increased growth and the proportional allocation of biomass to roots as well as elevating net carbon assimilation. As these changes affect the plant demand relative to nutrient supply, they are expected to alter discrimination. Indeed, elevated CO2 affected N allocation, which in turn altered foliar 8 1 5 N values in Phalaris arundinacea L. (Holdfast) and Physalis peruviana L. (Cape Gooseberry) (Stock and Evans 2006). 5 Root-to-shoot ratios influence the nutrient dynamics of the plant. In pruning experiments performed on tomatoes, decreasing the root biomass by pruning correspondingly decreased the total nitrogen uptake but increased the uptake per unit root weight (Bar-Tal et al. 1994). This alteration of the root-specific nitrogen uptake capacity should limit supply relative to demand, thus decreasing discrimination. 1.2 Research objectives 1.2.1 Research questions and purpose The work presented in this thesis sought to further clarify the role of the efflux/influx ratio in plant isotope discrimination by intentionally manipulating the N supply and demand dynamics of P. trichocarpa. I sought answers to the following questions: (1) Does genotypic variation in P. trichocarpa result in differential N isotope discrimination between populations? (2) Does manipulating the supply/demand regime result in changes in N isotope discrimination? The first objective was to determine whether there is genotypic variation in N isotope discrimination in poplar. I hypothesized that provenances with a high nitrogen demand, high nitrogen assimilation capacity, or a low uptake capacity would have a low substrate-to-enzyme ratio which, according to the efflux/influx model, should result in decreased discrimination as measured by increasing 5 1 5 N. Understanding variations in discrimination arising at the genotypic level provides insight into nutritional efficiencies and acts as a stepping stone for additional discrimination-based physiological experiments. The second objective was to alter discrimination experimentally. Three approaches were taken. The first approach was to physically alter the root-to-shoot ratio by pruning. The purpose of these surgical alterations was to specifically reduce either the capacity of the plant to supply N (root pruning) or to reduce the overall demand (shoot pruning). By manipulating the supply/demand physiology in this manner, my intent was to shift N discrimination by changing the efflux/influx ratios in a closed system. I hypothesized that reducing the root biomass would increase uptake rate and thus decrease discrimination. Conversely, reducing shoot biomass would decrease nutrient demand and thus reduce discrimination. M y second approach was to use CO2 elevation to provide a non-invasive 6 means of manipulating N demand and isotope discrimination, in a near-open system. Here I hypothesized that by increasing the growth demands with CO2, the discrimination would correspondingly decrease. Elevated atmospheric CO2 carries current ecological interest. Because of impending increases in atmospheric CO2 concentrations the knowledge gained here may be applicable to future research. The third approach specifically examined changing the steady-state N H 4 + media concentration, also in a near-open system. I hypothesized that when this alteration of the media N H 4 + increased the nutrient supply relative to demand, the discrimination would increase. The inverse was also hypothesized. Overall, the experiments laid out in the following thesis chapters seek to examine the relationship of supply relative to demand on N isotope discrimination. 1.2.2 Approach The following two manuscript chapters tackle the above objectives by measuring discrimination and/or isotope fractionation in either closed or open systems, respectively. The first chapter uses substrate depletion analyses to determine discrimination as a function of decreasing media N H 4 + concentrations. Attempts to achieve differences in supply/demand regimes were made through exploiting genotypic variation and by physical pruning of the roots or shoots. The second chapter uses steady-state conditions to obtain the time-averaged discrimination resulting from genotypic variation in interaction with the effects of CO2 concentration on a two-fold difference in nutrient media N H 4 + concentration. 7 1.3 References Bar-Tal, A. , Feigin, A . Rylski, I., and Pressman, E. 1994. Effects of root pruning and N-NO3 solution concentration on nutrient uptake and transpiration of tomato plants. Scientia Hort 58: 77-90. Benowicz, A. , Guy, R.D. and El-Kassaby, Y . A . 2000. Geographic pattern of genetic variation in photosynthetic capacity and growth in two hardwood species from British Columbia. Oecologia 123: 168-174. Broeker, W.S. and Oversley, V . M . 1971. Chemical Equilibria in the Earth. McGraw-Hill, New York. Comstock, J.P. 2001. Steady-state isotopic fractionation in branched pathways using plant uptake of N 0 3 - as an example. Planta 214: 220-234. Ehleringer, J.R. and Rundel, P.W. 1988. Stable isotopes: history, units, and instrumentation. Stable Isotopes in Ecological Research. (Ed. by P.W. Rundel, J.R. Ehleringer and K . A . Nagy) Springer-Verlag, New York.. 1-16. Evans, R.D. 2001. Physiological mechanisms influencing plant nitrogen isotope composition. Trends Plant Sci 6: 121-126. Evans, R.D., Bloom, A.J . , Sukrapanna, S.S. and Ehleringer, J.R. 1996. Nitrogen isotope composition of tomato (Lycopersicon esculentum mill. cv. T-5) grown under ammonium or nitrate nutrition. Plant Cell Environ 19: 1317-1323. Handley, L .L . and Raven, J.A. 1992. The use of natural abundance of nitrogen isotopes in plant physiology and ecology. Plant Cell Environ 15: 965-985. Handley, L .L . , Robinson, D., Forster, B.P., Ellis, R.P., Scrimgeour, C M . , Gordon, D . C , Nevo, E. and Raven, J.A. 1997. Shoot delta N-15 correlates with genotype and salt stress in barley. Planta 201: 100-102. Handley, L .L . and Scrimgeour, C M . 1997. Terrestrial plant ecology and 1 5 N abundance. Advances in Ecological Research 27: 133-212. Hoch M.P., Fogel, M . L . and Kirchman, D.L. 1992. Isotope fractionation associated with ammonium uptake by a marine bacterium. Limnol Oceanogr 37:1447-1459. Hogberg P., Hogberg, M . N . , Quist, M.E. , Ekblad, A . and Nasholm, T. 1999. Nitrogen isotope fractionation during nitrogen uptake by ectomycorrhizal and non-mycorrhizal Pinus sylvestris. New Phytol 142:569-576. Kolb K.J . and Evans, R.D. 2003. Influence of nitrogen source and concentration on nitrogen isotopic discrimination in two barley genotypes (Hordeum vulgare L.). Plant, Cell Environment 26:1431-1440. 8 Li B., McKeand, S.E., and Allen, H L . 1991. Genetic variation in nitrogen use efficiency of loblolly pine seedlings. For Sci 37: 613-626. Li B., Suzuki, J. and Hara T. 1998. Latitudinal variation in plant size and relative growth rate in Arabidopsis thaliana. Oecologia 115: 293-301. Needoba, J.A., Sigman, D.M., and Harrison, PJ . 2004. The mechanism of isotope fractionation during algal nitrate assimilation as illuminated by the N-15. Journal of Phycology 40: 517-522. Nier, A.O. 1950. A redetermination of the relative abundances of the isotopes of carbon, nitrogen, oxygen, argon, and potassium. Physical Review 77: 789-793. Pilon, J., Santamaria, L., Hootsmans, M. and van Vierssen, W. 2002. Latitudinal variation in life-cycle characteristics of Potamogeton pectinatus L.: vegetative growth and asexual reproduction. Plant Ecology 165: 247-262. Pritchard, E.S. and Guy, R.D. 2005. Nitrogen isotope discrimination in white spruce fed with low concentrations of ammonium and nitrate. Trees. 19: 89-98. Robinson, D. 2001. Delta N-15 as an integrator of the nitrogen cycle. Trends in Ecology & Evolution 16: 153-162. Shearer, G. and Kohl, D.H. 1989a. Estimates of N 2 fixation in ecosystems: the need for and basis of the 1 5 N natural abundance methods. Stable Isotopes in Ecological Research (Ed. by P.W. Rundel, J.R. Ehleringer and K.A. Nagy). New York, Springer-Verlag. 343-374. Shearer, G. and Kohl, D.H. 1989b. Natural 1 5 N abundance of N H 4 + , amide N, and total N in various fractions of nodules of peas, soybeans and lupins. Aust J Plant Physiol 16: 305-313 Stock, W.D. and Evans, J.R. 2006. Effects of water availability, nitrogen supply and atmospheric CO2 concentrations on plant nitrogen natural abundance values. Funct Plant Biol 33: 219-227. Taylor, G. 2002. Populus: Arabidopsis for forestry. Do we need a model tree? Annals of Botany 90:681-689. Tupker, K.A., Thomas, B.R. and Macdonald, S.E. 2003. Propagation of trembling aspen and hybrid poplar for agroforestry: Potential benefits of elevated CO2 in the greenhouse. Agrofor Syst 59: 61-71. Tuskan, G.A., et al. 2006. The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 313: 1596-1604. 9 Waser, N.A. , Y in , K. , Nielsen, B., Harrison, P.T. Turpin, D.H. and Calvert, S.E. 1999. Nitrogen isotopic fractionation during a simulated diatom spring bloom: importance of N-starvation in controlling fractionation. Mar Ecol Prog Ser 179: 291-296. Yoneyama, T., Kamachi, K. , Yamaya, T. and Mae, T. 1993. Fractionation of nitrogen isotopes by glutamine-synthetase isolated from spinach leaves. Plant Cell Physiol 34: 489-491. Yoneyama, T., Matsumaru, T., Usui, K. and Engelaar, W.M.H.G. 2001. Discrimination of nitrogen isotopes during absorption of ammonium and nitrate at different nitrogen concentrations by rice (Oryza sativa L.) plants. Plant Cell Environ 24: 133-139. Yoneyama, T., Omata, T., Nakata, S. and Yazaki, J. 1991. Fractionation of nitrogen isotopes during the uptake and assimilation of ammonia by plants. Plant Cell Physiol 32: 1211-1217. 10 2. 8 1 5 N Discrimination in Populus trichocarpa by the Substrate Depletion Method 2.1 Introduction Natural variation in plant nitrogen isotopic composition has the potential to provide information on plant nutrient dynamics and growth physiology. Discrimination against 1 5 N results from reaction kinetics where assimilation of the heavier isotope requires additional energy to break bonds with 1 5 N ; consequently, it is not incorporated into plant compounds as readily as the lighter isotope (1 4N). The factors that control the exact proportions of heavy to light nitrogen isotopes (8 1 5N) in plants are poorly characterized. Discrimination can only be expressed if there is opportunity for isotopically enriched N to return to the bulk medium through the process of efflux. If this possibility exists, fractionation of 1 4 N and 1 5 N could occur during net uptake from the medium into root cells or during enzymatic conversion into other N forms. Work on legumes by Shearer and Kohl (1989) demonstrated that fractionation of N H / during membrane transport does not occur, thereby leaving the primary opportunity for fractionation in ammonium assimilation with glutamine synthetase (GS). Discrimination by GS has been estimated in vitro at 16.5 ± 1.5%o (Yoneyama et al. 1993). Although not fully characterized, current opinion regarding whole plant N isotope discrimination (Dpiant) is that net discrimination depends primarily on nutrient supply relative to demand and its respective effects on ion efflux relative to influx (Pritchard and Guy 2005). To this end, if GS has free access to an infinite nitrogen source (i.e. influx=efflux) at a constant 8 1 5 N value, the resulting source-to-plant difference would approximate full discrimination by the enzyme. Most studies report much smaller differences in 8 1 5 N. In experiments where the substrate concentration varied, an increased nitrogen concentration resulted in greater differences in 8 1 5 N consistent with efflux/influx ratios approaching unity (Kolb and Evans 2003; Hoch et al. 1992; Hogberg et al. 1999; Yoneyama et al. 1991, 2001). Conversely, where influx is substantially greater than efflux, as in the case of a limited nitrogen supply, source to plant differences in 8 1 5 N should be proportionally smaller, as described by the following equation: [ 1 ] Dp^ = efflux/influx * Anzyme 11 where Dpiant is the whole plant 1 5 N isotope discrimination and the efflux/influx ratio is proportional to the growth demands and nutrition of the plant (Handley and Raven 1992). Anzyme refers to the discrimination constant for the enzyme of interest (GS in this case). Root-to-shoot ratios influence the nutrient dynamics of the plant by changing its supply/demand capacity. In pruning experiments performed on tomatoes, a reduction in root mass correspondingly reduced the total nitrogen uptake, but increased the uptake per unit root weight (Bar-Tal et al. 1994). In consideration of equation 1, this alteration of the root-specific nitrogen uptake rate carries the distinct potential of altering discrimination patterns because it should decrease the supply capacity relative to demand, thus decreasing discrimination. Genetic variation in components of nitrogen metabolism may also affect plant 8 1 5 N (Handley et al. 1997; Robinson et al. 2001; Pritchard and Guy 2005). Yoneyama (1991) documented changes in plant 8 1 5 N under N H / nutrition that were dependent on both media nitrogen concentration and rice cultivar. Pritchard and Guy (2005) proposed that genetic differences in discrimination are caused by different demands on assimilation and capacity for ion uptake, which in turn influence the balance between nitrogen influx and efflux. Having a large geographic range and ecological amplitude, P. trichocarpa provides a model for studying adaptive variation in trees. Genetic or environmental factors affecting growth rate or root-to-shoot ratios may control D; for example, in this species, there are latitudinal clines in photosynthetic capacity and other ecophysiological variables such as leaf N content which should influence N demand and be reflected in provenance D values (Gornall and Guy, 2002). In this experiment, I investigated the concentration dependence of Dpiant in the short-term, 1 5 N enrichment occurring during the progressive depletion of hydroponic media. The use of the draw-down technique provides the necessary data to calculate efflux and influx under natural abundance conditions. I hypothesized that net discrimination would decrease with decreasing media N H / concentration. Furthermore, I proposed that provenances with relatively high nitrogen demand (high rates of nitrogen assimilation) or relatively low capacity for uptake across the plasma membrane should all have a low substrate-to-enzyme ratio and therefore should discriminate less. Similarly, by altering the root-to-shoot ratio through pruning, I hypothesized that reducing the root biomass would decrease supply 12 relative to demand, therefore, decreasing discrimination. Likewise, reducing the demand relative to supply through shoot pruning should increase discrimination. 2.2 Methods 2.2.1 Plant material and growth Common garden stock was obtained from three latitudinally dispersed Populus trichocarpa populations: Jasper River (JASP), OR (44°00'), Quesnel River (QLKE), BC (52°58'), and Bell-Irving River (IRVD), BC (56°51'). For a complete description of the original population sources refer to Gornall and Guy (2002). Three distinct clones from each of these populations represented the provenance. The pruning experiments were restricted to a single clone from Quesnel River. Stem cuttings were rooted and grown in 200 uM NFLt+ + 1/10 modified Johnson's solution (Johnson et al. 1957) in a Conviron E-15 (Winnipeg, Canada) growth chamber with a 16 hr photoperiod (22°C:20°C, day:night). Regular media changes and tub cleaning limited algal contamination and ensured consistent plant growth. 2.2.2 Substrate depletion and diffusion techniques Discrimination was determined using a substrate depletion method, whereby the relative 1 5 N enrichment occurring during the partial uptake and assimilation of ammonium provided a measure of net discrimination. All of the experiments relied on this method in which the subject trees were placed in a known volume of stock solution (200 \iM NH 4 +) and samples were drawn from the nutrient solution at regular time intervals. The NH 4 + concentration remaining in each sample was measured using the colorimetric Phenol-Hypochlorite method (Soloranzo 1969), and N H 4 + was collected for isotope-ratio-mass-spectrometry by conversion to NH 3 for diffusion and deposition onto Teflon-encapsulated (Petro Tape, Jet Lube) acidified glass fiber filter disks (Holmes et al. 1998). In this way, the concentration and 5 1 5N of unassimilated substrate remaining in the samples was determined. Filters were packed in tin (Elemental Microanalysis Limited) for combustion and analysis in a Europa ANCA-GSL preparation module and a Europa Hydra 20/20 isotope ratio mass spectrometer, respectively (University of California at Davis Stable Isotope Facility). Isotopic composition is expressed as 8 1 5N values: 13 [2] 5= 1000 x [ R s a m p l e - R s t d ] / R s t d where R s a mpie is the isotope ratio ( 1 5 N/ 1 4 N) of the sample and R s td is the isotope ratio of the reference material (air). The N H 4 + salt composition was -0.96%o. The precautionary measures I followed to minimize and correct for N contamination were similar to those of Pritchard and Guy (2005). Prior to diffusion, the samples were stored at 4°C (Mulvaney and Khan 1999). In the diffusion process, all samples were brought to approximately equal liquid volumes (100 ml) and N concentrations (100 |oM) by dilution. A l l dilutions were made with N H 4 + - free distilled deionized water. Inside the diffusion flasks a constant surface area to volume ratio ensured standardized conditions for the conversion of N H 4 + to NH3 and gas transfer to the glass filters (Brooks et al. 1989). The diffusions were run for 8 days to ensure complete recovery of N (Nevena Ratkovich, pers. comm., UBC). Several initial experiments returned uncharacteristic N and 8 1 5 N values, prompting further methodological testing. I first tested our glass and Nalgene sample storage bottles for possible leaching contamination over long storage times. The 100 ml bottles were filled with standardized N H 4 + solutions (50 - 200 u.M N H 4 + ) and then subsequently tested for N H 4 + gain or loss over a period of 2 months. There were no significant differences between the bottles. I next examined the cleaning process of soaking all glassware in a 6N HC1 bath overnight on the pretext that the ions H + ions were not completely exchanging with N H 4 + on the glass exchange sites. To test this, the bottles were rinsed with an additional NaCl solution because Na + is a stronger cation and should more readily displace N H 4 + . 5 1 5 N and N values were only slightly affected by these cleaning treatments, but to ensure accuracy I instituted a complete acid bath change (Table 2.1). Following this, overall contamination decreased, but there continued to be N loss in -20% of the samples. Intermittent leakage of the Teflon packets containing the acidified glass fiber filters was tested next. Careful observation of the packets at the conclusion of the diffusions revealed that this may have been the case. The recent study by Sebilo et al. (2006) suggested using 'Mitex,' PTFE, hydrophobic filters. I switched to this product for subsequent data collection. The 8 1 5 N and N values were more consistent with the expected amount of N return. To compensate for the intermittent leakage in the earlier data sets, I applied a 20% error margin between the known N concentration of the samples, as determined by the Phenol-hypochlorite method, and the N content measured 14 during isotopic analysis. Any analyzed sample falling below its expected concentration was discarded. Table 2-1. Quantification of 8 1 5 N for the N H 4 + salt and for the contamination associated with glassware cleaning and Teflon. Test Expected (|u,g N) Apparent (|ig N) 5 1 5 N Acid wash 0 1.31 4.53 Acid wash 280 280 -1.07 Salt wash 0 1.44 -3.15 Salt wash 280 280 -1.29 Teflon 0 3 14.84 N H 4 + salt 101 107 -0.94 The Rayleigh distillation model was used to calculate discrimination factors. Application of this model allowed the calculation of enrichment (s) or discrimination (D), for or against 1 5 N , where D equals -s. Guy et al. (1989) define instantaneous D in per mil as: [3] D = (\ -<x)x 1000 where a is the isotope ratio of instantaneous product relative to the available substrate (Rproduct/Rsubstrate)- Henry et al. (1999) showed that the net D occurring during the progressive consumption of substrate in a closed system is given by: [4] D = -d In (1 + 5) / d In C x 1000 where 5=515N/1000 and C was the substrate concentration. The evaluation of D for several samples taken in succession is the regression of ln(l+8)xl000 against -In C when discrimination does not vary with substrate concentration (Henry et al. 1999). If, however, net N discrimination varied with substrate concentration as is typical of plant N uptake and assimilation, the relationship of ln(l+8)xl000 against -In C is a curve. As the precise form of this curve is not known, Eq. 4 can only be used to calculate the average discrimination occurring between successive samples on the curve. 15 2.2.3 Provenance experiments The provenance comparisons were conducted on three different clones from each of the three provenances. One ramet from each clone was analyzed in each replicate for a total of nine plants per draw-down series. A drawdown consisted of all nine plants each in a separate volume of 200 u M N H 4 + modified Johnson's nutrient solution. From this starting volume and concentration, samples were drawn at regular time intervals until the N H 4 + concentration was sufficiently reduced or the remaining volume became limiting. 2.2.4 Pruning experiments To examine the changes in the effect of root-to-shoot ratio, I conducted two pruning experiments. The first experiment pruned the shoots. A series of substrate depletion draw-downs were conducted on three control and three "pruned" rooted cuttings of one clone. A drawdown consisted of all six plants, each in a separate IL volume of 200 uM N H 4 + modified Johnson's nutrient solution. Samples were drawn at regular intervals as described above. Plants were tested in this manner for three consecutive days prior to the pruning treatment. Following two days of "rest," the three experimental trees then had half of their shoots removed less than 30 min prior to the start of three additional days of draw-downs. The pruning amount was arbitrarily determined by counting leaves and branches down from the primary apical meristem and cutting the stem at the halfway point. The growth media for all clones was changed the day before each draw-down series began. A l l clones were maintained in the same container when not in the drawdown flasks. The root pruning experiments ran six trees each day with three trees serving as controls and three as the pruned experimentals. The following day, the controls became the experimentals and new controls were introduced. This protocol controlled for day effects as well as specific tree effects. There were three of these paired draw-downs. A l l were run in the mid-afternoon with growth media solution changes occurring every day following the draw-downs. Volume was varied to accommodate for plant size while maintaining constant uptake rates (0.6-IL). Pruning involved gently dividing the root mass from tip to base lengthwise and removing half. Fresh mass was recorded for the root, shoot, and pruned portions of each ramet according to the water displacement method of Young and Werner (1984) for obtaining the root and shoot masses of intact, living plants. 16 2.3 Results 2.3.1 Substrate depletion analysis The uptake of nitrogen by the rooted cuttings caused the N H 4 + concentration in the media to decrease. Simultaneous with the decrease in residual NH4+, a progressive enrichment of 1 5 N with respect to 1 4 N occurred (Fig 2.1 A). At lower concentrations (<75 uM N H 4 + ) , the degree of enrichment per unit change in concentration was less than at higher concentrations (75-175 u.M N H 4 + ) . The slopes between the points for the re-plotted data in Figure 2.IB provide approximated values for the plant discrimination factor (Dpiam). Because these points yield a curvilinear relation, Dpiant is clearly influenced by the residual N H 4 + concentration. Plotting Dpia„, over the concentration range experienced between successive samples revealed a positive correlation between Dpiant and N H 4 + concentration (Figure 2.1C). 17 o o o o to 180 200 Average [NH4 ] F i g u r e 2.1 Data from a typical substrate depletion experiment showing the changes in residual N H 4 + 8 1 5 N as the substrate was consumed (A), and the same data plotted to show how discrimination is estimated as the slope of the line between two sample points (B). The relationship between the estimated discrimination from Panel B and N H 4 + concentration is shown in Panel C. 18 2.3.2 Efflux and influx The plant net uptake rate, as calculated from the consumption of N H 4 + from the growth media, decreased as the draw-down progressed and N H 4 + concentration decreased (Fig 2.2B). As is characteristic of substrate depletion experiments, the change in uptake rate was not linear. Rather, it decreased from 0.034 (xmol g"1 min"1 at 153 uM to 0.013 umol g"1 min"1 at 50 uM with the majority of the change between 154 u M and 121 uM N H 4 + . The efflux and influx rates were calculated from the net uptake (U) and knowledge of Dpiant according to a rearrangement of Eq. 1 and the relationship of net uptake to influx and efflux (U=I-E) where: [5] 1= \J/(l-DplaJDGS) or E=\J/DGS/Dplanrl). The calculated influx and efflux both followed the uptake rate pattern and decreased with decreasing media N H 4 + concentration as the draw-down progressed. At the start of the draw-down, influx rates were 0.072 [imol g"1 min"1 over the concentration range 174 uM to 133 [iM. N H 4 + . These fell sharply to 0.029 (imol g"1 min"1 in the next concentration interval (133 (xM to 109 \iM NFf 4 +) before stabilizing in a slowly descending pattern until the end of the draw-down. Over the same concentration ranges, efflux (and E/I) fell to nearly zero. At the beginning of the draw-down series, when the concentration was the highest (174 to 133 uM NH 4 + ) , the efflux (0.038 uM g"1 min"1) exceeded uptake (0.034 uM g"1 min"1) to comprise 53% of the gross influx. 19 0.6 0.5 0.4 -I S 0.3 0.2 0.1 0.06 ^ 0.04 E x 3 B Net uptake — Influx » — Efflux 0.02 0.00 -I -40 60 80 100 120 140 160 Average [NH4 +] Figure 2.2 The E/I ratio (A) was calculated from Eq. 1 and the discrimination factors in Figure 2.1C. Estimations of the net N H 4 + influx and efflux (B) were also made using the net N H 4 + uptake rate and the corresponding Dpiant according to Eq. 5. 2.3.3 Provenance comparisons The fresh-weight biomasses for the three provenances were not significantly different (Fig. 2.3). They ranged from 165 g for JASP to 218 g for Q L K E . Likewise, no differences were observed for the root-to-shoot ratio (Fig. 2.3). Root and shoot biomasses were nearly equal, with roots slightly out-weighing shoots, as evidenced by the ratios of 1.03, 1.09, and 1.01 for JASP, Q L K E , and IRVD respectively. 20 JASP QLKE IRVD Figure 2.3 The average provenance fresh weight biomasses (bars) and root-to-shoot fresh weight ratios (line) obtained at the time of the drawdown experiments. The three provenances were as follows: Jasper River (JASP), OR (44°00'), Quesnel River (QLKE), BC (52°58'), and Bell-Irving River (IRVD), B C (56°51'). Error bars represent the standard error of the mean. No latitudinal cline was observed in the discrimination factors, although JASP expressed considerable discrimination at the highest concentration range measured, 215 to 154 uM N H 4 + (Fig 2.4). For each provenance, Dpianl declined as the draw-down experiment progressed, in the same manner as described earlier. The range in discrimination for the three arbitrary N H 4 + concentration intervals was calculated from linear regression approximations of the curve generated from Eq. 4 (Henry et al. 1999). A direct approximation of the curve was not attempted since it is not clear, based on biophysical or physiological principles, what the form of the curve should be. Although this approach reduces calculation errors, more detailed differences may have been obscured due to the high levels of variation in the sampling method. 21 35 i 1 i i i 1 1 1 1 1 1 1 0 20 40 60 80 100 120 140 160 180 200 220 Average [NH 4 + ] Figure 2.4 The discrimination factors of the three provenances as calculated from approximations of the curve generated from Eq. 4. The provenance labels are identical to Figure 2.3. 2.3.4 Pruning effects Physical manipulation of the root-to-shoot ratio (R/S) by pruning back one-half of the shoot doubled the R/S ratio. This did not produce an observable corresponding increase or decrease in Dpiant (Fig 2.5). On the other hand, pruning the roots decreased the R/S ratio by one-half. This resulted in a general increase in net uptake per gram root mass (Fig 2.6) which corresponded to a decrease in DpiaM across all monitored N H / concentrations (Fig 2.7). 22 80 c o "co c 'E I _ o w b 60 40 20 -20 H -40 • Control o Prune 100 o o o o O p fo • .0) o o o 120 140 160 180 Average [NH 4 + ] 200 220 Figure 2.5 P. trichocarpa 1 5 N discrimination obtained via the drawdown technique for three days prior to and post shoot-pruning. 23 0.35 prune control Figure 2.6 The net uptake rate of N H 4 + per root mass in the root-pruned and unpruned P. trichocarpa (p=0.09). 24 Figure 2.7 The root-pruned and unpruned P. trichocarpa discrimination against 1 5 N . The correlation lines highlight the difference between the pruned (r2=0.203, p=0.341) and unpruned (r2=0.50, p=0. 019) treatments. 2.4 Discussion 2.4.1 5 1 5 Nand discrimination Plant discrimination is dependent on the balance between N supply and demand. In these drawdown experiments, the media became enriched in 1 5 N as the N was consumed (Fig 2.1 A). Fractionation in absolute terms was pronounced at high N concentrations, but was much less obvious at the low N H / concentrations reached towards the end of the draw-down. This pattern of reduced discrimination at lower nutrient concentrations is presumably associated with mineral ion utilization by the poplar plants. Because media solution and roots were not sterile, some of the N consumption and isotope enrichment may be due to associated root microorganisms. However, bacterial nitrification processes were not a likely 25 cause of media enrichment because the media solutions were changed regularly, reducing the opportunities for contamination and bacterial growth. As well, N H / uptake rates were within the expected range for poplars (Min et al. 1999). Another potential artifact would be the exudation of organic N from the roots. The efflux of organic N poses a concern as it could breakdown during sample preparation, thereby contaminating the inorganic fraction. However, since the rate of organic efflux is no more than 5% of the rate of inorganic influx (Merbach et al. 1999), I would not expect the accumulating organic N to have a significant effect on D even i f the organic N were to be significantly enriched (Pritchard and Guy 2005). Furthermore, N recovery methods similar to those used here showed that only 4.7% of the added organic N was broken down to N H 3 (Khan et al. 1997: Mulavaney and Khan 1999). The remaining opportunity for N enrichment lies with inorganic N efflux from the poplar roots. Net uptake of N H 4 + includes a substantial amount of inorganic N efflux, and isotopic discrimination should vary in direct proportion to the changing efflux to influx ratio (Handley and Raven 1992 and citations therein). Thus, when efflux approaches influx, as observed when external media concentrations are high or when assimilation rates are low, maximal fractionation occurs. Conversely, i f external concentrations are low or demand is high, most ions taken up will be immediately consumed and the efflux to influx ratio will approach zero. The results of the example substrate depletion experiment indicated decreasing discrimination with dropping N H 4 + supply concentration. This trend is consistent with the hypothesized effect of efflux relative to influx on discrimination. The numerical aspect of the E/I and Dpiant relationship is explored in the following section. The discrimination associated with the progressive consumption of substrate in a closed system decreased as N H 4 + was consumed from the growth media. This indicated a positive influence of media N concentration on discrimination (Henry et al. 1999). The highest Dpiant in this draw-down was 8.8%o indicating that the concentration range employed in this experiment did not permit the full expression of discrimination by GS (Pritchard and Guy 2005; Yoneyama et al. 1993). It does, however, compare favorably to the in vivo discrimination estimates from other studies on herbaceous plants (Yoneyama et al. 1991; Evans et al. 1996). 26 2.4.2 Estimating efflux and influx Estimated efflux/influx ratios increased as a function of external N concentration when they were calculated from a rearrangement of Eq. 1 (Fig 2.2A) (Kronzucker et al. 1995a, b). Calculations of the approximate rates of efflux and influx as they change with N H 4 + concentration were possible by using a simple model (Eq. 5) relating them to Dpi„nl and net N H 4 + uptake rate. Net uptake is the difference between what enters the root (influx) and what returns to the growth media (efflux) (i.e. net uptake is the portion of influx that remains in the plant, presumably because of assimilation by GS). The estimated E/I was comparable to, but somewhat higher, than what might be expected based on aspen measured at 100 uM N H 4 + and 1.5 m M N H 4 + (0.17 and 0.76 respectively) in flux-analysis experiments using 1 3 N radiolabelling (Min et al. 1999). Similarly, the actual flux estimates obtained through the draw-down technique and the application of Eq. 5 were well within the expected range for aspen flux rates as reported by Min et al. (1999). At similar N H 4 + media concentrations, the uptake rate and influx matched very closely while the efflux calculated by the draw-down technique was considerably higher than Min etal. (1999). There are several possible explanations for the differences between the numbers reported here and those of Min et al. (1999), not the least of which may be the differences in species. The two approaches also differ in that the compartmental analysis of Min et al. (1999) assumes steady-state conditions, unlike the substrate depletion analysis carried out here. Differences in growing conditions are also likely to have affected N demand in the two studies. Additionally, I have assumed no discrimination in membrane transport, but some slight contribution is possible, which, i f unaccounted for, would yield higher estimates for efflux. Finally, discrimination factors for GS have been measured only rarely and are poorly defined. The influx demonstrated the greatest numerical change, although the efflux had the greatest relative change. Since it is the proportion of these two fluxes against each other that determines discrimination in the proposed model, as their relative proportions change, so too should discrimination. In this case, the proportionate decrease in efflux was greater than that in the influx, resulting in a decreasing E/I ratio. This accounted for the decrease in discrimination as the E/I ratio changed in response to the media N H 4 + concentration. 27 The ability to calculate the efflux and influx values based on stable isotope discrimination within the substrate depletion technique provided a new method of approximating N fluxes in the roots. Previous work on N H 4 + root fluxes using radiolabeled N were complicated and limited in their applications due to the short half life of N (Min et al. 1999). By using the stable isotopes at natural abundance levels, the technique presented here offers a safe, simple tool for future work. 2.4.3 Genetic variation in N isotope discrimination The biomass and root-to-shoot ratios for the provenance comparisons in this study lacked a latitudinal cline (Fig 2.3). This contrasts with results from common garden experiments on the same populations where there were measurable differences in photosynthesis and foliar content (Gornall and Guy 2002). In not showing evidence of a latitudinal cline, the discrimination factors for the three provenances were consistent with the biomass and net uptake rate data. When Dpiant was calculated on a sample to sample basis, small imprecisions were exaggerated occluding the provenance Dpiant trends (data not shown). However, by approximating the Dpiant from three consecutive segments of the discrimination curve (Henry et al. 1999), JASP's Dpiant emerged at high N H 4 + concentrations (Fig 2.4). Since E/I should reflect the balance between the capacity for N uptake and assimilation (Evans 2001), and these are under genetic control (Li et al. 1991), I might have expected more genotypic variation in plant discrimination. If growth was limited by uptake, then the efflux/influx ratios would be low and the genetic variation in discrimination should mirror the uptake capacity. Contrastingly, i f assimilation is not limited by uptake, then the efflux/influx ratios should be higher and the discrimination differences would be determined by genetic assimilation capacity as controlled by growth demands. The former scenario may be significant to the JASP data as the changes in N H 4 + concentration appeared to have been the precipitating factors in JASP's change in DpiaM • In this case, supply needs of JASP were initially met (or exceeded) permitting DpiaM; however, as the N H 4 + concentration dropped, the supply dwindled and Dpiant was no longer feasible. Pritchard and Guy (2005) account for the lack of a correlation between tissue 5 1 5 N and biomass in the recurrent draw-down experiments they conducted as any such differences would be cancelled or scrambled due to the cycling between states of N H 4 + deficiency and 28 surplus. The present study is not subject to the same limitation as the root cuttings were maintained at constant N H 4 + prior to the draw-down and the medium, not the plant tissue, was analyzed in each draw-down. 2.4.4 Pruning as a means of altering N isotope discrimination Altering the plant's capacity to either supply or demand N through physically manipulating the root-to-shoot ratio affected Dv\snt only in the case of root pruning. Shoot pruning, which resulted in doubling the root-to-shoot ratio, did not produce an observable change in discrimination (Fig 2.5). The expectation was that by reducing shoot biomass, demand would be reduced relative to supply, thus increasing discrimination. The lack of any pattern over the three days of trials may indicate no real change in demand. In retrospect, partial shoot pruning might not necessarily reduce demand if re-growth is sufficiently stimulated. Net uptake rates for the shoot pruning were not calculated because root mass data was not obtained. Root pruning resulted in a general decrease in Dpian, across the draw-down N H 4 + concentrations (Fig 2.7). Thus, by reducing the root-to-shoot ratio by half, the plant's uptake capacity was correspondingly reduced. To compensate for the reduced root volume, net uptake per unit root weight (i.e. assimilation) increased (Fig 2.6). The increased N assimilation per unit root would have reduced efflux and resulted in the decreased discrimination as observed. 2.4.5 Conclusion Overall, my data are consistent with an interpretation of plant 8 1 5 N whereby discrimination parallels the efflux/influx ratio. I was able to manipulate discrimination by root pruning, but not by shoot pruning. There was insufficient precision to detect genotypic variation in isotope discrimination. By using the substrate depletion analysis, uptake as well as efflux and influx were calculated. In conclusion, with further development, the ability to accurately calculate efflux and influx using stable isotope discrimination methods at natural abundance levels provides an alternative approach for studies of nutrient uptake efficiency and in vivo controls on membrane transport and assimilation. 29 2.5 References Bar-Tal, A. , Feigin, A . Rylski, I., and Pressman, E. 1994. Effects of root pruning and N - N 0 3 solution concentration on nutrient uptake and transpiration of tomato plants. Scientia Horticulturae 58: 77-90. Brooks, P.D., Stark, J .M., Mclnteer, B.B. and Preston, T. 1989. Diffusion method to prepare soil extracts for automated nitrogen-15 analysis. Soil Sci Soc A m J 53: 1707-1711. Evans, R.D. 2001. Physiological mechanisms influencing plant nitrogen isotope composition. Trends Plant Sci 6: 121-126. Evans, R.D., Bloom, A.J. , Sukrapanna, S.S. and Ehleringer, J.R. 1996. Nitrogen isotope composition of tomato (Lycopersicon esculentum mill. cv. T-5) grown under ammonium or nitrate nutrition. Plant Cell Environ 19: 1317-1323. Gornall, J.L. and Guy, R.D. 2002. Geographic variation in ecophysiological traits of black cottonwood. (Populus trichocarpa) from western North America. Plant Physiology Canada. Calgary, A B . Guy, R.D., Berry, J.A., Fogel, M . L . and Hoering, T.C. 1989. Differential fractionation of oxygen isotopes by cyanide-resistant and cyanide-sensitive respiration in plants. Planta 177: 483-491. Handley, L .L . , and Raven, J.A. 1992. The use of natural abundance of nitrogen isotopes in plant physiology and ecology. Plant Cell Environ 15: 965-985. Handley, L .L . , Robinson, D., Forster, B.P., Ellis, R.P., Scrimgeour, C M . , Gordon, D . C , Nevo, E. and Raven, J.A. 1997. Shoot delta N-15 correlates with genotype and salt stress in barley. Planta 201: 100-102. Henry, B.K. , Atkin, O.K., Day, D.A., Millar, A .H . , Menz, R.I. and Farquhar, G.D. 1999. Calculation of the oxygen isotope discrimination factor for studying plant respiration. Aust J Plant Physiol 26: 773-780. Hoch M.P., Fogel, M . L . and Kirchman, D.L. 1992. Isotope fractionation associated with ammonium uptake by a marine bacterium. Limnol Oceanogr 37: 1447-1459. Hogberg P., Hogberg, M . N . , Quist, M.E. , Ekblad, A. and Nasholm, T. 1999. Nitrogen isotope fractionation during nitrogen uptake by ectomycorrhizal and non-mycorrhizal Pinus sylvestris. New Phytol 142: 569-576. Holmes, R .M. , McClelland, J.W., Sigman, D .M. , Fry, B. and Peterson, B.J. 1998. Measuring l 5 N - N H 4 + in marine, estuarine and fresh waters: an adaptation of the ammonia diffusion method for samples with low ammonium concentrations. Mar Chem 60: 235-243. 30 Johnson, C M . , Stout, P.R., Broyer, T.C. and Carlton, A.B. 1957. Comparative chlorine requirements of different plant species. Plant Soil 8: 337-353. Khan, S.N., Mulvaney, R.L., and Mulvaney, CS . 1997. Accelerated diffusion methods for inorganic nitrogen analysis of soil extracts and water. Soil Sci Soc Am J 61: 936-947. Kolb K.J. and Evans, R.D. 2003. Influence of nitrogen source and concentration on nitrogen isotopic discrimination in two barley genotypes (Hordeum vulgare L.). Plant, Cell Environment 26: 1431-1440. Kronzucker, H.J., Siddiqi, M.Y. and Glass, A.D.M. 1995a. Compartmentation and flux characteristics of ammonium in spruce. Planta. 196: 691-698. Kronzucker H.J., Siddiqi, M.Y., and Glass, A.D.M. 1995b. Compartmentation and flux characteristics of nitrate in spruce. Planta 196:674-682. Li , B., McKeand, S.E., and Allen, H.L. 1991. Genetic variation in nitrogen use efficiency of loblolly pine seedlings. For Sci 37: 613-626. Merbach Merbach, W., Mirus; E., Knof, G., Remus, R., Ruppel, S., Russow, R., Gransee, A. and Schulze, J. 1999. Release of carbon and nitrogen compounds by plant roots and their possible ecological importance. J Plant Nutr Soil Sci 162: 373-383. Min, X., Siddiqi, M.Y., Guy, R.D., Glass, A.D.M. and Kronzucker, H.J. 1999. A comparative study of fluxes and compartmentation of nitrate and ammonium in early-successional tree species. Plant Cell Environ. 22: 821-830. Mulvaney, R.L., Khan, S.A. 1999. Use of diffusion to determine inorganic N in a complex organic matrix. Soil Sci Soc Am J 63: 240-246. Pritchard, E.S. and Guy, R.D. 2005. Nitrogen isotope discrimination in white spruce fed with low concentrations of ammonium and nitrate. Trees 19: 89-98. Robinson, D. 2001. Delta N-15 as an integrator of the nitrogen cycle. Trends in Ecology & Evolution. 16: 153-162. Sebilo, M. , Mayer, B., Grably, M. , Billiou, D., and Mariotti, A. 2006. The use of the 'ammonium diffusion' method for 515N-NH4+ and 81 5N-N03_ measurements: comparison with other techniques. 1: 99-103. Solorzano, L. 1969. Determination of ammonia in natural waters by the phenolhypochlorite method. Limnol Oceanogr 14: 799-801. Shearer, G. and Kohl, D.H. 1989. Natural 1 5 N abundance of N H 4 + , amide N, and total N in various fractions of nodules of peas, soybeans and lupins. Aust J Plant Physiol 16:305-313 31 Yoneyama, T., Kamachi, K. , Yamaya, T. and Mae, T. 1993. Fractionation of nitrogen isotopes by glutamine-synthetase isolated from spinach leaves. Plant Cell Physiol 34: 489-491. Yoneyama, T., Matsumaru, T., Usui, K. and Engelaar, W.M.H.G. 2001. Discrimination of nitrogen isotopes during absorption of ammonium and nitrate at different nitrogen concentrations by rice (Oryza sativa L.) plants. Plant Cell Environ 24: 133-139. Yoneyama, T., Omata, T., Nakata, S. and Yazaki, J. 1991. Fractionation of nitrogen isotopes during the uptake and assimilation of ammonia by plants. Plant Cell Physiol 32: 1211-1217. Young E and Werner DJ. 1984. A nondestructive method for measuring root and shoot fresh weights. Hortscience 19: 554-555. 32 3. Tissue 5 1 5N of Populus trichocarpa Grown in Steady-State N H 4 + Nutrition 3.1 Introduction The current understanding of the ratio of the stable isotopes of nitrogen within plant tissues increasingly emphasizes the role of the relationship between plant growth and nitrogen nutrition in determining 8 1 5N. Variation in the 5 1 5 N of plant tissues arises through isotopic fractionation (Comstock 2001; Evans 2001; Robinson 2001). Fractionation is the kinetically determined process whereby the heavier isotope (1 5N) is "discriminated" against causing a relatively greater fraction of the lighter isotope (1 4N) to be incorporated into plant tissues (Handley and Raven 1992). In principle, fractionation of the nitrogen isotopes of ammonium could occur during nutrient uptake, assimilation, metabolism, or translocation. Uptake from the growth medium into the root cytosol is the first opportunity for plant-mediated discrimination. Studies based on media to whole plant isotopic signatures in rice (Yoneyama et al. 1991) and tomato (Evans et al. 1996) reported contradictory results with the former claiming discrimination during uptake and the latter not finding it. However, these studies did not clearly separate assimilation and uptake. Distinct separation was achieved in legumes using the membrane transport of NFL;* from the bacteroid to root cytosol, where no fractionation during transport was observed (Shearer and Kohl 1989). Ammonium is the central intermediate in the nitrogen metabolism of plants. It is assimilated by glutamine synthetase (GS) via the plastid GS2 or through a series of cytosolic GS1 isoforms (Lam et al. 1996). Due to the reaction kinetics of GS, in vitro estimates for discrimination (D) by glutamine synthetase (Das) are 16.5 +/- 1.5%o (Yoneyama et al. 1993). Contrastingly, in vivo discrimination estimates do not closely approach the in vitro estimates and often vary considerably (by up to 9%o) (Hoch et al. 1992; Hogberg et al. 1999; Robinson 2001; Yoneyama et al. 1991, 2001; Kolb and Evans 2003; Pritchard and Guy 2005). Marriotti et al. (1982) attributed the variability to changes in the substrate-to-enzyme ratio or supply relative to demand. When substrate levels were high relative to enzyme levels, discrimination increased. The inverse was also true. Several more recent studies have shown that significant isotope discrimination at the whole plant level can only occur when N influx exceeds N utilization and the surplus nitrogen returns to the bulk medium through efflux 33 (Waser et al. 1999; Pritchard and Guy 2005). Efflux and influx are controlled by the relative quantities of mineral nutrition (supply) as well as growth and genetic predispositions (demand). Thus, plant 8 1 5 N values are dependent on plant influx and efflux and the factors that influence these fluxes. The relationship of substrate-to-enzyme is analogous to supply and demand, which control influx and efflux. This leads to the following model, [1] Dpiant = efflux/influx * D Gs where Dpian, is net isotope discrimination at the whole plant level, and when ammonium is the sole N source (Handley and Raven 1992). It should be possible to increase or decrease discrimination by manipulating growth and nutrition to influence efflux/influx. Further opportunities for intra-plant discrimination arise from other metabolic processes and nutrient translocations within the plant. Differences in 8 1 5 N values of plant constituents and organs could, for example, result from enzymatic fractionation of organic nitrogen at branch points following assimilation (Yoneyama and Kaneko 1989). However, these differences wil l not affect whole plant 8 1 5 N unless specific constituents with marked enrichment or depletion of 1 5 N are processed (e.g. lost) at significantly different rates (Bergersen et al. 1988; Comstock 2001; Evans 2001). In many plants, when grown on NO3", a branch-point in N utilization occurs between direct assimilation in the root versus xylem translocation for assimilation in the shoot. Indeed, in NO3" grown plants, the 8 1 5 N of the leaves can be between 3-7%o greater than the roots (Bergersen et al. 1988; Yoneyama and Kaneko 1989; Evans et al. 1996). Translocation and leaf assimilation of mineral N03" in aspen (P. tremuloidies) resulted in distinct foliar 8 1 5 N enrichment, but there were no differences when grown on N H / (Buschhaus et al. in prep.). This process affects Dpiant in as much as xylem translocation competes with efflux removing N from a partially assimilated cytoplasmic pool. Similar affects would be expected i f xylem transport of N H / were to occur. The possibility of mineral N H / translocation to the shoot is controversial (Schjoerring et al. 2002). Min et al. (1998, 1999, 2000) used the 1 3 N radioisotope to examine N flux in aspen, lodge pole pine, and interior Douglas-fir. In no case was there evidence for significant translocation of unassimilated N H / to the leaves, and only in the aspen was there substantial movement of NO3" into the xylem (Min et al. 1999). In contrast, 34 Schjoerring et al. (2002) clearly linked xylem N H / concentrations to the nutrient media NFJ.4+ supply in several herbaceous species. Genetic variation in components of nitrogen metabolism may also affect plant 8 1 5 N (Handley et al. 1997; Robinson et al. 2001; Pritchard and Guy 2005). Yoneyama (1991) documented changes in plant 5 1 5 N under N H 4 + nutrition that were dependent on both media nitrogen concentration and rice cultivar. Pritchard and Guy (2005) proposed that genetic differences in discrimination are caused by different demands on assimilation and uptake capacity, which in turn influence the balance between nitrogen influx and efflux. Elevated atmospheric CO2 stimulates photo synthetic carbon reduction and competitively suppresses photorespiratory carbon oxidation (Hymus et al. 2001). This typically results in a marked increase in photosynthesis and growth, which must be matched by an increase in demand for mineral nutrients to sustain stimulated growth (Stitt and Krapp 1999). The demand is met by both increasing the nitrogen use efficiency (the rate of growth per unit of nitrogen in the plant) and by increasing the rate of uptake and assimilation. The latter requires sufficient nitrogen in the growth media to meet the increased demand (reviewed by Stitt and Krapp 1999). Within these studies, few if any examined NH4+ assimilation separate of NO3", thereby making direct conclusions regarding N H 4 + more challenging. In theory however, elevated CO2 could favor N H 4 + assimilation in the leaves because of the unused capacity normally associated with the photorespiratory recovery of N H 4 (Stitt and Krapp 1999). However, more carbohydrate is also available for root assimilation in C 0 2 enriched plants (Stitt and Krapp 1999). These potential changes in nitrogen uptake, assimilation and allocation under different CO2 regimes have been proposed to alter the plant 8 1 5 N in N O 3 " - fed plants (Stock and Evans 2006). However, no work has been completed on N H 4 + - grown plants, and separating soil and plant processes in the other studies on CO2 enrichment and 8 1 5 N makes them difficult to interpret (Billings et al. 2002; BassiriRad et al. 2003). Overall, there are strong interactions between nitrogen nutrition and growth in plants grown under elevated C 0 2 , which should be reflected in the plant 8 ! 5 N. The objective of this work was to elucidate the in-plant efflux/influx-mediated fractionation of nitrogen isotopes in Populus trichocarpa through manipulating the supply/demand regimes. To modify the supply, I conducted experiments at two, steady state NH4+ concentrations. M y intentions were two fold. First, a higher nitrogen supply should 35 increase discrimination as measured by decreasing 8 N . Second, it should provide the framework for further exploring the interaction between supply and demand. The demand was altered through elevating atmospheric CO2 and using natural genetic variation. I hypothesized that an increased nitrogen demand, created by the effect of elevated CO2 on growth rate, would decrease opportunities for discrimination, thereby increasing 8 1 5 N. Similarly, I hypothesized that genetic variation giving rise to differences in factors such as photosynthetic rate, nitrogen use efficiency and growth rate, which affect N demand, would result in differences in discrimination. For example, provenances with a high nitrogen demand, high nitrogen assimilation capacity, or a low uptake capacity would all have a low substrate-to-enzyme ratio and therefore should discriminate less, increasing the 8 1 5 N. In the course of these experiments, I observed unexpected relationships between foliar percent nitrogen and 8 1 5 N which prompted me to question assumptions regarding N H 4 + assimilation in the roots. 3.2 Methods 3.2.1 Plant material and maintenance I obtained common garden stock of three latitudinally dispersed Populus trichocarpa populations from Jasper River, OR (44°00'), Quesnel River, B C (52°58'), and Bell-Irving River, BC (56°51'). For a complete description of the original population sources, refer to Gornall and Guy (2002). Four rooted stem cuttings per treatment represented each of three clones per population for a total of 12 cuttings per population for each treatment. Uniform, 5 cm cuttings were rooted and grown in a modified Johnson's hydroponic medium for 6 weeks in two growth chambers (Conviron E-15) with either ambient (400 ppm) or enriched (800 ppm) C 0 2 (monitored via Vaisala GMM220) and with a 16 hr photoperiod (22°C:20°C, day: night), equivalent PPFD (450 umol»m" «s~) and constant humidity. Both growth chambers shared the same 500 L nutrient solution which was circulated through the media trays in each chamber. I conducted this experiment on separate occasions at either 200 uM N H 4 + or 400 uM NF£4 + ± 20% supplied as (NH 4 ) 2 S0 4 . The media solution was buffered at pH 6.4-6.8 by adding excess powdered CaC03. Regular concentration monitoring (phenol-hypochlorite method (Solorzano 1969)), limited N H 4 + additions, tub cleaning, and solution replacement 36 (every 2-3 days) maintained a nearly constant, un-enriched nutrient supply for the duration of the experiment. 3.2.2 Isotopic analysis After 6 weeks of growth, each tree was individually separated into leaves, stems and roots. It was then weighed for fresh mass, frozen in liquid nitrogen, and stored at -20 °C. Following this, the samples were then freeze dried and weighed for dry mass prior to pulverization using a ball-mill (Fritsch Laborgeratebau, Terochem Scientific). Relative growth rate was calculated using the mass of the initial cutting according to Hunt (1982). From the prepared leaf and root samples, 3 mg were packed into tin capsules (Elemental Microanalysis Ltd., 8x5mm, D1008) and analyzed for 5 1 5 N and elemental content on a Europa A N C A - G S L preparation module and a Europa Hydra 20/20 isotope ratio mass spectrometer (University of California at Davis Stable Isotope Facility). The 8 1 5 N for the ammonium salt used for the growth media was -0.96%o. Stems were subsequently analyzed on a ThermoFinnigan Delta plus and Delta plus X L stable isotope ratio mass spectrometer (University of British Columbia Stable Isotope Facility). 3.2.3 Statistical analysis I used analysis of variance with unequal numbers of observations per treatment to compare treatment means of relative growth rate, 8 1 5 N, % N and total N . The analysis of variance procedure was carried out with a General Linear Model (SAS 9.1.3; SAS Institute Cary, NC) to obtain estimates of the Least Squares Means followed by the F and T tests. To compare the regressions of 8 1 5 N with % N , I used covariance analysis within the General Linear Model (SAS 9.1.3; SAS Institute Cary, NC) adjusted for the 8 1 5 N variable. Differences between treatments described as significant are those where probability (P) was <0.05. 37 3.3 Results 3.3.1 Relative growth rate As a whole, elevated CO2 nearly doubled the total tree biomass in both nitrogen treatments (Table 3.1). In terms of relative growth rate, the CO2 effect was only significant for the 200uM N H 4 + treatment (0.0316 day"1 and 0.0469 day"1 at ambient and elevated C 0 2 respectively) as opposed to 400uM N H 4 + treatment which did not respond to the increased C 0 2 (0.041 day"1 and 0.046 day"1, respectively; Fig 3.1). There was a 2-way interaction effect with nitrogen treatment and provenance (p=0.0120). Two provenances failed to respond to high C 0 2 at high nitrogen (Fig 3.2). The root-to-shoot ratio was not affected by any of the treatments (data not shown). Table 3-1 Total plant tissue averages of P. trichocarpa for 6 weeks of growth in either 200 or 400 uM N H 4 + at either ambient or elevated C 0 2 . Averages represent three provenances: Bell Irving River, B C (IRVD); Quesnel Lake, B C (QLKE); and Jasper River, OR (JASP). RGR is the relative growth rate. 200 uM N H 4 + Ambient CO2 200 uM N H 4 + Enriched C 0 2 400 uM N H 4 + Ambient CO2 400 uM N H 4 + Enriched C 0 2 Total dry biomass 3.251 ± 0 . 7 2 4 8.404 ± 0 . 7 9 2 4.003 ± 0 . 8 5 1 7.119 ± 0 . 6 9 3 RGR 0.032 ± 0.003 0.047 ± 0.003 0.041 ± 0.004 0.046 ± 0.003 R/S ratio 0.143 ± 0 . 0 1 0.156 ± 0 . 0 1 0.145 ± 0 . 0 1 0.131 ± 0 . 0 1 Total %Npiant 3.76 ± 0 . 1 4 3.15 ± 0 . 1 5 3.97 ± 0 . 1 7 3.49 ± 0 . 1 4 Total 5 1 5 N p l a n t (%„) -3.98 ± 0 . 1 4 -3.38 ± 0 . 1 6 -3.37 ± 0 . 1 7 -3.37 ± 0 . 1 4 N H 4 + 8 1 5 N s a i t (%o) -0.96 -0.96 -0.96 -0.96 38 0.06 0.05 0.04 A CC O 0.03 H 0.02 A 0.01 0.00 200 Ambient 200 Elevated 400 Ambient 400 Elevated Figure 3.1 P. trichocarpa relative growth rate (RGR) values were for the first 6 weeks of ramet establishment on either 200 uM N H 4 + 400ppm C 0 2 (200 Ambient), 200 uM N H 4 + 800ppm C 0 2 (200 Elevated) , 400 uM N H 4 + 400ppm C 0 2 (400 Ambient) or 400 uM N H 4 + 800ppm C 0 2 (400 Elevated). RGR values are the averages of the three provenances: Bell Irving River, BC (IRVD); Quesnel Lake, BC (QLKE); and Jasper River, OR (JASP) (n=12). The letters (a, b) differentiate statistical significance of p<0.05. 39 0.08 0.06 or O 0.04 DC 0.02 0.00 200 Ambient 200 Elevated 400 Ambient 400 Elevated Figure 3.2 P. trichocarpa relative growth rate (RGR) values were for the first 6 weeks of ramet establishment. RGR values are the averages of the three clones representing each provenance. Treatment and provenance labels are identical to Figure 3.1. Statistical significances for the two N treatments were calculated separately and are indicated by the lower case letters (p<0.05). 3.3.2 Nitrogen content Across all treatments, plant tissue % N for both the roots and leaves was relatively high at 2.5-4.5%. Elevated C 0 2 resulted in significant whole plant percent nitrogen decreases across N treatments (Table 3.1; p=0.0032). Leaf % N was mainly responsible for this effect as it decreased from 3.4% to 2.5% in 200uM N H 4 + and 4.3% to 3.6% in 400uM N H 4 + (Fig C). The root % N was not affected by the atmospheric CO2 concentration (4.1% at 200uM N H 4 + to 3.5% at 400uM N H 4 + ) . Under increased N nutrition, the foliar and root % N content reversed position relative to the total plant % N . There were no significant differences between provenances. 40 Figure 3.3 P. trichocarpa root and leaf tissue % N were calculated as a percentage of the respective tissue's dry mass at the conclusion of the 6 week growing period. Treatment and provenance labels are identical to Figure 3.1. Statistical significances for the two N treatments were calculated separately and are indicated by the lower case letters (p<0.05). 3.3.3 5 1 5 N analysis Elevated C 0 2 increased whole plant 5 1 5 N from -4 to -3.4%0 in 200 u M NH 4 + ,but there was no apparent difference in 5 1 5 N in the higher N regime (Table 3.1). Looking across the N and CO2 treatments the provenance 8 1 5 N averages varied (Fig 3.4). JASP (x=-4.0%o) was significantly more depleted in 1 5 N than either IRVD (x=-3.4%0) or Q L K E (x=-3.2%o), and although the difference was not significant between IRVD and Q L K E , Q L K E was the most enriched of the three provenances. Within these trends, increasing the media N concentration resulted in greater differences between the provenance whole plant 5 1 5 N values. 41 Figure 3.4 P. trichocarpa 8 1 5 N values measured for the first 6 weeks of ramet establishment. 8 1 5 N values are the averages of the three clones representing each provenance. Treatment and provenance labels are identical to Figure 3.1. Statistical significances for the two N treatments were calculated separately and are indicated by the lower case letters (p<0.05). On average, whole-plant 5 1 5 N values (leaves, roots and stems combined) (average 815Npiant - -3.5%o) were lower than the supplied N H / (-0.96%o;Table 3.1). Across all treatments the leaves were significantly enriched (average 515Nieaves = -2.6%o) compared to the roots (average S , 5N r 0ots = -4.5%©) (Fig 3.5). The stems were comparable with the roots. Comparing foliar 8 1 5 N versus foliar % N revealed a trend where the low N and high C O 2 treatment diverged from the other treatment relationships. As the foliar % N increased, so too did the 8 ! 5 N (Fig 3.6). A similar pattern in the roots was not observed. 42 •6 200 Amb ien t 200 E levated 400 Amb ien t 400 E levated Figure 3.5 P. trichocarpa root and leaf tissue 8 1 5 N measured at the conclusion of the 6 week growing period. Treatment and provenance labels are identical to Figure 3.1. Statistical significances for the two N treatments were calculated separately and are indicated by the lower case letters (p<0.05). 43 Leaves -1 H o o v v » V V • 200N Ambient C 0 2 O 200N Elevated C 0 2 • 400N Ambient C 0 2 V 400N Elevated C 0 7 Roots Tissue %N Figure 3.6 Leaf and root 8 1 5 N values are plotted as a function of tissue % N . The correlation is for the 200 u M N H 4 + elevated C 0 2 treatment (r2=0.150, p=0.035). Treatment and provenance labels are identical to Figure 3.1. 44 To further explore the foliar 8 1 5N, I ran a multiple linear regression accounting for media nitrogen and CO2 treatments, total biomass, relative growth rate, R/S ratio, total plant %N, leaf %N, root % N and provenance latitude. There were significant relationships with provenance latitude (p=0.000T) and relative growth rate (p=0.0015), whereby, leaf 815N=-5.62 + 0.0771 (provenance latitude) -20.817 (relative growth rate). The root-to-shoot 8 1 5N differences were consistent for all treatments with the exception of the low N high CO2 treatment where the root to leaf 8 1 5 N difference diminished with decreasing leaf % N (Fig 3.7). Similar to leaf 8 1 5N, a multiple linear regression analysis found provenance latitude (p=0.0096), relative growth rate (p=0.0058) and leaf % N (p=0.0229) to all have a significant effect on 8 1 5 N ((leaf-root 8 1 5 N = 1.818+0.0386(provenance)-15.221(relative growth rate) +0.581 (leaf %N)) (r2 =0.1813). 5 O O z 3 -^ — ro • CD _ i in 2 - O 0 • s> ' t o •*-* O Ro 1 - ^5o 0 o» • 200N Ambient C 0 2 O 200N Elevated C 0 2 • 400N Ambient C 0 2 v 400N Elevated C 0 2 V ° Q O o o o v y v * 7 Leaf % N Figure 3.7 Root to leaf 8 1 5 N differences are plotted as a function of leaf %N. The correlation highlights the 200 uM N H 4 + elevated C 0 2 treatment (r2=0.372, p=0.0003). Treatment and provenance labels are identical to Figure 3.1. 45 3.4 Discussion 3.4.1 Growth rate and biomass accumulation Overall, there was a positive growth response to elevated atmospheric CO2 (Fig 3.1). The growth response likely resulted from concurrent stimulation of photosynthesis and suppression of photorespiration (Hymus et al. 2001). Similar responses have been seen in other studies, especially where nutrient supply was sufficient (Stitt and Krapp 1999). For the positive growth response to persist, mineral nutrient acquisition must keep pace with demand (Stitt and Krapp 1999). Two provenances failed to respond to elevated CO2 concentrations even under high N H / conditions. While this was not expected, Tupker et al. (2003) found significant variation in the CO2 growth responses of different poplar provenances. 3.4.2 Tissue % N Despite the low media N H 4 + concentration, the plants in both nitrogen treatments in this study had relatively high tissue nitrogen concentrations (Fig. 3.3) compared to other studies (DesRochers et al. 2006). This confirms that they contained sufficient, i f not surplus, nitrogen. The high tissue N may be due to the constant supply of nitrogen in the circulating hydroponic system preventing local rhizosphere depletion. Foliar % N was lower under elevated CO2, likely reflecting a decline in photosynthetic protein content (e.g. Rubisco) coupled with N dilution due to increased growth and starch accumulation (Stitt and Krapp 1999). Root % N was not affected by the shift in CO2 levels. Consequently, although total plant % N did not change between the two nitrogen treatments, the nitrogen concentration of roots and shoots reversed relative to the total plant values. 3.4.3 Whole plant 8 1 5 N Whole plant discrimination varied between the treatment groups. Under low N conditions and ambient CO2 I observed maximal discrimination (-2%o). Elevating the CO2 enriched the whole plant 5 1 5 N, suggesting that increasing mineral nutrient demand through stimulating growth reduced discrimination (-1.5%0). This is consistent with the proposed model where discrimination is dependent on the supply/demand regime. Unexpectedly, there 46 was no apparent difference in 8 1 5 N between the CO2 treatments under the higher N regime. The increased supply at 400N was predicted to change the 8 1 5 N values toward greater depletion. Since this did not occur, it suggested that demand must have also decreased. Such a scenario is supported by the treatment results for relative growth rate because the plants did not greatly increase their growth (demand) under the high N and high CO2 treatment. 3.4.4 Provenance 8 1 5 N Whole plant 8 1 5 N varied with provenance when averaged across the N and C 0 2 treatments. This provenance-level genetic variation in isotope discrimination did not directly correlate with relative growth rate or provenance latitude. However, JASP, the most southerly provenance, was significantly more depleted in 1 5 N than both IRVD and Q L K E . Increased discrimination in JASP must arise from a genetically controlled physiological adaptation that presumably resulted in a greater efflux/influx ratio. Either a decrease in assimilation rate (N demand) relative to uptake or an increase in rate of uptake relative to demand is implied. Increasing the media N concentration resulted in greater differences between the provenance 8 1 5 N values. Under low N supply the plants were presumably all discriminating against 1 5 N to the same low degree because the substrate supply was limiting. At higher N , the substrate supply was not uniformly limiting across all provenances. Put another way, at low N the plants all discriminate similarly because efflux/influx is uniformly low; however, as more N is supplied, their inherent discrimination is no longer limited by supply but rather controlled by differences in uptake (influx) or the plant's nitrogen demand physiology. In the present data, JASP had a lower 8 1 5 N resulting from greater discrimination and thus lower N demand relative to uptake. The others had a higher demand relative to uptake and thus discriminated less. 3.4.5 Plant tissue component 8 1 5 N In every case, the P. trichocarpa ramet discriminated against 1 5 N , resulting in lower whole plant 8 1 5 N relative to the supplied N H / salt. The roots and stems were consistently more depleted than the leaves by an average 2%o difference. No distinct 1 5 N enrichment of leaves has previously been reported for any plant species grown on N H 4 + . A root-to-shoot 47 difference in 8 1 5 N would not be expected i f all plant N H 4 + assimilation took place in the roots. (Buschhaus et al. in prep; Min et al. 1999; Wang et al. 1993). Previous research on aspen (Populus tremuloides) found that foliar 1 5 N enrichment occurred when NO3" was used as the nitrogen source but not when N H 4 + was used (Buschhaus et al. in prep). When the source nitrogen was radiolabeled ( 1 3N), it was shown that NO3" was assimilated in both roots and shoots, while NFL* assimilation was restricted to the roots (Min et al. 1999). These data were obtained at rates of N supply that resulted in low tissue N concentrations. Taken together, these previous observations suggest that the foliar enrichment seen in the present study resulted from direct translocation of N H 4 + to the leaves because the rate of N supply was sufficiently high to result in high tissue N . Although N H 4 + is primarily root-assimilated, where root N is replete or in excess, some N H 4 + moves directly to the shoot prior to assimilation (Schjoerring et al. 2002). This movement of leftover unassimilated root N H 4 + would result in foliar enrichment. As GS discriminates against 1 5 N , preferentially assimilating 1 4 N H 4 + , the remaining pool of N H 4 + in the root cytoplasm becomes enriched. Efflux of unassimilated N H 4 + is from this enriched pool, but it is also the source of N for direct transport to the shoot. If all N H 4 + assimilation occurred in the roots, the root and shoot tissue 8 1 5 N values should be comparable. If, on the other hand, some of the enriched N H 4 + is transported to the leaves for assimilation, the leaves will become proportionately more enriched. Alternative explanations for the foliar N enrichment involve the potential loss of isotopically enriched 1 5 N from the roots or depleted 1 4 N from the shoots. Robinson (2001) predicted some efflux of organic N in a nitrate-based model, while Merbach et al. (1999) estimated the rate of organic N efflux to be no more than 5% of the rate of inorganic N influx. The large difference between the roots and leaves would require that the effluxed organic N be exceedingly enriched given that it represents such a small fraction of plant assimilated N . This seems unlikely. The loss of depleted foliar N through the escape of ammonia in a plant-atmosphere exchange may also be a factor accounting for the root to leaf 8 1 5 N differences. However, these losses would have to be significantly larger than they are currently understood to be (Farquhar et al. 1982). Furthermore, in species where NO3" assimilation appears restricted to the roots, there was no root to leaf 8 1 5 N difference even when grown at relatively high concentrations (1.5 48 mM NO3") (Kronzucker et al. 1995; Min et al. 1999; Buschhaus et al. in prep). Contrastingly, species such as aspen, which assimilate NO3" in the leaves, had a distinct root to leaf 8 1 5 N separation. Since the turnover of N H / associated with photorespiration occurs in the leaves and root to leaf 8 1 5 N differences in NO3" grown aspen imply mineral N translocation, the observed tissue 8 1 5 N in this study may be interpreted as coming from N H / translocation. 3.4.6 Comparative analysis An overall correlation between whole plant % N and 8 1 5 N was not observed across or within most treatments. However, in the leaves of the low N , high CO2 treatment there was a positive relationship with increasing % N and 8 1 5 N. No other treatments demonstrated a similar pattern. This was also the only treatment to have low foliar % N . The root 8 1 5 N of this treatment remained consistent with the other treatments leading to a root-to-shoot difference that diminished with decreasing leaf % N (Fig 3.7). I would expect this i f the demand were high relative to supply causing leaf N to be increasingly root-assimilated and thereby decreasing the shoot-root difference. Multiple linear regression analysis revealed a composite effect of relative growth rate, provenance latitude, and % N on root to leaf 8 1 5 N. The effects of these three interacting factors were all as expected. Downward trends in relative growth rate increased the root-to-shoot 8 1 5 N difference, further suggesting that the supply/demand regime partially controls the assimilation locations for N H / . The increasing foliar % N was matched with increasing root-to-shoot 8 1 5 N differences implying changing sites of N H / assimilation. 49 Figure 3.8 Modeled diagram of N fluxes during the uptake of NFL; + from the source pool in the growth media and assimilation into the organic sink pools in both root and leaf tissues. The cytoplasmic intermediate pool is considered to be in a steady state with respect to both total mass and isotopic composition. Arrows refer to the total unidirectional fluxes of both isotopes with labeling according to the specific flux: E = efflux, I = influx, GS = glutamine synthetase, T 0 = organic N translocated to the shoot, Tj = inorganic N translocated to the shoot, and T t = total N translocated to the shoot. 3.4.7 NHV" transport and assimilation model Accepting that root-leaf differences in 8 1 5 N are mediated by xylem transport of NHV", I examined the data using a modified version of Comstock's (2001) NO3" transport and assimilation isotopic composition model (Fig. 3.8). Unlike Comstock, I assume no re-translocation of shoot-assimilated N to the roots. This simplifying assumption is quite reasonable for N H 4 + , especially i f leaf assimilation is limited as appears to be the case. Also, the close similarity between stem and root 8 I 5 N values suggest little or no export of leaf-assimilated N . The proportion of total plant N found in the leaves (Niea^Ntotai) is a function of plant tissue N concentration and tissue mass: [2] Nieaf/N t o t ai= ([N]ieaf x massif)/ (([N]i e af x massif) x ([N]root+stem x mass r o o t + s t em) 50 Like Comstock, I assume that all N H / translocated to the shoot (flux Tj) enters the xylem in isotopic equilibrium with the root cytoplasmic pool. It is also assumed that root-assimilated N H / is depleted in 1 5 N by 16.5%o (i.e. DGS) relative to this same cytoplasmic pool. Additionally, the organic N delivered to the shoot (flux T 0) does not differ isotopically from root-assimilated organic N . I can then calculate the proportion of total leaf N translocated to the leaves as inorganic N H / (Tj/T t) from the leaf-(root+stem) difference in isotopic composition: [3] T i /Tt = 8 1 5 N,eaf - 8 1 5 N r o o t *tem/ Ass-The proportion of total plant N that was assimilated in the leaves is simply the product of Nieaf/Ntotai x Tj /T t . The remaining fraction of plant N must then be assimilated in the roots (J): [4] /=l-(N,eaf/NtotalX Tj/Tt) Because Tj removes N from the cytoplasmic pool that then becomes unavailable to root assimilation or efflux, the expression of DGS at the whole-plant level is proportional to both E/I and/: [5] ( 5 1 5 N s o u r c e - 5 , 5 N p l a n t ) = E/I (Z)GS*/) where ( 8 1 5 N source- 8 1 5 N p i a nt ) is D in equation [1] and D G s * / accounts for the D associated with GS root assimilation. By rearranging equation [5], I can obtain E/I from: [6] E/I= ( 8 1 5 N s o u r c e - 815Npiant)/(£>Gs7) Knowledge of/also allows calculation of 8 1 5 N c y t o p iasm from a rearrangement of equation 23 in Comstock (2001): [7] 8 1 5 N c y , o p l a s r n =/* J DGs + 5 1 5 N p l a n t where 8 1 5 N c y t o p iasm is analogous to Comstock's 8 1 5 N r o o t N 0 3 - -Table 3.2 presents the model results based on treatment data summarized in table 3.3. The estimated portion of leaf N transported to the leaves as inorganic N H / (Tj) ranged from 10.3% to 12.5%. This range is consistent with direct xylem analysis reported by Schjoerring et al. (2002). They found that N H / constituted up to 11% of the total translocated N in the xylem at their highest hydroponic media concentration, 10 m M (in sand/hydroponics), in several plant species. M y media N concentration was much lower; but as previously noted, the actual rate of supply was more than sufficient as demonstrated by the high leaf % N because the growth media was maintained at a steady concentration in constant circulation. The significant link between relatively high N availability, direct translocation, and thus 51 enriched leaf material may explain why Buschhaus et al. (in prep) did not find foliar enrichment in N H 4 + grown aspen under sand culture. Their media concentrations were higher, but the leaf N content was presumably lower because of root zone nutrient depletion. Table 3-2 Model input data obtained from the P. trichocarpa steady-state growth experiment with the specific treatment conditions as noted. The treatment averages are inclusive of all three tested provenances. 2 0 0 uM N H 4 + Ambient CO2 2 0 0 uM N H 4 + Elevated C 0 2 4 0 0 n M N H 4 + Ambient CO2 4 0 0 uM N H 4 + Elevated C 0 2 D G s 1 6 . 5 % o 1 6 . 5 % o 1 6 . 5 % o 1 6 . 5 % o x 1 5xr ° ^ source - 0 . 9 6 % o - 0 . 9 6 % o - 0 . 9 6 % o - 0 . 9 6 % o [ N ] leaves 0 . 0 3 3 8 0 . 0 2 4 8 0 . 0 4 3 0 0 . 0 3 5 7 [Njroot+stem 0 . 0 4 0 8 0 . 0 3 9 6 0 . 0 3 6 2 0 . 0 3 4 0 5 1 5 N , e a f - 2 . 7 2 % 0 - 2 . 3 2 % 0 - 2 . 6 8 % o - 2 . 6 0 % o § N(r0ot+stem) - 4 . 7 8 % o - 4 . 1 3 % o - 4 . 3 8 % o - 4 . 3 0 % o 8 1 5 N p iant - 3 . 8 8 % o - 3 . 2 5 % o - 3 . 3 1 % o - 3 . 2 5 % o Root mass 0 . 4 4 4 g 1 . 2 2 8 g 0 . 5 5 6 g 0 . 9 4 5 g Stem mass 1 . 2 4 g 2 . 1 0 g 1 . 0 9 g 1 . 8 6 g Table 3-3 Model outputs for the P. trichocarpa treatment data given in Table 3.2. Tj/Tt equals the proportion of foliar N translocated to the leaves as inorganic NH4; / equals the fraction of total plant N assimilated in the roots; E/I equals the ratio of efflux to influx in the root-growth medium continuum; 8 1 5N c ytopiasm equals the isotopic composition of the N in the root cytoplasm. 200 u M N H 4 + 200 u M N H 4 + 400 u M N H 4 + 400 u M N H 4 + Ambient C Q 2 Elevated C Q 2 Ambient C Q 2 Elevated C Q 2 Tj/Tt 0.125 ±0.02 0.109 ±0.02 0.103 ±0.02 0.103 ±0.02 F 0.95 ±0.01 0.96 ±0.01 0.94 ±0.01 0.94 ±0.01 E/I 0.19 ±0.01 0.15 ±0.01 0.16 ±0.01 0.15 ±0.01 6 1 5 N c y t 0 pi a sm 12.7 ±0.20 12.4 ±0.20 12.1 ±0.20 12.2 ±0.20 The calculated values for / E/I, and 8 1 5 N cytoplasm were consistent with our expectations (Table 3.3). Across treatments, approximately 94% of total plant N was assimilated in the roots. The root to leaf difference in tissue 8 1 5 N values can be accounted for 52 by the remaining mere 6% of assimilation occurring in the leaves. The relatively limited source to plant 8 1 5 N differences I observed are consistent with the low E/I ratios this model predicts. The relatively stable 5 1 5 N values between treatments corresponded to the calculated E/I ratios which did not vary significantly between treatments. The enriched cytoplasm 8 1 5 N estimates were in coherence with the limited data available on cytoplasm 8 1 5 N values (Needoba et al. 2004). 3.4.8 Conclusion The uptake and assimilation capacity of P. trichocarpa worked within the efflux/influx model to determine plant nitrogen discrimination. Growth medium N H / and atmospheric C O 2 concentrations interacted to change the efflux/influx balance. The discrimination of the P. trichocarpa provenances could be manipulated by changing the supply/demand regimes. Plant tissue 8 1 5 N revealed a distinct foliar enrichment unprecedented in isotopic literature. From this data, I developed a model which accounted for the translocation and subsequent assimilation of N H 4 + in the leaves. The data and insights of this work enhance the overall understanding of NH4 +transport and assimilation. 53 3.5 References BassiriRad, H. , Constable, J.V.H., Lussenhop, J., Kimball, B.A. , Norby, R.J., Oechel, W.C., Reich, P.B., Schlesinger, W.H., Zitzer, S., Sehtiya, H.L. and Silim, S. 2003. Widespread foliage delta N-15 depletion under elevated CO2: Inferences for the nitrogen cycle. Global Change Biol 9: 1582-1590. Bergersen, F.J., Peoples, M . B . and Turner, G.L. 1988. Isotopic discriminations during the accumulation of nitrogen by soybeans. Aust J Plant Physiol 15: 407-420. Billings, S.A., Schaeffer, S.M., Zitzer, S., Charlet, T., Smith, S.D. and Evans, R.D. 2002. Alterations of nitrogen dynamics under elevated carbon dioxide in an intact mojave desert ecosystem: Evidence from nitrogen-15 natural abundance. Oecologia 131: 463-467. Buschhaus, H.A.E. , and Guy, R.D. in prep. Drawdown method allows measuring D and I/E for P. trichocarpa. Trees Buschhaus, H.A.E. , Min , X . and Guy, R.D. in prep. Within-plant variation of nitrogen isotope composition and its relationship to the site of nitrogen assimilation in trees. Trees Comstock, J.P. 2001. Steady-state isotopic fractionation in branched pathways using plant uptake of N 0 3 - as an example. Planta. 214: 220-234. DesRochers, A. , van den Driessche, R and Thomas, B. 2006. N P K fertilization at planting of three hybrid poplar clones in the boreal region of Alberta. Forest Ecology and Mangement 232: 216-225. Evans, R.D. 2001. Physiological mechanisms influencing plant nitrogen isotope composition. Trends Plant Sci 6: 121-126. Evans, R.D., Bloom, A.J . , Sukrapanna, S.S. and Ehleringer, J.R. 1996. Nitrogen isotope composition of tomato (Lycopersicon esculentum mill. cv. T-5) grown under ammonium or nitrate nutrition. Plant Cell Environ 19: 1317-1323. Farquhar, G.D., Oleary, M . H . and Berry, J.A. 1982. On the relationship between carbon isotope discrimination and the inter-cellular carbon-dioxide concentration in leaves. Aust. J. Plant Physiol. 9: 121-137. Gornall, J., and Guy, R.D. (2002) Geographic variation in ecophysiological traits of black cottonwood. (Populus trichocarpa) from western North America. Plant Physiology Canada. Calgary, A B . Handley, L .L . , and Raven, J.A. 1992. The use of natural abundance of nitrogen isotopes in plant physiology and ecology. Plant Cell Environ 15: 965-985. 54 Handley, L .L . , Robinson, D., Forster, B.P., Ellis, R.P., Scrimgeour, C M . , Gordon, D . C , Nevo, E. and Raven, J.A. 1997. Shoot delta N-15 correlates with genotype and salt stress in barley. Planta. 201: 100-102. Hoch, M.P., Fogel, M . L . and Kirchman, D.L. 1992. Isotope fractionation associated with ammonium uptake by a marine bacterium. Limnol Oceanogr 37: 1447-1459. Hogberg, P., Hogberg, M . N . , Quist, M.E. , Ekblad, A . and Nasholm, T. 1999. Nitrogen isotope fractionation during nitrogen uptake by ectomycorrhizal and non-mycorrhizal Pinus sylvestris. New Phytol 142: 569-576. Hymus, G.J., Baker, N.R. and Long, S.P. 2001. Growth in elevated CO2 can both increase and decrease photochemistry and photoinhibition of photosynthesis in a predictable manner. Dactylis glomerata grown in two levels of nitrogen nutrition. Plant Physiol 127: 1204-1211. Kolb, K.J . , and Evans, R.D. 2003. Influence of nitrogen source and concentration on nitrogen isotopic discrimination in two barley genotypes (Hordeum vulgare L.). Plant Cell Environ 26: 1431-1440. Kronzucker, H.J., Siddiqi, M . Y . and Glass, A . D . M . 1995. Compartmentation and flux ' characteristics of ammonium in spruce. Planta 196(4): 691-698. Lam, H .M. , Coschigano, K.T., Oliveira, I .C, MeloOliveira, R. and Coruzzi, G .M. 1996. The molecular-genetics of nitrogen assimilation into amino acids in higher plants. Annu. Rev. Plant Physiol. Plant Mol Biol 47: 569-593. Mariotti, A. , Mariotti, F., Champigny, M.L . , Amarger, N . and Moyse, A . 1982. Nitrogen isotope fractionation associated with nitrate reductase-activity and uptake of N03- by pearl-millet. Plant Physiol 69: 880-884. Merbach, W., Minis, E., Knof, G., Remus, R., Ruppel, S., Russow, R., Gransee, A. and Schulze, J. 1999. Release of carbon and nitrogen compounds by plant roots and their possible ecological importance. J Plant Nutr Soil Sci 162: 373-383. Min, X . , Siddiqi, M . Y . , Guy, R.D., Glass, A . D . M . and Kronzucker, H.J. 1998. Induction of nitrate uptake and nitrate reductase activity in trembling aspen and lodegpole pine. Plant Cell Environ 21: 1039-1046. Min, X . , Siddiqi, M . Y . , Guy, R.D., Glass, A . D . M . and Kronzucker, H.J. 1999. A comparative study of fluxes and compartmentation of nitrate and ammonium in early-successional tree species. Plant Cell Environ 22: 821-830. Min, X . , Siddiqi, M . Y . , Guy, R.D., Glass, A . D . M . and Kronzucker, H.J. 2000. A comparative kinetic analysis of nitrate and ammonium influx in two early-successional tree species of temperate and boreal forest ecosystems. Plant Cell Environ 23: 321-238. 55 Needoba, J.A., Sigman, D .M. , Harrison, P.J. 2004. The mechanism of isotope fractionation during algal nitrate assimilation as illuminated by the N-15. Journal of Phycology 40: 517-522. Pritchard, E.S., and Guy, R.D. 2005. Nitrogen isotope discrimination in white spruce fed with low concentrations of ammonium and nitrate. Trees. 19: 89-98. Robinson, D. 2001. Delta N-15 as an integrator of the nitrogen cycle. Trends in Ecology & Evolution 16: 153-162. Shearer, G. and Kohl, D.H. 1989. Natural 1 5 N abundance of N H 4 + , amide N , and total N in various fractions of nodules of peas, soybeans and lupins. Aust J Plant Physiol 16: 305-313 Schjoerring, J.K., Husted, S., Mack, G. and Mattsson, M . 2002. The regulation of ammonium translocation in plants. J Exp Bot. 53: 883-890. Solorzano, L. 1969. Determination of ammonia in natural waters by the phenol-hypochlorite method. Limnol Oceanogr 14: 799-&. Stitt, M . , and Krapp, A . 1999. The interaction between elevated carbon dioxide and nitrogen nutrition: The physiological and molecular background. Plant Cell Environ 22: 583-621. Stock, W.D., and Evans, J.R. 2006. Effects of water availability, nitrogen supply and atmospheric C O 2 concentrations on plant nitrogen natural abundance values. Functional Plant Biol 33: 219-227. Tupker, K . A . , Thomas, B.R. and Macdonald, S.E. 2003. Propagation of trembling aspen and hybrid poplar for agroforestry: Potential benefits of elevated C O 2 in the greenhouse. Agrofor Syst 59: 61-71. Wang, M . Y . , Siddiqi, M . Y . , Ruth, T.J. and Glass, D . M . 1993. Ammonium uptake by rice roots. Plant Physiol 102:1249-1258. Waser, N.A. , Yin , K. , Nielsen, B. , Harrison, P.T. Turpin, D.H., Calvert, S.E. 1999. Nitrogen isotopic fractionation during a simulated diatom spring bloom: importance of N -starvation in controlling fractionation. Mar Ecol Prog Ser 179: 291-296. Yoneyama, T., and Kaneko, A . 1989. Variations in the natural abundance of N-15 in nitrogenous fractions of komatsuna plants supplied with nitrate. Plant Cell Physiol 30: 957-962. Yoneyama, T., Kamachi, K. , Yamaya, T. and Mae, T. 1993. Fractionation of nitrogen isotopes by glutamine-synthetase isolated from spinach leaves. Plant Cell Physiol 34: 489-491. 56 Yoneyama, T., Matsumaru, T., Usui, K. and Engelaar, W.M.H.G. 2001. Discrimination of nitrogen isotopes during absorption of ammonium and nitrate at different nitrogen concentrations by rice (Oryza sativa L.) plants. Plant Cell Environ 24: 133-139. Yoneyama, T., Omata, T., Nakata, S. and Yazaki, J. 1991. Fractionation of nitrogen isotopes during the uptake and assimilation of ammonia by plants. Plant Cell Physiol 32: 1211-1217. 57 4. Conclusion Nutrient supply and demand command a central role in determining isotopic discrimination in Populus trichocarpa. The substrate depletion experiments of Chapter 2 clearly demonstrated that the manipulation of N supply achieved through root pruning controlled the efflux/influx ratio and, in turn, plant N discrimination. The decrease in supply corresponded to a decrease in discrimination. Shoot pruning and genetic variation did not appear to play a role in plant N discrimination when assessed via the substrate depletion method. The substrate depletion experiments also allowed us to calculate for the first time root N influx and efflux from 8 1 5 N values and the net uptake rate. This novel application of the efflux/influx model for discrimination returned efflux and influx values consistent with literature values obtained using radiolabeled 1 3 N . The ability to accurately calculate efflux and influx using stable isotope discrimination methods at natural abundance levels provides a new, non-radioactive approach for further nutrient-uptake efficiency studies. In the steady-state experiments of Chapter 3, the uptake and assimilation capacity of P. trichocarpa worked within the efflux/influx model to determine plant nitrogen discrimination. Within the growth medium N H / and atmospheric C O 2 concentration treatments, discrimination at the whole plant level corresponded to the relative growth rate responses, signifying the dependence of Dpiatu on plant physiological factors. Genotypic differences in the discrimination values of P. trichocarpa provenances could be manipulated by changing the supply/demand regimes. Plant tissue 8 1 5 N revealed a distinct foliar enrichment unprecedented in isotopic literature. From this I developed a revised model that accounts for translocation and subsequent assimilation of N H / in the leaves. This newest model now provides testable hypotheses for future N translocation and assimilation studies. The data and insights of this work enhance the overall understanding of N H / transport and assimilation. 4.1 Recommendations for future research In completing the experiments for the current research, many further experiments present themselves. These range from continuing to refine the substrate depletion method to teasing apart the physiological controls of efflux/influx and discrimination. Building on the 58 current study, the potential results from the proposed experiments in the following paragraphs hold the promise of verifying the theoretical models of this thesis and pushing forward our knowledge of plant N physiology. Methodological refinements are still required to optimize the use of isotopic discrimination as a physiological tool. The substrate depletion experiment relies on the membrane diffusion method of Holmes et al. (1998) with subsequent modifications by Pritchard and Guy (2005) and Nevena Ratkovich (UBC, personal communication). As noted in Chapter 1, I experienced significant contamination and leakage from sources that I was unable to completely resolve. This reduced the size of the data set and thus reduced the power to resolve some of the physiological questions. To maximize future data sets, the method must be completely dependable. Preliminary trouble-shooting points to pillow failure in the permeability of the Teflon itself. One way to avoid this is through suspending the filter away from the sample solution. Suspending the pillow above the diffusion solution has been attempted in the past for significantly larger volumes of sample and it may be necessary to refine this method for the smaller sample size I used. Alternately, direct analyses of liquid or gaseous samples would bypass the problematic pillow step and decrease sample preparation time by nearly two weeks. These possible improvements must also be pursued. On a physiological level, further experiments are needed to clarify the assumptions implicit in the findings of this work. First, the discrimination factor for glutamine synthetase (GS) should be verified as it has been defined only rarely and this with limited confidence (Pritchard and Guy 2005). Literature discrimination values vary widely depending on enzyme activity and experimental design for in vivo experiments (Robinson 2001). Only the data published by Yoneyama et al. (1993) provides GS discrimination values in vitro to date. Since the calculations of efflux (E) and influx (I) require the enzymatic discrimination constant for glutamine synthetase (DGS) according to the equation: [1] Dpiant - E/I * DGS where Dpia„, is the plant discrimination, a verified value for DGS would increase the accuracy and confidence of efflux and influx values. Second, in studying the mineral N H 4 + fluxes in the root-growth medium continuum, organic root N efflux should be assessed as a potential sink for plant assimilated N . To 59 quantify the relative proportion of organic N efflux and its isotopic signature, experiments could be conducted similarly to compartmental efflux analysis methods but with 1 5 N instead of radiolabeled 1 3 N (Kronzucker et al. 1995; Min et al. 1999). Knowing the 8 1 5N of the organic root efflux is especially important for the substrate depletion experiments where the 5 1 5N of the remaining solution is used to calculate plant discrimination. The experiments in this study began under the assumption, based on earlier reports, that N H / was solely assimilated in the roots (Min et al. 1999; Wang et al. 1993). However, the foliar 5 1 5N enrichment data presented in Chapter 3 provided evidence for N H / translocation to and assimilation in the shoot. This raises many questions that can now be answered from the framework provided by the N H / translocation and assimilation model: (1) Quantitatively, how much N H / is translocated from the roots to the leaves? Is the translocated N H / destined for all leaves, certain leaf development stages, or specific locations within the leaf? What is the isotopic signature of the translocated inorganic NH/? (2) What is the effect of changing the supply/demand regime on N H / translocation? (3) What is the metabolic destiny of the translocated NH/? (4) Is there discrimination associated with the multiple branch-points in N H / metabolism and can this discrimination be quantitatively determined? (5) What is the discrimination factor associated with the foliar NH3 compensation point for N movement in either direction? The foundation laid by the current thesis opens up further avenues for research and provides a platform from which to push the boundaries of plant nitrogen physiology research. 60 4.2 References Holmes, R.M., McClelland, J.W., Sigman, D.M., Fry, B. and Peterson, B.J. 1998. Measuring 15N-NH4+ in marine, estuarine and fresh waters: an adaptation of the ammonia diffusion method for samples with low ammonium concentrations. Mar Chem 60: 235-243. Kronzucker, H.J., Siddiqi, M.Y. and Glass, A.D.M. 1995. Compartmentation and flux characteristics of ammonium in spruce. Planta 196: 691-698. Min, X., Siddiqi, M.Y., Guy, R.D., Glass, A.D.M. and Kronzucker, H.J. 1999. A comparative study of fluxes and compartmentation of nitrate and ammonium in early-successional tree species. Plant Cell Environ 22: 821-830. Pritchard, E.S., and Guy, R.D. 2005. Nitrogen isotope discrimination in white spruce fed with low concentrations of ammonium and nitrate. Trees 19: 89-98. Robinson, D. 2001. Delta N-15 as an integrator of the nitrogen cycle. Trends in Ecology & Evolution 16: 153-162. Wang, M.Y., Siddiqi, M.Y., Ruth, T.J. and Glass, D.M. 1993. Ammonium uptake by rice roots. Plant Physiol 102:1249-1258. Yoneyama, T., Kamachi, K., Yamaya, T. and Mae, T. 1993. Fractionation of nitrogen isotopes by glutamine-synthetase isolated from spinach leaves. Plant Cell Physiol 34: 489-491. 61 

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